BLOOD DISEASES

1. Blood Cells Production

This is the amount of red cells. Neutrophils, a type of WBCs and platelets that are produced every day. The
production of cells must be very active and coordinated and all the cells are produced in the bone marrow.

Stem cells must be able to renew themselves otherwise the cell pool will be soon exhausted and must be
able also to differentiate into cells that eventually will lead to production of mature RBCs.

Hematopoiesis: (αἷμα, "blood" and ποιεῖν "to make”) is the formation of blood cellular components. All
cellular blood components are derived from hematopoietic stem cells. In a healthy adult person,
approximately 1011- 1012 new blood cells are produced daily in order to maintain steady state levels in the
peripheral circulation. Hematopoietic stem cells (HSCs) reside in the bone marrow and have the unique
ability to give rise to all of the different mature blood cell types and tissues.

HSCs are self-renewing cells: when they proliferate, at least some of their daughter cells remain as HSCs, so
the pool of stem cells does not become depleted. This phenomenon is called asymmetric division. The other
daughters of HSCs (myeloid and lymphoid progenitor cells), can commit to any of the alternative
differentiation pathways that lead to the production of one or more specific types of blood cells, but cannot
self-renew.

All blood cells are divided into three lineages:

1. Erythroid cells: are the oxygen carrying red blood cells. Both reticulocytes and erythrocytes are functional
and are released into the blood. In fact, a reticulocyte count estimates the rate of erythropoiesis.
Reticulocytes are immature red blood cells, typically composing about 1% of the red cells in the human
body. They develop and mature in the bone marrow and then circulate for about a day in the blood
stream before developing into mature red blood cells. Reticulocytes do not have a cell nucleus. They are
called reticulocytes because of a reticular (mesh-like) network of ribosomal RNA that becomes visible
under a microscope with certain stains such as new methylene blue. If you count reticulocytes, they
could be approximately 2 (the number of reticulocytes) in 120 (the total number of RBCs) x100 giving the
result which is between 1-2%. When there is an increased production of red blood cells to overcome
chronic or severe loss of mature red blood cells, such as in a haemolytic anemia, people often have a
markedly high number and percentage of reticulocytes. A very high number of reticulocytes in the blood
can be described as reticulocytosis. Abnormally low numbers of reticulocytes can be attributed to
chemotherapy, aplastic anemia, pernicious anemia, bone marrow malignancies, problems of
erythropoietin production, various vitamin or mineral deficiencies (B9, B12, iron), disease states (anemia
of chronic disease) and other causes of anemia due to poor RBC production.

2. Lymphocytes: are the cornerstone of the adaptive immune system. They are derived from common
lymphoid progenitors. The lymphoid lineage is primarily composed of T-cells and B-cells (types of white
blood cells). This is lymphopoiesis. Pathologies in lymphopoiesis lead to any of various
lymphoproliferative disorders, such as lymphomas and lymphoid leukemias.

3. Myelocytes: include granulocytes, megakaryocytes and macrophages. They are derived from common
myeloid progenitors, are involved in such diverse roles as innate immunity, adaptive immunity, and blood
clotting. This is myelopoiesis. The myeloid progenitor can differentiate in the bone marrow into
granulocytes, macrophages (mature monocytes), mast cells (whose blood-borne progenitor is not well
defined), and dendritic cells of the innate immune system. The granulocytes, give rise to three short lived
cell types including eosinophils, basophils, and neutrophils. A granulocyte differentiates into a distinct cell
type by a process called granulopoiesis. In this process it first transforms from a common myeloblast
(myeloid progenitor) to a common promyelocyte. This promyelocyte gives rise to a unique myelocyte that
for the first time can be classified as an eosinophil, basophil, or neutrophil progenitor based on the
histological staining affinity (eosinophilic, basophilic, or neutral granules). The unique myelocyte next
differentiates into a metamyelocyte and then a band cell, with a "C" shaped nucleus, before becoming a
mature eosinophil, basophil, or neutrophil. Macrophages come from monoblast progenitors that
differentiate into promonocytes, which mature into monocytes. Monocytes eventually enter the tissues
and become macrophages.

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Megakaryocytopoiesis is the haematopoiesis of megakaryocytes. Megakaryocytes in turn produce
platelets.

At any division, the hemopoietic stem cell HSC can produce 2 stem cells or 1 stem cell and 1 differentiated
cell or 2 differentiated cells. The system becomes imbalanced when the differentiation doesn’t happen: the
patient will develop a disease due for example to the exhaustion of stem cells. This happens in acute
leukemia, where the mutation prevents the production of a number of cells, and leads to accumulation of
immature cells that are called blasts.

Exhaustion of stem cells leads also to a specific type of anemia: Aplastic anemia, in which the production of
RBCs and WBCs is affected. The most dramatic example of destruction of stem cells that led to Aplastic
anemia was in 1945 due to the atomic bombs of Hiroshima and Nagasaki. The subjects surviving the
explosion died in the next weeks or months due to the exposure to ionizing radiations that completely
destroyed the HSCs. The subjects that didn’t die of aplastic anemia, died of Leukemia or other cancers due
to the mutation effect of ionizing radiations.

The main feature of HSCs is the expression of an antigen called CD34. Autologous hematopoietic stem cells
transplantation is based on the use of stem cells of the same patient while Allogeneic hematopoietic stem
cells transplantation is used based on the use of stem cells of a donor.

Autologous HSCT: is done mainly in patients with Multiple Myeloma (MM) in which there is the proliferation of
malignant plasma cells in the bone marrow. In these patients there is the need to destroy all the abnormal
plasma cells and so a chemotherapy or additional therapy is performed, but in order to be sure to eliminate
completely the abnormal plasma cells, a very high dose of chemotherapy should be used. This will destroy
also the normal stem cells. The Multiple Myeloma would be treated, but there would be Aplastic Anemia. We
need to mobilize the stem cells of the patient from the bone marrow to peripheral blood where we will collect
the CD34 cells (which are stem cells) and we will try to conserve them for a while. At this point we can give a
very high dose of chemotherapy or radiation to destroy completely the bone marrow including the abnormal
plasma cells. Subsequently we will use the CD34 HSCs for repopulating the bone marrow.

Allogeneic HSCT: can be performed in two cases. The first one is β Thalassemia Major, the second one is
Acute Myeloid Leukemia. If a young boy with Thalassemia major has a compatible sibling, a brother or a
sister, we can again treat the patient with an approach that attacks his/her bone marrow and we can then
use the stem cells of the donor to repopulate the bone marrow of the patient. The same happens in a patient
with acute myeloid leukemia. What is crucial for the transplantation of stem cells from one person to another
is the HLA system: allogeneic HSCT can be don only between HLA compatible subjects. The donor can be a
sibling, a family relative or someone compatible with the recipient.

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Red and white blood cell production is regulated with great precision in healthy humans, and the production
of leukocytes is rapidly increased during infection. The proliferation and self-renewal of these cells depend
on Growth factors.
One of the key players in self-renewal and development of haematopoietic cells is stem cell factor (SCF).
Absence of this factor is lethal. There are also other important glycoprotein growth factors, which can
regulate the proliferation and maturation, such as IL-2, IL-3, IL-6, IL-7.
Other factors, termed colony-stimulating factors (CSFs), specifically stimulate the production of committed
cells. These stimulate granulocyte formation and are active on either progenitor cells or end product cells.
Three CSFs are:
- granulocyte-macrophage CSF (GM-CSF),
- granulocyte CSF (G-CSF)
- macrophage CSF (M-CSF).
Erythropoietin is required for a myeloid progenitor cell to become an erythrocyte. On the other hand,
thrombopoietin makes myeloid progenitor cells differentiate to megakaryocytes (thrombocyte-forming cells).

2. Bone Marrow and Peripheral Blood

Performing bone marrow aspirate or biopsy, peripheral blood smear and then
sometimes in the lab by looking with the microscope, we can define the disease of
the patient.

Bone marrow aspiration is the aspiration of blood from inside the bone marrow. This
blood is then used for preparing the smear.

For bone marrow biopsy, we use the Jamshidi Needle which is a hollow needle with
a stylet to penetrate in the bone and the bone marrow. The stylet is then removed
and you proceed only with the hollow needle. This procedure is performed at the
posterior superior iliac spine where the bone is about 6-7 cm deep and there is not
the risk of causing any damage. The bone marrow is then examined.

A normal bone marrow has a cellularity of around 50%: about half of the space is
occupied by fat which can be seen as the empty spaces (fat is dissolved by excision
of bone marrow biopsy). In patients who have. Aplastic Anemia, almost all of the
bone marrow is occupied by fat without HSCs or small residual amount of HSCs. An
example of the importance of bone marrow biopsy is the diagnosis of hematologic
diseases.

Erythropoiesis

We have, as in any cell line, the HSC which starts to differentiate. The “colony
forming unit - granulocyte, erythrocyte, megakaryocyte and monocyte” (CFU-
GEMM) is an hematopoietic progenitor able to give rise to different cell types.

The “burst forming unit-erythroid” (BFU-E), cannot be identified at the microscope

but, using specific culture systems, we can identify these burst. It was thus seen
that hundreds of erythroid cells are derived from a single cell called BFU-E.

1. a multipotent hematopoietic stem cell, becomes

2. a common myeloid progenitor or a multipotent stem cell, and then
3. a unipotent stem cell, then
4. a pronormoblast (or pro-erythroblast), which has also nucleoli, this becomes
5. a basophilic or early erythroblast, which is actively synthesizing globins, then
6. a polychromatophilic or intermediate erythroblast, polychromatic due to the fact
that globins are combined with HEME in hemoglobin molecules, then
7. an orthochromatic or late erythroblast, which loses the nucleus, becoming
8. a reticulocyte, which has residual RNA

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The cell initially is having a dark blue cytoplasm and then it acquires different colors eventually terminating
with an orange cytoplasm. The cytoplasm which is initially dark blue because of the high amount of RNA (for
the basophilic activity) becomes polychromatophilic and this means that it can be stained with different or
more than one stain. Eventually the color of the cytoplasm becomes light orange (ortho- means “light color”)
because the erythroid cells contain Hb which, using the regular means of staining, stains red/orange.

Erythropoietin, also known as EPO, is a glycoprotein hormone that controls erythropoiesis. It is a cytokine
(protein signalling molecule) for erythrocyte (red blood cell) precursors in the bone marrow.
It is produced by interstitial fibroblasts in the kidney in close association with peritubular capillary and
proximal convoluted tubule. It is also produced in perisinusoidal cells in the liver. While liver production
predominates in the fetal and perinatal period, renal production is predominant during adulthood.
In addition to erythropoiesis, erythropoietin also has other known biological functions. For example, it plays
an important role in the brain's response to neuronal injury. EPO is also involved in the wound healing
process.
Erythroblasts proliferate and differentiate within specialised
microenvironmental compartments called Erythroblastic islands (Isolotto
Eritroblastico). These islands consist of a central macrophage that
extends cytoplasmic protrusions to a ring of surrounding erythroblasts.
The interaction of cells within the erythroblastic island is essential for
both early and late stages of erythroid maturation.
It has been proposed that early in erythroid maturation the
macrophages provide nutrients, proliferative and survival signals to the
erythroblasts, and phagocytose extruded erythroblast nuclei at the
conclusion of erythroid maturation. There is also accumulating evidence
for the role of macrophages in promoting enucleation itself. Both
macrophages and erythroblasts display adhesive interactions that
maintain island integrity. Such interactions enable regulatory feedback
within islands via cross talk between cells and also trigger intracellular signalling pathways that regulate
gene expression.
An additional control mechanism for cellular growth within the erythroblastic islands is through the
modulation of apoptosis via feedback loops between mature and immature erythroblasts and between
macrophages and immature erythroblasts.
Abnormalities in erythroblastic islands may cause Anemia.
When the orthochromatic erythroblast loses the nucleus in the bone marrow, the cell for a couple of days is
still presenting RNA residues in the cytoplasm and they can be stained. By using for example cytochrome
staining of the RNA residues, these cells can be stained and counted and finally the diagnosis for Anemia
can be made.

Granulopoiesis (or granulocytopoiesis) is hematopoiesis of granulocytes. It occurs primarily within bone

marrow and involves the following stages:
1. Pluripotential hemopoietic stem cell
2. Myeloblast
3. Promyelocyte (in Acute Myeloid Leukemia, the mature cells are mainly Myeloblasts and Promyelocytes)
4. Eosino/neutro/basophilic myelocyte
5. Metamyelocyte
6. Band cell (Stab cell)
7. Granulocytes (Eosino/neutro/basophil)

Acute promyelocytic leukemia is caused by a chromosomal translocation, involving chromosome 15 and

chromosome 17. The fusion of PML (15) and RARA (17) results in the expression of a hybrid protein with
altered functions. This fusion protein binds with enhanced affinity to sites on the cell's DNA, blocking
transcription and differentiation of granulocytes. This fusion gene blocks the differentiation of promyelocytes
so leads to their accumulation. These granules of the abnormal granulocytes activate coagulation and lead to
a condition called what coagulopathy. The final result will be hemorrhage because the activation of
coagulation leads to disseminated intravascular coagulation (DIC), consumption of coagulation factors,
consumption of platelets, and hemolysis.

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———Clinical case.—————————————————————————————————————— —
A young English patient who had a nasal hemorrhage, came to the hospital. He was found to have anemia,
leukopenia, thrombocytopenia and evidence of disseminated intravascular coagulation. In peripheral blood,
but most importantly in the bone marrow, most of the cells were abnormal promyelocytes. We now treat
these patients with agents that could reduce the differentiation and maturation of these abnormal
promyelocytes: All Trans Retinoic Acid ATRA, similar to Vitamin A, which is also reasonable from the
scientific point of view because of the retinoic acid receptors involved and they found that combination of
therapy with ATRA was able to rapidly reduce the incidence of relapse. Arsenic Trioxide was also able to
reduce the differentiation of the abnormal promyelocytes and eventually, in Italy we conduct a study in which
we use ATRA and Arsenic Trioxide without any chemotherapy, so just 2 compounds which are not
chemotherapy. This approach leads to complete remission in about more than 90% of cases.

In myeloproliferative diseases, including Chronic Myeloid Leukemia or Primary Myelofibrosis in which the
cells that can be found in peripheral blood are these cells close to mature cells (myeloblasts and
promyeloblasts).

The platelets are derived from Megakaryocytes. The

relationship between platelets and megakaryocytes was
discovered by Bizzozzero. Platelets are just fragments of the
cytoplasm of Megakaryocytes and the process is regulated
by many factors but in particular by Thrombopoietin (TPO).
Thrombopoietin is a protein that in humans is encoded by the
THPO gene. Thrombopoietin is a glycoprotein hormone
produced by the liver and kidney. It stimulates the production
and differentiation of megakaryocytes.

What can you see in Peripheral Blood? In peripheral blood

we can see RBCs and WBCs. WBCs can have different types
of Granules (neutrophils, eosinophils or basophils). The cells
without many granules are what we call Monocytes
(mononuclear, uni-lobar nuclei while the previous ones had
segmented nuclei). In order to do differential WBCs count,
you should count at least 100 WBCs in peripheral blood and
then from this you will get the proportion of these cells.

Granulocytes:

- Neutrophils: the granules don’t have any specific staining, they are not eosinophil or basophil, they are
intermediate and the number of the nucleus segments (lobes) is between 2 and 4. These cells are the
most abundant and prominent WBCs in peripheral blood. There will be different types of mature or less
mature neutrophils. The higher the number of segmentations, the greater the maturity of the neutrophil.
They defend against bacterial or fungal infection. They are usually first responders to microbial infection;
their activity and death in large numbers forms pus. These cells are not able to renew their lysosomes
(used in digesting microbes) and die after having phagocytosed a few pathogens. Neutrophils range
between 45-75% under normal conditions, during infection or inflammation the neutrophils specifically
increase. The life span of a circulating human neutrophil is about 5.4 days

- Eosinophils: normally have fewer segmentations and bigger granules, which appear orange with eosin
staining. In general, their nucleus is bi-lobed, and the lobes are connected by a thin strand. Eosinophils
range between 1-20%, and they increase in many disorders typically in allergic disorders. This count
fluctuates throughout the day, seasonally, and during menstruation. It rises in response to allergies,
parasitic infections, collagen diseases, and disease of the spleen and central nervous system. They are
rare in the blood, but numerous in the mucous membranes of the respiratory, digestive, and lower urinary
tracts. They primarily deal with parasitic infections. Eosinophils are also the predominant inflammatory
cells in allergic reactions. The most important causes of eosinophilia include allergies such as asthma, hay
fever, and hives; and also parasitic infections.

- Basophils: are rare, 1% or less in peripheral blood, big granules shown in the cytoplasm with the nucleus.
They can be recognized by several coarse, dark violet granules, giving them a blue hue. The nucleus is bi-
or tri- lobed, but it is hard to see because of the number of coarse granules that hide it. The final

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 destination of basophils is to leave peripheral blood, enter tissues and become Mast cells. Basophils are
chiefly responsible for allergic and antigen response. They excrete two chemicals that aid in the body's
defenses: histamine and heparin. Histamine is responsible for widening blood vessels and increasing the
flow of blood to injured tissue. It also makes blood vessels more permeable so neutrophils and clotting
proteins can get into connective tissue more easily. Heparin is an anticoagulant that inhibits blood clotting
and promotes the movement of white blood cells into an area. Basophils can also release chemical signals
that attract eosinophils and neutrophils to an infection site.

Mono-nucleated cells:

- Lymphocytes: may be activated during a number of infections, including viral infections. Lymphocytes are
much more common in the lymphatic system than in blood. They can be distinguished because they have
a deeply staining nucleus that may be eccentric in location, and a relatively small amount of cytoplasm.
Lymphocytes are between 20-40%.

- Monocytes: are the biggest WBCs in peripheral blood. They have different types of nuclei. Typically the
nucleus is folded, having a kidney-like aspect. The monocytes have small cytoplasmic granules.
Monocytes are between 3-7%. Monocytes share the "vacuum cleaner" (phagocytosis) function of
neutrophils, but are much longer lived as they have an extra role: they present pieces of pathogens to T
cells so that the pathogens may be recognized again and killed. Monocytes eventually leave the
bloodstream and become tissue macrophages, which remove dead cell debris as well as attacking
microorganisms. Unlike neutrophils, monocyte are able to replace their lysosomal contents and are
thought to have a much longer active life. Once monocyte move from the bloodstream out into the body
tissues, they undergo changes (differentiate) allowing phagocytosis and are then known as macrophages.

WHAT IS PRECISION MEDICINE

The Precision medicine approach is to use sequencing of genome for defining molecular base of a disease
and elaborate therapies and molecular drugs specifically targeting the founding genetic lesion.

Precision medicine is an emerging field of medicine that takes into account individual differences in people's
genes, microbiomes, environments, and lifestyles, allowing the development of more precisely targeted
treatments for diseases like cancer and diabetes.

This is incredibly important because right now, most medical treatments are designed for the average patient
however, treatments that are very successful for some patients don't work for others.

President Obama has now unveiled the Precision Medicine Initiative, a bold new enterprise to revolutionize
medicine and generate the scientific evidence needed to move the concept of precision medicine into every
day clinical practice.

We could face thousands of types of myeloid leukemia for example and the information can be used for
defining a precise treatment.

How can we use the information from the codification of a genome? For instance one simple use does not
directly concern intervention but just the follow-up. What can be done when these genes are found mutated
at diagnosis? In acute myeloid leukemia it is especially important that chemotherapy drugs to destroy all
leukemic cells. One potential use is thus to sequence or to look for these specific mutations during follow-up
(after treatment) in order to decide if the effects and the remission was radical. This can be done looking at
the presence or absence of these clones: if one of these clones survives to chemotherapy the risk of relapse
is increased. The patient should then immediately undergo allogenic transplantation in order to replace the
whole hematopoietic stem cells.

We now have specific inhibitors of mutated IDH1. One possible use of this information is that we combine
conventional chemotherapy with a drug which is a specific inhibitor of mutated IDH1. Knowing specific drugs
that will target these specific mutated genes responsible for the malignancy, we will able to personalize the
treatment of the patients. But these genes, which are mutated mainly in patients with a history of
myelodysplastic syndrome, make this acute myeloid leukemia very high risk by definition and we already
know that this patients should receive a high risk treatment and when a donor is available, this treatment is
allogenic stem cell transplantation in order to replace the whole bone marrow with donor bone marrow.

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Complete Blood cell Count CBC

• WBC (white blood cell) leukocyte count: 4,300 to 10,800 cmm. High WBC count: leukocytosis (infections,
inflammation, leukemia, tissue necrosis, allergic responses). Low WBC count: leukopenia (bone marrow damage,
bone marrow disorders such as myelodysplastic syndrome, vitamin B12 or folate deficiency, lymphoma, autoimmune
disorders, sepsis, diseases of the immune system.

• WBC (white blood cell) differential count:

- Neutrophils: 40% to 60% of the total. May indicate bacterial or acute vital infection

- Lymphocytes: 20% to 40%. Higher with some viral infections, or chronic lymphocytic leukemia (CLL). Can be
decreased by HIV infection.

- Monocytes: 2% to 8%. May be raised in bacterial infection, tuberculosis, malaria, monocytic leukemia.

- Eosinophils: 1% to 4%. Increased in parasitic infections, asthma, or allergic reaction.

- Basophils: 0.5% to 1%. May be increased in bone marrow related conditions (leukemia or lymphoma).

• RBC (red blood cell) erythrocyte count: 4.2 to 5.9 million cmm. Low RBC count (anemia): trauma, RBCs
destruction (hemolytic anemia, hemoglobinopathies as sickle cell anemia, thalassemia, hereditary spherocytosis,
enzyme defects as G6PD deficiency), acute or chronic bleeding from GI, nutritional deficiencies (iron, Vitamin B12,
folate deficiencies), bone marrow damage or disorders (leukemia, MM, myelodysplasia, lymphoma), chronic
inflammatory diseases, kidney failure (decrease in erythropoietin production). High RBC count (polycythemia):
polycythemia vera, dehydration, lung disease, congenital heart disease, kidney tumor, smoking, genetic causes.

• Hematocrit (Hct): 45% to 52% for men; 37% to 48% for women. Anemia: abnormally low hematocrit (if
also low MCV and high RDW: chronic iron-deficient anemia. Also leukemia). Polycythemia: abnormally high
hematocrit. Hematocrit is an important parameter only in Polycythemia Vera, in which there is an increase in RBCs and
WBCs production in the bone marrow. A type of therapy called Venesection of blood volume of 400 ml every week or
month and its target is to bring the hematocrit levels (RBC percentage) below 45.

• Hemoglobin (Hgb): 13 to 18 g/dL for men; 12 to 16 g/dL for women.

• Mean corpuscular volume (MCV): 80 to 100 femtoliters. Anemia types can be normocytic, macrocytic (up
to 150 femtolitres: all the Megaloblastic Anemias that are associated with vitamin B12 or folate-deficiency, pernicious
anemia) or microcytic (as low as 60 to 70 femtoliters: iron deficiency, thalassemia, sideroblastic anemia or chronic
disease.

• Mean corpuscular hemoglobin concentration (MCHC): 28% to 36%. MCHC is diminished

("hypochromic") in microcytic anemias, and normal ("normochromic") in macrocytic anemias (due to larger cell size,
though the hemoglobin amount or MCH is high, the concentration remains normal). MCHC is elevated
("hyperchromic") in hereditary spherocytosis, sickle cell disease and homozygous hemoglobin C disease.

• Mean corpuscular hemoglobin (MCH): 27 to 32 picograms.

• Red cell distribution width (RDW or RCDW): 11% to 15%. When anemia presents with normal RDW, think
about Thalassemia. When anemia presents with high RDW: Iron Deficiency Anemia (low MCV), folate and vitamin
B12 deficiency anemia (high MCV), mixed deficiency (Iron + B12 or folate) anemia, recent hemorrhage (normal MCV).

• Platelet count: 150,000 to 400,000 mL. Thrombocytopenia, low pc (< 150,000): cancer treatments such as
drugs or chemotherapy, as well as radiation, drugs, autoimmune disorders. Thrombocytosis, high pc (> 400,000):
anemia in which blood cells are destroyed earlier than normal, infections, surgery, trauma, cancer, medicines, CML,
polycythemia vera, primary thrombocythemia, recent spleen removal.

• Mean Platelet Volume (MPV): 7.5 to 11.5 femtoliters. High MPV: destruction of platelets (inflammatory
bowel disease, immune thrombocytopenic purpura (ITP), myeloproliferative diseases and Bernard-Soulier syndrome. It
may also be related to pre-eclampsia, and recovery from, transient hypoplasia). Low MPV (thrombocytopenia when it
is due to impaired production as in aplastic anemia).

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The Reticulocyte count is very important because an increase of Reticulocyte count in a patient with anemia,
by definition, means Hemolytic Anemia. The reverse condition in a patient with anemia but with a normal
reticulocyte count, excludes the diagnosis of Hemolytic Anemia.

Red cell transfusion is sometimes used for donation of RBCs in a patient with RBC lifespan less than 120
days. The average lifespan of peripheral blood red cells is 4 months (120 days) in the donor subject so if the
donor donates 400 ml of blood, the average lifespan of newly transfused blood can’t be 120 days so the
efficacy of the transfusion will not be 4 months but will be less than 2 months. Normally the efficacy of
transfusion is 1 month. Platelets’ lifespan is 10 days which means that the transfused platelets’ lifespan will
be 5 days so the efficacy of platelets transfusion is only a few days and most of the platelets die throughout
the procedure. RBCs, at the end of the 4 months, are taken up by macrophages which are found in the bone
marrow, spleen and liver. RBCs are just containers of Hb. After RBC destruction, globin is taken to the
amino acid pool, iron will be removed and part of it will be stored in ferritin within the macrophage and the
other part will be released to Transferrin protein in peripheral blood. Transferrin protein also carries iron from
macrophages to blood. Iron is released from this side of the macrophage back to transferrin through
Ferroportin protein. Protoporphyrin 9, in the macrophage is turned into Biliverdin and after than transformed
into bilirubin. Bilirubin will be eventually transported to the liver.

2. Anemias

Anemia or anaemia (ἀναιμία, from ἀν- + αἷμα) is usually defined as a decrease in the amount of red blood
cells (RBCs) or hemoglobin in the blood. It can also be defined as a lowered ability of the blood to carry
oxygen. There are three main types of anemia:

1. the one due to blood loss (for example trauma and gastrointestinal bleeding),

2. the one due to decreased red blood cell production (iron deficiency, vitamin B12 deficiency, thalassemia
and a number of neoplasms of the bone marrow),

3. the one due to increased red blood cell breakdown (genetic conditions such as sickle cell anemia,
infections like malaria and some autoimmune diseases).

Anemias can also be classified based on the size of red blood cells and amount of hemoglobin in each cell.
Diagnosis in men is based on a hemoglobin of less than 13 to 14 g/dL while in women it must be less than
12 to 13 g/dL.

The physiological response to anemia is overproduction of erythropoietin. HIF-1: hypoxia inducible factor 1,
transcription factor for erythropoietin. As Hb decreases and hypoxia develops, it is sensed at renal level, and
the specialized fibroblasts of the kidney under the effect of HIF start producing higher amounts of
Erythropoietin. There is thus higher expression of the cell type we saw initially: BFU-E.

JAK 2 is a very important protein that mediates transduction of erythropoietin signalling. CFU-E is one of the
progenitors with the highest amount of receptors for EPo. The action of EPo makes CFU-E differentiate in
erythroblasts, leading to the production of RBCs. In the absence of erythropoietin, there is apoptosis.

Apoptosis is a physiological phenomenon, not pathological like necrosis. We have more hematopoietic stem
cells than we need, and only a fraction of them and of progenitors eventually results in production of RBCs. If
the system wouldn’t be redundant it would be more exposed to the risk of exhaustion.

So if that is the system, we have many types of general mechanisms of Anemia.

• Hypoproliferative: there is an impaired proliferation of erythroid cells in the bone marrow. In all these
anemias, the bone marrow is very rich in erythroid cells but for different reasons these cells are not able to
go through the maturation process and eventually die in the bone marrow. The erythropoiesis is there, but
the final result is poor. There are many examples for this but the most important ones are Megaloblastic
Anemias (due to vitamin B12 or folic acid deficiency) and Thalassemia Syndrome (in particular: β
Thalassemia Major also called Cooley’s disease).

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Hypoproliferative anemias may be due to:

- absence or low number of HSCs (Aplastic Anemia).

- defective iron supply to the bone marrow (Iron Deficiency Anemia).

- inhibition of bone marrow erythroid by pro-inflammatory cytokines (Chronic Diseases Anemia).

- defective production of erythropoietin (Renal Anemia).

• Hemolytic Anemias: in some cases the life span of red cells decreases because of pathological reasons so
instead of surviving 4 months, they will survive 4 days or even less. These are Hemolytic anemias typically
associated with an increase in Reticulocyte count (which allows us to make a distinction between
hemolytic and non-hemolytic anemias). The most prevalent type of hemolytic anemia is malaria, which is
one of the leading causes of death, in particular in children under the age of 3.

• Acute Blood Loss Anemia: it is seen in patients with venous stenosis and blood loss through esophageal
varices.

Our approach to patients affected by anemia includes:

1. The use of Hb for establishing the presence of anemia and its reason.

2. The MCV to differentiate between microcytic, normocytic and macrocytic anemias.

3. The reticulocyte count to distinguish between hemolytic or non hemolytic anemia.

2.1 Iron deficiency anemia

Iron deficiency anemia and anemia of inflammation are two very common conditions worldwide.

Iron deficiency (sideropenia or hypoferremia) is the most common nutritional deficiency in the world. Iron is
present in all cells in the human body and has several vital functions, such as: carrying oxygen to the tissues
from the lungs as a key component of the hemoglobin protein; acting as a transport medium for electrons
within the cells in the form of cytochromes; facilitating oxygen use and storage in the muscles as a
component of myoglobin and as an integral part of enzyme reactions in various tissues.

Total body iron averages approximately 3.8 g in men and 2.3 g in women.

In blood plasma, iron is carried tightly bound to the protein transferrin. There are several mechanisms that
control human iron metabolism and safeguard against iron deficiency. The main regulatory mechanism is
situated in the gastrointestinal tract. When loss of iron is not sufficiently compensated by adequate intake of
iron from the diet, a state of iron deficiency develops over time. When this state is uncorrected, it leads to
iron deficiency anemia. Before anemia occurs, the medical condition of Iron Deficiency without anemia is
called Latent Iron Deficiency (LID) or Iron-deficient erythropoiesis (IDE). Iron deficiency anemia occurs when
the body lacks sufficient amounts of iron, resulting in reduced production of the protein hemoglobin.

Most of the iron is found in circulating red blood cells. In a 70 kg man, there are about 2gr of iron in
circulating RBC as hemoglobin iron. In men, but not for instance in young women, there is about 1 g of iron
stored in the liver. Young females probably have no iron in liver because they just finished to grow so most of
the iron has been used for expanding the body and increasing the body mass. Then the menstrual cycle
starts and they are losing iron during periods. Children, pre-menopausal women (women of child-bearing
age) and people with poor diet are most susceptible to the disease.

The peculiarity of the iron metabolism in men is that the entrance of the iron in the system is very small, and
absorption is of 1-2 mg per day. Human beings do not have clearance of iron, so iron is lost only by chance
(for example during menstruation or loss of iron in cells that we lose during desquamation). The fact that we

9
do not have an active recruitment of iron and we do not have a system for eliminating the iron in excess,
explains why human being are exposed to 2 opposite risks: iron deficiency and iron overload.

Iron overload is also associated with mortality. This is the case of patients with genetic hemochromatosis and
with thalassemia major, which receive regular blood transfusion and are exposed to iron toxicity because of
iron overload.

———Clinical case.——————————————————————————————————————
This is a 70 years old man who had a transient ischemic attack 3 years before. The treatment was
successful and in order to prevent relapse and maybe irreversible damage he was taking low dose aspirin
(100mg/day). Low dose aspirin is a drug that inhibits platelets aggregation, which is mediated by fibrinogen.
It does so by altering the balance between prostacyclin and other molecules, reducing the rate of platelets
aggregation and therefore the risk of thrombosis. After few months, the man developed dyspnea. Probably
he was also pale and the family doctor decided to perform a complete blood count. The patient had 9.7 g/dL
of Hb (normal man between 13-17 g/dL). By definition this man was anemic. Anemia was microcytic because
the mean cell volume was low (69.7 fL). We could thus exclude some anemia types that are macrocytic
(MCV > 100 fL) or normocytic (MCV between 80-100 fL). The anemia was also hypochromic. These red
blood cells were smaller, contained less hemoglobin than normal. The RBCs count was almost normal or
slightly reduced. RDW (Red Blood Cell Distribution Width) was increased.

Looking at the peripheral blood smear of this patient it was possible to see that the cells were smaller.
Moreover RBCs were almost empty. They had only a rim of cytoplasm but inside there was almost no
hemoglobin: anulocytes.

The red blood cell of a patient with iron deficiency anemia, not only is smaller but it also contains less
hemoglobin. In fact iron deficiency limits the ability of erythroblast to produce hemoglobin.

One of the side effects of low dose aspirin is hemorrhagic gastritis, which means that everyday this subject
may lose 10-20 mL of blood in the stools, initially without symptoms. Losing blood every day in the stools
however means that the patient is losing iron and over the years iron deficiency anemia may develop.

———Clinical case.——————————————————————————————————————
Here we have a 22 years-old female student. She had a complete blood cell count and now she knows why
she had progressive fatigue and reduced exercise tolerance. Her periods are irregular but most importantly
they last 8-9 days with heavy flow, meaning that she is losing a lot of blood. The normal amount of blood lost
during periods may be 30-40 mL but in case like these one it is about 80-100 mL. Regular blood loss means
loss of hemoglobin and so loss of iron: she developed iron deficiency anemia. In this case iron deficiency
has developed quicker because this person didn’t have enough time in her life to develop consistent iron
stores. She is pale and anemic. Not surprisingly the WBCs count is normal, the platelet count is normal but
let’s say marginally elevated. Iron deficiency is normally associated with high platelets count. Her anemia is
microcytic and hypochromic: RBCs have low hemoglobin concentration.



The iron enters the system through the gut and it’s mainly heme iron and the iron contained in the meat.
Since meat is the main source of iron in human beings, by definition vegetarian people are at risk of iron
deficiency. 1 mg of iron is the amount of iron absorbed by the gut. Everyday 1 in 120 of RBCs is destroyed
so every day 20 mg of iron enters the macrophages because senescent RBCs are phagocytized. This
means that iron in the cells must be given to transferrin and the molecule that does this is ferroportin. Most of
the iron circulating in blood derives from catabolism of senescent RBCs and not from iron storage: 1mg of
iron from the gut and about 20 mg from this second process.

The evaluation of the body iron status it is done by assessing:

- Serum iron
- Ferritin
- Transferrin
- Total iron-binding capacity (TIBC)
- Transferrin saturation (Iron saturation of transferrin)
- Unsaturated iron binding capacity (UIBC)
- Transferrin receptor (TfR)

10
Proteins of iron metabolism:

1. Transferrin: it is responsible for providing all cells of the body with iron. Transferrins are iron-binding
blood plasma glycoproteins that control the level of free iron in biological fluids. The function of transferrin
is to carry iron from the gut or from the liver to every tissue, in particular to erythroid bone marrow.
Erythroblasts are in fact the cells that use more iron for hemoglobin synthesis. Human transferrin is
encoded by the TF gene. Transferrin glycoproteins bind iron tightly, but reversibly. Although iron bound to
transferrin is less than 0.1% (4 mg) of total body iron, it forms the most vital iron pool with the highest rate
of turnover (25 mg/24 h). Transferrin has a molecular weight of around 80 KDa and contains two specific
high-affinity Fe(III) binding sites. The affinity of transferrin for Fe(III) is extremely high, but decreases
progressively with decreasing pH below neutrality. When not bound to iron, transferrin is known as
"apotransferrin". When a transferrin protein loaded with iron encounters a transferrin receptor on the
surface of a cell, it binds to it and, as a consequence, is transported into the cell in a vesicle by receptor-
mediated endocytosis. The pH of the vesicle is reduced by hydrogen ion pumps (H+ ATPases) to about
5.5, causing transferrin to release its iron ions. The receptor (with its ligand, transferrin, bound) is then
transported through the endocytic cycle back to the cell surface, ready for another round of iron uptake.
Each transferrin molecule has the ability to carry two iron ions in the ferric form (Fe3+) and we can have
apotransferrin, monoferric transferrin and diferric transferrin. Medical professionals may check serum
transferrin level in iron deficiency and in iron overload disorders such as hemochromatosis. The liver is
the main site of transferrin synthesis but other tissues and organs, including the brain, also produce
transferrin. The receptor helps maintain iron homeostasis in the cells by controlling iron concentrations.

2. Ferritin: it is an ubiquitous intracellular protein that stores iron and releases it in a controlled fashion. In
humans, it acts as a buffer against iron deficiency and iron overload. The ranges for ferritin can vary
between laboratories but are usually between 30–300 ng/mL (=μg/L) for males, and 18–115 ng/mL (=μg/
L) for females. Ferritin is found in most tissues as a cytosolic protein, but small amounts are secreted into
the serum where it functions as an iron carrier. Ferritin is found mainly in the liver hepatocytes and more
generally in reticulo-endothelial cells, so macrophages, which are mainly in liver, bone marrow and
spleen. Ferritin is synthesized inside these cells but a fraction of the ferritin that is synthesized inside the
cells is exported outside through the Golgi apparatus. Plasma ferritin is also an indirect marker of the
total amount of iron stored in the body, hence serum ferritin is used as a diagnostic test for iron deficiency
anemia. Ferritin is a globular protein complex consisting of 24 protein subunits and is the primary
intracellular iron-storage protein, keeping iron in a soluble and non-toxic form. Ferritin that is not
combined with iron is called apoferritin. Ferritin serves to store iron in a non-toxic form, to deposit it in a
safe form, and to transport it to areas where it is required. The presence of iron itself is a major trigger for
the production of ferritin. Free iron is toxic to cells as it acts as a catalyst in the formation of free radicals
from reactive oxygen species via the Fenton Reaction. Hence vertebrates evolve an elaborate set of
protective mechanisms to bind iron in various tissue compartments. Within cells, iron is stored in a
protein complex as ferritin or hemosiderin. Apoferritin binds to free ferrous iron and stores it in the ferric
state. As ferritin accumulates within cells of the reticuloendothelial system, protein aggregates are formed
as hemosiderin. Iron in ferritin or hemosiderin can be extracted for release by the RE cells although
hemosiderin is less readily available. Under steady state conditions, the serum ferritin level correlates
with total body iron stores; thus, the serum ferritin FR5Rl is the most convenient laboratory test to
estimate iron stores. Circulating ferritin or serum ferritin reflects the amount of ferritin in the body iron
stores, thus serum ferritin is one main indicator for the measurement of body iron stores. The higher the
storage iron, the higher serum ferritin. So in a patient with iron deficiency anemia serum ferritin will be
very low. Also for young female serum ferritin will be around 10 μg/L, while in men over the age of 50 the
iron stores are considerable and serum ferritin will be around 100 μg/L.

3. Hepcidin: is a protein that in humans is encoded by the HAMP gene. Hepcidin is a key regulator of the
entry of iron into the circulation in mammals. In states in which the hepcidin level is abnormally high such
as inflammation, serum iron falls due to iron trapping within macrophages and liver cells and decreased
gut iron absorption. This typically leads to anemia due to an inadequate amount of serum iron being
available for developing red cells. Hepcidin is a small peptide produced by the hepatocytes. The
synthesis of hepcidin is regulated at different levels: (i) iron stores, (ii) inflammatory cytokines (IL-6) and
(iii) erythroid activity. If the body iron stores, so the iron in the hepatocytes where hepcidin is produced,
are low, the result will be an decreased production of hepcidin, an increased absorption of iron and
increase in the release of iron from macrophages. In iron overload instead, due for example to genetic
hemochromatosis or to repeated blood transfusion, hepcidin production is increased, iron absorption is
decreased and iron release from macrophages is decreased. Why repeated blood transfusions cause

11
 iron overload? Humans do not have a clearance mechanism for iron. If in the unit of RBC we use there
are about 200 mg of iron, if the patient needs about 2 unit per month he receives 400 mg per month
which means about 5 g per year. So after one year of regular blood cell transfusion the patient will have
his iron content doubled. When body iron is above 100mg per kg (so in a 70 kg man are 7g) the first
signs of iron toxicity may develop. When the hepcidin level is abnormally low such as in
hemochromatosis, iron overload occurs due to excessive ferroportin mediated iron influx. Hepcidin is a
regulator of iron metabolism. Hepcidin inhibits iron transport by binding to the iron export channel
ferroportin which is located on the basolateral surface of gut enterocytes and the plasma membrane of
reticuloendothelial cells (macrophages). Hepcidin ultimately breaks down the transporter protein in the
lysosome. Inhibiting ferroportin, it prevents iron from being exported and the iron is sequestered in the
cells. By inhibiting ferroportin, hepcidin prevents enterocytes from allowing iron into the hepatic portal
system, thereby reducing dietary iron absorption. The iron release from macrophages is also reduced by
ferroportin inhibition. Increased hepcidin activity is partially responsible for reduced iron availability seen
in anemia of chronic inflammation. Any mutation in hepcidin results in juvenile hemochromatosis. The
majority of juvenile hemochromatosis cases are due to mutations in hemojuvelin. Mutations in TMPRSS6
can cause anemia through deregulation of Hepcidin. There are many diseases where failure to
adequately absorb iron contributes to iron deficiency and iron deficiency anaemia. The treatment will
depend on the hepcidin levels that are present, as oral treatment will be unlikely to be effective if hepcidin
is blocking enteral absorption, in which cases parenteral iron treatment would be appropriate. Beta-
thalassemia is one of the most common congenital anemias arising from partial or complete lack of β-
globin synthesis. Excessive iron absorption is one of the main features of β-thalassemia and can lead to
severe morbidity and mortality. The overload of iron is associated with low levels of hepcidin. It was found
in fact that patients with β-thalassemia have low hepcidin levels.

4. Ferroportin: is responsible for exportation of iron from inside macrophages and mucosal cells to
transferrin. Ferroportin is inhibited by hepcidin, which binds to ferroportin and internalises it within the
[Link] results in the retention of iron within enterocytes, hepatocytes, and macrophages with a
consequent reduction in iron levels within the blood serum. This is especially significant with enterocytes
which, when shed at the end of their lifespan, results in significant iron loss. This is part of the
mechanism that causes anaemia of chronic disease; hepcidin is released from the liver in response to
inflammatory cytokines, namely interleukin-6, which results in an increased hepcidin concentration and a
consequent decrease in plasma iron levels. The ferroportin expression is also regulated by the IRP
regulatory mechanism. If the iron concentration is too low, the IRP concentration increases, thus
inhibiting the ferroportin translation. Ferroportin is responsible for transporting iron from enterocytes to
plasma.

5. Divalent Metal Transporter 1 (DMT1): also known as natural resistance-associated macrophage protein 2
(NRAMP 2), and divalent cation transporter 1 (DCT1), is a protein that in humans is encoded by the
SLC11A2 (solute carrier family 11, member 2) gene. DMT1 represents a large family of orthologous metal
ion transporter proteins. As its name suggests, DMT1 binds a variety of divalent metals including
cadmium (Cd2+), copper (Cu2+), and zinc (Zn2+) however it is best known for its role in transporting
ferrous iron (Fe2+); DMT1 expression is regulated by body iron stores to maintain iron homeostasis.
DMT1 is also important in the absorption and transport of manganese (Mn2+). The divalent metal
transporter 1 (DMT1) is responsible for transporting iron from the gut lumen to the enterocytes.

If in a unit we have 10 molecules of transferrin, we can have 6 molecules of apotransferrin with no binding
sites occupied by iron, 3 molecules of mono ferric transferrin and 1 molecule of diferrin transferrin. Serum
iron atoms bound would be 5. The number of total number of binding sites is 20, so if all of them were
occupied the total iron binding capacity would be 20. 5 is serum iron, 20 is the potential TIBC, and 5/20
(25%) is transferrin saturation. In the lab the units used are μg/dL. When body iron stores are reduced serum
iron is reduced and the number of transferrin molecules increases (liver tries to maximize the binding of the
little amount of iron available), so the TIBC is increased and saturation is reduced.

Major causes of iron deficiency in South and East of the world: Some people have a diet which is inadequate
in terms of iron supply. Children and young female in many countries do not have a type of diet rich in meat,
so they have an iron intake which is inadequate. Moreover young females may have many pregnancies. A
pregnancy involves about 500-700 mg of iron because the iron is given to the baby and it is also needed in
the subsequent months. So, in conclusion, the subgroups of population that are at higher risk of iron
deficiency are children and teenagers, woman in fertile age.

12
Major causes of iron deficiency in West of the world: chronic blood loss from the gut in adults or elderly
people.

Diagnosis of full blown iron deficiency anemia is quite easy: we have an anemia that is microcytic,
hypochromic and serum iron is low; the production of transferrin increases and serum ferritin decreases.
During the transition from normal subject to subject affected by full blown iron deficiency anemia, other
manifestations of iron deficiency anemia could be present (especially when iron deficiency is very prolonged
we can have abnormalities of tissue of the nails).

Searching for a cause is extremely important. If iron deficiency anemia is present, the first lab examination
you ask for is total blood count, but in order to make diagnosis you need to know serum iron, transferrin
saturation, TIBC and serum ferritin. If this patient has a serum iron of 30 mg for dL (< 60 μg); low serum
ferritin, let’s say 10 μg/dL, high TIBC, low transferrin saturation, we know that this patient has iron deficiency
anemia. We suspect that this patient may have for months or years hemorrhagic gastritis due to abuse of low
dose aspirin. The approach is gastroscopy. We also need to remember that the risk of developing any tumor
increases with age. So a 70 years old man may also have a gastric carcinoma. If there is a carcinoma that
we do not diagnose and we treat this patient with iron deficiency anemia giving him iron or even with
intravenous iron, the patient would be initially satisfied but we will have misdiagnosed the cancer so it would
be a huge problem. In a young boy or woman with heavy periods we can assume it is the age or the period
to be the cause of the iron deficiency anemia and to correct anemia with iron is ok. In an elderly patient,
identifying the cause of this type of anemia, excluding a tumor is especially important. Colon carcinoma is a
very common tumor. Nowadays starting from 55, especially in subjects with family history, colonoscopy is
performed, and any abnormality is removed and analyzed in order to prevent the formation of colon
carcinoma. So, in elderly people with iron deficient anemia, we should first suspect that there is chronic
blood loss from the gut and then other multiple causes. Defining the cause is as important as treating the
patient.

Treatment is quite easy. The first option is to start treatment with oral iron in tablets. However it is very
difficult to correct completely the anemia when it is due to very heavy periods, because the loss of blood
involves a loss of iron. The doctor could thus give pills in order to control the periods, to reduce their length
and amount. We rarely use IV iron but it is used in many women who cannot tolerate oral iron because of
side effects. This should be done in the hospital because IV iron may involve important side effects and so
the patients should be monitored.

2.2 Anemia of inflammation or anemia of chronic disease

———Clinical case.——————————————————————————————————————
Here we have a 54 years old woman complaining about abdominal pain, fever (> 38°), night sweating and
weight loss. Fever, night sweating and weight loss are also called systemic symptoms and many conditions
both malignant and not malignant are associated with these traits. Family doctor suggests a complete blood
count: Hb is low (8.4), so she’s anemic; MCV is borderline and marginally reduced as well as the mean
corpuscular hemoglobin. WBC count is increased and there is a condition called neutrophilia and platelet
count is marginally increased. CRP is C-reactive protein and it is markedly increased. Infection is one
possibility. Since there is abdominal pain you consider something in the abdomen and you perform
ultrasonography. This patient had low serum iron but serum ferritin was markedly increased. Transferrin
saturation was low: this is compatible with iron deficiency. But TIBC which was decreased (and not
increased), and this was not compatible with iron deficiency. So we have a situation in which the iron is
reduced in peripheral blood but probably not in the tissues. And this situation is called anemia of
inflammation or anemia of chronic disease.

Anemia of chronic disease, or anemia of chronic inflammation, is a form of anemia seen in chronic infection,
chronic immune activation, and malignancy. These conditions all produce massive elevation of Interleukin-6,
in response to which the liver produces increased amounts of hepcidin. Hepcidin in turn causes increased
internalisation of ferroportin molecules on cell membranes which prevents release from iron stores.
Inflammatory cytokines also appear to affect other important elements of iron metabolism, including
decreasing ferroportin expression, and probably directly blunting erythropoiesis by decreasing the ability of
the bone marrow to respond to erythropoietin.

13
In addition to effects of iron sequestration, inflammatory cytokines promote the production of white blood
cells. Bone marrow produces both white blood cells and red blood cells from the same precursor stem cells.
Therefore, the upregulation of white blood cells causes fewer stem cells to differentiate into red blood cells.
This effect may be an important additional cause for the decreased erythropoiesis and red blood cell
production seen in anemia of inflammation, even when erythropoietin levels are normal, and even aside from
the effects of hepcidin. Macrophages secrete lots of ferritin, but do not release iron from circulating blood. So
we have low serum iron but high serum ferritin. Nonetheless, there are other mechanisms that also
contribute to the lowering of hemoglobin levels during inflammation: (i) Inflammatory cytokines (TNF, IL-1,
INF-gamma) suppress the proliferation of erythroid precursors in the bone marrow; (ii) inflammatory
cytokines (TNF, IL-1, INF-gamma) inhibit the release of erythropoietin from the kidney; and (iii) the survival of
circulating red cells is shortened.

In the short term, the overall effect of these changes is likely positive. However, if inflammation continues, the
effect of locking up iron stores is to reduce the ability of the bone marrow to produce red blood cells. These
cells require iron for their massive amounts of hemoglobin which allow them to transport oxygen. Anemia of
chronic disease may also be due to neoplastic disorders and non-infectious inflammatory diseases.
Neoplastic disorders include Hodgkin’s disease, lung and breast carcinoma and non-infectious inflammatory
diseases include rheumatoid arthritis and systemic lupus erythematosus. Renal cell carcinoma, is a condition
typically associated with a high production of pro-inflammatory cytokines. Endocarditis, is probably the most
common infection leading to anemia of chronic diseases. Tuberculosis is another.

In order to distinguish between iron deficiency anemia and anemia of chronic diseases two parameters are
fundamentally important (serum iron in fact doesn’t distinguish between the two conditions because it
decreases in both):

- TIBC is increased in iron deficiency anemia and normal to low in anemia of chronic disease or of
inflammation.

- Serum ferritin is low in iron deficiency anemia and normal or increased in anemia of chronic diseases.

Rheumatoid arthritis is an autoimmune disease so there is inflammation by definition, but treatment, which is
based on cortisol or non-steroid anti-inflammatory drugs, may lead to chronic blood loss and so it can lead to
a combination of anemia of inflammation and anemia due to iron deficiency. The primary approach is to try
with iron administration to see if there is an improvement or not.

2.3 Genetic hemochromatosis

Body Iron storage is mainly in the Liver (hepatocytes and reticuloendothelial cells). Normal body iron stores
are up to 30-40 mg/kg body weight, so in a 70 kg man, up to 2 grams. In most young people, and especially
in female subjects, body iron stores are negligible. Iron overload is by definition 100 mg/kg body weight, but
clinical manifestations of iron overload become apparent over 200 mg/kg body weight, so always looking at
the 70kg man it is about 10 grams.

One peculiarity about the iron metabolism in men is that iron absorption is quite small, 1-2 mg/day, but the
system lacks a clearance. So, almost all iron atoms that enter the body will stay there for life and the only
loss is by chance, by desquamation of mucosal cells. In other words, if the iron that enters the body is
exceeding the normal amounts, iron overload will be essentially inevitable.

Hepcidin was defined as the main regulator of body iron metabolism. It is synthesized by hepatocytes and
the main regulators of its synthesis are the iron stores in hepatocytes, inflammatory cytokines and erythroid
cells.

The higher the amounts of iron in the hepatocyte, the higher the synthesis of hepcidin to limit the amount of
iron reaching the cells. Ferroportin is internalized and degraded. Iron absorption decreases and iron release
from macrophages also decreases. On the opposite side, if iron is scarce in the hepatocytes, there is a need
to absorb more iron, so hepcidin synthesis will be suppressed.

14
In Anemia of inflammation, especially because of overproduction of Il6, hepcidin synthesis is increased, iron
absorption is suppressed and reticuloendothelial iron mobilization is also suppressed. In subjects with
increased erythroid activity (high number of erythroblasts), which need iron for the activity, hepcidin is also
suppressed since there is the need for increased iron absorption and, especially, increased iron released
from reticuloendothelial cells.

If there is something wrong with the production of hepcidin, inevitably there will be an increase in iron
absorption. One condition associated with iron overload is genetic hemochromatosis. Hemochromatosis
(also HFE hereditary hemochromatosis or HFE-related hereditary hemochromatosis) is a hereditary disease
characterized by excessive intestinal absorption of dietary iron resulting in a pathological increase in total
body iron stores. Excess iron accumulates in tissues and organs disrupting their normal function. The most
susceptible organs include the liver, adrenal glands, heart, skin, gonads, joints, and the pancreas; patients
can present with cirrhosis, polyarthropathy, adrenal insufficiency, heart failure or diabetes. The disease is
inherited in an autosomal recessive pattern, which means both copies of the gene in each cell must have the
mutation in order for the subject to manifest the disease. The basis for the disease is a general mutation in
one the genes that control hepcidin production. Subjects can be heterozygous or homozygous.

The most common type of hemochromatosis is HFE gene related, and there are 2 types of mutations related
to the HFE gene that can cause this condition:

I. Juvenile genetic hemochromatosis: characterized by onset of severe iron overload occurring typically in
the first to third decades of life. Males and females are equally affected. Prominent clinical features
include hypogonadotropic hypogonadism, cardiomyopathy, arthropathy, and liver fibrosis or cirrhosis. The
main cause of death is cardiac disease. If juvenile hemochromatosis is detected early enough and if
blood is removed regularly through the process of phlebotomy to achieve iron depletion, morbidity and
mortality are greatly reduced. In southern Europe, we noticed a prevalence of juvenile (most common
features are hypogonadism at 17 years old and cardiac failure around the age of 20).

II. Genetic hemochromatosis related to the loss of function mutations of the ferroportin gene: is associated
instead with a germiline mutation of ferroportin. So hepcidin synthesis is normal, it is the ferroportin that
is abnormal and this leads to a particular type of genetic hemochromatosis with accumulation of iron in
the macrophages of reticuloendothelial cells.

In addition to these genetic types of hemochromatosis, a number of conditions are associated with iron
overload. The most common type is transfusion iron overload and the pragmatic example is Thalassemia
major. These patients depend on regular transfusions, so they can receive 1-2 blood units every 2-3 weeks.
1 unit contains 200 mg of iron, so in a few years iron accumulation will be responsible for morbidity and also
mortality.

All different conditions leading to genetic hemochromatosis are responsible for decreased production of
hepcidin. There is a mutation in the hepcidin gene, for instance introducing a stop codon, resulting in a
defective production of hepcidin. A mutation in the HFE gene is also responsible for defective hepcidin
production.

The C282Y mutation takes place in more patients with genetic hemochromatosis at the heterozygous state
and is particularly common in patients from northern Europe. 5% of all HFE alleles carry the mutation and
homozygous are almost 10%. Completely normal subjects are about 90% of total population. The proportion
of patients, which are heterozygous for genetic hemochromatosis is about 9.5%. The proportion of
homozygous subjects is 2-3 per thousand. So it’s a quite common genetic disease, probably the most
common in western countries.

The second mutation is more common, about 25 percent of subjects in the population carry the mutation,
and about 2% are homozygous for the mutation. But this mutation is quite weak, so even homozygous
subjects don’t have problems of iron overload (low penetrance).

In practice, the subjects with HFE genetic hemochromatosis are subjects homozygous for a strong mutation
or double heterozygous for the 2 mutations while subjects homozygous for the weak mutation normally don’t
have iron overload.

15
The other feature of these conditions is the low penetrance of the mutations and different variable phenotypic
expression. For instance, the statement says that the disease is modified by acquired factors like alcohol
consumption and dietary iron intake, blood loss associated with menstruation and pregnancy, and blood
donation. Patients with alcoholic liver cirrhosis have an increased iron content, so if one combines this
tendency to accumulate iron with consumption of large amounts of alcohol, the risk of developing this
disease is increased. If one homozygous subject is vegetarian and another one eats a lot of meat, the
second subject is more likely to develop iron overload. Also, it is more likely that the male develops the
disease, since in the fertile age for females the regular blood loss with menstruation prevents the
accumulation of iron. So there are really a lot of factors that can influence the phenotype. Only few
individuals really develop iron overload.

By studying the frequency of the homozygous subjects in Europe, it is quite clear that the strong association
between iron overload and the homozygosity for the mutation is found mainly in Celtic regions, especially in
France and Britain. Why now only 10% of subjects carry the mutation? 10,000 years ago, if a subject was
heterozygous for the mutation, had an increased iron absorption. In general, if you have an increased iron
absorption the risk of developing iron deficiency anemia is much lower. Iron deficiency anemia is an easily
treatable condition now, but it was not easily manageable many years ago. Then, probably, for women not to
have iron deficiency was an advantage. Women with heterozygosity for the mutation were thus more likely to
have babies and this justifies the prevalence of the mutation in the population. The disadvantages instead
(hypogonadotropic hypogonadism and heart failure, at least in homozygous subjects) become apparent at
the age of 40-50 and at that time the subjects had already reproduced, therefore this had no impact on the
selection.

The potential clinical manifestations, which are not found simultaneously in most patients, include:

- liver fibrosis and cirrhosis, which should be prevented because once cirrhosis has established, the risk of
hepatocellular carcinoma is very high and most patients die because of this.

- change in skin pigmentation, which becomes of a bronze-like color.

- diabetes, due to the iron accumulation in the pancreas

- hypogonadotropic hypogonadism, due to the iron accumulation in the anterior pituitary

- arthritis, due to accumulation of iron in the joints

- heart failure, which once it occurs, it is very difficult to treat and most of the young patients die because of
this complication.

When all these manifestations (hepatomegaly, skin pigmentation, diabetes mellitus, heart disease, arthritis
and hypogonadism) are present, diagnosis is easy. But significant overload may exist with none, or only
some, of these manifestations which can however be associated with many different disorders. The major
parameters for diagnosis are:

- Serum iron: increased

- Total iron binding capacity: decreased

- Transferrin saturation: markedly increased

- Serum creatinine: increased

These are just the opposite of what we would see in iron deficiency patients! In particular cases, if liver
biopsy is indicated (for instance to evaluate the degree of liver damage) we can use the first stain for ferritin
and hemosiderin, and show increased amounts of iron in the hepatocytes.

If you have a 20 year old man in which a high serum iron has already led to the diagnosis of genetic
hemochromatosis, what kind of treatment would you start in order to prevent accumulation of iron and organ
damage? Treatment: flebotomy, removal of 400 - 500 ml of blood once a week or twice a week. Therefore,
removing iron, the body will utilize the iron in storage. The patient can undergo 2 flebotomies per week.

16
Since the disease is not transmittable because it’s a genetic disease, these individuals (at least the ones
without organ damage) should become blood donors. The objective is to reach a normal serum iron, a
normal transferrin saturation and a serum ferritin under 50mg/L. Once the normal body iron status has been
achieved we should follow the patient, and consider regular flebotomies of 3-4 months in order to prevent
iron accumulation. The best blood donors today are subjects with genetic hemochromatosis: they can donate
one unit per week or one per month. While the average blood donor donates 1 every 3 months.

Why is chronic blood loss associated with aspirin hemorrhagic gastritis not responsible for hemorrhagic
anemia but instead for iron deficiency anemia? Hemorrhagic anemia is the anemia that develops when the
iron is lost in a considerable amount and in a relatively short time. For instance, if a patient has esophageal
varices and there is a rupture of these, the amount of iron that can accumulate in the stomach may be as
much as 1 or 2 liters in a relatively short period (40 min-1 hour). In a patient taking aspirin and having
hemorrhagic gastritis the amount of blood that is lost is in the order of 10-20 ml per day (a small amount over
a long period). This small amount is initially compensated by increased production, by the bone marrow, but
it involves loss of iron and in the long run iron deficiency.

Heterozygosity for mutation in the ferroportin gene can leads to the accumulation of iron in Kupfer cells. This
condition will have no clinical manifestations apart from high serum creatinine. In classical hemochromatosis,
there is accumulation of iron in hepatocytes, here only in Kupfer cells or reticuloendothelial cells.

2.4 Megaloblastic anemias and Pernicious anemia

———Clinical case.——————————————————————————————————————
On old man, who has had vitiligo since many years, now presents with anorexia, weight loss, paresthesias
and ataxia. A CBC shows: Hb 9.8 g/dl, MCV 115 fL, reticulocyte 35x10^9/L, WBC 3.5x10^9/L, PLT 80x10^9/
L. Serum bilirubin is equal to 2.8 mg/dl. Which is the most likely diagnosis?

A. Aplastic anemia (has similar features but serum bilirubin is low, because there is no production of red
cells, or low destruction of red cells)

B. Iron deficiency anemia (regularly microcytic, therefore associated with a low MCV)

C. Pernicious anemia (associated with vitamin B12 deficiency)

D. Chronic lymphocytic leukemia (is associated with high WBC, while here it is low)

E. Chronic myeloid leukemia (characterized by high blood count)

What causes vitiligo? Depigmented skin. It can be typically found around mouth or eyes or hand. It is an
autoimmune disease. The patient had anorexia, weight loss, paresthesia and ataxia (problems in stability/
uncertainty in walking). Hb is low (anemia), mean cell volume is very high (macrocytic anemia). Reticulocyte
count is normal, which excludes hemolytic anemia. Tendency towards low value of WBC and platelets count.
Serum bilirubin is increased.

If you look at the peripheral blood smear of this patient, you can see a neutrophil with 8 segmentation
nucleus! (normally there are only 2 - 5). In a patient with macrocytic anemia, finding one neutrophil in 100
WBC is highly suggestive of megaloblastic anemia and in this case of pernicious anemia. Pernicious
because it is indicated by the fact that this patient has vitiligo.

———Clinical case.——————————————————————————————————————
Patient at age of 65 was diagnosed with gastric carcinoma and underwent a gastrectomy, which was
successful (7 years ago). He has developed severe dyspnea and severe anemia, which is macrocytic,
associated with pancytopenia, thrombocytopenia and increased bilirubin. What is the cause of anemia in this
patient? We need to remember that the major function of the stomach is production of the intrinsic factor, a
protein essential for subsequent absorption of vitamin B12 in the ileum. Even in this case we had neutrophils
with 8 segmentations. Nowadays a patient undergoing gastrectomy like this one, would receive regular
administration of vitamin B12 by intramuscular injections. The main feature of megaloblastic anemia is that
mechanism is usually cellular, and the anemia is due to ineffective erythropoiesis or premature death of the
erythroblasts in the bone marrow. Which of the parameters indicates that this patient has an ineffective
erythropoiesis? Bilirubin increase! The cause of Megaloblastic anemia is here vitamin B12 deficiency.

17
Megaloblastic anemia is an anemia of macrocytic classification that results from inhibition of DNA synthesis
during red blood cell production. When DNA synthesis is impaired, the cell cycle cannot progress from the
G2 growth stage to the mitosis (M) stage. This leads to continuing cell growth without division, which
presents as macrocytosis. Megaloblastic anemia has a rather slow onset, especially when compared to that
of other anemias. The defect in red cell DNA synthesis is most often due to hypovitaminosis, specifically a
deficiency of vitamin B12 and/or folic acid. Loss of micronutrients may also be a cause. Megaloblastic
anemia not due to hypovitaminosis may be caused by antimetabolites that poison DNA production directly,
such as some chemotherapeutic or antimicrobial agents. The pathological state of megaloblastosis is
characterized by many large immature and dysfunctional red blood cells (megaloblasts) in the bone marrow
and also by hypersegmented neutrophils (those exhibiting five or more nuclear lobes, with up to four lobes
being normal). These hyper-segmented neutrophils are found in the "peripheral blood" (i.e., a diagnostic
smear of a blood-sample taken from the circulation).

Causes of Megaloblastic anemia:

• Vitamin B12 (cobalamin) deficiency: vitamin B12 is synthesized exclusively by microorganisms and its only
source is food of animal origin. Vegetables do not contain it. The body stores are of 2-5 mg, thus they are
sufficient for 3/4 years. Absorption is mediated by binding with intrinsic factor and patients that undergo
gastrectomy will no longer be able to absorb it. If there is selective destruction of these cells producing
intrinsic factor, there will be this same absorption problem. Daily requirements of vitamin B12 are 1-3 µg
per day. Causes of Vitamin 12 deficiency can be:

- Malabsorption

- Nutritional (vegans)

- Gastric causes (functional abnormality of gastrectomy)

- Intestinal causes (ileal resection, fish tapeworm, etc)

• Folate deficiency: Folic acid is mainly found in liver, yeast, spinach… Normally the amount of folate that we
need (100-400 µg x day) derives from the vegetables we eat. Prolonged cooking destroys most folate. The
body stores of it are relatively small (500–20,000 µg) meaning that with a daily requirement the body stores
are sufficient only for 3/4 months. Folate deficiency is responsible for this anemia because it results in
ineffective erythropoiesis and hematopoiesis, and also hyperhomocysteinemia, associated with increased
risk of arterial thrombosis and thrombophilia. Supplement is important during pregnancy together with iron
supplementation, to prevent neural tube defects in the fetus.

Many patients with folate deficiency are elderly who live alone and don’t have a proper diet, people that don’t
eat vegetables (mainly cheese, burgers, etc) so a diet with low folate content, homeless people, alcoholics.
Alcoholism is associated with folate recipiency in the diet and so leads quite easily to folate deficiency. Also,
people with gluten-induced enteropathy may be affected (this condition also leads to iron deficiency). So if a
patient has typical iron deficiency anemia, this will be a microcytic anemia. If a patient has folate deficiency
anemia, this will be a typical macrocytic anemia. But if there is a combination of folate and iron deficiency the
anemia may be normocytic, this will be more difficult to understand and more difficult to recognize (an
example is the combination of folate and iron deficiency associated to gluten-induced enteropathy).

The blood film can point towards vitamin deficiency showing:

- decreased red blood cell (RBC) count and hemoglobin levels

- increased mean corpuscular volume (MCV, >95 fl, most commonly between 110 and 120 fL) and mean
corpuscular hemoglobin (MCH)

- normal mean corpuscular hemoglobin concentration (MCHC, 32–36 g/dL)

- decreased reticulocyte count due to destruction of fragile and abnormal megaloblastic erythroid precursor

- possible decreased platelet count

18
- hyper-segmentation of neutrophil granulocytes ("senile neutrophil"). This is thought to be due to decreased
production and a compensatory prolonged lifespan for circulating neutrophils. This is useful for differential
diagnosis because in myeloid dysplastic syndromes there is hypo-segmentation

- macrocytes (larger than normal RBCs) are present

- most patients have also mild thrombocytopenia and leukopenia: pancytopenia (one potential differential
diagnosis is with aplastic anemia)

Nowadays, the bone marrow aspirate is no longer done for diagnosis, but when it was performed the typical
pattern was a very cellular bone marrow with many erythroblasts (morphological representation of ineffective
erythropoiesis). The bone marrow contains a lot of immature red cells, most of these cells die because of
apoptosis and do not give rise to circulating red cells. The typical combination of ineffective erythropoiesis is.
reticulocytes (not increased), serum bilirubin (increased) and lactate dehydrogenase (increased).

The way we evaluate the cause in megaloblastic anemia is to assess circulating serum vitamin B12, serum
folate and serum homocysteine. The Schilling test is performed rarely and it is performed to understand if
vitamin B12 is absorbed or not. It involves the use of radio-labelled vitamin B12. The doctor then checks if
the labeled vitamin is absorbed or lost in the urine. The objective of the test is process the urinary loss and
so urinary radioactivity. If there is no urinary radioactivity it is the proof that the vitamin is not absorbed. How
do we treat these patients? administration of Vitamin B12. The only available drug in Italy involves 500mg of
vitamin B12 through intramuscular injection and we start with 1 injection/day and once anemia has been
corrected we discontinue treatment (initially one per week, then one per month, and so on) until 1 every 3-4
months in patients wit pernicious anemia in order to prevent the reappearance of the deficiency. It is easier
for patients with folate deficiency where the therapy involved administration of a tablet of 5mg of folic acid.

Pernicious anemia

Pernicious anemia is one of many types of the larger family of megaloblastic anemias. It is more common in
the case of relatives with this problem or other organ-specific autoimmune diseases (vitiligo, hypothyroidism,
Addison disease). One way pernicious anemia can develop is by loss of gastric parietal cells, which are
responsible, in part, for the secretion of intrinsic factor, a protein essential for subsequent absorption of
vitamin B12 in the ileum. Since the absorption from the gut of normal dietary amounts of vitamin B12 is
dependent on intrinsic factor, the loss of intrinsic factor leads to vitamin B12 deficiency. The loss of ability to
absorb vitamin B12 is the most common cause of adult B12 deficiency. Impaired absorption of vitamin B12
may be due to a loss of intrinsic factor or to a number of other conditions that decrease production of gastric
acid, which also plays a part in the absorption of B12 from foods.

Modern tests that specifically target B12 absorption can be used to diagnose the disease before it becomes
apparent. Replacement of vitamin stores does not correct the defect in absorption from loss of intrinsic
factor. Since the defect defines the disease, people without the ability to absorb B12 in this way will have
pernicious anemia for the remainder of their lives. However, unless the patient has sustained permanent
peripheral nerve damage before treatment, regular B12 replacement will keep PA in check, with no anemia
and no further symptoms. If one starts the proper treatment, which is vitamin B12 replenishment, life
expectancy is quite normal in women but not in men. This because the risk of gastric carcinoma is higher
than expected in the reference population, so, these people, especially men, have a condition that not only
involves defective intrinsic factor production, but also higher risk of developing this carcinoma.

2.6 Aplastic anemia

Aplastic anemia is a disease in which the bone marrow, and the blood stem cells that reside there, are
damaged. This causes a deficiency of all three blood cell types (pancytopenia): red blood cells (anemia),
white blood cells (leukopenia), and platelets (thrombocytopenia). Aplastic refers to inability of the stem cells
to generate the mature blood cells.
Symptoms and signs are:
• Anemia with malaise, pallor and associated symptoms such as palpitations
• Thrombocytopenia (low platelet counts), leading to increased risk of hemorrhage, bruising and petechiae
• Leukopenia (low white blood cell count), leading to increased risk of infection
• Reticulocytopenia (low counts of reticulocytes, that is, immature red blood cells)

19
It is most prevalent in people in their teens and twenties, but is also common among the elderly. It can be
caused by exposure to chemicals, drugs, radiation, infection, immune disease, and heredity; in about half the
cases, the cause is unknown.
The definitive diagnosis is by bone marrow biopsy; normal bone marrow has 30-70% blood stem cells, but in
aplastic anemia, these cells are mostly gone and replaced by fat.
First line treatment for aplastic anemia consists of immunosuppressive drugs, typically either anti-lymphocyte
globulin or anti-thymocyte globulin, combined with corticosteroids and cyclosporine. Hematopoietic stem cell
transplantation is also used, especially for patients under 30 years of age with a related, matched marrow
donor.

2.6 Hemolytic anemia

Hemolytic anemia is a form of anemia due to hemolysis either in the blood vessels (intravascular hemolysis)
or elsewhere in the human body (extravascular). It has numerous possible causes. The general classification
of hemolytic anemia is either inherited or acquired. Treatment depends on the cause and nature of the
breakdown.
Symptoms of hemolytic anemia are similar to other forms of anemia (fatigue and shortness of breath), but in
addition, the breakdown of red cells leads to jaundice and increases the risk of particular long-term
complications, such as gallstones and pulmonary hypertension.
In a healthy person, a red blood cell survives 90 to 120 days in the circulation, so about 1% of human red
blood cells break down each day. The spleen (part of the reticulo-endothelial system) is the main organ that
removes old and damaged RBCs from the circulation. In healthy individuals, the breakdown and removal of
RBCs from the circulation is matched by the production of new RBCs in the bone marrow.
In conditions where the rate of RBC breakdown is increased, the body initially compensates by producing
more RBCs; however, breakdown of RBCs can exceed the rate that the body can make RBCs, and so
anemia can develop. Bilirubin, a breakdown product of hemoglobin, can accumulate in the blood, causing
jaundice. In normal subjects 90% of erythrocytes are phagocytized by macrophages (in the spleen and bone
marrow) in the process of extravascular hemolysis, and 10% is in the process of intravascular hemolysis.
Hepatocytes function as a clearance mechanism for bilirubin. In hemolytic anemia, the bilirubin is always
increased.
In general, hemolytic anemia occurs as a modification of the RBC life cycle. That is, instead of being
collected at the end of its useful life and disposed of normally, the RBC disintegrates in a manner allowing
free iron-containing molecules to reach the blood. The distinguishing feature of intravascular hemolysis is the
release of RBC contents into the blood stream. Free hemoglobin can bind to haptoglobin, or it may oxidize
and release the heme group that is able to bind to either albumin or hemopexin. The heme is ultimately
converted to bilirubin and removed in stool and urine. Hemoglobin may be cleared directly by the kidneys
resulting in fast clearance of free hemoglobin but causing the continued loss of hemosiderin loaded renal
tubular cells for many days.
In intravascular hemolysis, there's production of free Hb in plasma and haptoglobin can bind Hb. The
complex is taken up by the receptor CD163+. The complex is internalized and destroyed into lysozymes and
converted into iron and biliverdin and from there into bilirubin. However, if too much intravascular hemolysis
is present, the heme group in then attached to hemopexin and the complex then is taken up by CD91 cells
(hepatocytes and macrophages) and from there it is transported inside the cell to be destroyed by the
lysozyme and still converter into Fe2+ and bilirubin.
What happens when both extravascular and intravascular hemolysis are markedly increased? On physical
examination is possible to see jaundice (main sign of hemolytic anemia). In many cases the spleen is
enlarged and there is splenomegaly. When red cell destruction is compensated by bone marrow increased
production, the patient displays signs of hemolysis but not anemia. These conditions are therefore called
hemolytic disorders. The MCV in most conditions is mildly elevated and there is an higher reticulocyte count.
Reticulocytes compared to all the red cells have higher MCV (>100 fl). One enzyme present both in mature
and non-mature red blood cells is LDH. The combination of high reticulocyte count, LDH and bilirubin is
typical of anemic patients. Haptoglobin is measured as free haptoglobin in blood. If there's red cell
destruction, most of the Hb will bind haptoglobin which will be reduced to absent.
In order to understand the various subclasses of hemolytic anemias, it is important to measure serum
bilirubin and LDH. High values are suggestive of hemolytic anemia. More importantly also the reticulocytes
are to be assessed.
Ineffective erythropoiesis: serum bilirubin and LDH increased.
Hemolytic anemias: the reticulocytes are increased as well. The cutoff is more than 3% or in absolute terms
more than 100x10^9/ L.

20
To understand the origin of this hemolytic anemia (extravascular or intravascular), it's important to assess
haptoglobin: if hemoglobin is present in peripheral blood, it can go through the renal filter, inside the tubule
and it can appear in the urine.
Both hemoglobin and heme that are bound (to haptoglobin or hemopexin) can pass the filter and enter the
renal tubules and both of them can be absorbed by tubular cells, which process hemoglobin and heme just
like the remaining cells. They use iron which is deposited in hemosiderin and since these cells have short
time span, this is present as hemosiderinuria.
Reticulocytes count normally is low. But in the case of anemic patient their number increases.
Types of hemolytic anemia:
1. Extravascular hemolysis can be due to macrophages or red blood cells membrane disorder (hereditary
spyrocytosis), or hemoglobinopathies (sickle-cell anemia), enzymatic defects(G6PD or pyruvate kinase
deficiency) or immune mediated hemolytic anemias, typically autoimmune.
2. Intravascular hemolysis can be due mechanical fragmentation, thrombotic microangiopathies, immune-
mediated hemolytic anemia, malaria, and also paroxysm nocturnal hemoglobinuria.

Red cell's membrane disorders:

The cytoskeleton of red blood cells is very complex. The main proteins are ankyrin, spectrin, band 3 and
band 4.2. These diseases are inherited disorders and are germline mutations (most of them are mis-sense).
Different mutations in the same gene can give and lead to slightly different disorder. For instance a mutation
in the gene encoding the b chain or the spectrin can both create Hereditary spherocytosis or Hereditary
Elliptocytosis. The contrary is also true. In fact, a single disorder as HS can come from other genes (b chain
of spectrin or gene encoding ankyrin).

Hereditary spherocytosis
It is an autosomal dominant abnormality of erythrocytes caused by mutations in genes relating to membrane
proteins that allow for the erythrocytes to change shape. The abnormal erythrocytes are sphere-shaped
(spherocytosis) rather than the normal biconcave disk shaped. This difference in shape also makes the red
blood cells more prone to rupture. Cells with these dysfunctional proteins are taken for degradation at the
spleen. This shortage of erythrocytes results in hemolytic anemia. The clinical severity of HS varies from
symptom-free carrier to severe hemolysis because the disorder exhibits incomplete penetrance in its
expression.
Symptoms include anemia, jaundice, splenomegaly, and fatigue.

On a blood smear, Howell-Jolly bodies (basophilic nuclear

remnants, aka clusters of DNA) may be seen within red
blood cells. Primary treatment for patients with
symptomatic HS has been total splenectomy, which
eliminates the hemolytic process, allowing normal
hemoglobin, reticulocyte and bilirubin levels.
The peripheral blood cells are typically seen by their lack
of the pale central part which characterizes instead the
normal red blood cells.

Hereditary spherocytosis is caused by a variety of molecular defects in the genes that code for the red blood
cell proteins spectrin (alpha and beta), ankyrin, band 3 protein, protein 4.2 and other red blood cell
membrane proteins. These proteins are necessary to maintain the normal shape of a red blood cell, which is
a biconcave disk. The integrating protein that is most commonly defective is ankyrin which is responsible for
incorporation and binding of spectrin, thus in its dysfunction cytoskeletal instabilities ensue.
All these proteins are involved in the cytoskeleton. The phenotype may be similar in all of these conditions
even if the genotype is different.
Anemia is found in many of the patients affected by HS, but not in all of them. This because the bone marrow
can compensate the excessive destruction of RBCs by increasing their production, and thus the hemoglobin
levels will be normal. You may thus have a patient with Hb of 14 and bilirubin of 3.5 mg/dl (while normally is
up to 1mg/dl). In a patient like this the next to ask for in order to know if he has hemolytic anemia, before
haptoglobin is the reticulocyte count (if you have a number of 200x10^9/L you already have anemia).
As in non-hereditary spherocytosis, the spleen destroys the spherocytes. This process of red blood cells
rupturing directly results in varying degrees of anemia (causing a pale appearance and fatigue), high levels
of bilirubin in the blood (causing jaundice), and splenomegaly.

21
Acute cases can threaten to cause hypoxia through anemia and acute kernicterus through high blood levels
of bilirubin, particularly in newborns.
Chronic symptoms include anemia, increased blood viscosity, and splenomegaly. Furthermore, the detritus of
the broken-down blood cells – unconjugated or indirect bilirubin – accumulates in the gallbladder, and can
cause pigmented gallstones to develop. In chronic patients, an infection or other illness can cause an
increase in the destruction of red blood cells, resulting in the appearance of acute symptoms: a hemolytic
crisis. Spherocytosis patients who are heterozygous for a hemochromatosis gene may suffer from iron
overload despite the hemochromatosis genes being recessive.
One of the possible complications could be aplastic crisis, with dramatic fall in hemoglobin level and
(reticulocyte count) decompensation. This is usually due to maturation arrest and is often associated with
megaloblastic changes; it may be precipitated by infection, such as influenza, notably with parvovirus B19. It
does this by selective erythroid infection, can be very severe and can lead to transfusion dependence. Most
of the patient will have splenomegaly because the site of excessive destruction is predominately the spleen.

In HS the MCV is marginally low. This is due to both the elevated presence of reticulocytes which have high
MCV and the spherocytes which have low MCV. MCHC is poorly useful in most anemias. Its normal values
are 32-36 g/dl, but it tends to be higher in this cases.
In longstanding cases and in patients who have taken iron supplementation or received numerous blood
transfusions, iron overload may be a significant problem. This is a potential cause of heart muscle damage
and liver disease. Measuring iron stores is therefore considered part of the diagnostic approach to hereditary
spherocytosis.
An osmotic fragility test can aid in the diagnosis. In this test, the spherocytes will rupture in liquid solutions
less concentrated than the inside of the red blood cell. This is due to increased permeability of the
spherocyte membrane to salt and water, which enters the concentrated inner environment of the RBC and
leads to its rupture. Although the osmotic fragility test is widely considered the gold standard for diagnosing
hereditary spherocytosis, it misses as many as 25% of cases.

There exists no cure for the genetic defect that causes hereditary spherocytosis. Current management
focuses on interventions that limit the severity of the disease.

Treatment options include:

• Splenectomy: As in non-hereditary spherocytosis, acute symptoms of anemia and hyperbilirubinemia
indicate treatment with blood transfusions or exchanges and chronic symptoms of anemia and
splenomegaly indicate dietary supplementation of folic acid and splenectomy. Splenectomy is
indicated for moderate to severe cases, but not mild cases. Splenectomy cannot be performed in
young children, but only in patients that are older than 5 years. To decrease the risk of sepsis, post-
splenectomy spherocytosis patients require immunization against the pneumococcus bacterium,
influenza virus, and prophylactic antibiotic treatment.
• Partial splenectomy: Since the spleen is important for protecting against encapsulated organisms, as
streptococcus pneumoniae, neisseiria meningiditis and hemophilus Influenzae, sepsis caused by
encapsulated organisms is a possible complication of splenectomy. The option of partial
splenectomy may be considered in the interest of preserving immune function.
• Cholecystectomy may be necessary.
22
The patients undergo vaccination against the encapsulated organisms before the splenectomy. If this is not
possible, for instance when the spleen is damaged and the splenectomy must be done immediately, there is
first the administration of antibiotics and then of vaccines.

Hereditary elliptocytosis
It is an inherited blood disorder in which an abnormally large number of the patient's erythrocytes are
elliptical rather than the typical biconcave disc shape. In its severe forms, this disorder predisposes to
haemolytic anaemia. HE is characterized by a morphologically different abnormality than HS, but the
mechanism of extravascular hemolysis is similar.
A number of genes have been linked to common hereditary elliptocytosis. These mutations have a common
end result: they destabilise the cytoskeletal scaffold of cells. This stability is especially important in
erythrocytes as they are constantly under the influence of deforming shear forces. As disc-shaped
erythrocytes pass through capillaries, which can be 2-3 micrometres wide, they are forced to assume an
elliptical shape in order to fit through. Normally, this deformation lasts only as long as a cell is present in a
capillary, but in hereditary elliptocytosis the instability of the cytoskeleton means that erythrocytes deformed
by passing through a capillary are forever rendered elliptical. These elliptical cells are taken up by the spleen
and removed from circulation when they are younger than they would normally be, meaning that the
erythrocytes of people with hereditary elliptocytosis have a shorter than average life-span (a normal person's
erythrocytes average 120 days or more).

Hemolytic anemia can be caused, other than by a membrane/cytoskeleton dysfunction or hemoglobin

dysfunction, also from enzymatic problems. The most important enzymes in red blood cells which can give
this pathology are pyruvate kinase, hexokinase phosphate and G6PD.
Clinical presentation: most afro-americans with G6PD have mild chronic hemolysis, a few hemolytic crisis
and severe intravascular hemolysis for different reasons. The the Mediterranean G6PD deficiency (favism)
instead is associated with chronic hemolysis and severe hemolytic crisis. Since the gene responsible for this
deficiency is located on chromosome X, females can be carriers, while males cannot. All female babies of an
affected male (hemizygous) are carriers or affected (depending on the mother). Theoretically therefore
there's the possibility that a female could be homozygous for the condition. Hemoglobinuria will be present
during hemolytic crisis due to destruction of the red blood cells. The haptoglobin system will in fact be unable
to bind every hemoglobin molecule. Hb will not be absorbed by the tubular cells and free Hb will be present
then in the urine.

Immunological hemolytic anemia can be due to alloantibodies (antibodies generated by different individuals),
autoantibodies, and antibodies related to drugs.
The typical example of a reaction to alloantibodies is the hemolytic condition of the newborn. The classical
example is when the mother (Rh-) has a second child Rh+. This leads to production of alloantibodies
directed to an antigen and this causes then an intravascular hemolysis in the child.
The most present condition of immune hemolytic anemia however is the autoimmune hemolytic anemia. The
typical classification is based on distinction of warm and cold antibodies: warm antibodies are typically IgG
and they cause extravascular hemolysis. The cold ones are typically IgM and they cause normally
complement-mediated intravascular hemolysis. There are also biphasic antibodies but are mostly a curiosity.
Why does a patient with autoimmune hemolytic anemia due to warm antibodies have excessive hemolysis?
The normal mechanism of antibodies is that there's an antibody binding the pathogen and since
macrophages express an Fc receptor, the complex can be bound to it and it's internalized. This happens also
in autoimmune hemolytic anemia: many red cells have autoantibodies bound to the surface.
In warm type autoimmune hemolytic anemia these are unable to bind the complement so there is no
hemolysis, but when the red cells circulate in the spleen, where many macrophages express the Fc receptor,
the whole complex (antibodies and red cells) is internalized causing extravascular hemolysis. A way to block
this extravascular hemolysis is to block the receptor that binds the antibodies and the immunoglobulins.
The cold autoantibodies disease derives from IgM due to infection (mycoplasma pneumoniae) or
lymphoproliferative disorders. The hemolysis is normally not severe. The process of RBCs agglutination after
exposure to cold causes cyanosis of the extremities and sometimes ischemic necrosis. This kind of patients
are normally symptomatic in winter seasons.
The condition is called primary in the case of unknown origin, and secondary when associated to other
autoimmune conditions. These conditions can be SLE, lymphoproliferative diseases (chronic lymphocytic
leukemia), infectious diseases associated with autoimmune hemolytic anemia and a number of neoplasms.
Full blood count is needed in these patients. A specific test for these conditions is the Coombs test. The
direct test is for detection for Ab or complement fraction of red cells surface, while the indirect is for detection

23
of possible Abs in the serum. In the Coombs test the autoimmune Abs generate agglutination with creation of
bridges between RBCs. The direct Coombs test detects Abs on the surface of the patient RBCs and is the
typical test for detection for diagnosis of autoimmune hemolytic anemia wild type. 

For the indirect test instead we have Abs in the
serum of the patient which will be added to the
normal blood cells. Again the reaction will create
agglutination.
Treatments: drugs to suppress the B
lymphocyte, corticosteroids to lower the
production of autoantibodies (effective only in a
number of patient and only for a short period).
The second-line treatment is splenectomy. Also
large amounts (grams) of immunoglobulins can
be used in order to block the Fc receptor. These
however require some time, so they are not
used in patients with very severe anemia and
cannot be used in pregnant women. Recently
Rituximab (anti-CD20 antibody) has been
introduced for killing B lymphocytes expressing
CD20. This can be considered when dealing
with autoimmune hemolytic anemia if no other
treatment is effective. The side effects are
many, so it 's not the first line treatment.
Transfusion therapy must be handled with care
because autoantibodies can have an effect on
the transfused red cells.

We can also have hemolytic anemia due to fragmentation. Fragmented red blood cells (schistocytes) can be
seen in the peripheral blood smears. After an acute hemolytic crisis there are circulating erythroblasts due to
expansion of bone marrow. The hemolytic crisis can be due to infection, oxidants, drugs, toxins.
Some patients with cardiac valve prothesis, or aortic stenosis or aortic aneurysm have just a mechanical
cause of fragmentation of red blood cells, therefore a mechanical fragmentation causing hemolytic anemia.
Disseminated intravascular coagulation leads to fibrin formation in vivo and this again can lead to
fragmentation. In the case of thrombotic thrombocytopenic purpura, due to abnormal adhesion of platelets to
endothelium, there's excessive formation of platelet thrombi and irregular surface of vessels and
fragmentation of red cells. These patients will have low level of haptoglobin, even hemosiderinuria when
intravascular hemolysis is considerable. Also irregular surface of the vessels and fragmentation of RBCs.
The most common type of hemolytic anemia due to fragmentation of red blood cells is Malaria. 90% of the
children affected by malaria are anemic, while this number is about 80% in pregnant women. The main
mechanism of anemia in these patients is intravascular hemolysis but also additional types of hemolysis do
exist. These are extravascular hemolysis, anemia of chronic disease and ineffective erythropoiesis
(plasmodium can infect the immature red cells in the bone marrow). Patients will have an anemia of variable
degree. The Hb will be below 5 g/dl and there will be an high reticulocyte count and an increased serum
bilirubin.

Paroxysmal nocturnal hemoglobinuria (PNH)

It is a rare, acquired, life-threatening disease of the blood characterized by destruction of red blood cells by
the complement system. This destructive process is a result of a defect in the formation of surface proteins
on the red blood cell, which normally function to inhibit such immune reactions. Since the complement
cascade attacks the red blood cells throughout the circulatory system, the hemolysis is considered an
intravascular hemolytic anemia.
PNH is the only hemolytic anemia caused by an acquired (rather than inherited) intrinsic defect in the cell
membrane (deficiency of glycophosphatidylinositol leading to absence of protective proteins on the
membrane). It may develop on its own ("primary PNH") or in the context of other bone marrow disorders
such as aplastic anemia ("secondary PNH”).
All cells have proteins attached to their membranes, often serving as a mode of communication or signalling
between the cell and the surrounding extracellular milieu. These signalling proteins are physically attached to
the cell membrane in various ways, commonly anchored by glycolipids such as glycosyl

24
phosphatidylinositols (GPI). PNH occurs as a result of a defect in the assembling of these glycolipid-protein
structures on the surface of blood cells. The most common defective enzyme in PNH is phosphatidylinositol
glycan A (PIGA), one of several enzymes needed to make GPI. The gene that codes for PIGA is located on
the X chromosome, which means that only one active copy of the gene for PIGA is present in each cell
(initially, females have two copies, but one is silenced through X-inactivation). A mutation in the PIGA gene
can lead to the absence of GPI anchors expressed on the cell membrane. When this mutation occurs in a
hematopoietic stem cell in the bone marrow, all of the cells it produces will also have the defect. Several of
the proteins that anchor to GPI on the cell membrane are used to protect the cell from destruction by the
complement system, and, without these anchors, the cells are more easily targeted by the complement
proteins. The main proteins that protect blood cells from destruction are decay-accelerating factor (DAF/
CD55), which disrupts formation of C3-convertase, and protectin (CD59/MIRL/MAC-IP), which binds the
membrane attack complex and prevents C9 from binding to the cell.
Allogeneic bone marrow transplantation is the only curative therapy, but has significant rates of both mortality
and ongoing morbidity. Patients affected by paroxysmal nocturnal hemoglobinuria have severe
pancytopenia.
Now we have a drug against one of this components (C5). There's a strong correlation between aplastic
anemia (T cells against hematopoietic cells) and PNH. PNH is almost a survival escape from aplastic
anemia.

3. Thalassemia syndromes and sickle cell disease

Thalassemia, as well as genetic hemochromatosis and sickle cell anemia had an evolutionary
advantage.
Germline mutations are present already in the sperm or oocyte and are inherited. Somatic
mutations occur in a tissue or in an already formed body or during embryogenesis.
Thalassemia, sickle cell disease and genetic hemochromatosis are all conditions associated with
a germline mutation while for instance acute leukemia, malignant lymphomas, chronic
leukemias, cancer, are disorders caused by somatic mutations.
A mutation is normally found in less than 1% of the reference population. A mutation is always
characterized by an abnormal allele, and you can also define the normal allele .
Polymorphisms are variations in the DNA sequence, not necessarily associated with any clinical
consequence. It’s almost impossible with many polymorphisms to define which one is the normal
allele or the abnormal allele.
Thalassemia can be due to a mutation in a gene, meaning any abnormality in the primary
nucleotide sequence of DNA. Some mutations may be of little significance: may have no impact,
no change in phenotype or may even be associated with a gain of function.

3.1 Thalassemia syndromes

Thalassemia is a form of inherited autosomal recessive blood disorder characterized by

abnormal formation of hemoglobin. The abnormal hemoglobin formed results in improper oxygen
transport and destruction of red blood cells. Thalassemia is caused by variant or missing genes
that affect how the body makes hemoglobin. People with thalassemia make less hemoglobin and
have fewer circulating red blood cells than normal, which results in mild or severe anemia.
Thalassemia can cause complications, including iron overload, bone deformities, and
cardiovascular illness. However, this same inherited disease of red blood cells may confer a
degree of protection against malaria (specifically, malaria caused by the protozoan parasite
Plasmodium falciparum), which is or was prevalent in the regions where the trait is common. This
selective survival advantage of carriers (known as heterozygous advantage) may be responsible
for perpetuating the mutation in populations. In that respect, the various thalassemias resemble
another genetic disorder affecting hemoglobin, sickle-cell disease.

A complete blood count of a subject who is heterozygous for beta thalassemia, is characterized
by marginally elevated RBC count, combined with a marginally reduced Hb level, a severe
microcytosis and hypochromia.
In order to understand thalassemia it’s fundamental to know that we have 4 types of hemoglobin.
The hemoglobin molecule is composed by 4 globin chains, 2 pairs, and each globin chain binds
a heme group.
Hemoglobin has a quaternary structure which comes from its four subunits disposed in tetrahedral
arrangement. In most vertebrates, the hemoglobin molecule is an assembly of four globular protein subunits
25
and each subunit is composed of a protein chain tightly associated with a non-protein heme group. The
folding pattern contains a pocket that strongly binds the heme group.

In adult humans, we can have three different types of hemoglobin types:

• Hemoglobin A (α2β2): it is the most common, with a normal amount over 95%. It is a tetramer
(which contains 4 subunit proteins) consisting of two α and two β subunits non-covalently
bound, each made of 141 and 146 amino acid residues, respectively.
• Hemoglobin A2 (α2δ2): δ chain synthesis begins late in the third trimester and, in adults, it has
a normal range of 1.5 - 3.5%.
• Hemoglobin F (α2γ2): in adults Hemoglobin F is restricted to a limited population of red cells
called F-cells. However, the level of Hb F can be elevated in persons with sickle-cell disease
and beta-thalassemia. In human infants, the hemoglobin molecule is made up of 2 α chains
and 2 γ chains. The gamma chains are gradually replaced by β chains as the infant grows.

The laboratory exam needed in order to distinguish the type of hemoglobin the patient has is
electrophoresis (the most useful being High Performance Liquid Chromatography).

Both α- and β-thalassemias are often inherited in an autosomal recessive manner, thus both
parents must be carriers for a child to be affected. If both parents carry a hemoglobinopathy trait,
the risk is 25% for each pregnancy for an affected child. Genetic counseling and genetic testing
are recommended for families who carry a thalassemia trait.

There are really many genes responsible for globin synthesis but some of them like Z, Psi and
Epsilon are active only in embryonic life, so just in the first weeks or moths, while the genes that
are active in adulthood are alpha, beta, delta and gamma.
One initial observation is that we have 2 alpha genes so the alpha gene is duplicated in humans
and there may be a reason for that: initially, many thousands of years ago there probably was
only one alpha gene and then a mutation occurred in a single individual and this mutation
conferred an evolutionary advantage and so now all human beings have 2 alpha genes. The
evolutionary advantage is linked to the fact that having 2 alpha genes the deletion of the gene is
less likely. We also have 2 gamma genes but they differ just in 1 base and 1 amino acid.
It is very important to understand how these genes are expressed during life.
In the yolk sac erythropoiesis is active and hematopoiesis takes place in liver and spleen. It is
just before birth that erythropoiesis ad hematopoiesis move from the spleen to the bone marrow.

The definition of thalassemia syndromes is a defective synthesis of one or more globin chains
but the conditions we will examine together are associated with the defective synthesis of only
one globin chains.
The general mechanism of anemia in thalassemias is:
- Ineffective erythropoiesis in beta thalassemia
- Peripheral hemolysis in alpha thalassemia.
Basically when the synthesis of one chain is defective there is an imbalance between the two
chain types.
In beta thalassemia there is an excessive amount of alpha chain while in alpha thalassemia
there is excessive amount of beta chain and this imbalance results in damage to immature
erythroblasts in beta thalassemia causing excessive apoptosis and ineffective erythropoiesis or
an imbalance in circulating red blood cells in alpha thalassemia leading to peripheral hemolysis.

Alpha thalassemia:
The α-thalassemias involve the genes HBA1 and HBA2, and are inherited in a Mendelian recessive fashion.
α thalassemias result in a decreased alpha-globin production, therefore fewer alpha-globin chains are
produced, resulting in an excess of β chains in adults and excess γ chains in newborns. The excess β
chains form unstable tetramers (called hemoglobin H or HbH of 4 beta chains), which have abnormal oxygen
dissociation curves.

If you look at the distribution of alpha thalassemia in the different regions of the world, it is mainly found in
South East Asia and also in those regions where malaria was common, as well as in Sub-Saharan Africa
where it is still common. The reason for this localized prevalence is that, being carriers of alpha thalassemia
provided more resistance to the Plasmodium, giving thus evolutionary advantage to the affected patients in
regions where malaria was frequent.

26
There are two genetic loci for α globin, and thus four genes in diploid cells. Two genes are maternal in origin
and two genes are paternal in origin. The severity of the α thalassemias is correlated with the number of
affected α globin genes: the greater, the more severe will be the manifestations of the disease.

The molecular mechanism causing gene deletion is unequal crossing over of 2 chromatids.

• One allele affected: this is known as alpha thalassemia minima. Here there is minimal effect on hemoglobin
synthesis. Three α-globin genes are enough to permit normal hemoglobin production, and there are no
clinical symptoms. Patients may have a slightly reduced MCV and MCH. Genotype: -/α α/α

• Two alleles affected: called alpha thalassemia minor. Two α genes permit nearly normal production of red
blood cells, but there is a mild microcytic hypochromic anemia. The disease in this form can be mistaken
for iron deficiency anemia. Genotype: -/- α/α or -/α -/α. Alpha thalassemia minor can exist in two forms:

- alpha-thal-1, (αα/--), associated with Asians, involves cis-deletion of both alpha genes on the same
chromosome;

- alpha-thal-2 (α-/α-), associated with Africans, involves trans-deletion of alpha genes on different
(homologous) chromosomes.

• Three alleles affected: The condition is called Hemoglobin H disease. Two unstable hemoglobins are
present in the blood: Hemoglobin Barts (tetrameric γ chains which are due to a defective production of
alpha chains) and Hemoglobin H (tetrameric β chains). Both of these unstable hemoglobins have a higher
affinity for oxygen than normal hemoglobin, resulting in poor oxygen delivery to tissues. There is a
microcytic hypochromic anemia with target cells and Heinz bodies (precipitated HbH) on the peripheral
blood smear, as well as hepatosplenomegaly. The patients have Hb: 8-10 g/dL, which is compatible with
normal life without the need of transfusion, maybe during an infection or any complication hemoglobin can
decrease and they might need a transfusion during life. The disease may first be noticed in childhood or in
early adult life, when the anemia and hepatosplenomegaly are noted. The mechanism of anemia is
peripheral hemolysis so they have high reticulocytes count. Genotype: -/- -/α

• Four alleles affected: The fetus cannot live once outside the uterus and may not survive gestation: most
such infants are stillborn with hydrops fetalis (severe edema of the whole body due to the severe anemia),
and those who are born alive die shortly after birth. They are edematous and have little circulating
hemoglobin, and the hemoglobin that is present is all tetrameric γ chains (hemoglobin Barts). Genotype: -/-
-/-

Beta thalassemia:
β thalassemias are a group of inherited blood disorders caused by reduced or absent synthesis of the beta
chains of hemoglobin. This results in variable outcomes ranging from severe anemia to clinically
asymptomatic individuals. It is caused by mutations in the HBB gene on chromosome 11, inherited in an
autosomal recessive fashion. The molecular mechanism responsible for most cases of beta thalassemia is
point mutation which introduces a stop codon, which is TAG (Thymidine in DNA, Uracil in RNA). The severity
of the disease depends on the nature of the mutation. Beta thalassemia is found in the same regions of the
world as alpha thalassemia but with higher prevalence and in all countries facing the Mediterranean Sea so
Italy, south of Spain, south of France, Greece, Turkey and north Africa.

HBB blockage over time leads to decreased Beta-chain synthesis. This leads to the
underproduction of HBA. Microcytic anemia ultimately develops in respect to inadequate HBB for
sufficient red blood cell functioning. Due to this factor, the patient must undergo a blood
transfusion for survival to make up for the blockage in the Beta-chains. However, repeated blood
transfusions lead to build-up of iron overload, ultimately resulting in iron toxicity. This iron toxicity
produces myocardial siderosis and heart failure leading to the patient’s death.
If beta thalassemia is due to mutations that prevent the synthesis of the beta chain (for example a stop
codon in the beta gene, which results in no production of the beta chain), and the subject is homozygous for
this mutation, there will be no synthesis of beta chains. Will the baby with this homozygous mutation have
any clinical manifestation at birth? No. These babies are fully normal at birth because they have fetal
hemoglobin, which is characterized by gamma and alpha chains (alpha2gamma2). It will be only at the time
of the hemoglobin switch (6-12 months) that the disease will become apparent. For this reason thalassemia
major has been considered for many years as an acquired disease.

27
• Thalassemia minor: Only one of β globin alleles bears a mutation. Individuals will suffer from microcytic
anemia. Detection usually involves lower than normal MCV value (<80 fL). Genotype: β+/β or βo/β

• Thalassemia intermedia: Affected individuals can often manage a normal life but may need occasional
transfusions, e.g., at times of illness or pregnancy, depending on the severity of their anemia. Genotype
β+/β+ or βo/β+

• Thalassemia major: Occurs when both alleles have thalassemia mutations. This is a severe microcytic,
hypochromic anemia. Untreated, it causes anemia, splenomegaly and severe bone deformities. It
progresses to death before age 20. Treatment consists of periodic blood transfusion; splenectomy for
splenomegaly and chelation of transfusion-caused iron overload. Genotype: βo/βo

———Clinical case.——————————————————————————————————————
CBC of a 20-year-old man:

- Hb=13 normal
- MCV=65 microcytosis
- MCH=21.2 hypochromia
Hemoglobin analysis:
- Hb A=93.6% (normally >97%) slightly reduced
- Hb A2=5.5% (normally <3%) increased
- Hb F=0.9% (normally <1%)

The patient is affected by heterozygous beta thalassemia. The reason for the increase of Hb A2
is that there is a defective production of beta chains so more alpha chains are available.
By showing an increased amount of hemoglobin A2 we can conclude for heterozygous beta
thalassemia and we refer the patient to a center in which the beta globin gene can be sequenced
and molecular abnormalities can be detected.
It is important to make diagnosis because if this patient marries a carrier woman the risk of
having an affected child is 25%.

Patients have skeletal abnormalities like:

- Brush skull (“cranio a spazzola”), because the erythroid bone marrow is so expanded that
the bone has this aspect because erythroblast are 20 times more than normal but most of
them die of apoptosis.
- Hepatosplenomegaly, because there is the activation of hematopoiesis in organs in which it
was active only during fetal life (“Addome Batraciano”= frog-like abdomen).
The patient will be treated with regular transfusion every 2-3 weeks (depending on the body
weight). Initially it may be 1 red cell unit every 2 weeks or, in older individuals, 2 red cell units
every 3 weeks. The pre-transfusion hemoglobin shouldn't be lower than 9 because the lower the
hemoglobin the more severe the anemia and the bigger the dysfunction of the erythropoiesis
(thus greater skeletal abnormalities and so on).
With regular transfusion all of this patients develop hypogonadotropic hypogonadism at the time
of puberty due to transfusion iron overload because of the accumulation of iron in the liver,
anterior pituitary, pancreas and heart.
The next step in trying to improve the outcome of these patients with regular transfusion and iron
chelation therapy.
Survival has improved dramatically also because we are able to prevent the transfusion iron
overload. In fact now we have 2 iron chelators (with oral administration). These 2 compounds
have different effects: one is active on cardiac iron overload while the other one on liver iron
overload.
These are effective treatments, but they do not cure definitively the disease. The only effective
treatment in terms of cure is the allogeneic bone marrow transplantation: if you replace the
hematopoiesis of the patient with the hematopoiesis of a donor/sibling, the mutation will still be
present in any cell of the body except in hematopoietic stem cells therefore the disease will really
be cured.
The best results are obtained when transplantation is done before iron overload develops.

28
Gene therapy (replacing the mutant gene with a normal gene) has also been performed but it’s
not very effective.
The best approach remains to prevent the disease (it is a controversial issue) through prenatal
diagnosis (using DNA from chorionic villi or fetal DNA present in maternal plasma).

3.2 Sickle cell disease

Sickle-cell disease (SCD), also known as sickle-cell anaemia (SCA), is a hereditary blood
disorder, characterized by an abnormality in the oxygen-carrying haemoglobin molecule in red
blood cells. This leads to a propensity for the cells to assume an abnormal, rigid, sickle-like
shape under certain circumstances. Sickle-cell disease is associated with a number of acute and
chronic health problems, such as severe infections, attacks of severe pain ("sickle-cell crisis"),
and stroke, and there is an increased risk of death. Sickle-cell disease occurs when a person
inherits two abnormal copies of the haemoglobin gene, one from each parent. Several subtypes
exist, depending on the exact mutation in each haemoglobin gene.
Normally, humans have haemoglobin A (two alpha and two beta chains), haemoglobin A2 (two alpha and two
delta chains), and haemoglobin F (two alpha and two gamma chains) in their bodies. Of these, haemoglobin
F dominates until about 6 weeks of age then A dominates throughout life.
Sickle-cell conditions have an autosomal recessive pattern of inheritance from parents. The types of
haemoglobin a person makes in the red blood cells depend on what haemoglobin genes are inherited from
her or his parents. If one parent has sickle-cell anaemia and the other has sickle-cell trait, then the child has
a 50% chance of having sickle-cell disease and a 50% chance of having sickle-cell trait. When both parents
have sickle-cell trait, a child has a 25% chance of sickle-cell disease, 25% do not carry any sickle-cell alleles,
and 50% have the heterozygous condition.

In people heterozygous for HgbS (carriers of sickling haemoglobin) the normal allele is able to produce over
50% of the haemoglobin. In people homozygous for HgbS, the presence of long-chain polymers of HbS
distort the shape of the red blood cell from a smooth doughnut-like shape to ragged and full of spikes,
making it fragile and susceptible to breaking within capillaries. Carriers have symptoms only if they are
deprived of oxygen (for example, while climbing a mountain) or while severely dehydrated. The sickle-cell
disease occurs when the sixth amino acid, glutamic acid, is replaced by valine to change its structure and
function; as such, sickle-cell anemia is also known as E6V. Valine is hydrophobic, causing the haemoglobin
to collapse on itself occasionally. The structure is not changed otherwise. When enough haemoglobin
collapses on itself the red blood cells become sickle-shaped.

Haemoglobin S with this mutation is referred to as HbS, as opposed to the normal adult HbA. The genetic
disorder is due to the mutation of a single nucleotide, from a GAG to GTG codon on the coding strand, which
is transcribed from the template strand into a GUG codon. This is normally a benign mutation, causing no
apparent effects on the secondary, tertiary, or quaternary structures of haemoglobin in conditions of normal
oxygen concentration. What it does allow for, under conditions of low oxygen concentration, is the
polymerization of the HbS itself.

A person who receives the defective gene from both father and mother develops the disease (clinical
manifestations are highly variable due to the genetic background which can be characterized by many
polymorphisms. Overall the patient is anemic, but not transfusion dependent, and since the anemia is
hemolytic, the reticulocyte count is increased).
A person who receives one defective and one healthy allele remains healthy, but can pass on the disease
and is known as a carrier or heterozygote (no hematologic phenotype and no symptoms, normal CBC). You
can screen for sickle cell carriers using high performance liquid chromatography (the only abnormality you
can detect is the presence of hemoglobin S). Heterozygotes are still able to contract malaria, but their
symptoms are generally less severe.
Due to the adaptive advantage of the heterozygote, the disease is still prevalent, especially among people
with recent ancestry in malaria-stricken areas, such as Africa, the Mediterranean, India, and the Middle East.
Without endemic malaria, the sickle-cell mutation is purely disadvantageous, and tends to decline in the
affected population by natural selection, and now artificially through prenatal genetic screening.
The synthesis of beta chains is normal but the beta chains that are produced are qualitatively
abnormal.

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———Clinical case———————————————————————————————————————
• Very sick, 2 years old, Senegalese child, he comes to the pediatric ER with fever, severe anemia, tender
abdomen, and splenomegaly. The cause of this condition is acute venous obstruction of the spleen: splenic
sequestration crisis. This causes a condition of shock and can be treated only with emergency transfusion
and most often splenectomy.
• Patient with chest pain, Hb level 8.5, normocytic with leukocytosis, high platelets count, high reticulocytes
count (indicating peripheral hemolysis), and elevated bilirubin: hemolytic anemia. From the blood smear you
can see normal red cells and neutrophils but also sickle cells.
• Sicilian man with anemia, reduced MCV, increased platelet count and WBC count: sickle cell anemia is
usually associated with inflammation.

The mutant gene responsible for beta thalassemia and sickle cell disease is the same gene (HBB, encoding
globin beta) but with two different mutations:
- Beta0 thalassemia: the mutation introduces a premature stop codon leading to defective production of a
normal globin.
- Sickle Cell Anemia: the mutation leads to an amino acid substitution leading to normal production of a
slightly modified globin chain.

The abnormal sickle cells express integrins, that allow the linking between endothelial cells and the red cells,
mediating the adhesion of the sickle cells to the endothelial cells.
The result may be the occlusion of a vessel, and this can happen in any part of the body (including brain,
kidney, thorax, and bones). One of the typical manifestations of sickle cell anemia is the vasocclusive crisis,
which is a type of thrombosis mediated by abnormal red cells and not by platelets (like in others
thromboembolic disorders).

Any human being has 2 beta genes, one can have the mutation for sickle cell disease and the second one
can have the mutation for beta thalassemia.
If one parent is heterozygous for Beta thalassemia and the other parent is heterozygous for sickle cell
disease they have 25% of chances to have a baby who is a genetic compound and the result will be a child
affected by a condition called Micro-drepanocytosis.
The marker of microdrepanocytosis is a low MCV, because it has manifestation of both the diseases
(Microcytosis= beta thalassemia; Drepanocytosis= sickle cell disease).

We have two possibilities:

1. If the mutation is beta0: the individual will have no production of normal beta chain so the hemoglobin
composition will be almost identical to the classical sickle cell disease
2. If the mutation is beta+: the hemoglobin composition will be hemoglobin S, hemoglobin A, hemoglobin A2
and hemoglobin F.

———Clinical case—————————————————————————————————— —————

54 years old lady who came to the ER because of pain in the thorax and in the previous days she had flu,
which probably was the provocative factor of the vasocclusive crisis. Vasocclusion may involve also the bone
and there might be necrosis as a result of this, it may also be complicated by an overlapping infection so the
final result can be osteomyelitis. Typically osteomyelitis is due to Salmonella.
If vasocclusion involves the fingers in a young person, the subsequent development is that one finger may
be shorter than the remaining ones and this is called hand-foot syndrome.
Main clinical manifestation:
- Acute splenic sequestration
- Sepsis (particular pneumococcal sepsis), this because repeated infarctions of the spleen lead to functional
asplenia, therefore the spleen cannot prevent pneumococcal infection
- Acute chest syndrome: vasocclusion in any part of the chest
- Stroke: at the age of 10-20 years old.

Two treatments are recommended for these patients depending on the severity of the disease:
1. Hydroxyurea: it is a cytoreductive drug. It improves the production of hemoglobin F reducing the formation
of sickle cells and the vasocclusive crisis.

30
In case the disease is more severe, with a tendency to stroke:
2. Allogeneic hematopoietic stem cell transplantation. The mutation is still there but there is production of
normal RBCs. The problem is that transplantation is not always safe. It is safe in young people and less safe
in elderly people, this because the immunologic system developing after transplantation is the donor’s one
so the most difficult complication to treat is the Graft versus Host disease which means that the graft attacks
the recipient body. Moreover in order to do the transplant you need chemotherapy and radiotherapy to
destroy the patient’s bone marrow, they can have an impact on the future of the patients specially if they are
young in terms of gonadal function and so on.

4. Hematologic malignancies

Tumors of the hematopoietic and lymphoid tissues or hematopoietic and lymphoid malignancies are tumors
that affect the blood, bone marrow, lymph, and lymphatic system. As those elements are all intimately
connected through both the circulatory system and the immune system, a disease affecting one will often
affect the others as well, making myeloproliferation and lymphoproliferation (and thus the leukemias and the
lymphomas) closely related and often overlapping problems.
While uncommon in solid tumors, chromosomal translocations are a common cause of these diseases. This
commonly leads to a different approach in diagnosis and treatment of haematological malignancies.
Not all haematological disorders are malignant (“cancerous”).

Hematological malignancies may derive from either of the two major blood cell lineages:

I. myeloid cell line, which normally produces granulocytes, erythrocytes, thrombocytes, macrophages and
mast cells. Acute and chronic myelogenous leukemia, myelodysplastic syndromes and myeloproliferative
diseases are myeloid in origin.

II. lymphoid cell line, which normally produces B, T, NK and plasma cells. Lymphomas, lymphocytic
leukemias, and myeloma are from the lymphoid line.

A subgroup of them are more severe and are known as haematological malignancies or blood cancer. They
may also be referred to as liquid tumors.

For the analysis of a suspected hematological malignancy, a complete blood count and blood film are
essential, as malignant cells can show in characteristic ways on light microscopy. When there is
lymphadenopathy, a biopsy from a lymph node is generally undertaken surgically. In general, a bone marrow
biopsy is part of the "work up" for the analysis of these diseases.
A number of these diseases can now be classified by cytogenetics (AML, CML) or immunophenotyping
(lymphoma, myeloma, CLL) of the malignant cells.

Historically, hematological malignancies have been most commonly divided by whether the malignancy is
mainly located in the blood (leukemia) or in lymph nodes (lymphomas).
However, the influential WHO Classification (published in 2001) placed a greater emphasis on cell lineage.

• Leukemias:
- Acute lymphoblastic leukemia (ALL)
- Acute myelogenous leukemia (AML)
- Chronic lymphocytic leukemia (CLL)
- Chronic myelogenous leukemia (CML)
- Acute monocytic leukemia (AMoL)
- Other leukemias
• Lymphomas
- Hodgkin's lymphomas (all four subtypes)
- Non-Hodgkin’s lymphomas (all subtypes)
• Myelomas

Our understanding of the molecular basis of cancer has dramatically improved in the last 2 years, this mainly
thanks to the introduction of techniques of whole genome sequencing, allowing us to get an overview of
genetic sequences in cancer cells.
The output of our sequencing will be from 200 to 600 mutations and then we have to pick the significant
ones. Not all the mutations in fact are relevant in the pathogenesis of a cancer.

31
There are several types of mutations:
I. Founding mutations: are the ones occurring in the gatekeeper genes. These are genes that when
mutated initiate the process of tumorigenesis.
II. Passenger mutations are the mutations that don’t have any direct or indirect effect on the biology of the
cell. Passenger mutations alter the structure of the protein, but do not affect its function. The target of a
therapy must be the founding mutation, not the passenger mutation.
III. Sub-clonal mutations are all the mutations that occur only in a subset of cells of the neoplasm. These
mutations are useful in order to identify a sub clone of the original clone. Again this may have important
therapeutic implications because if we target a sub clonal mutation we only delete a part of the original
neoplastic clones.
IV. Driver mutations, any mutation that confers selective growth advantage to the cells. Usually both
founding and sub clonal mutations are driver mutations because they’re able to give a growth advantage
to the cells. Passenger mutations instead don’t drive the expansion, and thus are not driver mutations.
V. Silent mutation, is one that doesn’t affect the protein structure: nucleotidic changes that don’t alter the
amino acid formation.


Hematopoietic progenitor stem cells even in a normal subject may carry a certain number of age dependent
passenger mutations. If one of the hematopoietic stem cells of the individual acquires a functional relevant
mutation (a relevant driver founding mutation) this mutation will drive the expansion of mutated cells by
conferring a growth advantage. Then each of the cells belonging to the neoplastic clone may accumulate a
number of passenger mutations. This is the first source of clonal tumor heterogeneity. We are in fact dealing
with clonal cells, but all the cells may carry different mutations (passenger or sub clonal), in addition to the
original mutation.
One of the clones may acquire a driver mutation driving the expansion of the sub clone and another cell may
acquire different sub-clonal driver mutations, giving rise to another sub clone.
Most human cancers are due to some genetic alterations that may develop at any stage, and each of these
alterations is increasing the proliferative rate of cells, and is conferring a selective growth advantage.
We know about 140 genes that may confer advantage (driver genes).


Intrametastatic heterogeneity exists as well: different metastatic lesions of the same patient differ from the
others. Intrametastatic heterogeneity can occur with the same mechanism: the cell of the individual
metastasis acquire additional passenger or driver mutations, and their behaviour becomes equal to that of
the original clone.

Each metastasis differs from the other in the end.


Finally we have inter patient heterogeneity: even two types of the same tumor in two patients may have a
different genetic background. They share the founding mutation but then the genetic background and the
disease progression may differ.


Personalized or precision medicine implies that we might be able to identify all the specific relevant somatic
mutations in the patients, and then develop therapeutic strategies. Most of these data derived from
hematologic tumors, this because hematologic tumor cells can be isolated and collected, so we might have
samples of very clear cancer cells available for analysis from Bone Marrow. This is not the case of most
tumors, since from biopsies we find tumor cells, but also stroma and endothelial cells, rendering results more
difficult to interpret.


Myeloid neoplasms are tumors originating from hematopoietic stem cells or progenitor cells that are giving
origin to hematopoiesis in the bone marrow. Myeloid neoplasms are providing an interesting model of
carcinogenesis mechanisms, particularly because all the steps are evident, and we’re able to follow, identify
and maybe treat them. This doesn’t occur instead with solid tumors.

There are 3 major types of myeloid neoplasms:

- myeloproliferative neoplasms: the control of proliferation is lost (usually as a consequence of gain of
function mutations in cell signalling pathways), but differentiation capacity is kept, therefore resulting in
tumor cell population represented by mature cells, that may be red cells, granulocytes or platelets.
Characterized by:
• uncontrolled proliferation of cells in granulocytic lineage (resulting in increased number of granulocytes): chronic
myeloid leukemia
• uncontrolled production of red cells: polycytemia vera
• uncontrolled production of platelets: thrombocytopenia


32
- myelodysplastic neoplasms: the cell has lost the ability to differentiate (as a consequence of different kinds
of mutations) but it maintains the control on cell proliferation, so the result is the ineffective hematopoiesis,
in which bone marrow cells are unable to complete the maturation process. Myelodysplastic diseases are
classified in :
• Refractory anemia, characterized by less than 5% primitive blood cells (myeloblasts) in the bone marrow and
pathological abnormalities primarily seen in red cell precursors
• Refractory anemia with ring sideroblasts (RARS), also characterized by less than 5% myeloblasts in the bone
marrow, but distinguished by the presence of 15% or greater red cell precursors in the marrow being abnormal iron-
stuffed cells called "ringed sideroblasts”
• Refractory anemia with excess blasts (RAEB), characterized by 5-20% myeloblasts in the marrow
• Refractory anemia with excess blasts in transformation (RAEB-T) characterized by 21-30% myeloblasts in the
marrow (>30% blasts is defined as acute myeloid leukemia)
• Chronic myelomonocytic leukemia (CMML), not to be confused with chronic myelogenous leukemia or CML,
characterized by less than 20% myeloblasts in the bone marrow and greater than 1*109/L monocytes (a type of
white blood cell) circulating in the peripheral blood.

- acute myeloid leukemias: the mutation results in loss of control on proliferation and also on differentiating
capacity. This results in complete maturation arrest and abnormal proliferation of immature progenitor
cells.


Since 10 yrs we also recognize a category of overlapping disorders sharing myelodysplastic and
myeloproliferative characteristics: myelodysplastic-myeloproliferative neoplasms.

The useful info from next generation sequencing is the frequency of mutations: we’re now able to define the
temporal sequence of mutations. The most represented mutation is in the original clone, while mutations with
a smaller mutation borden are those which occurred during the clonal evolution. Based on the proportion of
mutations in the sample, we have an idea of the clonal evolution of the cells. This may be very helpful in
clinics and research, where we try to define the founding mutation.
The number of somatic mutations resulting in clonal hematopoiesis depends on age (are nearly absent in
subjects up to 15, but then progressively increase). No less than 10% of 70 yrs subjects have the clone in
their blood, and most of these subjects have a normal blood count, suggesting that these mutations are a
clonal advantage of the cells which does not interfere with hematopoiesis.
Subjects with evidence of one or more clones have a higher risk of developing hematologic malignancies in
the following 10 yrs compared with subjects with no mutations.

———Clinical case.——————————————————————————————————————
74 years old man, retired, he was former worker in a factory. In his medical history he mentioned that in the
last two months there has been the occurrence of fatigue and dyspnea during moderate efforts. His GP
prescribed him a blood count which showed Hb 10.2 g/dL, MCV 106 fL (slightly high). WBC count was 3.8
billion cells/dL (slightly low) and platelet count 97 billion cells/dL (low). A reduction of the cells of all three
lineages is defined as pancytopenia. The absolute number of reticulocytes was normal, but in a patient with
anemia this number is inadequate for the Hb level. So we are facing the anemia of Ineffective erythropoiesis
or hypoproliferative anemia. If reticulocyte count was increased, we were facing anemia of hemolysis or
hemorrhage. Folic acid and vitamin B12 were normal, slightly macrocytic anemia, with no sign of hemolysis.
Having a macrocytic anemia without evidence of vitamin deficiency is an indication to proceed to BM
investigation. BM aspirate showed an increase in cellularity with gross hyperplasia and morphological
abnormality: dysplasia involving erythroid, myeloid and megakaryocytic cells. Conventional cytogenic
analysis showed deletion of the long arm of chromosome 11. Conclusion: myelodysplastic syndrome.

The disease is strongly age related and overall incidence is about 4-5 new cases per 100.000 people/year,
more than 40% in patients more than 70 yrs old. It is a typical disease of aging and this is not surprising
because the accumulated number of mutations results in an increasing probability of having a founding
mutations.
Looking at the peripheral blood smear, you can find normal granulocytes as well as hypo-lobulated
granulocytes.
The situation is different in megaloblastic anemia: the granulocytes are hyper-lobulated. In the bone marrow
there is hypercellularity, since the hematopoiesis tries to overcome the differentiation inefficiency and
therefore progenitors are usually hyper-plastic.
Ring sideroblasts are erythroid precursors, with iron loaded mitochondria. They are typical of a subtype of
myelodysplastic syndrome: refractory anemia with ring sideroblasts.

33
In 2011 the molecular basis of refractory anemia with ring sideroblasts was discovered. Recurrent somatic
mutations in genes encoding for splicing factors were detected in a high proportion of these patients (the
spliceosome is a complex mechanism of nucleoproteins, whose aim is to remove the non coding introns from
mRNA).
TEX2, DNMT8, ASXL1 are the three most frequent genes found in normal subjects with clonal
hematopoiesis, these genes provide the cells with proliferative advantage, without major impact on function
and differentiation of these cells.

In patients with low risk and early disease without clonal evolution, we might stimulate the BM to increase its
production through recombinant human erythropoietin. If there is clonal evolution we might try to eradicate
the clone by more intensive treatment. If the patient is young, but this situation is not frequent in MDS, we
might try to cure the disease by allogeneic stem cells transplantation.
This is what we define individual risk based therapeutic strategy: adapt the therapy to the risk of each
patient. We are now in a phase where we have a rather clear idea of molecular basis, but none of the
therapies target molecular lesions involved in the progression of the disease. There are however some
splicing modulators in vitro studies going on and these could be available for patients carrying the mutation
in the future.

5. Myeloproliferative neoplasms

The myeloproliferative neoplasms (MPNs), are a group of diseases of the bone marrow in which excess cells
are produced. They are related to, and may evolve into, myelodysplastic syndrome and acute myeloid
leukemia, although the myeloproliferative diseases on the whole have a much better prognosis than these
conditions.
The increased numbers of blood cells may not cause any symptoms, but a number of medical problems or
symptoms may occur. The risk of thrombosis is increased in some types of MPN.
Although not a malignant neoplasm like other cancers, MPNs are classified within the hematological
neoplasms.
There are four main myeloproliferative diseases, which can be further categorized by the presence of the
Philadelphia chromosome. The Philadelphia chromosome is a specific abnormality of chromosomal
chromosome 22, which is unusually short. It is the result of a reciprocal translocation between chromosome
9 and chromosome 22. This gives rise to a fusion gene, BCR-ABL, that juxtaposes the Abl1 gene on
chromosome 9 to a part of the BCR ("breakpoint cluster region") gene on chromosome 22. The presence of
this translocation is a highly sensitive test for CML, since 95% of people with CML have this abnormality.
However, the presence of the Philadelphia (Ph) chromosome is not sufficiently specific to diagnose CML,
since it is also found in acute lymphoblastic leukemia (ALL, 25–30% in adult and 2–10% in pediatric cases)
and occasionally in acute myelogenous leukemia (AML).

Philadelphia chromosome positive

• Chronic myelogenous leukemia (CML)
Philadelphia chromosome negative
• Essential thrombocytosis (ET)
• Polycythemia vera (PV)
• Myelofibrosis (MF)
All MPNs arise from precursors of the myeloid lineages in the bone marrow. The lymphoid lineage may
produce similar diseases, the lymphoproliferative disorders (acute lymphoblastic leukemia, lymphomas,
chronic lymphocytic leukemia and multiple myeloma).
Most Philadelphia chromosome negative cases have an activating JAK2 or MPL mutation.

5.1 Chronic Myeloid Leukemia

Chronic myelogenous (or myeloid or myelocytic) leukemia (CML), is a cancer of the white blood cells. It is a
form of leukemia characterized by the increased and unregulated growth of predominantly myeloid cells in
the bone marrow and the accumulation of these cells in the blood. CML is a clonal bone marrow stem cell
disorder in which a proliferation of mature granulocytes (neutrophils, eosinophils and basophils) and their
precursors is found. It is a type of myeloproliferative disease associated with a characteristic chromosomal
34
translocation called the Philadelphia chromosome. CML is now largely treated with targeted drugs called
tyrosine kinase inhibitors (TKIs) which have led to dramatically improved long-term survival rates. These
drugs have revolutionized treatment of this disease and allow most patients to have a good quality of life
when compared to the former chemotherapy drugs.
CML was the first cancer to be linked to a clear genetic abnormality, the chromosomal translocation known
as the Philadelphia chromosome. In this translocation, parts of two chromosomes (the 9th and 22nd) switch
places. As a result, part of the BCR ("breakpoint cluster region") gene from chromosome 22 is fused with the
ABL gene on chromosome 9. This abnormal "fusion" gene generates a protein. Because ABL carries a
domain that can add phosphate groups to tyrosine residues (a tyrosine kinase), the BCR-ABL fusion gene
product is also a tyrosine kinase.
The fused BCR-ABL protein interacts with the interleukin 3beta(c) receptor subunit. The BCR-ABL transcript
is continuously active and does not require activation by other cellular messaging proteins. BCR-ABL
activates a cascade of proteins that control the cell cycle, speeding up cell division. Moreover, the BCR-ABL
protein inhibits DNA repair, causing genomic instability and making the cell more susceptible to developing
further genetic abnormalities. The action of the BCR-ABL protein is the pathophysiologic cause of chronic
myelogenous leukemia. With improved understanding of the nature of the BCR-ABL protein and its action as
a tyrosine kinase, targeted therapies (the first of which was imatinib mesylate) that specifically inhibit the
activity of the BCR-ABL protein have been developed. These tyrosine kinase inhibitors can induce complete
remissions in CML, confirming the central importance of bcr-abl as the cause of CML.

———Clinical case.——————————————————————————————————————
This was a young woman who did a complete blood count before deciding to take the pill. The white blood
cell count was found to be considerably increased. The platelet count was also considerably increased, and
within the increased WBC, it was mainly myeloid cells (neutrophils, monocytes) that were increased.
Lymphocytes were in proportion decreased, but the absolute number was normal. There were also immature
granulocytes (precursors of neutrophils, eosinophils and basophils), that are normally found only in the bone
marrow but in this condition can circulate in the peripheral blood. The woman thus had an overall increase in
WBCs and in peripheral blood there were also cells that normally shouldn’t be present, such as myelocytes,
that are cells found in the bone marrow and are granulocytic precursors. Also promyelocytes could be seen,
thus the condition was not only characterized by increased numbers of WBC, but also by the presence of
immature WBC in peripheral blood. These are not the cells we call “blast”, which are the most immature
ones, like myelobasts, these are granulocytic precursors, their presence in peripheral blood is abnormal and
suggests that there is a myeloproliferative disorder. Leukocytosis and thrombocytosis suggest expansion of
granulopoiesis and megakariocytopoiesis and so a myeloproliferative disorder.

Nowadays more than half of cases are diagnosed through a routine CBC before any clinical manifestation
appears. This CML was called “chronic” many years ago, when Acute Myeloid Leukemia had a survival of a
few weeks or a few months, so this condition with a median survival at that time of a few years, was defined
as chronic. After the chronic phase typically the condition progresses to what we call accelerated phase, this
time anemia develops, blastocytes like myeloblasts or lymphoblasts appear in peripheral blood or bone
marrow, there is shift from thrombocytosis to thrombocytopenia, systemic symptoms appear like fever, and
skeletal pain is very common due to the expansion of the hematopoietic bone marrow. Finally this condition
rapidly progresses to a condition similar to Acute Myeloid / Lymphoid Leukemia, called “blast-crisis” which
even nowadays inevitably leads to death if there is no way of performing an allogeneic stem cell
transplantation. The bone marrow pattern in myeloid blast crisis is mostly constituted by blastocysts there is
one neutrophil here, but most of the cells are poorly differentiated, this illustrates the inability of this bone
marrow to differentiate, maturate and produce peripheral blood cells. Sometimes the cells have a lymphoid
pattern and we describe this condition as “lymphoid blast crisis”.

So initially there is leukocytosis, but the granulocytic marrow is still able to undergo differentiation and
produce mature cells, like neutrophils, eosinophils or basophils. Erythropoiesis is normal while
megakariocytopoiesis is expanded and there is thrombocytosis.
With the progression of the disease, erythropoiesis becomes inefficient, so anemia develops. There is
increasing inability to produce mature cells and so blast cells accumulate in peripheral blood and bone
marrow and also megakariocytopoiesis declines and there is a lower production of platelets and eventually
when blast crisis develops, most of the cells in the peripheral blood and bone marrow are blast cells
incapable of differentiation and maturation.
In the chronic phase we do not find blast cells in the peripheral blood, we find precursors of neutrophils.

35
The mutated stem cells (with BCR-ABL translocation) have a lower propensity to apoptosis, which means
that they have an advantage over normal stem cells and quite easily generate a clone. One of the cellular
consequences of the oncoprotein is that the hematopoietic precursors have lower adhesion to the bone
marrow matter, and this explains why some immature granulocytes are found in peripheral blood. Genomic
instability means that these cells have a higher propensity to acquire additional mutations, the so called sub-
clonal events that lead to the progression of the disease and to blast crisis.
The additional events are not associated with a gain of function, but loss of function. So these cells are less
able to differentiate and mature. The initial mutation forms the clone and the clone undergoes additional
mutations that make it unable to produce normal amounts of blood cells. But this is exactly what happens
also in myelodysplastic syndromes.

Chronic myeloid leukemia represents the first example of target therapy: at least 4 drugs that specifically
inhibit the erythroid activity and selectively killing mutant cells were developed.

Interferon-alpha was produced as a recombinant molecule, it was shown to be able to suppress cell
proliferation. Clinical trials on the use of IFN-alpha compared to hydroxyurea showed that there was a
significant prolongation of survival and also significant cytogenetic response. The drug was able to inhibit
selectively the mutant cells and to allow the emergence of normal stem cells and restoration of normal
hematopoiesis in some patients. Median survival was 8-9 years, so significantly longer, but most patients
again underwent accelerated phase and blast crisis. And at that time the only curative treatment was
allogeneic stem cell transplantation: if you remove the whole bone marrow by chemotherapy or radiotherapy
or by a combination of both and infuse the donor stem cells, you restore the donor hematopoiesis. However
this treatment has a high morbidity and a mortality of about 10-30%, depending on the patients age and
essentially because of GVHD.
The idea was then to develop a drug that could occupy the ATP pocket, preventing the ATP entrance and
deleting any ability of the cell to have energy for tyrosine kinase activity. Imatinib was eventually developed,
which is very effective and has a much higher affinity for this pocket compared to ATP and so is able to
suppress cell proliferation.
Observing the Kaplan Meyer estimator curve, Imatinib is much better than the best available treatment at
that time, which included Interferons. Another interesting point is the actual risk of having a complete
cytogenetic response, which means specific suppression of the mutant clone and restoration of normal
polyclonal hematopoiesis, and as you see here Imatinib is effective in about 90% of cases in doing that,
compared to at that time best available treatment.
In 2001 one group that was very active in studying Imatinib, reported that there may be a clinical resistance
to treatment because of BCR-ABL gene mutation or amplification. And this side-effect is exactly the same
that has been described many years ago with antibiotics and bacteria, so if there is a mutation in the ATP
pocket of the oncoprotein, a mutation that prevents binding of Imatinib to the oncoprotein, Imatinib is no
longer effective.

There are 2 possibilities:

- the mutation is present already in the beginning
- the mutation develops during treatment

If the mutation is present initially, lets say we have 1000 cells with a BCR-ABL fusion gene and 1 of these
1000 has an additional mutation that prevents binding of Imatinib, what is the mechanism of resistance?
Pressure selection. So 999 without the mutation will be inhibited and eliminated, the only one with the
additional mutation survives because it is not inhibited and expands. So even if the number of mutant cells is
low initially, inevitably they will expand.
In order to overcome this problem, additional specific inhibitors have been developed:
- Dasatinib
- Nilotinib
These new drugs however have additional effects which were not clear initially in clinical trials, but which are
relevant in clinical practice and one of them includes cardiac failure.
For patients carrying the BCR-ABL gene with this type of mutation, allogeneic Stem Cell transplantation is
still the only curative way.
This is a rare disease.

In summary, in MDS, the funding mutation is usually the one affecting genes of DNA methylation (TET2,
DNMT3a) or genes of DNA splicing machinery (SR3B1 and SRSF2). There is then a disease because the

36
hematopoiesis became clonal, but this occurs without a phenotypic expression of it. Probably we need at
least one gene for creating a morphological abnormalities and ineffective hematopoiesis: the clinical
manifestation of MDS are anemia, leukopenia and most commonly thrombocytopenia although there is one
specific MDS that associated with 5q-deletion that is thrombocytosis.

In CML, the initiating event is 9 —> 22 chromosomes translocation with the formation of the BCR-ABL1
fusion gene. This somatic mutation by itself is able to form a clone and give rise to the clinical manifestation
of the disease, because it leads to a hyper-proliferative bone marrow with overproduction of leukocytes
(leukocytosis) and thrombocytes (thrombocytosis). In this case the additional somatic mutation events are
those responsible for progression to blast crisis or acute myeloid leukemia but in some cases we saw also
acute lymphoblastic anemia. In AML instead, there is a defect in the differentiation capacity of stem cells so
there is accumulation of blast cells and bone marrow failure with overproduction of immature blastocyst.
Most of the mutation responsible for AML start to be acquired at the initiating event which may cause
genomic instability and drive clonal evolution.
CML is a disease associated with a single initiating event, the BCR-ABL fusion gene. The BCR-ABL fusion
gene is detected starting from a CBC. Karyotyping is effective, it shows the translocation. Then Fluorescence
in situ hybridization is performed, and this allows to show the fusion gene in all cells. PCR is then the typical
approach to show the fusion gene. And RT-PCR so reverse-transcriptase PCR, is now used for defining the
response, in the sense that it estimates the fusion gene at RNA level, so the expression of the fusion gene.
The lower the amount of fusion gene RNA, the better the response to treatment.

Overall PV and ET and PMF are due to a gain of function mutation of the JAK2 gene. These cells have lower
tendency to apoptosis and advantage on the normal stem cell and this leads to formation of a clone. Initially
this patient had an heterozygous clone but in one stem cell mitotic recombination occurred and one of the
daughter cell became homozygote for the mutation. The homozygous clone then has an additional
advantage, so the marrow becomes full with cells characterized by this mutation. Patient with a small number
of cells that are heterozygous for this mutation, mainly have thrombocytosis so ET. Homozygous patients
have also erythrocytosis, so PV. Over time a high mutant allele burden leads to fibrotic bone marrow and
abnormal cell trafficking and so to PMF.

Essential thrombocythemia (ET) is mainly associated with isolated thrombocytosis but it involves a high risk
of thromboembolic complications. ET may progress to PV or directly to myelofibrosis and AML.

Primary myelofibrosis (PMF) is characterized by bone marrow fibrosis, splenomegaly, abnormal cell
trafficking (a high number of stem cells are exiting the bone marrow and circulate in the peripheral blood and
colonize spleen and liver leading to myeloid metaplasia). AML is very likely in patient with PMF.

5.2 Polycythemia Vera

Polycythemia vera is a neoplasm in which the bone marrow produces too many red blood cells. It may also
result in the overproduction of white blood cells and platelets.
Most of the health concerns associated with polycythemia vera are caused by the blood being thicker as a
result of the increased number of red blood cells. It is more common in the elderly and may be symptomatic
or asymptomatic.
Clinical manifestation: aquagenic pruritus, wide range of vascular symptoms, head ache, dizziness, buzzing,
acrobat ataxia, ocular disturbance, and Raynaud's phenomenon observed in winter when the cold hands
become painful and red. Sometimes there is bleeding of the nose, gingiva and GI but the most common
complication is thrombosis, myocardial infarction, ischemia, deep vein thrombosis, arthropathy, and
superficial vein thrombosis. Patients with polycythemia vera are more likely to have gouty arthritis.
The main objective of treatment is to prevent thrombosis, which is responsible for considerable morbidity. In
some patients basophils are increased and probably this is related to the vascular symptoms. To decide
which treatment should be given, we decide if the patient is at high or low risk for thromboembolism
complication.
Low risk patients are those below the age of 50 (because thrombosis is less common), and those who had
no previous thrombosis and bleeding. In a low risk patient we perform regular phlebotomy (because
hemoglobin >17 and hematocrit 50%), lowering the hematocrit bellow 45%. The hematocrit should be always
bellow 45%, so the risk for thrombosis will be also lower. Low dose aspirin can be administered for the rest of
their life.

37
High risk patients are those who are 60 or older and had previous bleeding/thrombosis. these patients
should also do cytoreductive treatment (hydroxyurea or interferon alpha), in order to reduce RBC, WBC and
palates.
In order to diagnose polycythemia vera in a patient you do CBC, peripheral blood smear, bone marrow
biopsy (shows high cellularity and is the only way to diagnose fibrosis) and PCR for JAK2 V617F mutation
(95% of the patients, the remaining 5% are exon 12 mutations).
Polycythemia vera (PCV) is caused by neoplastic proliferation and maturation of erythroid, megakaryocytic
and granulocytic elements to produce what is referred to as panmyelosis. In contrast to secondary
polycythemias, PCV is associated with a low serum level of the hormone erythropoietin (EPO). PCV cells
have a mutation in the tyrosine kinase (JAK2), which acts in signalling pathways of the EPO-receptor,
rendering those cells hypersensitive to EPO.
Polycythemia vera (PT) is characterized by erythrocytosis but what does polycythemia mean? All of the cells
are overproduced and there is erythrocytosis, thrombocytosis and leukocytosis. Most patient have
splenomegaly and most patients develop thromboembolic complications. PV over many years tends to
progress to myelofibrosis and then to AML.

———Clinical case.——————————————————————————————————————
man who presented to the ER with chest pain that started one hour earlier while sleeping. The pain was
severe substernal, radiating to the left arm, and was accompanied by nausea and vomiting. In the past he
had experienced intermittent chest pain with similar features but less intense. The doctor in the ER asked for
electrocardiogram, which shows erasing of the ST segments in V2 to V6. This is diagnostic of acute
myocardial infarction. The patient underwent coronary angiogram and the anterior and left descending artery
was found to be closed. After removal of the thrombus and insertion of the stent the flow was restored. So
the infarction was due to occlusion. After this initial treatment the patient started double antiplatelet
aggregation with low dose aspirin and another drug for at least 1 year in order to prevent occlusion of the
stent. Blood count showed high hemoglobin levels (above 17), leukocytosis and thrombocytosis. Normally
acute myocardial infarction involves inflammation, thus after the infarction there is a decrease in hemoglobin.
In this case we had also leukocytosis and thrombocytosis and diagnosis was easier. The patient had
polycythemia vera and the reason for erythrocytosis is somatic mutation in hematopoietic stem cells.

There are families with gain of function mutation of the erythropoietin receptor. The receptor is shorter and
has better transduction efficiency of the signal so these families have hemoglobin levels around 19-20
without organ damage.
Secondary erythrocytosis is the most common type and occurs in individuals that have many types of
hypoxia and appropriately increased plasma erythropoietin. The cells responsible for RBCs production sense
the oxygenation levels. If this is low, there is an increase erythropoietin production which causes expansion
of the erythroid marrow and leads to over production of RBC. The condition is due for example to high
altitude. Secondary erythrocytosis may also occur in patients with right to left shunt (blood goes from left to
right without passing the lung circulation), chronic bronchitis, pulmonary emphysema, variants of hemoglobin
with high affinity for oxygen and lower release of oxygen to tissue, patients with inherited mutation of the
hypoxia inducible factor pathway of oxygen cells. The erythrocytosis secondary to pulmonary disease is the
most common.
PV is rare condition and the average diagnosis is made when patients are about 60 years. In 2005 it was
found a gain of function somatic mutation of the JAK2 gene, which encodes for a protein that has the
following function: when erythropoietin binds its receptor there is dimerization of the receptors and then
activation of JAK2 with phosphorylation and signal transduction. So binding of erythropoietin activates these
proteins and then activates those genes. Erythropoietin main function on the nephroid cells is the prevention
of apoptosis. So this transduction means that genes responsible for apoptosis are silenced. But this receptor
and JAK2 are not specific for erythropoietin receptor but they also true for G-CSF the main factor responsible
for neutrophil production and thrombopoietin receptor that is responsible for megakaryocyte differentiation
maturation and platelet production.
So JAK2 plays a major role in transduction of erythropoietin G-CSF signal and thrombopoietin signal.
It was found that the most common chromosome abnormality in PV is acquired uniparental disomy of
chromosome 9 (called also copy neutral loss of heterozygosity). This results when the cell duplicates and
there is exchange of chromatids, which leads to uniparental disomy. From heterozygous cell you will get one
normal cell and one homozygous cell. Mutation was found in about 95% of PV, 50-70% of ET and same in
PMF. The mutation occurs in the pseudokinase domain of JAK2. So the protein has a tyrosine kinase domain
which is responsible for phosphorylation but in order to limit the activity of the protein there is also a
pseudokinase function that suppresses the activity of the protein. If this domain is mutated there is less

38
limitation of the activity and so transduction is more affective. In this sense this is a gain of function mutation
which leads to over transduction of erythropoietin signal but also of G-CSF and Thrombopoietin activity. This
explains why this patient have typically erythrocytosis, thrombocytosis, and leukocytosis. 95% of patient with
PV have this unique mutation the remaining 5% have different mutations in the same gene but in different
exons.

5.3 Essential Thrombocythemia

Essential thrombocythemia is a rare chronic blood disorder characterised by the overproduction of platelets
by megakaryocytes in the bone marrow. It may develop into acute myeloid leukaemia or myelofibrosis. It is
one of four myeloproliferative disorders (disorders characterised by increased production of a particular line
of blood cell). It is an indolent condition. ET shares with the developing red cells in polycythaemia vera the
fact that developing megakaryocytes are more sensitive to growth factors. Platelets derived from the
abnormal megakaryocytes are activated, which, along with the elevated platelet count, contributes to the
likelihood of thrombosis. The increased possibility of bleeding when the platelet count is over 1 million is due
to von Willebrand factor (vWF) sequestration by the increased mass of platelets, leaving insufficient vWF for
platelet adhesion. A mutation in the JAK2 kinase (V617F) is present in 40–50% of cases.
Hemorrhagic complications are mainly observed in patients with very high platelet count (in the order of 1.5
or 2 billions), because when the platelet count is very high, circulating platelet bind to von Willebrand factor
and this process lead to low levels of vWF. Since vWF is a protein responsible for adhesion of platelets to the
endothelium, low levels of vWF impair platelets adhesion which is the first step of hemostasis, thus
hemorrhage is a typical complication. So, paradoxically, the higher the platelet count, the lower the risk of
thrombosis and the higher the risk of a hemorrhage. In AML with disseminated intravascular coagulation this
level is combined with low platelet count because of acute leukemia. So the combination of
thrombocytopenia and low coagulation factors is responsible for the hemolysis.
There is also a genetic disease which is due to the defective production of vWF but the final result platelet
adhesion is less efficient and hemolysis is more likely.
In order to understand why there is overproduction of platelets in ET, we should remember that platelets are
produced by megakaryocytes. The pro-platelets are expansion of the megakaryocytes cytoplasm, which
enter the vessel. The main factor responsible for differentiation and maturation of megakaryocyte is
thrombopoietin, and the receptor of thrombopoietin is called c-MPL. Thrombopoietin is produced by liver
cells, it circulates, reaches the bone marrow and binds the thrombopoietin receptor that is a dimeric receptor
(very similar to the erythropoietin receptor) and signal transduction is again mediated by JAK2. So, gain of
function mutations of JAK2 involve also a higher production of platelets by megakaryocytes.
Also ET is a rare disorder, more commonly found in females, and more commonly in adults or elderly
patents.
It was found that the somatic mutations of MPL, the receptor of thrombopoietin, were responsible for ET or
PMF (primary myelofibrosis). A gain of function mutation of the same gene, can lead to deferent phenotypes
depending on the heterozygous or homozygous state.
However, 1/3 of patient with PV and PMF did not carry any of these mutations. After whole gene sequencing,
the mutation was found to be in the transmembrane part of the receptor, and to lead to a more efficient
transduction of TPO. So it is a gain of function mutation that leads to ET. Can this patient have also PV? No,
because the receptor is specific and leads only to reproduction of platelets and not of red cells.
A whole sequencing of peripheral blood granulocytes and T lymphocytes was performed as well. T
lymphocytes were the control tissue because T lymphocytes do not derived from myeloid cells. A somatic
mutation of calreticulin was found. This is a gene encoding a protein that controls cytoplasmic calcium. 2/3 of
patient have a gain of function of JAK2 which leads to better transduction of TPO (thrombopoietin) 5% of
patients have a somatic mutation of c-MPL exon 10 ,which involves again a better transduction of TPO signal
and for reasons we don’t know somatic mutation of calreticulin, a protein which operates in the cytoplasm of
the megakaryocytes, leads again to ET or PNF in about 1/4 -1/3 of patients.
The reason why many patients develop myelofibrosis is that the activated megakaryocyte not only produces
an excess of platelets but also of collagen fibers. So there is a deposition of these fibers in the bone marrow
matrix.
These patients have a normal life expectancy, while the polycythemia vera patients have a decrease life
expectancy, mainly because of a progression to myelofibrosis and acute myeloid leukemia. The ET has a
higher risk of thromboembolic complication, hemolysis and there may be progression to secondary
myelofibrosis. Progression to AML is normally low and it is due to additional somatic mutations that are
responsible of defective differentiation of hematopoietic cells.
We use regularly low dose aspirin as an antiplatelet drug with the only exception of patients with very high
platelets count and acquired vWF disease: in these patients using a low dose aspirin is contra indicating
because the major risk is hemorrhage.

39
The risk of progression to polycythemia vera is about 25-30% in 15 years, and is mainly related to the
occurrence of mitotic recombination.

4.4 Primary Myelofibrosis

Myelofibrosis, also known as osteomyelofibrosis, is a rare bone marrow cancer. It is currently classified as a
myeloproliferative neoplasm, in which the proliferation of an abnormal clone of hematopoietic stem cells in
the bone marrow and other sites results in fibrosis, or the replacement of the marrow with collagenous
connective tissue fibers. The term myelofibrosis alone usually refers to primary myelofibrosis (PMF) (chronic
idiopathic myelofibrosis [cIMF]), the idiopathic form of the disease, in contrast with myelofibrosis secondary
to polycythemia vera or essential thrombocythaemia.
Myelofibrosis is a clonal neoplastic disorder of haematopoiesis, and is one of the myeloproliferative
disorders. Production of cytokines such as fibroblast growth factor by the abnormal hematopoietic cell clone
(particularly by megakaryocytes) leads to replacement of the hematopoietic tissue of the bone marrow by
collagen fibrosis, impairing the patient's ability to generate new blood cells and resulting in a progressive
pancytopenia. However, the proliferation of fibroblasts and deposition of collagen is a secondary
phenomenon, and the fibroblasts are not themselves part of the abnormal cell clone.
In primary myelofibrosis, progressive scarring, or fibrosis, of the bone marrow occurs, for the reasons
outlined above. The result is extramedullary hematopoiesis, or blood cell formation occurring in sites other
than the bone marrow, as the haemopoetic cells are forced to migrate to other areas, particularly the liver
and spleen. This causes an enlargement of these organs (hepatomegaly and splenomegaly), which also
contributes to causing pancytopenia, particularly thrombocytopenia and anemia. Another complication of
extra-medullary hematopoiesis is poikilocytosis, or the presence of abnormally shaped red blood cells.
Kaplan Meier curve suggests that patients with calreticulin mutation have a median of 18 years of survival, 9
for JAK2 and MPL, and 3 years only for those without these mutations, indicating that it is a more aggressive
disease with genetic basis that we don’t know at the moment. If a patient will come without any of these
mutation and with clinical picture of PMF he will need an allogeneic stem cell transplantation, while in
calreticulin mutation we can wait because the survival is about 18 years. If the only problem is anemia we
can use recombinant human erythropoietin or in some patient prednisone. If the main problem is
splenomegaly, severe thrombocytosis or leukocytosis, until recently we used hydroxyurea but now we are
starting to use the JAK2 inhibitor. When splenomegaly is really massive, there might be the need to perform
splenectomy.

6. Myeloid neoplasms

6.1 Myelodysplastic syndromes

The myelodysplastic syndromes are hematological medical conditions characterized by ineffective
production (or "dysplasia") of all blood cells.
Patients with MDS can develop severe anemia and require blood transfusions. In some cases, the disease
worsens and the patient develops cytopenias (low blood counts) caused by progressive bone marrow failure.
Many people live normal lifespans with MDS. Often, people are asymptomatic and are unaware they even
have MDS until it shows up in a routine blood test.
In MDS, hematopoiesis is disorderly and ineffective. The number and quality of blood-forming cells decline
irreversibly, further impairing blood production.
The median age at diagnosis of a MDS is between 60 and 75 years; a few patients are younger than 50;
MDS diagnoses are rare in children. Signs and symptoms are nonspecific and generally related to the blood
cytopenias:
• Anemia: chronic tiredness, shortness of breath, chilled sensation, sometimes chest pain;
• Neutropenia: increased susceptibility to infection;
• Thrombocytopenia: increased susceptibility to bleeding and ecchymosis (bruising), as well as
subcutaneous hemorrhaging resulting in purpura or petechia.

Many individuals are asymptomatic, and blood cytopenia or other problems are identified as a part of a
routine blood count:
• neutropenia, anemia and thrombocytopenia (low cell counts of white and red blood cells, and platelets,
respectively);
• splenomegaly or rarely hepatomegaly;
• abnormal granules in cells, abnormal nuclear shape and size; and/or
• chromosomal abnormalities, including chromosomal translocations and abnormal chromosome number.

40
There are additional myeloproliferative disorders which are very rare, and additional types of fusion genes.
For instance there is a rare myeloproliferative disorder associated with a translocation 5-12 and the
generation of a fusion gene. In this case there is overproduction of eosinophils which accumulate in the skin.
Investigators showed that the oncoprotein generated by this fusion gene has an ATP pocket similar to that of
the BCR-ABL oncoprotein and so they treated these patients with Imatinib and the efficacy is clearly visible
here, because all the abnormal accumulation of eosinophils disappears with treatment.

Since at least 1974, the deletion in the long arm of chromosome 5 has been associated with dysplastic
abnormalities of hematopoietic stem cells. Chromosome 5q deletion syndrome is an acquired, hematological
disorder characterized by loss of part of the long arm (q arm, band 5q31.1) of human chromosome 5 in bone
marrow myelocyte cells. This chromosome abnormality is most commonly associated with the
myelodysplastic syndrome.
The 5q-syndrome is characterized by macrocytic anemia, often a moderate thrombocytosis,
erythroblastopenia, megakaryocyte hyperplasia with nuclear hypolobation, and an isolated interstitial deletion
of chromosome 5.
The 5q- syndrome is found predominantly in females of advanced age. Several genes in the deleted region
appear to play a role in the pathogenesis of 5q-syndrome. Haploinsufficiency of RPS14 plays a central role,
and contributes to the anemia via both p53-dependent and p53-independent tumor suppressor effects. Other
genes at this region include miR-145 and miR-146a, whose deletion is associated with the megakaryocytic
dysplasia and thrombocytosis seen in 5q- syndrome; SPARC, which has antiproliferative and antiangiogenic
effects; and the candidate tumor suppressors EGR1, CTNNA1, and CDC25C.

———Clinical case.——————————————————————————————————————
Here we have a patient with a combination of anemia which is quite macrocytic, normal WBC count and a
marginally elevated platelet count. If you perform a blood smear you will see few abnormal granulocytes with
few granules. In these patients we regularly perform a bone marrow aspiration. Cellularity is quite good, but
there is a high number of these cells which are much greater than the remaining bone marrow cells:
megakaryocytes. But these megakaryocytes are abnormal because they are mono-nucleated. Typical
megakaryocytes have multiple nuclei. Performing a cytogenetic examination, deletion 5q was observed.

Neutrophils had hypo-segmented nuclei are a finding typical of myelodysplastic syndromes.

The condition typically associated with hyper-segmentation is instead Megaloblastic anemia, which is very
similar as clinical presentation and CBC to myelodysplastic syndromes. So this single morphological
parameter allows you to suspect a megaloblastic anemia, compared to the previous finding which is typical
of myelodysplastic syndrome. An additional abnormality includes giant platelets.
In the bone marrow there are immature red cells with mitochondria, loaded with iron and having a
perinuclear distribution.
A part from deletion 5q other cytogenetic abnormalities in patients with myelodysplastic syndromes are:
deletion 5q, trisomy 8, monosomy 7, deletion 1. Deletion 5q is associated with a very good prognosis, while
monosomy 7 is associated with a very bad prognosis.
Until recently the only abnormalities seen in myelodysplastic syndromes were cytogenetic abnormalities,
which are important but the only one that represent the cause of the disease is deletion 5q. SF3B1 is a
recurrent mutation, and the gene has a role in the pathophysiology of the disease. SF3B1 encodes for RNA
splicing. So SF3B1 is a gene of the RNA splicing, and mutations in splicing factors have been found in
40-80% of cases with myelodysplastic syndrome, particularly in those with ringed sideroblasts.

In the bone marrow we have the first mutation, and the mutation gives advantage to the mutated cells which
form clones. These gradually replace all the normal clones in the bone marrow. But the bone marrow is not a
single tissue, and there is no anatomical continuity between its different sections. In order for the mutant
clone to become dominant, the mutant stem cells must also circulate in peripheral blood and reach the other
bone marrow sites. At a certain point all the stem cells will be replaced by the mutated stem cells, and in all
bone marrow we will have a single clone, which is still able to differentiate and maturate and to produce
normal red cells, but it’s less efficient. There will thus be anemia, leukopenia, thrombocytopenia and
morphological abnormalities, such as immature red cells with 2 nuclei instead of one. There can then be the
occurrence of additional mutations, like the ones we saw in CML which leads to increasingly lower ability to
produce, differentiate and maturate, so anemia leukopenia and thrombocytopenia become more severe, and
there is a lower ability to differentiate until the development of acute myeloid leukemia.

41
6.2 Acute Myeloid Leukemia
Acute myeloid leukemia (AML), is a cancer of the myeloid line of blood cells, characterized by the rapid
growth of abnormal white blood cells that accumulate in the bone marrow and interfere with the production of
normal blood cells. AML is the most common acute leukemia affecting adults, and its incidence increases
with age.
The symptoms of AML are caused by replacement of normal bone marrow with leukemic cells, which causes
a drop in red blood cells (fatigue, shortness of breath), platelets (easy bruising and bleeding), and normal
white blood cells (increased risk of infection).
As an acute leukemia, AML progresses rapidly and is typically fatal within weeks or months if left untreated.
AML has several subtypes; treatment and prognosis varies among subtypes. AML is treated initially with
chemotherapy aimed at inducing a remission; patients may go on to receive additional chemotherapy or a
hematopoietic stem cell transplant.
———Clinical case.——————————————————————————————————————
English patient who was travelling by train between Milan and Genoa. He had severe epistaxis on the train
so he stopped in in Pavia and went to ER at San Matteo hospital. Doctors called us because he had anemia,
leukopenia, severe thrombocytopenia, a prolonged prothrombin time and findings suggesting disseminated
intravascular coagulation. When we saw the peripheral blood with few WBC we saw abnormal
promyelocytes characterized by Auer rods or “corpo di Auer” in Italian. Auer rods are clumps of azurophilic
granular material that form elongated needles seen in the cytoplasm of leukemic blasts. They can be seen in
the leukemic blasts of acute myeloid leukemia with maturation and acute promyelocytic leukemia (known as
acute myeloid leukemia M2 and M3, in the FAB classification, respectively) and in high grade
myelodysplastic syndromes and myeloproliferative syndromes. They are composed of fused lysosomes/
primary neutrophilic granules and contain peroxidase, lysosomal enzymes, and large crystalline inclusions.
The patient was thus diagnosed with acute promyelocytic leukemia. He was then admitted for acute
promyelocytic leukemia and disseminated intravascular coagulation. Induction therapy was effective.

———Clinical case.——————————————————————————————————————
Similar patient, a little bit older, with severe haemorrhages and WBC count greater than 200.000. The patient
had severe leukocytosis. He was somewhat slow in answering the questions. He deteriorated very rapidly
and died during the night because of cerebral hemorrhage. This is a variant of acute promyelocytic leukemia.
The cells have a typical nucleus which is called from French lunettes cells because they remind glasses. It is
a variant of acute promyelocytic leukemia in which leukocytosis almost inevitably leads to severe
disseminated intravascular coagulation and especially several haemorrhages.

———Clinical case.——————————————————————————————————————
Elderly woman with history of myocardial infarction, diabetes, peripheral vascular disease, shortness of
breath, pancytopenia. The number of circulating WBCs in AML is really variable, so we can have patients
with low WBC count and patients with extremely high WBC count. What is always low is Hb level and
platelets count. Bone marrow aspiration and bone marrow biopsy were performed showing about 40%
myeloblasts.

AML belongs to myeloid neoplasms. As chronic myeloid leukemia and myelodysplastic syndrome, AML is a
clonal proliferation of multipotent stem cells. In all this conditions we have an initiating molecular event which
in CML is the fusion gene, in MDS can be many different events, for instance somatic mutations of SF3B1,
but in any case the mutant stem cells have an advantage on normal stem cells and they form a clone.
Formation of this clone is associated with suppression of residual normal stem cells and what is different
here, what distinguish AML for instance from CML, is that in AML the ability to differentiate and maturate is
lost so there is an accumulation of leukemic blasts. As a consequence of this inability to differentiate we have
the defective production of mature cells, such as red cells, granulocyte, monocytes and platelets. The typical
feature will be pancytopenia, but when the blast cells start to proliferate in the bone marrow and circulate in
peripheral blood the WBC count increases.

Individuals with Down syndrome are more likely to develop acute myeloid leukemia. The same is true for
Fanconi anemia and other inherited disorders. After Hiroshima and Nagasaki, Chernobyl and now also
Fukushima, the number of cases of AML have increased considerably. Exposition to benzene is associated
with high risk of aplastic anemia and AML. The use of chemotherapeutic drugs, especially alkylating agents,
is associated with higher risk of acute myeloid leukemia. Conditions like MDS and CML involve high risk of
progression to a condition very similar to AML.

42
Probably at least 2 mutations are required for progression to disease. One single mutation may cause clone
hematopoiesis. So you may have subjects with normal blood count but with the bone marrow which is totally
clonal instead of being polyclonal. But in order to lose the ability to differentiate and maturate you need
additional mutations.

There are specific cytogenetic abnormalities which are associated with AML:

- acute promyelocytic leukemia, which is associated with translocation 15-17. in this case 1 event is able to
produce acute leukemia, the translocation which generates a PML/RARα fusion gene is able to produce
the disease. Like the BCR/ABL fusion gene it is able to produce chronic myeloid leukemia. Physiological
functions of PML are not clear but RARα encodes for the retinoid acid receptor alpha. So patients having
this mutant gene have impaired differentiation of promyelocytes leading to accumulation of promyelocytes
initially in the bone marrow, so there is leukopenia, but when the accumulation in bone marrow is massive
these cell circulate in peripheral blood and leukocytosis develops. Since the abnormal promyelocytes
release their granules in peripheral blood and these granules have pro-coagulant activity, this bursts
intravascular coagulation and generates disseminated intravascular coagulation. This means consumption
of coagulation factors and so the clinical manifestations are haemorrhages.

Retinoic acid receptor alpha is involved in the translocation and generation of the fusion gene. If you
incubate the abnormal promyelocytes with retinoic acid in vitro, retinoic acid is able to induce differentiation
and this is what happen after administration of all-trans retinoic acid to a patient with acute promyelocytic
leukemia. So after 7- 14- 30 days the abnormal promyelocytes are replaced by neutrophils. The current
treatment for acute promyelocytic anemia is indeed a treatment based on the combination of all-trans retinoic
acid and a chemotherapeutic drug called idarubicin. This combination is called AIDA.

- acute myeloid leukemia associated with somatic mutation in NPM1, the gene encoding nucelophosmin. In
2005 Brunangelo Falini, was working on the gene NMP1 (which was also responsible for a subtype of
malignant lymphoma) and he found out that some patients with AML and normal karyotypes (so having no
cytogenetic abnormalities), showed an accumulation of nucelophosmin in the cytoplasm of aplastic cells.
Nuclephosmin is normally found in the nucleoli, but the protein is clearly produced at cytoplasmic level and
then it must go to the nucleus and reach the nucleoli. What Brunangelo was able to show was that the
different mutations in the gene NMP1 deleted the signals responsible for the nuclear destination. The
mutation removes this signal and makes the protein unable to reach the nucleus so the protein
accumulates in the cytoplasm of the hematopoietic cells and this accumulation leads to the inability of the
cell to differentiate. This eventually leads to AML.

- a group of researchers found a somatic mutation in the gene IDH1 which encodes isocitrate
dehydrogenase 1, a very common enzyme, and they found out that this mutant enzyme produces a
catabolite that is different from the normal one and leads to abnormal proliferation of stem cells.

Anemia is common to all cases. Most patients have neutropenia and infectious complications that derive
form neutropenia. Many patients have haemorrhages associated to thrombocytopenia. Many patients have
tissue infiltration of leukemic blasts and when leukocytosis is severe in the order of hundreds thousands
there may be leukostasis in the lung or CNS. The white blood cell count is highly variable and ranges from
leukopenia to severe leukocytosis. The blasts cell count is always increased in the bone marrow and by
definition according to the WHO classification we can make a diagnosis of AML when the blasts count is in
equal or grater than 20%. Some patients, in particularly those with acute promyelocytic leukemia have
disseminate intravascular coagulation and so an additional cause of haemorrhages after the
thrombocytopenia.
Diagnosis and prognosis are evaluated through a complete blood count in peripheral smear, and bone
marrow aspirate. If there is a condition called “dry tap” (aspiration gives no blood cells), bone marrow biopsy
is performed. The cells can also be studied through flow cytometry. Cytogenetic analysis is always
performed.
So in every patient in Boston, the doctors not only perform bone marrow biopsy and mutation analysis of few
genes, but they are currently sequencing 96 genes and they are considering moving on to the whole genome
sequence in every single patient with AML.

Age and comorbidity are important factors that need to be considered when defining prognosis. Elderly
patients have a bad prognosis and the presence of co-morbidities limits the efficacy of chemotherapy and
represents a negative prognostic factor.

43
Cytogenetics is important because some abnormalities are associated with good outcome while complex
karyotypes represent a negative prognostic factor. Somatic mutations are important: NMP1 and CEPBα
mutations are favourable prognostic factors while FLT3 duplication is a negative prognostic factor.
There are 2 phases for what concerns treatment:
1. induction of remission
2. consolidation of remission.
There are 2 treatments, one for acute promyelocytic leukemia and one for AML different from acute
promyelocytic leukemia. The induction therapy for acute promyelocytic anemia is the combination of all-trans
retinoic acid and idarubicin (AIDA). It is extremely important in the first days of treatment also to specifically
treat disseminated intravascular coagulation in order to prevent haemorrhages. Fresh plasma is
administered in order to provide patients with coagulation factors since they are destroyed by intravascular
coagulation.
Once remission has been achieved it is consolidated with additional idarubicin. During the follow-up there is
regular monitoring of the PML/RARα fusion gene by PCR. As long as the patient remains in complete
remission there is nothing else to do. If there is molecular evidence of relapse, i.e. the appearance of PML/
RARα fusion gene, there is the need for additional treatments. One is arsenic trioxide, the second one and
the most effective one is allogeneic stem cell transplantation. It has been shown that a combination of all-
trans retinoic acid and arsenic trioxide, so two drugs which are not chemotherapeutic, is able to induce
complete remission in acute promyelocytic leukemia. A number of studies are currently ongoing in order to
define which patients should be treated with this combination of drugs.
In AML different form promyelocytic leukemia we use a combination of idarubicin or daunorubicin with
cytarabin. It consists of three days of idarubicin and seven days of cytarabin. Then the consolidation is made
with high dose cytarabin.
Allogeneic stem cell transplantation should be considered when the risk of relapse is very high. Only 10% of
patients with acute myeloid leukemia survive at 10 years.

There are many causes of AML from chromosomal translocations to point mutations but the final result is
always the accumulation of myeloid blasts.

7 Lymphoid neoplasms

Lymphoid tissue is a fundamental part of the immune system and is present in several organs and tissues.
You can find lymphoid tissue in lymph nodes, which are the organs where the lymphoid tissue is in most part
represented, but also in extra nodal sites that include mucosal surfaces such as GI tract, lungs, bronchi, but
also the Waldeyer’s ring in the oral cavity, the spleen and so on. The lymphoid tissue that you can find in
association with mucosal surfaces is also called mucosal associated lymphoid tissue or MALT.
Lymph nodes may undergo expansion, an increase in size and dimension following several conditions. The
most frequent cause of lymph node enlargement is a reactive lymphoid proliferation following antigenic
stimulation. This is the case of reactive lymphadenopathies. However there may be also involvement of
neoplastic proliferation. Helicobacter infection in the lymphoproliferative disorder of the stomach is the
prototype example of lymphoma developing from a chronic infection.

Lymph nodes
The architecture of the normal lymph node account for 3 main areas: the
cortical area, the para-cortical area and the sinus.
The cortical area, as you can see in the picture, is containing aggregates
of lymphoid cells called lymphoid follicles which contain the germinal
centers that are the results of follicular centers stimulation by antigens
and reactive process.
The para-cortical area surrounds the cortical area and mainly contains T-
lymphocytes.
The sinus drains the lymphatic system and contains mainly histocytic
cells.

The cortical area comprises lymphoid follicles with germinal centers. The
germinal center contains centrocytes and centroblasts that are the
proliferating B-cell stimulated by the antigenic trigger.
The germinal center is circumscribed by a peripheral zone consisting of
small lymphocytes, called mantle zone area. This area contains

44
unstimulated virgin B-cells that give rise to the germinal center reactions.

In the periphery of the mantle zone we have an area that

consists of clear cells with monocytolic nuclei that is called
marginal zone area. The marginal zone area contains mainly
B- cells. It is important to remember this anatomic
compartments, i.e. the germinal centers or follicles, the mantle
zone and the marginal zone, because the different lymphoid
populations present in this area give rise to different subtypes
of lymphomas, such as follicular lymphoma, mantle zone
lymphoma and marginal zone B-cell lymphoma.
Lymph nodes may be enlarged during infections or
autoimmune disorders because of a reactive process. Reactive
conditions are the most frequent causes of nodal enlargement.
In few other cases, the increase in size of lymph nodes is
related to a primary or secondary metastatic malignant
disease.
The nodal stage may be involved either in primary lymphoid tumors and in secondary localization of solid
organ tumors (solid tumors may spread throughout the lymphatic system and the first localization of
metastasis are lymph nodes).

The immunological staining usually used are anti T-cell (specific for CD3, expressed by T lymphocytes)
markers and anti B-cell markers (specific for CD79, a molecule that is specifically expressed by B
lymphocytes). The stain for CD79 strongly reacts with B-dependent areas, i.e. germinal center, mantle zone
and marginal zone.
The immunological staining marker directed against T lymphocytes (CD3) is a specular image, and as you
can see it highlights the presence of small T lymphocytes in the para-cortical area.

7.1 Malignant Lymphomas

Lymphoma is the name applied to a group of blood cell tumors that develop from lymphatic cells. The name
often refers to just the cancerous ones rather than all such tumors.
Symptoms may include enlarged lymph nodes (usually painless), fever, drenching sweats (mostly at night),
weight loss, itching, and tiredness.
The two main categories of lymphomas are Hodgkin lymphomas (HL) and the non-Hodgkin lymphomas
(NHL). About 90% of lymphomas are non-Hodgkin lymphomas. Lymphomas and leukemias are a part of the
broader group of tumors of the hematopoietic and lymphoid tissues.
Risk factors for Hodgkin lymphoma include infection with Epstein–Barr virus and a history of the disease in
the family. Risk factors for common types of non-Hodgkin lymphomas include autoimmune diseases, HIV/
AIDS, infection with human T-lymphotropic virus, eating a large amount of meat and fat, immunosuppressant
medications, and some pesticides.
Diagnosis, if enlarged lymph nodes are present, is usually by lymph node biopsy. Blood, urine, and bone
marrow testing may also be useful in the diagnosis. Medical imaging may then be done to determine if and
where the cancer has spread. Spread is most often to lungs, liver, and/or brain.
Treatment may involve one or more of the following: chemotherapy, radiation therapy, targeted therapy, and
surgery.
In some non-Hodgkin lymphomas, an increased amount of protein produced by the lymphoma cells causes
the blood to become so thick that plasmapheresis is performed to remove the protein.
The outcome depends on the subtype with some being curable and treatment prolongs survival in most. The
five-year survival rate in the United States for all Hodgkin lymphoma subtypes is 85%, while that for non-
Hodgkin lymphomas it is 69%.

Based on pathogenesis, lymphomas may be subdivided in primary lymphomas and secondary lymphomas.
Secondary lymphomas are the transformation of low grade small cell lymphomas that progress to high grade
malignancies.
According to their site of origin, lymphomas may be subdivided into:
- Primary nodal or systemic lymphomas, characterized by primary involvement of nodal regions
- Primary extra nodal lymphomas, characterized by involvement of extra nodal sites, including the
gastrointestinal tract, the skin, the thyroid the lungs and so on. The definition of primary extra nodal

45
 lymphoma is based on the fact that, 6 month following diagnosis of lymphoma, the patient will not develop
any nodal focalisation of the disease.

There is a difference between the definition of lymphoma and leukemia. Both lymphomas and leukemia may
derive from lymphoid cells.
Lymphoma: lymphoid proliferation clinically presented as a tumor in nodal or extra nodal sites. Lymphomas
mainly involve solid proliferation of nodal or extra nodal sites
Leukemia: neoplasm with a prevalent bone marrow and peripheral involvement in the absence of solid tumor
masses. Leukemias are characterized by infiltration of bone marrow and presence of circulating neoplastic
cells as peripheral blood.

Lymphomas account for 7% of all malignancies in Western countries. They are the 5th cause of death
among neoplastic diseases. The peak of incidence of lymphoma is between 45 and 60 years old. In the last
20 years it was observed an increased incidence of lymphomas.
Lymphomas are the malignancies that show the highest incidence after malignant melanoma and lung
cancer in women. In Western countries a sharp increase in non-Hodgkin lymphoma has been observed while
in developing countries Hodgkin’s lymphoma incidence is increasing.
The explanations for this increase in incidence are not completely understood but epidemiological and
pathologic studies allowed us to identify several risk factors.
First of all, primary and secondary immunodeficiencies represent a well-defined condition that is associated
with an increased risk of developing lymphoproliferative disorders. Depending on the type of
immunodeficiency the defect in the immune response is a risk factor for development of deregulated
proliferation that may apply oncogenic lesions and transform this deregulated proliferation in full developed
malignancies such as lymphomas.
Primary immunodeficiency include congenital disease such as Wiscott-Aldrich’s disease, Common Variable
Immunodeficiency.
Secondary immunodeficiency may be related to viral infection such as HIV infection, or to iatrogenic
conditions such as post-transplant immunosuppression or immunosuppressive therapies such as
methotrexate treatment in rheumatoid arthritis and so on.
Some bacterial and viral infections may be responsible for an increased risk of lymphomas development.
The most frequent agent that has been associated with increased risk of lymphoma development are
Helicobacter pylori and the HCV infection for specific B cells lymphomas subtypes.
Chronic infections result in chronic stimulation of the immune system. In the setting of chronic immune
stimulation a clonal proliferation may emerge and acquire oncogenic lesion that transform this proliferation in
lymphoma.

Classifications
Lymphomas are represented by a broad spectrum of diseases. More than 50 entities with several variants
have been included in more recent classifications. Lymphomas are heterogeneous in terms of
- Clinical presentation: nodal sites or extra nodal sites.
- Biology and histogenesis: each lymphoma subtype derive from specific lymphoid subpopulation.
- Prognosis: very different according to lymphoma subtype.

Lymphomas may derive either from B or T lymphocytes. In Western countries the large majority of
lymphomas derives from B lymphocytes. Lymphomas of B-cell origin account for 85% of cases. 15% of
cases derive from T lymphocytes or accessory immunopopulations.

The current classification is the WHO classification developed in 2001 and released in 2003.

The first classification was proposed by Rappaport in

the 1966. This classification was mainly based on the
pattern that lymphomas showed in nodal biopsies.
Lymphomas growing in nodules resembling follicles
have a follicular pattern.
Lymphomas that result in a complete effacement of
nodal architecture are called diffuse lymphomas
because you cannot recognize any normal structure
or any structure resembling follicles. In some cases
you may have a mixed follicular diffused pattern.

46
In some cases, the para-cortical compartment is mainly involved in neoplastic proliferation: these cases have
a follicular or para-cortical pattern. Finally, some cases are characterized by a prominent expansion of the
marginal zones and this is called marginal zone like pattern.

In the follicular proliferation pattern, the lymphoid cells form numerous follicular nodules. Some aggregates of
lymphoid cells that resemble the follicle of normal lymph nodes may be recognized.
In the diffuse pattern, the histological observation of the infiltrate does not reveal any nodal aggregate, and
we can see only a diffuse sheath of proliferating neoplastic cells. There is a diffuse effacement of normal
architecture without evidence of any follicle structure.
Follicular nodular lymphomas have a indolent course while diffuse lymphomas tend to have an aggressive
clinical course.
According to Rappaport, the different subtypes were subsequently classified according to the composition of
infiltrates without establishing a correlation between the different lymphomas subtypes and the normal
lymphoid populations that comprises the lymphoid system and the immune response. He used merely
descriptive criteria.

Kiel classification was first published in 1974 and it was quite different compared to Rappaport classification.
It combined morphology to new biological issues. In the 70s immunology and immunophenotypic
characterization of lymphoid population begun providing info about the histogenesis and about the
phenotypic characteristics of normal lymphoid cells and of lymphoma proliferation.

Karl Lennert was the first author to classify lymphoproliferative disorders according to their histogenetic
origin. In other words, he proposed an attempt to link each lymphoma subtypes to a specific normal
counterparts: proliferative lymphomas derive form germinal centers, mantle cell lymphomas derive from other
zones of lymphomas and so on.

The different lymphomas subtypes were then classified according to the site of lymphoma cells and
according to the degree of atypical changes in these cells. Lymphomas consisting of small cells with low
proliferative index were considered low grade indolent lymphoma, whereas lymphomas mainly consisting of
large cells with an high proliferative index were classified as high grade lymphomas.

This cartoon summarizes possible histogenetic

relationships between the normal lymphoid cells
and their lymphomas counterparts.

Naïve B-cells are unstimulated B-cells and carry a

mutated immunoglobulin rearrangement gene.
These cells when stimulated by antigenic
processes enter the germinal center reaction and
two main molecular events take place in the
germinal center: somatic hypermutation and class
switch.

1. Somatic hypermuation aims to select B-cells that carry B-cells receptors with an increased affinity for the
antigen.
2. Class switching consists in the switch of constant regions of immunoglobulin receptors.

Virgin B-cells carry IgG and IgM receptors, whereas B-cells that enter germinal center reaction acquire IgA,
IgE or IgG immunoglobulin receptors. The cells that are selected by the germinal center reaction exit the
germinal center and become plasma cells or memory cells. Plasma cells are responsible for the production
of large amount of immunoglobulins whereas memory B-cells are able to give rise to a quicker immune
response when human organism encounter the same antigen.
Each of these physiological B-cell compartments may give rise to a specific lymphoma subtype:
- virgin B-cells comprised in the mantle zone: mantle cell lymphomas;
- lymphoid cells comprised in the germinal center: germinal center lymphomas;
- post germinal center B-cells: wider series of lymphomas subtypes.

47
In 1982, the American oncologists and pathologists proposed a new classification that was called Working
Formulation of Non-Hodgkin’s Lymphomas. This classification included concepts deriving both form Rapport
and Kiel classifications. Advances in the knowledge of immunology and immunohistochemistry confirmed
that any lymphoma subtype probably represented the neoplastic counterpart of physiological B and T cell
populations. This classification was called Revised European American Lymphoma (REAL) Classification,
and was the result of the integration of morphological, phenotypic, molecular and clinical data. This
classification included the best aspects of the Kiel classification and defined criteria useful to define a
lymphoma entity.
Each lymphoma entity was classified based on:
- the clinical presentation
- the morphologic characteristics
- the immune-phenotype (each lymphoma subtype is characterized by the expression of specific antigens)
- data provided by molecular investigations (some lymphomas subtypes are characterized by specific
genetic alterations, mainly chromosomal substitutions).

The REAL classification was reviewed in 1997 by the WHO committee and the result of this work was the
publication of WHO Lymphoma Classification in 2001.
This classification followed the principles of the REAL classification and each lymphoma is classified based
on clinical presentation, morphologic characteristics of lymphoma cells, phenotypes and cytogenetic
properties. This classification includes myeloproliferative diseases, myelodysplastic syndrome, myeloid
leukemia and lymphomas.
Lymphomas are classified into 3 large groups based on their histogenesis:
• B-cell neoplasms (i.e. lymphomas that derive from B-lymphocytes);
• T and NK cells neoplasms (that include the lymphomas deriving from T- lymphocytes and Natural Killer
cells);
• Hodgkin’s lymphoma (whose histogenesis was not completely deigned at the time of WHO lymphoma
classification).

Primary Mediastinal Diffuse Large B-cell Lymphoma (PMLBCL)

PMLBCL is a distinct type of diffuse large B-cell lymphoma involving the mediastinum. It arises from a
putative thymic peripheral B cell.
It affects primarily young adults (the median age is 37 years) and is more common in females.
At initial presentation, PMLBCL is usually confined to mediastinum, but its bulk can sometimes be palpated
at the low neck.

The definition of this condition is based on the combination of morphological, immune-histochemical,

molecular and clinical data. This lymphoma subtype has very typical clinical features because it primarily
arises in the mediastinum and it is characterized by a large B-cell infiltrate that defines a stenosis.
It is a B-cells neoplasm because it expresses the CD20 marker.
It may represent a clinical emergency because it has a very aggressive clinical course: it may infiltrate the
pericardium.

Typical symptoms are related to compression and infiltration of mediastinal structures by lymphoma cells.
The patient presents with cough, chest pain, dyspnea and dysphonia, thrombosis of SVC may occur and in
some cases lymphoma localizes in supraclavicular lymph nodes. Pleural-pericardial effusion may also occur.
Involvement of bone marrow is very rare.
This lymphoma subtype accounts for 5% of all aggressive NHL.
Histology is characterized by angiotropism: the walls of the vessels are infiltrated by these large lymphoma
cells that migrate and disrupt the walls of the vessels. This result in hemorrhagic changes and necrosis.
Lymphoma cells are characterized by a large clear cytoplasm. The neoplastic cells have an abundant clear
cytoplasm. The third feature is the presence of a fibrotic reaction that may be in large bands or in fine bands.

The phenotype of this lymphoma is characteristic. It is characterized by the expression of cell markers that
are shared with all B cell lymphomas, but it is also characterized by the expression of specific markers such
as CD30 and CD23. Cytogenetic studies revealed that primary mediastinal B cells lymphomas are mainly
characterized by alteration of chromosome lines that have been amplified in 70% of cases and this
chromosomal addition contains c-rel gene oncogene. On the contrary Bcl-2 and Bcl-6, which are primary
involved in other B-cell lymphoma subtypes, are usually not targeted by genetic alterations in mediastinal B-
cell lymphomas.

48
In the last 20 years it has been also identified the possible normal counterpart of this lymphoma subtype. The
thymus is the site of maturation of T cells. So why do B cell lymphomas primarily arise in the thymus? In
recent year it has been identified a subpopulation of B-cells that specifically localizes in the thymus. They are
called medullary CD20+ B cells and they represents the normal counterpart of primary mediastinal B-cells
lymphomas.
The primary site of involvement is considered of fundamental importance in WHO classification because
clinical behaviours of morphological identical lymphomas may be very different according to the site of
presentation.

In 2008 a new edition of WHO lymphoma classification was published and this represent current
classification used by pathologists and hematologists.
This lymphoma classification recognizes a main distinction between Non-Hodgkin’s lymphomas (NHL) and
Hodgkin’s lymphomas (HL).

• The histogenesis of HL has been defined in recent years but it probably arises from B-lymphocyte. It is
however considered a distinct entity from NHL because the therapeutic approach to HL is very different in
terms of therapy and it is based on specific chemotherapeutic agents.

• NHL derived from B and T-lymphocytes. Extra nodal lymphomas are characterized by peculiar features
different from nodal lymphomas in terms of clinical presentation, biology and prognosis. They account for
30% to 40% of NHL. Primary extra nodal lymphomas are most frequently T-cell lymphomas and most
frequent sites are the GI tract, skin, mediastinum, lung, spleen, the soft tissue and bones and CNS.
Considering NHL, another distinction is between neoplasms arising from precursor lymphoid cells and
between neoplasm deriving from mature B cells.
- Neoplasms deriving from precursor lymphoid cells include the cases in which precursor have not rearranged B -cell
receptors and T-cell receptors. This cases mainly present as leukemic diseases and are lymphoblastic leukemias.
Precursor lymphoid neoplasms are highly aggressive diseases. They affect mainly children and derive from
precursor lymphoid cells. The main diagnostic marker used to identify this neoplasm is TdT enzyme (Terminal
deoxynucleotidyl Transferase) that is an enzyme active in precursor B-cells and required in the process of
immunoglobulin and T-cell receptors rearrangements.
- Mature B-cell neoplasms derive from B-cells that have rearranged their TCR and BCR genes and include
neoplasms deriving from virgin B cells, from germinal center cells and from post-germinal center cells. Mature B-
cell lymphoma. Mature B cell lymphomas account for 85% of NHL and they are characterized by specific genetic
and immunophenotypic features.

7.2 Hodgkin’s Lymphoma

Hodgkin lymphoma (Hodgkin’s lymphoma or Hodgkin's disease), is a type of lymphoma in which cancer
originates from white blood cells called lymphocytes.
A history of infectious mononucleosis due to infection by Epstein–Barr virus (EBV) may increase risk of HL,
but the precise contribution of Epstein–Barr virus remains largely unknown.

Hodgkin lymphoma is characterized by the orderly spread of

disease from one lymph node group to another and by the
development of systemic symptoms with advanced disease.
When Hodgkins cells are examined microscopically,
multinucleated Reed–Sternberg cells (RS cells) are the
characteristic histopathologic finding.
Reed–Sternberg cells are different giant cells found with
light microscopy in biopsies from individuals with Hodgkin's
lymphoma. These cells are binucleated cells characterized
by two prominent nucleoli with a clear perinuclear area.
They are usually derived from B lymphocytes, classically
considered crippled germinal center B cells, meaning they
have not undergone hypermutation to express their
antibody.
RS cells do not resemble any normal lymphoid cell and usually do not express phenotypic markers. They do
not express neither B or T cell markers. In other words they are negative for both CD20, CD78 and so on. So
the histogenesis of RS cells was considered obscure and this is the reason why it was initially called
Hodgkin’s disease and not Hodgkin’s lymphoma.

49
General data:
- 2-3 cases x 100.000 persons.
- ratio M/F= 1,5:1
- 2 peaks: 15-30 years and often 60 years.

Hodgkin lymphoma may be treated with radiation therapy, chemotherapy, or hematopoietic stem cell
transplantation, with the choice of treatment depending on the age and sex of the patient and the stage, bulk,
and histological subtype of the disease.

Hodgkin’s lymphoma was the first malignancy for which chemotherapy was developed. At the beginning 4
drugs were used together (MOPP), before that, Hodgkin’s lymphoma was treated with radiotherapy. The 4
drugs were shown to be highly effective but one of these agent was an alkylating agent, causing secondary
malignancies such as acute myeloid leukemia.
The side effects of this chemotherapy were not just secondary malignancies but also (in young men or
women) gonadal toxicity and infertility.
In order to prevent secondary tumors and infertility the oncologists developed an alternative treatment using
drugs that were not mutagens and did not have gonadal toxicity. This treatment (ABVD: A= Adriamycin, B=
Bleomycin; V= Vinblastine; D= Decarbazine) was highly successful in terms of complete remission and in
terms of no gonadal toxicity.
The next evolution was just aimed at defining the optimal treatment for a single individual patient. So, based
on the extension of the disease and on the subtype of Hodgkin’s lymphoma the number of chemotherapy
cycles were defined.
Patients nowadays receive the minimum effective therapy.
Autotransplant is the second line treatment. It is the modality through which we define the high dose
treatment with very high doses of chemotherapy. We sustain it with hematopoietic stem cells from the same
patient. The patient can be treated with high doses thanks to the fact that after the treatment we will inject the
hematopoietic stem cells that we
collected and frozen before starting it. These cells in 10-20 days permit the hematologic recovery of the
patient.
some patients can be refractory and we there are now new drugs acting on Hodgkin’s lymphoma: one of
them is the CD 30 antibodies. CD 30 is a tumor factor and in this case if you link the drug to the receptor it
will kill the cell with the lymphoma. The drug is linked to the antigen and the surface of the cell, after this link
there is the release of cytotoxic drug in the cell.

Clinical features at diagnosis:

- at diagnosis the disease is supra-diaphragmatic in 90% of cases
- in 60-80% of cases there is cervical-supraclavicular adenopathy with mediastinal mass
- isolated mediastinal disease in 10%
- other lymph nodes: axilla, inguinal regions
- rare extra-nodular involvement
- 25-30% of patients have systemic symptoms at presentation
1. fever > 38° without apparent cause
2. weigh loss > 10% in the last 6 months
3. Profuse night sweats
Itching is a quite frequent unspecific symptom of presentation but is not formally included among the
systemic symptoms.
Lymph nodes appearance (also in non-Hodgkin’s lymphoma): different sizes, not painful, hard-elastic
consistency, well isolated, non-adherent to surrounding structures (you can move them).

It is not possible to distinguish Hodgkin or non-Hodgkin lymphoma without histological analysis. Diseases
that do not require an histological diagnosis are quite rare (acute lymphoblastic leukemia, B cell leukemia,
chronic lymphocytic leukemia).
Not all the mediastinal masses or lymphomas in the neck and chest are Hodgkin’s lymphomas, thus it is
important to do differential diagnosis with:
- Non-Hodgkin’s lymphomas
- Infections
- Sarcoidosis
- Metastasis
Infections and solid cancers can be distinguished by the clinical appearance and clinical symptoms.

50
Staging
HL and NHL are staged according to Ann Arbor Staging but in HL the staging is a very important prognostic
factor because this lymphoma spreads in a known manner from a nodal region to another nodal region.
Staging is a major prognostic factor in HL.
After Hodgkin lymphoma is diagnosed, a patient will be staged: he/she will undergo a series of tests and
procedures that will determine what areas of the body are affected.
These procedures may include documentation of their histology, a physical examination, blood tests, chest
X-ray radiographs, computed tomography (CT)/Positron emission tomography (PET)/magnetic resonance
imaging (MRI) scans of the chest, abdomen and pelvis, and usually a bone marrow biopsy.
Positron emission tomography (PET) scan is now used instead of the gallium scan for staging. The PET
scan is a functional imaging technique (CT is anatomical) thanks to which it is possible to
have 3D images of the body. Fluorodeoxyglucose (FDG) molecule, is an analogue of glucose, which is a
tracer that concentrates in the highly metabolic tissues of the lymphomas.
Why is lymphoma so avid of this tracer and so active? Hodgkin’s lymphoma is characterized by heavy
inflammatory background, and the neoplastic component is the minor component of the tissue. This activity
and avidity are due to the non neoplastic component. A practical point is not to use too much steroids before
performing the PET because they could modify the results.

On the basis of this staging, the patient will be classified according to a staging classification (the Ann Arbor
staging classification scheme):
I. involvement of a single lymph node region (mostly the cervical region) (I) or single extra-lymphatic site
(Ie);
II. involvement of two or more lymph node regions on the same side of the diaphragm (II) or of one lymph
node region and a contiguous extra-lymphatic site (IIe);
III. involvement of lymph node regions on both sides of the diaphragm, which may include the spleen (IIIs)
and/or limited contiguous extra-lymphatic organ or site (IIIe, IIIes);
IV. disseminated involvement of one or more extra-lymphatic organs.

The absence of systemic symptoms is signified by adding 'A' to the stage.

The presence of systemic symptoms is signified by adding 'B' to the stage.
The major systemic symptoms associated with both HL and NHL are intermittent fever, sweating and weight
loss.
For localised extra-nodal extension from mass of nodes that does not advance the stage, 'E' is added.
Splenic involvement is signified by adding 'S' to the stage.
The inclusion of "bulky disease" is signified by ‘X'. Bulky mediastinum is defined as mediastinum
enlargement up to 1/3 of its size or a nodal mass larger than 10 cm in maximal diameter. Patients presenting
with bulky disease, especially in mediastinal location, has a worse prognosis.

51
At the beginning of the 21th century a new technology became available. It was based on micro dissection of
single cells. Researchers were able to dissect from wall adhesion tissue the typical RS cell and to study the
molecular features of these micro dissected cells. Analysis of immunoglobulin gene rearrangement in these
micro-dissected cells documented a clonal rearrangement of immunoglobulin gene in RS cells demonstrating
for the first time that the large majority of cases (95%) of HL actually derives from B cells.

In order to establish prognosis, different criteria can be used:

- For the early stage we use the “EORTC” prognostic criteria, based on the stage, age, bulky, erythrocyte
sedimentation rate (marker of the inflammatory status) and the presence of B symptoms.
- For the advanced stage we use the Hasenclever score: it is a quite complicated score.

Classification
The current classification of Hodgkin’s Lymphoma recognizes 2 main subtypes. These subtypes are
distinguished mainly according to histological and phenotypic features of atypical cells.

1. Classical Hodgkin’s Lymphoma (CHL). In CHL the neoplastic cell is RS cell. Reed–Sternberg cells are
usually of B cell origin. Some T cell markers (such as CD2 and CD4) are occasionally expressed. CHL is
sub-classified in 4 variants based on the composition of the reactive background that accompanies
Reed–Sternberg cells.
- Nodular sclerosing HL: Is the most common subtype and is composed of large tumor nodules showing scattered
lacunar classical RS cells set in a background of reactive lymphocytes, eosinophils and plasma cells with varying
degrees of collagen fibrosis/sclerosis.
- Mixed-cellularity subtype: Is a common subtype and is composed of numerous classic RS cells admixed with
numerous inflammatory cells including lymphocytes, histiocytes, eosinophils, and plasma cells without sclerosis.
This type is most often associated with EBV infection and may be confused with the early, so-called 'cellular' phase
of nodular sclerosing CHL.
- Lymphocyte-rich or Lymphocytic predominance: Is a rare subtype, shows many features which may cause
diagnostic confusion with nodular lymphocyte predominant B-cell Non-Hodgkin's Lymphoma (B-NHL). This form
also has the most favourable prognosis.
- Lymphocyte depleted: Is a rare subtype, composed of large numbers of often pleomorphic RS cells with only few
reactive lymphocytes which may easily be confused with diffuse large cell lymphoma. Many cases previously
classified within this category would now be reclassified under anaplastic large cell lymphoma.

2. Nodular Lymphocytes Predominant Hodgkin’s Lymphoma (NLPHL). In NLPHL the neoplastic cell is
called L&H cell or popcorn cell. Nodular lymphocyte predominant Hodgkin's lymphoma expresses CD20,
and is not currently considered a form of classical Hodgkin's.

Classic Hodgkin’s Lymphoma

CHL accounts for 95% of HL. It has a typical bimodal distribution. It involves both superficial and visceral
lymph nodes, including mediastinal lymph nodes and abdominal lymph nodes.
In the 55% of cases it presents at stage I and II, while the remaining present at a higher disease stages. 60%
of cases in particular the nodular subtype present with mediastinal involvement; splenic location is detected
in 20% of patients and presents a higher chance of extra nodal spread. Bone marrow involvement is very
rare since it is documented in 5% of patients.
About 40% of patients affected by HL present with symptoms such as fever, sweating and weight loss; itch is
also documented in some patients.
A fraction of CHL is related to chronic EBV infection whose role is lymphomagenic transformation of tumor
cells.
Classic HL may be diagnosed along with a setting of primary and secondary immunodeficiencies and in
these cases it have a more aggressive clinical behaviour and a less favourable prognosis.

CHL comprises 4 variants based on the characteristics of the reactive background: nodular sclerosis, mixed
cellularity, lymphocyte rich variant and lymphocyte depleted variant. All these variants share the nodal
architecture effaced by a polymorphic cellular infiltrate. They also share the atypical cell, the neoplastic cell
that is the Reed-Sternberg cell.
Reed-Sternberg cell account for less than 10% of lymphoma infiltrate and the remaining infiltrates comprises
the reactive background.
RS cells are characterized by a strong expression of cellular markers CD15 and CD30. These markers are
normally expressed by immune cells engaged in immune response and producing cytokines and
interleukins.

52
CHL Reed-Sternberg cells are negative for common B-cells markers such as CD20 and CD79 and they do
not express the common leukocytic antigen. The only B-cell marker that is expressed by CHL cells is PAX5,
also called B-cell specific activator protein, and remember that its typical of RS cells.

Also CHL carry monoclonal rearrangement of Ig, and this rearrangement is detectable only by using micro-
dissection techniques. Also in HL the rearranged genes are characterized by the presence of somatic
hypermutation that means that they do not have ongoing mutations. CH is histogenetically related to B-cells
that are in the post-germinal center stage of maturation.
Another important difference form PHL is that HL cells carry rearrangement immunoglobulin genes but do not
synthesize Ig genes. In other words insight in the investigation of the expression of Ig genes have resulted to
be negative in CHL RS cells.

1. Nodular sclerosis variant, is most frequent. The hallmark of this variant is the presence of diffuse
capsular thickening of the lymph node with intraparenchymal extension of fibrosis that gives to the
lymphoma infiltration a clear nodular pattern. The large band of fibrosis is delimitated by large lymphoid
nodules containing RS cells. The fibrosis may be composed of collagen and these collagen stripes
present intense birefringence at polarized light examination. The nodules are composed of
lymphocytes, histiocytes, eosinophils, Hodgkin and Reed-Sternberg cells. In nodular sclerosis variant,
RS cells have a peculiar feature: they have a clear cytoplasm and they are also called lacunar cells. In
nodular sclerosis lymphoma also we can find also mummified cells that are RS cells with hyper-
chromatic nuclei that represent apoptotic RS cells. In the 80s a grading system for nodular sclerosing
HL was proposed, and it was based on the number of RS cells.
-­‐ Grade I: cases in which more than 75% of nodules are characterized by scattered RS cells. RS cells have a
scattered distribution.
-­‐ Grade II: cases in which more than 50% of nodules are characterized by the presence of large amount of RS
cells. RC cells are more abundant and form sheets of cells. Further analysis showed the worst prognosis was in
grade II nodular sclerosis.
3 possible factors related to a more aggressive clinical behaviour in nodular sclerosis have been identified
and they are:
-­‐ eosinophilia: when more than 5% of cellularity consists of eosinophils;
-­‐ high degree of atypic RS cells: when bizarre or anaplastic elements are more than 25%;
-­‐ the reduction in depletion of lymphocytes with a fraction of small lymphocytes accounting for less than
33% of cellularity.
When patients affected by nodular sclerosis HL present one of these unfavourable prognostic factors they
are classified as high risk patients.
2. Mixed cellularity variant. In classic HL mixed cellularity, the fibrosis is only mild and the background is
very prominent with large amounts of eosinophils and histiocytes. Mixed cellularity CHL is a subtype
most frequently associated with EBV infection. EBV positive cases may account up to 75% of cases.
3. Lymphocyte-rich variant. It is a recent controversial entity. The background consists only of small
lymphocytes and should be distinguished from NLPHL because of the presence of RS cells. The
background is similar to that of NLPHL but the atypical cells are RS cells and not popcorn cells.
4. Lymphocyte-depleted variant. It accounts for less than 5% of CHL. The Hodgkin RS cells are very
abundant with fibrotic background and this characterized by lymphocyte depletion. This type of CHL is
diagnosed mainly in immunosuppressive patients and it appears to have a more aggressive clinical
behaviour but this aggressive behaviour is probable more related to immunological characteristics of the
patients than to underlying biologic features.

The final slides are devoted to the biological aspects that are responsible for the features of HL.
Differential features between Reed-Sternberg cells and L&H cells:
-­‐ both RS and H&L carry rearrangement of Ig genes.
-­‐ RS cells do not express B-cell markers or immunoglobulin gene receptors. Molecular studies and gene
expression profiling in RS cells documented the general loss of this specific genetic expression of B-cell
related genes and the most important method responsible for the silencing of B-cell program in classical
RS cell seems to be promoter cell hypermethylation.

B-cells that do not express functional B cell receptors should not survive. Which mechanisms allow RS cells
survival without functional B-cell receptor expression? It has been hypothesized that apoptosis pathways
may be involved. Actually both apoptosis intrinsic and extrinsic pathways may be targeted by genetic
alterations in classic HL.
Apoptosis extrinsic pathway is controlled by CD95 and in HL has been reported an upregulation of CD
proteins that inhibits activity of CD95. In 90% of cases inhibiting mutation of Fas gene has been reported.
53
Apoptosis intrinsic pathway is controlled by Bcl-2 proteins and RS cells are characterized by hyper-
expression of anti-apoptotic proteins such as inhibitors of IAP proteins and of survivin.
EBV may promote the over-expression of these proteins, so, also EBV infection may explain survival of RS
cells lacking functional B-cell receptors.
Clonal EBV infection is found in 10% to 75% of cases and the two major oncogenic proteins are LMP1 and
LMP2A. LMP1 may activate expression of Bcl2 protein but also activates NFkB intracellular signalling.
LMP2A is able to simulate the BCR signalling activation. It has been hypothesized that EBV infection makes
RS-cells BCR “signalling” independent so that it expands independently from the stimulation of BCR
signalling and even from the presence of a functional BCR.

RS cells may secrete large amounts of cytokines and chemokines and these may recruit inflammatory
population. This explains why CHL is characterized by an inflammatory rich background in which RS cells
represents only a minority. This reactive background probably provides survival signalling to RS cells. So the
interaction between RS cells and the reactive infiltrate probably promote the expansion and survival of RS
cells.
The background composition is also explained by the kind of chemokines and cytokines secreted by RS
cells.
RS cells produce molecules with inhibitory activity on Th1 and CD8+ lymphocytes. So the background of
reactive infiltrating HL is characterized by prevalence of T-helper 2 cells and this create a favourable
microenvironment for RS cells, limiting immune system activity against RS cells.

Nodular Lymphocytes Predominant Hodgkin’s Lymphoma (NLPHL)

NLPHL account for 5% of all HL. It mainly occurs in male patients and the peak of incidence is between 30
and 50 years of age. It involves superficial lymph nodes such as latero-cervical, axillary and inguinal lymph
nodes, while mediastinum, spleen and bone marrow involvement is rare. Most cases present at stage 1.
Only 5-20% of cases are diagnosed in a more advanced stage. The course is more indolent compared to
CHL but recurrence may occur. It is responsive to chemotherapy and overall survival is >80% in 10 years.
NLPHL may progress to a high graded lymphoma, meaning that lymphoma is characterized by a dramatic
increase in the number of large cells and it transforms into diffuse large B-cell lymphoma. This may happen
in 3-5% of cases and it is a bad prognostic indicator because this transformed lymphomas result to be
refractory to chemotherapy and are invariably fatal.
The morphology of NLPHL is characterized by the effacement of nodular architecture by a nodular or diffuse
proliferation of lymphoid cells that mainly consists of small lymphocytes and epithelioid histiocytes (having
eosinophilic large structure).
Scattered among these reactive background consisting of small T-lymphocytes, you can find atypical cells
called L&H cells (“Lymphocytic & Histiocytic”) or popcorn cells (have polylobated irregular nuclear contour
that resemble popcorns). These cells have large vesicular nuclei with prominent peripherally located nucleoli.
I have said that also the phenotype of H&L cells is atypical. In contrast with CHL cells, they are characterized
by strong expressions of B-cell markers and leukocyte antigen common (CD45).
Popcorn cells are positive for CD20 and CD79 and they are uniformly negative for CD30 and CD15. CD30
and CD15 co-expression is the diagnostic marker of CHL in RS cells.
Another characteristic of NLPHL is the presence of dendritic meshwork that is highlighted by immunological
staining for specific follicular dendritic cell markers such as CD21, CD22 and CD 35.
NLPHL is characterized by the presence of a high number of CD57 positive T-lymphocytes, called T-follicular
lymphocytes. These are abundant in germinal centers and are responsible for the germinal center reaction.
The typical feature of NLPHL are the CD75 cell rosettes around atypical popcorn cells.
Popcorn cells carry rearrangement immunoglobulin gene and show presence of somatic hypermutation and
ongoing mutations.
These data support the hypothesis of derivation of popcorn cells from B-cells at the germinal center stage of
their maturation. The immunoglobulin gene rearrangements are active and cyto-hybridization of mRNA
immunoglobulin gene documented the presence in the cytoplasm of popcorn cells of immunoglobulins. They
are also positive for transcriptional factors or Oct2 and BOB1 which are two molecules required for the
stimulation of the transcriptional promoter factors of immunoglobulin genes. These molecules are highly
expressed in NLPHL and this can explain the expression of immunoglobulin genes in this subtype of
lymphoma.

7.3 Non-Hodgkin’s lymphomas (NHL)

In the most recent WHO lymphomas classification, each lymphoma entity is defined according to the
combination of clinical, morphologic, phenotypic and molecular features. Incidence, biology and clinical
representation differentiate Hodgkin lymphomas and NH lymphomas. While Hodgkin lymphomas are a

54
primary nodal basal disease, NHL may primarily arise at extra nodal sites, often showing unique clinic-
pathologic and genetic features. Moreover NHL prognosis depends more on histotype than on stage, while
the opposite is true for Hodgkin lymphomas.
The non-Hodgkin lymphomas are diverse group of blood cancers that include any kind of lymphoma except
Hodgkin's lymphomas. Types of NHL vary significantly in their severity, from slow growing to very aggressive
types.
Lymphomas are types of cancer derived from lymphocytes, a type of white blood cell.
Lymph-node biopsy of other extra-nodal sites is mandatory for the diagnosis of lymphomas. Fine needle
aspiration just suggests the lymphoma, it is not sufficient for diagnosis.
In diagnosis of a lymphoma we need to perform both bone marrow biopsy and bone marrow aspiration.
Lymphomas are treated by combinations of chemotherapy, monoclonal antibodies (CD20), immunotherapy,
radiation, and hematopoietic stem cell transplantation.
The many different forms of lymphoma likely have different causes.
• Infectious agents:
- Epstein-Barr virus: associated with Burkitt's lymphoma, Hodgkin's lymphoma, follicular dendritic cell sarcoma,
extranodal NK-T-cell lymphoma
- Human T-cell leukemia virus: associated with adult T-cell lymphoma
- Helicobacter pylori: associated with gastric lymphoma
- HHV-8: associated with primary effusion lymphoma, multicentric Castleman disease
- Hepatitis C virus: associated with splenic marginal zone lymphoma, lymphoplasmacytic lymphoma and diffuse
large B-cell lymphoma
- HIV infection: aggressive NHL, often extra-nodal
• Chemicals, like polychlorinated biphenyls (PCBs), diphenylhydantoin, dioxin, and phenoxy herbicides.
• Medical treatments, like radiation therapy and chemotherapy.
• Genetic diseases, like Klinefelter's syndrome, Chédiak-Higashi syndrome, ataxia telangiectasia syndrome.
• Autoimmune diseases, like Sjögren’s syndrome, celiac sprue, rheumatoid arthritis, and systemic lupus
erythematosus. The autoimmune diseases present a prolonged auto-antigenic stimulation.
• Immunodeficiency status, such as the post- transplant situation (PTLD) of a solid organ as lung, liver or
kidney.

The majority of lymphomas are however without any evident cause.

We have a clinical distinction between Indolent NHL and aggressive NHL, and we have also highly
aggressive lymphomas.
Based on the clinical behaviour, NHLs may be subdivided in :
– indolent: characterized by long term survival (overall survival of 10 yrs in about 50% cases), however no
patient is cured, and in the end death predominantly occurs by lymphoma complications.
– aggressive: when untreated, they display a very short overall survival (almost all patients would die in 5
years). However, treated patients may be cured. In high grade or aggressive lymphoma, the disease is most
often localized. Chest localization is typical, while it is quite rare in indolent lymphoma.

– highly aggressive: include for example Burkitt’s lymphoma. They are characterized by a high proliferative
index. However, they can be cured with an intensive chemotherapeutic regimen.

The classification of neoplasm is between the B and T cell neoplasms and between the neoplasm of the
precursors and those of the peripheral cells. This approach has been used also in WHO classification in
1997 and in 2008.

We may distinguish between primary and secondary lymphoid organs:

• primary lymphoid organs: thymus and bone marrow. Bone marrow is the site of maturation of normal B
lymphocytes, here the recombination of genes encoding for the Ig chains takes place and it is controlled by
two enzymes, RAG 1 and RAG 2 ( RAG stands for Recombination Activating Gene).
• secondary lymphoid organs: lymph nodes, spleen and mucosa-associated lymphoid tissue. These are the
site of encounter between antigens and lymphoid cells. Here the antigen dependent maturation of B cells
occurs, virgin B cells enter the germinal centre, where they proliferate. Then we have the appearance of
centroblasts and centrocytes. The aim of the germinal center reaction is to select B cells carrying a BCR
with high affinity for the antigens. Cells carrying a BCR with an increased affinity for the antigen exit the
germinal center and survive and give rise to memory B cells and plasma cells, while cells carrying a BCR
with a decreased affinity for the antigen undergo apoptosis (this is why germinal center cells are
physiologically negative for Bcr2 proteins which are anti- apoptotic proteins). The main molecular events
that take place within the germinal centers are:

55
 - generation of somatic hypermutation in the immunoglobulin genes sequences. The presence of somatic
hypermutation in the sequences of genes encoding for immunoglobulin heavy and light chains may be employed
as a histogenetic marker of B cells through the germinal centre reaction.
- class switch from IgM to IgG, IgD and IgE. These molecular events are mediated by a specific enzymatic
machinery, which is called AID.


Most B cell lymphomas derive from B cells carrying hyper mutated rearranged Ig genes:
–  about 50% of B-CLLs and the majority of mantle cell lymphomas derive from virgin B cells
–  follicular lymphomas derive from the germinal center B cells
–  Burkitt's lymphoma derive from crossed germinal center B cells 


Neoplastic transformation of human cells requires the activation of proto-oncogenes: many lymphomas are
associated with specific chromosomal translocations, which alter the expression, structure and function of
specific oncogenes. This might places the oncogene below a regulatory sequence of the Ig genes, which are
constitutively expressed in a normal cell, or can result in the juxtaposition of two genes to form a chimeric
gene encoding for a fusion protein with novel function (this is the case of many AML and CML ). In the
second case, usually the portion of the protooncogene is responsible for the regulation of proliferative
mechanisms of cells carrying the translocations, while the cells deriving from the partner gene giving to the
chimera the capability to autoactivate. 

Alternatively oncogenes may be activated by point mutation or gene amplification.


Why translocations are so frequent in lymphoproliferative lymphomas? This depends on physiological

maturation of these cells: molecular events in B lymphocytes requires maturation of RAG and AID genes. It
has been thought that translocation takes place following aberrant enzymatic machinery. 


The first distinction is between small and large B cells lymphomas.


Small B cells lymphomas: consist of an infiltration of small to medium sized B cells whose histology
corresponds to clinical indolent lymphomas. Small lymphomas include many subtypes: CLL/SLL,
lymphoplasmacytic lymphoma, hairy cell leukemia, myeloma/plasmacytoma, marginal zone nodal. Peripheral
small B cell lymphomas are a heterogeneous group of NHLs, they share small cell cytology, but may be
divergent in histogenesis, biology, someway in clinical behaviour (mantle cell lymphoma has an aggressive
clinical course differently from other small B cells lymphomas) and therapy.
Large B cells lymphomas: lymphomas with large immunoblastic and centroblastic cells result in aggressive
lymphomas. They include diffuse large B cell lymphoma, mediastinal B-cell lymphoma, primary effusion
lymphoma and Burkitt's lymphoma.

Small B cells lymphomas have a long clinical course, in which they may accumulate secondary genetic
alterations. These might then be responsible for progression of low grade indolent lymphomas into
aggressive diseases, showing a diffuse large cell histology.

Indolent lymphomas
These types of lymphoma grow very slowly. As a result, people with indolent NHL may not need to start
treatment when it is first diagnosed. They are followed closely, and treatment is only started when they
develop symptoms or the disease begins to change. This is called watchful waiting.
When indolent lymphoma is located only in one area, it is called localized disease (stages I and II). For
people with localized disease, radiation therapy may eliminate the NHL.
However, most patients with indolent NHL have later-stage disease, stage III or IV, at the time of diagnosis.
There are many effective treatments for these stages of indolent NHL, but it may come back months or years
after treatment has finished, requiring additional treatment.
Indolent NHLs are rare in young people. They have their typical peak around 60 years. This is the general
rule but in the clinical practice it can be diagnosed in patients of 40 years.
Presentation is with diffuse adenopathy, hepatosplenomegaly, frequent bone marrow involvement and
leukemic disease in PB (circulating lymphoma cells).
Indolent or low grade NHL are:
- lymphocytic lymphoma: it is the counterpart of chronic lymphocytic leukemia.
- lymphoplasmacytic lymphoma: the counter part of Waldenström's macroglobulinemia.
- mantle cell lymphoma: subtype of B-cell lymphoma, due toCD5 positive antigen-naive pre-germinal center
B-cell within the mantle zone that surrounds normal germinal center follicles.
- follicular lymphoma: most common of the indolent NHL, and the second-most-common form of NHL overall.
- marginal zone lymphoma

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It is important to evaluate the prognosis of any indolent lymphoma because is important to detect the
patients that have an high risk.

Aggressive lymphomas
These types of lymphoma may develop rapidly, and treatment is usually started immediately. These types of
lymphoma usually need more intensive chemotherapy. Radiation treatment may be recommended in addition
to chemotherapy, especially in patients with limited stage disease (stage I or II). Many forms of aggressive
lymphoma may be cured with effective treatment.
The most frequent lymphoma of this category is the Diffuse large B cell lymphoma. Another lymphoma of this
class is the Primary mediastinal B-cell lymphoma. Moreover, one of the most aggressive is the Peripheral T-
cell lymphoma but also highly aggressive lymphomas such as Burkitt’s lymphoma and the lymphoblastic
lymphoma. Burkitt’s and mediastinal lymphomas are quite rare.

7.3.1 Small B cells lymphomas



B-cell chronic lymphocytic leukemia (B-CLL) / SLL
B-cell chronic lymphocytic leukemia (B-CLL), also known as chronic lymphoid leukemia (CLL), is the most
common type of leukemia (a type of cancer of the white blood cells) in adults. It’s the most frequent leukemia
in Europe and in the USA, on the other hand is quite rare in Asia such as in Japan. Is a disorder of the
elderly, and it is quite rare before 40 years: it’s not impossible to make diagnosis of CLL in a 25-30-40 years
patients but it’s impossible to make it in a child. The peak of the disease is around 60-70 years and there is
also a male prevalence.
CLL affects B cell lymphocytes, which originate in the bone marrow, develop in the lymph nodes, and
normally fight infection by producing antibodies.
In CLL, B cells grow out of control and accumulate in the bone marrow and blood, where they crowd out
healthy blood cells. CLL is a stage of small lymphocytic lymphoma (SLL), a type of B-cell lymphoma, which
presents primarily in the lymph nodes. CLL and SLL are considered the same underlying disease, just with
different appearances.
CLL is a disease of adults. Most (>75%) people newly diagnosed with CLL are over the age of 50, and the
majority are men. However, in rare cases, it can occur in teenagers and occasionally in children. Some of
these may relate to an inherited predisposition.
Most people are diagnosed without symptoms as the result of a routine blood test that returns a high white
blood cell count, but, as it advances, CLL results in swollen lymph nodes, spleen, and liver, and eventually
anemia and infections. Early CLL is not treated, and late CLL is treated with chemotherapy and monoclonal
antibodies.
CLL, differently from ALL, may be asymptomatic. In the case of symptomatic patients, symptoms are related
to the anemia, infection or are systemic. A high symptomatic patient is not so common in CLL. As a
consequence, the physical evaluation depends on the stage of the patient: in those with only high
lymphocyte level, the physical examination is negative: no nodes, no organomegaly. On the other side, if the
disease is advanced we have adenopathies and organomegaly. After that, we can have symptoms, of bone
marrow failure for bone marrow infiltration. A typical symptom of CLL is the frequent infections related to low
gamma globulin level. This is quite typical of chronic lymphoid disorders.
Many patients, especially older ones, with slowly progressing disease can be reassured and may not need
any treatment in their lifetimes.
CLL is a neoplasm involving peripheral blood and bone marrow, spleen and lymph nodes. CLL is the name
used when it involves peripheral blood and BM. SLL is represented by non leukemic cases with tissue
morphology and immunophenotype of CLL. These are different clinical presentations of the same biological
disease.
It’s not necessary to perform bone marrow aspiration or biopsy and lymph node histology in order to make
the diagnosis because analysis of peripheral blood is sufficient. On the other side, the lymphoid cells are
present also in the bone marrow with the same morphological features and in some cases there is a
complete lymphoid marrow metaplasia, so all the bone marrow is represented by these lymphoid cells.
In the past we performed the bone marrow biopsy because there is an infiltration that progresses from
nodular to spread, diffuse infiltration, and this was of prognostic relevance in the past. Now we have many
prognostic factors for CLL that are better than the bone marrow picture.

Histological picture of a bone marrow biopsy from a patient with CLL will show infiltration of small cells,
nodular interstitial and diffused pattern, lymphoma cells with a rounded nuclear contour, rounded chromatin,
inconspicuous nucleoli. 30% of the bone marrow cellularity is constituted by lymphocytes.

57
Bone marrow examination in CLL is not required, but it may be useful in order to assess the residual
hematopoiesis.
If there is a patient with high level of lymphocytes, performing a morphological evaluation of the peripheral
blood we will be able to observe small lymphocytes of mature appearance with very low cytoplasmic halo
and no sign of aggressive cells. A typical finding of CLL is the presence of the nuclear shadows
(Gumprecht’s shadows). These are not circulating cells but arise from the breaking of the cells during the
trauma of the smear. These are not diagnostic for CLL but if I see a patient with these I can say that the
patient may be affected by CLL. However the shadows can be present in other similar chronic
lymphoproliferative disorders, thus diagnosis shouldn’t be based on this finding alone.
Lymph node involvement by small lymphocyte lymphomas is characterized by diffuse infiltrate of three
different cell populations:
- Monomorphic, small round lymphocytes, similar to mature lymphocytes with clumped chromatin and
scanty cytoplasm. These are the most represented ones.
- Pro-lymphocytes, small cells with relatively clumped chromatin and small nucleoli.
- Paraimmunoblasts (larger cells with round to oval vesicular nuclei with central prominent nucleoli and
dispersed chromatin), they resemble immunoblasts. They represent the proliferative compartment of
chronic lymphocytic leukemia.

The diagnosis is made if there is the presence in peripheral blood of more than 5.000 monoclonal B
lymphocytes/microL for at least three months and a typical immune-phenotypic profile (CD19, CD20, CD23,
CD5) and monoclonal. So the diagnosis is based on flow cytometry.
CLL is characterized by expression of B cell markers (a common feature of all B cells lymphomas). B cells
display the same B antigens as ALL (CD19, CD20, CD23) and there is co-expression of a T cell associated
antigen CD5.
When seeing a patient at the flow cytometry we need to ask for CD23 and CD5 expression because the
differential diagnosis with other disorders is based on expression of these antigens.
CLL is CD5+ and CD5 is a T cell antigen, but it is now known that CD5+ B cells are involved in autoimmune
disorders: autoimmune manifestations are quite typical because there is a low control of the emergence of
autoimmune clones, so in this disorders is quite typical to have autoimmune hemolytic anemia or
autoimmune thrombocytopenia. In other words, CLL anemia can be related to the bone marrow involvement,
to the chronic disease anemia or to hemolytic anemia, so when you have a patient with CLL and at the CBC
there is anemia you need to rule out the hemolytic anemia, so you ask for Coombs test and reticulocytes.
In order to demonstrate that the B cell population is monoclonal, we can observe the immunoglobulin
expression on the cells at low density (of k and lambda light chain) that will be monoclonal.
Around 50% CLLs carry unmutated Ig gene rearrangement (meaning a high degree of homology with
germline sequences >90%), 50% carry mutated rearrangement.  
BCR in CLL shows biased V region usage, this suggests the possible relation between CLL and chronic
stimulation by specific antigens.
CLL is an indolent disease, characterized by gradual leukemia cell accumulation rather than by rapid cell
proliferation.
Median survival times range from less than 10 to more than 25 yrs. The disease course is best predicted by
combining clinical and pathological prognostic factors:
- Clinical prognostic factors: Rai and Binet clinical staging system. Younger patients are at higher risk of
transformation. The Binet system is based on 3 stages:
- stage A patients are those with less than 3 lymph nodes involved, no anemia and no thrombocytopenia.
- stage B is characterized by no anemia, no thrombocytopenia but more than 3 lymph nodes involved.
- stage C stage is characterized by anemia and thrombocytopenia.
The Rai system is a bit more complicated.
- rai I with lymphocytosis and adenopathy,
- rai II with lymphocytosis and organomegaly with or without adenopathy,
- rai III is lymphocytosis with anemia,
- rai IV is lymphocytosis with thrombocytopenia.

- Pathological prognostic factors: analysis of IgHV mutational status (mutated patients have a better
prognosis), CD38 expression (causes adverse prognosis). Several cytogenetic abnormalities have been
reported, the most frequent is 13q14 deletion which occurs in >50% of cases, it is associated with good
prognosis, 11q22-23 deletion is found in 20% cases, associated with a higher risk of disease progression,
trisomy 12 in 20% with atypical morphology and phenotype and with NOTCH 1 mutations. The most
unfavourable prognostic factor is the presence of 17p13 deletion, which is very rare, occurring in less than
10% cases, and associated with a very poor prognosis.

58


Talking about CLL progression into high grade lymphomas, mainly DLBCLs (diffuse large b cell lymphoma,
consisting of immunoblastic cells) occur into 2 to 8 % patients with CLL.

Transformed cases show a poor prognosis, median survival of less 1 yr, very aggressive behaviour and high
degree of resistance to chemotherapy. Unmutated CLLs are at higher risk of transformation into DLBCLs.

The two most common genetic events for transformation are abnormalities in TP53 and MYC genes (in
particular rearrangement of MYC). Chronic lymphocytic leukemia (CLL).

The most important aspect of the survival is that we have a very wide variability: we have some patients that
are going to die with the CLL and not due to it, and some patients are going to die due to the CLL. But many
patients are going to die due to another reason and not to the CLL itself. For chronic, indolent, somewhat
benign disorders such as CLL it is necessary to identify patients with bad prognosis, especially young
patients with less than 60 years.

The FISH panel is extremely important in the clinical practice. Through the FISH panel we can study four
defined chromosomal aberrations: 17p deletion, 11q deletion, trisomy of chromosome 12, deletion of
chromosome 13.
According to the cytogenetical abnormalities in CLL we have very different outcomes. Patients with the
isolated deletion of chromosome 13q and patients with 17 deletion show completely different overall survival.
Patients with mutated status show a good prognosis, on the other side patients with unmutated Ig genes
show a bad prognosis, because, as I explained you for ALL, all the diseases of mature B calls are better than
the diseases of immature B cells. This is the explanation of the overall survival of the patients with mutated
and unmutated status: the median of survivals are around 80 months and almost 300 months.

It is one of the tumor with the lowest background of mutation: we have very few mutations in CLL, but we
have at least 4 genes that are mutated in more than 5% of cases: TP53, NOTCH1, SF3B1 and ATM. These
alterations are related to the prognosis of the patients. This is important because if we add these mutations
to the information that we obtain from the FISH analysis, we perform a better prognostic analysis of these
patients.
Another important abnormality to evaluate in patient with CLL is the TP53 abnormality because the presence
of this abnormality is related to the refractoriness to treatment. So we have different markers, prognostic and
predictive markers. A prognostic mark is a baseline mark, if I have this mark the survival of the patient is
more or less good. The predictive marker is a marker that is able to predict the response to the treatment.
Why is it clinically important to understand if there is a mutation in a cell? Because if I have a subclone with
this mutation and then I treat the patient harbouring this mutation, the chemotherapy is able to remove the
wild type cells but the cells with the mutation progress and are resistant to the chemotherapy. This is an
interesting model of the behaviour of a cancer, quite similar to the behaviour of acute myeloid leukemia: you
treat the good cells but the bad ones are selected by the action of chemotherapy, and then the disease is
complicated because you select the bad cells that expand after the treatment.

Many patients are not treated at the beginning: many patients are going to die with CLL but not due to it.
Then there are patients in which CLL progresses to a symptomatic disorder, and thus there is the need to
perform a risk assessment in each patient evaluating the age and comorbidities.
The most recent therapeutic approach is the combination of rituximab, that is the monoclonal antiCD20
antibody that we use also for non-Hodgkin lymphoma, coupled with other drugs.
This immunochemotherapy approach is the best one but it’s not good for everyone: the complete remission
rate is more than 40%.

Monoclonal B cell lymphocytosis

Before the CLL there is monoclonal cell lymphocytosis: flow cytometry is quite good in detecting B cell
clones, and there is some situation in which subjects present with a clonal B cell population in peripheral
blood whose level is not sufficient to make a diagnosis of CLL: we have small clones, no adenopathies, no
cytopenia. We call it monoclonal B cell lymphocytosis. It can progress to CLL. Patients with monoclonal B
cell lymphocytosis have a normal cell count and differential leukocyte count. Lymphocytosis may be evident
but not sufficient to make CLL diagnosis.
Richter syndrome: diffuse B cell lymphocytosis with the appearance of systemic symptoms. There are
patients with a typical clinical picture of CLL, but have also systemic symptoms such as night sweats, fever
and so on that are not typical of CLL. Masses, bulky adenopathies and so on can be found as well. In this
conditions we can hypothesize Richter syndrome and it is important to perform the lymph node biopsy,
because the diagnosis is based on the histological evaluation.
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Mantle cell lymphoma
Mantle cell lymphoma (MCL) is a type of non-Hodgkin's lymphoma, comprising about 6% of NHL cases. MCL
is difficult to treat and seldom considered cured. Median survival is of 3-5 yrs.
Mantle cell lymphoma derives from a subset of unmutated naive B cells characterized by CD5+ expression.
Patients present with generalized adenopathy, splenomegaly and bone marrow involvement.
Extra-nodal involvement occurs in GI, lungs and Waldeyer's ring.

It is one of the most aggressive diseases since it combines the worst features of aggressive and indolent B
cell lymphomas: it has an extra nodal localization, aggressive clinical presentation, but it is not curable like
indolent lymphomas, not as sensitive to chemotherapy as aggressive lymphomas, and it results in frequent
relapses.

This disease occurs because of a specific translocation 14-18 responsible for CDT1 translocation, providing
a selective advantage responsible for the aggressive pattern. However, in most cases this is not the only
cytogenetic alteration: they acquire many other cytogenetic events interfering with pathways which are
responsible for chemoresistance .

Histology: quite large cells, may be characterized by a mantle zone pattern. With disease progression, the
neoplastic mantle cells colonize and infiltrate the germinal centre, and may appear with a nodular
appearance. Nodal architecture is obtained in most aggressive cases. It may have a mantle, nodular or
diffused pattern .

Histologically the most classical variant of mantle cell is made of small to medium sized cells, irregular nuclei
with condensed chromatin and scanted cytoplasm. The most typical feature is the presence of nuclei
resembling centrocytes: it was called centrocytic lymphoma, with at least 3 variations:
- small cell variant (which enters differential diagnosis with lymphocytic leukemia)
- blastoid variant
- pleomorphic variants, characterized by middle sized cells.

Their distinction may have a prognostic relevance: usually these variants show a higher proliferative index,
characterized by a more aggressive clinical course with median survival of even less of 5 yrs.


When MCL at extra-nodal site presents in the GI, it may have a classical presentation called lymphomatous
polyploids in small and large bowel. Endoscopically you can find a large number of polypoid masses which
are resembling adenomatous polyps of the large bowel. If performing a biopsy, histologic examination
reveals a neoplastic lymphoid infiltration and features consistent with MCL.
MCL is characterized coexpression of CD5 and Cyclin D1+, and in contrast with CLL, CD23 is negative.
Germinal centre markers are negative as well. A recently discovered marker is SOX 11, a transcription factor
which seems to be even more sensitive than CD5 to identify mantle cell lymphoma cells.
MCL is associated with specific translocations: a large fraction of MCLs (95%) carries a translocation
involving chromosome 11 and 14, which places the CCND1 gene under the control of the IgH locus
enhancer. This results in the over-expression of cyclin D1, which is a controller of the cell cycle, having as
ultimate effect the shift from G1 to S phase of the cell cycle. 

Cyclin D1 translocation is not the only molecular abnormality that may occur in MCL, many other genes are
involved with mutations, deletions and rearrangements. Many of these genes are involved in the control of
apoptosis and proliferative cell pathways.
Variants of MCLs presenting with leukemic and minimal or non nodal disease seem to have an indolent
clinical course.

Marginal zone lymphoma

Marginal zone is a B cell area surrounding mantle zone.
From the immunologic point of view most MZ B cells are memory B cells able to organize a quick response
to antigenic stimuli. MZ is not apparent in normal lymph node, except in some particular conditions like
toxoplasmic lymphadenitis. MZ is mainly represented in extra nodal lymphoid sites, and MZ lymphomas are
the most frequent primary extra nodal small B cells lymphomas.
MZLs according to WHO classification published 2008 includes 4 subtypes:
• extra-nodal, accounts for 7-8% of all NHLs. Most cases are associated with chronic immune stimulation:
these lymphoma subtypes arise in people with chronic autoimmune disease, who are at higher risk of
developing MZL of salivary and thyroid glands. Another subset of primary extra-nodal MZL is associated
with chronic infections. The prototype for instance is in the stomach, and there is a strict association with
[Link] in the stomach and MZL development
• nodal, which independently from site and pathogenesis show common morphological features. When the
pathologist provides you a diagnosis of MZLs in a nodal biopsy you should always exclude the possibility of

60
 a secondary localization of MZL primary arising at extra nodal site. Analysis should always include
endoscopy, othorino-laringoiatric consultation and so on in order to exclude any other primary site of MZL.
• splenic, there is a strict association between HCV and development of MZL in spleen.
• primary cutaneous MZL.


Nodal MZL and extra-nodal biopsy shows a pattern that may mimic the growth pattern of normal MZN,
circumscribing residual germinal centers, when neoplastic cells colonize the GC the infiltrate has a nodular
and then diffuse appearance, like mantle cell.
Cytologically, the infiltrate consists of cells with varying morphological features: the most typical population is
medium sized clear cells which are called monocytoid B cells, with a relatively abundant cytoplasm, slightly
irregular nuclei similar to that of monocytes.
Morphologic features of cells comprising MZLs: medium sized cells, slightly dislocated cytoplasm,
monocytoid nuclei, lymphocytic features. In some cases you can also find Dutcher bodies intranuclear
inclusions.
Immunophenotype: MZL do not show specific immunophenotype markers. From the immunophenotypic point
of view this is mainly an exclusion diagnosis. The cells are positive for B cells markers, but negative for CD5,
CD23 and cyclin D1.

Molecular genetics of MZL: nodal MZL do not show specific molecular alterations, while wild type MZL may
have different recurrent chromosomal translocations. 4 recurrent translocations have been reported in wild
type MZL, 3 of these deregulate the same molecular pathway (NF-kB, which becomes always active and
induces deregulated cellular proliferation). The incidence of these translocations varies according to the site
of lymphoma presentation

Lymphoplasmacytic lymphoma (LPL)


it is a disseminated B cell lymphoproliferative disorder characterized by a spectrum of small B cells,
plasmacytoid lymphocytes and plasma cells. This mainly involves BM, although splenic and nodal disease
can be seen as well. LPL is typically associated with an IgM gammopathy.
The main attributing antibody is immunoglobulin M (IgM). It is a type of lymphoproliferative disease, which
shares clinical characteristics with the indolent non-Hodgkin lymphomas.
The disease is characterized by an uncontrolled increase of B-cells, i.e., white blood cells formed in the bone
marrow and lymph nodes. The proliferation of B-cells interferes with the production of red blood cells,
resulting in anemia. A unique characteristic of the disease is that the B-cells produce excess amounts of
immunoglobulin protein (IgM), thickening the blood, and requiring additional treatment. While the disease is
incurable, it is treatable.

LPL is a disease that involves bone marrow mainly, where the disease may have nodular, interstitial,
paratrabecular, and diffuse patterns. The infiltrate cytologically consists of small lymphocytes, plasma cells,
plasmacytoid lymphocytes. Dutcher bodies are frequently found
LPL is associated with a specific lymphoma genic alteration: a MYD88 mutation. This mutation results in
deregulating activation of NF-kB, resulting in deregulated proliferation of lymphoma cells.

Follicular lymphoma (FL)

it is a prototype of lymphomas of germinal centre B cells, with typicaly show at least partially partially
follicular patterns centrocytes and centroblasts .
Follicular lymphoma is the most common of the indolent non-Hodgkin's lymphomas, and the second-most-
common form of non-Hodgkin's lymphomas overall. It is defined as a lymphoma of follicle center B-cells
(centrocytes and centroblasts), which has at least a partially follicular pattern. It is positive for the B-cell
markers CD10, CD19, CD22, and usually CD20, but almost always negative for CD5.
It often involves lymph nodes, spleen, Waldeyer’s ring, and bone marrow. Lymphadenopathies are often
found. It is a non curable, indolent lymphoma and the median survival of the patients affected is of 8-10
years.

New treatments including Rituximab have markedly improved the prognosis for FL, resulting in increased
survival rates and longer times to re-treatment.

Transformation to diffuse large B cell lymphoma occurs in 25-35 % of patients and it is followed by an
aggressive disease course.
The tumor is composed of follicles containing a mixture of centrocytes, "small cells", and centroblasts, "large
cells". These follicles are surrounded by non-malignant cells, mostly T-cells. In the follicles, centrocytes
typically predominate; centroblasts are usually in minority.

Grading of follicular lymphomas is based on the ratio between centroblasts and centrocytes:
• grade 1 (< 5 centroblasts per high-power field (hpf))
• grade 2 (6-15 centroblasts/hpf)

61
• grade 3 (> 15 centroblasts/hpf).
Grade 3 is further subdivided into:
• grade 3A (centrocytes still present)
• grade 3B (the follicles consist almost entirely of centroblasts)
The WHO 2008 update classifies grades 1 and 2 now as low grade follicular lymphoma, grade 3A as high
grade follicular lymphoma, and grade 3B as Diffuse Large B Cell Lymphoma (DLBCL).

Follicular lymphomas show expression of CD10 and Bcl 6, in the absence of CD5 and CD23. Follicular
lymphoma are characterized by over-expression of Bcl 2, an important diagnostic criterium to differentiate FL
from polyactive follicular hyperplasia.
A translocation between chromosome 14 and 18 results in the overexpression of the bcl-2 gene. As the bcl-2
protein is normally involved in preventing apoptosis, cells with an overexpression of this protein are basically
immortal. The bcl-2 gene is normally found on chromosome 18, and the translocation moves the gene near
to the site of the immunoglobulin heavy chain enhancer element on chromosome 14.
Histological grade and growth pattern are prognostic factors, high histological grade and diffuse pattern are
associated with higher risk of bad progression.

Microenvironment composition seems to have prognostic relevance, but histological transformation into large
cells is the most relevant prognostic factor.

For evaluation of prognosis of follicular lymphomas we have the FLIPI score. The prognostic factors that are
considered are: age, stage, number of lymph node areas involved, serum hemoglobin level, and serum LDH.
One point is assigned for each of the following adverse prognostic factors:
• Age > 60 years
• Stage III or IV disease
• > 4 lymph node groups involved
• Serum hemoglobin < 12 g/dL
• Elevated serum LDH

The sum of the points allotted correlates with the following risk groups:
• Low risk (0-1 points): 5 and 10 year survivals of 91% and 71%, respectively
• Intermediate risk (2 points): 5 and 10 year survivals of 78% and 51%, respectively
• High risk (3-5 points): 5 and 10-year survivals of 53% and 36%, respectively

Another feature of the follicular lymphoma is the duration of the remission. At each treatment line the
duration of the response is shorter. The disease is less and less responsive to the treatment.
The best treatment approach is allogeneic transplant. The standard approach is the combination of
chemotherapy with monoclonal antibodies. In this case we talk about anti CD20 antibodies. It has been
demonstrated that the combination of Rituximab and chemotherapy ameliorates the survival of the patient
and the mortality risk of patients with follicular lymphoma.
The combination of Rituximab and Lenalidomide is effective in the control of indolent lymphoma such as
follicular lymphoma.

7.3.2 Large B cells lymphomas

Diffuse large B cell lymphomas ( B cell lymphoma with aggressive behaviour ) DLBCL
It is a cancer of B cells. It is the most common type of non-Hodgkin lymphoma among adults. Diffuse large B
cell lymphoma constitutes 1/3 of all non-Hodgkin’s lymphomas. This cancer occurs primarily in older
individuals, with a median age of diagnosis at approximately 70 years of age, though it can also occur in
children and young adults in rare cases.
DLBCL is an aggressive tumour which can arise in virtually any part of the body (40% are primary extra
nodal), and the first sign of this illness is typically the observation of a rapidly growing mass, sometimes
associated with fever, weight loss, and night sweats.
The causes of diffuse large B-cell lymphoma are not well understood. Usually DLBCL arises from normal B
cells, but it can also represent a malignant transformation of other types of lymphoma or leukemia. All
indolent or low grade lymphomas can transform in an aggressive lymphoma. An underlying
immunodeficiency is a significant risk factor. Infection with Epstein-Barr virus has also been found to
contribute to the development of some subgroups of DLBCL.
Diagnosis of DLBCL is made through biopsy. Several subtypes of DLBCL have been identified, each having
a different clinical presentation and prognosis. However, the usual treatment for each of these is
chemotherapy, often in combination with an antibody targeted at the tumour cells. Through these treatments,

62
more than half of patients with DLBCL can be cured, and overall survival for older adults at five years is
around 58%. DLBCL is highly heterogenous in its histological appearance.
Prognosis is estimated through the IPI (= International Prognostic Index).
One point is assigned for each of the following risk factors:
• Age > than 60 years
• Stage III or IV disease
• Elevated serum LDH
• ECOG/Zubrod performance status of 2, 3, or 4
• > 1 extra-nodal site
The sum of the points allotted correlates with the following risk groups:
• Low risk (0-1 points): 5 year survival of 73%
• Low-intermediate risk (2 points): 5 year survival of 51%
• High-intermediate risk (3 points): 5 year survival of 43%
• High risk (4-5 points): 5 year survival of 26%
A better distinction between good prognosis and bad prognosis can be done through the gene expression
profiling. Gene expression profiling was able to define and study in the same time gene expressions and
hyper-expressions or down-expression of many genes; this was able to define 2 categories: the germinal
and the activated. This technique is better than IPI but it is expensive and not easy to perform.
The standard therapeutic approach is immunochemotherapy and we have the combination of Rituximab and
R-CHOP chemotherapy.

DLBCL may arise :

- de novo
- as progression/transformation from an underlying low grade/indolent lymphoma. 20% DLBCL derive from
progression of low grade B cell lymphomas.

DLBCL includes 4 morphologic variants:

- centroblastic, mainly consist of centroblasts, which are large cells with vestibular nuclei, multiple peripheral
nucleoli, and different from immunoblasts, which have a single central nucleolus and a more abundant
basophilic cytoplasm.
- immunoblastic, infiltrated mainly made of immunoblasts, which must be more than 90% of lymphoma
infiltrate. Immunoblastic are most common in immunosuppressed and have a worse prognosis than
centroblastic.
- anaplastic, characterized by a peculiar growth pattern, bizarre pleomorphic phenotype. A characteristic
phenotypic feature is the coexpression of Cd20-cd30. It is characterized by a prevalent background of
small reactive T cells and histiocytes, among these populations some large B cells are scattered, appear
like binucleated, with centroblasts and immunoblasts.
- T cell/histiocyte rich lymphoma.
Independently from morphology, the most frequent alteration occurring in DLBCL is a rearrangement or a
translocation.
Many variances have been reported in clinics, some according to extra nodal clinical presentation: primary
mediastinal B cell lymphoma is the most typical example, then there are intravascular B cell lymphoma
(lymphoma cells only detectable in vascular spaces), primary effusion lymphoma (rare subtype that occurs
specifically in cavities, such as peritoneum), primary DLBCL of the CNS, primary cutaneous DLBCL leg-type
(90% cases occur on the legs).
Primary mediastinal B-cell lymphoma is an aggressive lymphoma with a very peculiar presentation. It is a
quite strange disease because it derives from the neoplastic transformation of the B cells of the thymus (in
the thymus we have T cells). In these cases patients are young and are typically female. The clinical
presentation is similar to the Hodgkin’s lymphoma but is more aggressive, we have a very massive
mediastinal mass with compression of the vascular structure and this is not frequent in the Hodgkin’s
lymphoma as well.
This is a very great emergency in haematology but it has a good prognosis because 70% of these patients
are cured with chemotherapy. The approach is R-CHOP or a similar approach with Rituximab with weekly
administration of chemotherapy.
Some variants have been also defined based on the etiology: EBV positive DLBCL (in Eastern countries, has
a very aggressive clinical course), large B cell lymphoma in Castleman disease (associated with HHV8,
detected by specific immune staining) and DLBCL associated with chronic inflammation (pyothorax).


Multi agent chemotherapy is the treatment and 40 - 50% of the patients are cured.

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Burkitt's lymphoma ( BL )
Burkitt lymphoma is a cancer of the lymphatic system, particularly of B lymphocytes found in the germinal
center.
Currently, Burkitt lymphoma can be divided into three main clinical variants:
• The endemic variant (also called "African variant”), most commonly occurs in children living in malaria
endemic regions of the world (e.g., equatorial Africa, Brazil, and Papua New Guinea). Epstein-Barr virus
(EBV) infection is found in nearly all patients. The disease characteristically involves the jaw or other facial
bone, distal ileum, cecum, ovaries, kidney, or breast.
• The sporadic type of Burkitt lymphoma (also known as "non-African") is the most common variant found in
places where malaria is not holoendemic. The tumor cells have a similar appearance to the cancer cells of
classical endemic Burkitt lymphoma. Sporadic lymphomas are rarely associated with the Epstein–Barr
virus. Non-Hodgkin lymphoma, which includes Burkitt's, accounts for 30–50% of childhood lymphoma. The
jaw is less commonly involved, compared to the endemic variant. The ileocecal region is the common site
of involvement.
• Immunodeficiency-associated Burkitt lymphoma is usually associated with HIV infection or occurs in the
setting of post-transplant patients who are taking immunosuppressive drugs. Burkitt lymphoma can be one
of the diseases associated with the initial manifestation of AIDS.
By morphology (i.e. microscopic appearance) or immunophenotype, it is almost impossible to differentiate
these three clinical variants. Immunodeficiency-associated Burkitt lymphoma may demonstrate more
plasmacytic appearance or more pleomorphism, but these features are not specific.


Seen under microscopy, the tumor consists of sheets of a monotonous (i.e. similar in size and morphology)
population of medium-sized lymphoid cells with high proliferative activity and apoptotic activity. The "starry
sky" appearance seen under low power is due to scattered tingible body-laden macrophages (macrophages
containing dead apoptotic tumor cells). The tumor cells are mostly medium in size (i.e. tumor nuclei size
similar to that of histiocytes or endothelial cells). Tumor cells possess small amount of basophilic cytoplasm
with three to four small nucleoli. The cellular outline usually appears squared off.

The tumor cells in Burkitt lymphoma generally strongly express markers of B cell differentiation (CD20,
CD22, CD19), as well as CD10 and BCL6. The tumour cells are generally negative for BCL2 and TdT. The
high mitotic activity of Burkitt lymphoma is confirmed by nearly 100% of the cells staining positive for Ki67.

Normal B cells of a germinal center possess rearranged immunoglobulin heavy and light chain genes, and
each isolated B cell possesses a unique IgH gene rearrangement. Since Burkitt lymphoma and other B-cell
lymphomas are a clonal proliferative process, all tumor cells from one patient are supposed to possess
identical IgH genes. When the DNA of tumor cells is analyzed using electrophoresis, a clonal band can be
demonstrated, since identical IgH genes will move to the same position. On the contrary, when a normal or
reactive lymph node is analyzed using the same technique, a smear rather than a distinct band will be seen.
This technique is useful since sometimes benign reactive processes (e.g. infectious mononucleosis) and
malignant lymphoma can be difficult to distinguish.


Sporadic and endemic variants are highly aggressive but potentially curable. An intensive combination of
chemotherapy regiments results in cure of up to 90% of patients.

7.4 Acute Lymphoid Leukemia

Acute lymphoblastic leukemia, also known as acute lymphocytic leukemia or acute lymphoid leukemia (ALL),
is an acute form of leukemia, or cancer of the white blood cells, characterized by the overproduction and
accumulation of cancerous, immature white blood cells, known as lymphoblasts. In patients with ALL,
lymphoblasts are overproduced in the bone marrow and continuously multiply, causing damage and death by
inhibiting the production of normal cells, such as red and white blood cells and platelets, in the bone marrow
and by spreading (infiltrating) to other organs.
It is a clonal disorder of lymphoid progenitors with accumulation of cells called blasts (in this case we have
lymphoid blasts). As a consequence we have the suppression of the normal hematopoiesis.
ALL patients are always symptomatic, it is not possible to have a patient with acute leukemia without
symptoms. On the other side, in patients with chronic disorders many times the patients are without any
symptoms.
The symptoms of ALL are indicative of a reduced production of functional blood cells, because the leukemia
wastes the resources of the bone marrow, which are normally used to produce new, functioning blood cells.
These symptoms can include fever, increased risk of infection (especially bacterial infections like pneumonia,

64
due to neutropenia), increased tendency to bleed (due to thrombocytopenia) and signs indicative of anemia
including pallor, tachycardia, fatigue and headache.
"Acute" refers to the relatively short time course of the disease to differentiate it from chronic lymphocytic
leukemia, which has a potential time course of many years.
It was one of the first cancers for which an effective chemotherapeutic treatment was developed.

The first step during the investigation is the evaluation of the morphology: you can make a diagnosis of acute
leukemia observing the morphology. The differences in the blasts can concern the dimension, chromatin,
nucleus and nucleolus, cytoplasm, presence of basophilia and vacuoles. However we now have the flow
cytometry. The diseases that can be diagnosed with flow cytometry are very few: we have ALL and CLL.
In the clinical practice, in order to perform a diagnosis of lymphoma, we have the morphology and the flow
cytometry. After that we have other investigations, like cytogenetics: we need to know the abnormalities in
the genome because some disorders are associated with different chromosomal abnormalities (Philadelphia
translocation), or some chromosomal abnormalities can be prognostically relevant for treatment definition.
Similarly, we have the molecular analysis with PCR for molecular detection of events such as BCR-ABL
rearrangement, and also the FISH analysis for typical translocation such as t(11:14) for mantle cell
lymphoma, t(14:18) for follicular lymphoma and so on.

Subtyping of the various forms of ALL used to be done according to the French-American-British (FAB)
classification.
• ALL-L1: small uniform cells. L1 type is very frequent in children and less frequent in adult patients.
• ALL-L2: large varied cells. It is the prevalent form in the old patients.
• ALL-L3: large varied cells with vacuoles (bubble-like features). It is very rare in young people and is
more frequent in the adult.

Each subtype is then further classified by determining the surface markers of the abnormal lymphocytes,
called immunophenotyping. There are 2 main immunologic types: pre-B cell and pre-T cell. The mature B-
cell ALL (L3) is now classified as Burkitt's lymphoma/leukemia. Subtyping helps determine the prognosis and
most appropriate treatment in treating ALL.

For the B cells we need panB markers (CD19, CD20, CD22, CD79a) and also a marker of lymphoid
precursor.
On the other side for the T cell leukemia: CD2, CD3, CD5, CD7 as panT markers and then we need to define
if the disease is CD4 or CD8 according to helper or cytotoxic cells.
Based on these markers we can make the distinction between different types of B and T cells, but the most
important message is that the B cell ALL are the most frequent type: more or less ¾ of the cases are of B
lineage. On the other side, we have only ¼ of cases of T lineage.
Within this category of B cell and T cell we can distinguish different types of ALL.
So we have the Pro-B, the Pre-B and the B cell.
The CD10+ is the Common ALL and is the most frequent subtype of B cell.
         
Which is the difference between precursor B lymphoblastic leukemia and precursor B lymphoblastic
lymphoma? It’s a very nosological distinction, not a biological one, because the biology is different but in the
lymphoma cases we have not the leukemic picture and the bone marrow infiltration is limited. Biologically we
are talking about the same disorder, but with different clinical presentation. When we talk about ALL we talk
about a very rare disorder, a minority of lymphoid disorders.

ALL is a disorder of the lymphoid precursor cells in the bone marrow.

When we talk about the neoplasia of the peripheral blood and node T cells, we are talking about cutaneous T
cell lymphoma, mycosis fungoides and T cell peripheral lymphoma, that are very different disorders with
respect to ALL of T cell lineage.

The precursor ALL is characterized by different cytogenetic abnormalities. T(9:22) is popular, that is BCL-
ABL translocation of CLL.
A clonal alteration is present in the majority of cases. On the other side we have aberrations in number and
structure of the chromosomes. A high number of chromosomes is more common than a low number, but
what’s important is that we have distinct translocations in ALL, and the most important one is t(9:22).

The clinical appearance of a patient with ALL is very similar to that of acute myeloid leukemia: we have
symptoms related to anemia, thrombocytopenia and low levels of granulocytes. We can have infections,
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bleeding of skin and mucosae, weakness and fatigue related to the anemia. These are non-specific
symptoms, so the patient can be diagnosed with a big delay, especially children, because there can be many
differential diagnosis with infectious disorders.
On the other side, ALL is characterized by silent symptoms related to the infiltration: we can have
adenopathy in ALL that are not present in acute myeloid leukemia. If you have a patient with a very high
count of leukocytes and at physical evaluation you find adenopathies, the diagnosis of acute myeloid
leukemia is less probable.
Another characteristic of ALL is the possibility to detect a mass in the chest. We saw Hodgkin lymphoma, the
primary mediastinal lymphoma with sclerosis, and then also the ALL (or, to be precise, the T cell ALL) is
frequently associated with a mass in the chest. Another typical feature of this leukemia is the involvement of
CNS and gonads. These sites are called sanctuaries, so we need to perform a prophylaxis to avoid
involvement of these sites during the history of the disease.

We are talking about a very important and aggressive disorder, so we need to distinguish between patients
with a standard risk and with high risk disease. There are some features related to the biology of the disease,
the cytogenetic abnormalities and some immuno-phenotypic features (such as Philadelphia chromosome,
rearrangement of the MLL gene on chromosome 11, immune-phenotype pro-B or pre-T or with expression of
myeloid antigen). These are features intrinsic to the biology of the disease.
High-risk patients are those with more than 30-35 years or with more than 30.000 leucocytes/microL for B
cell ALL.
We saw before that the distribution of the morphological subtypes is different according to the age. Also risk
categories are different according to the age: children are commonly at low risk, while adult patients are
commonly at high risk.
Philadelphia chromosome is a very bad prognostic factor and as expected the presence of this chromosome
is quite rare in children, is present in around 20% of young adults and is quite frequent in the elderly patients.

In a patient with ALL, before starting with the treatment we need to have the blood count (to see the
morphological features of peripheral blood), the coagulation, liver and kidney function, and uric acid level.
After that you perform the bone marrow aspirate for morphological examination and cytogenetic analysis.
Which is the most important cytogenetic analysis in ALL? Is the detection of Philadelphia chromosome
because it’s pivotal in the choice of treatment because we have the specific inhibitor. So the most important
information is about the BCR-ABL rearrangement.
Treatment cornerstone is chemotherapy, but for starting chemotherapy we need to make a good support
treatment with transfusion, to lower the acid uric level and so on. After that, we need to make an induction
therapy whose aim is the reduction of the blast below 5% of bone marrow cells. This is only a complete
remission of the disease, is not the end of the therapy. After the induction of the remission I need to
consolidate it, maybe in some cases with allogeneic transplant, it depends from the biological and prognostic
features of the patient.
Another important part of the treatment is the meningeal prophylaxis with chemotherapy in CNS. The
prophylaxis is mandatory because the risk of meningeal relapse is almost 50% within 2 years. We have
some risk factors for CNS involvement: high level of leukocytes, T cell phenotype and the tumor syndrome,
so very high level of uric acid.

Minimal residual disease is an important method to detect the disease in the bone marrow after treatment.
The minimal residual disease in ALL can be evaluated through PCR, that will detect the rearrangement of the
Ig gene (we are talking about B cells so each B cell carries a distinct Ig rearrangement).
The other method is the flow cytometry, but it has a lower sensibility than the PCR for Ig gene and TCR.

It is important to detect the minimal residual disease because we can have a complete remission, but the
disease may be still prevalent at the molecular level. Only if the treatment is able to reduce the disease this
does not appear again. On the other side we can also follow this signal (flow cytometry, PCR) and then
decide to transplant the patient if minimal residual disease is present and increasing in level. This is a
dynamic definition of prognosis.
What happens if you can’t get the disease to complete remission? It means that one is a refractory patient
and you have to change the treatment. You may need to perform a salvage treatment. Response is quite
common, the problem is to maintain it. Also when you have a response the disease is still prevalent. This
applies also to follicular lymphoma, CLL, more or less to acute myeloid leukemia, but there we are talking
about indolent, low-grade, quite good prognosis disorders, in this case we are talking about a very

66
aggressive disorder with a bad outcome so in this situation with a young patient I need to consolidate the
hematological response.

Treatment results are different according to the age of the patient: complete remission is very frequent in
children and is not so bad also in adults. The results are quite bad instead in the elderly patients.
It’s important to decide risk groups to decide whether to perform or not an allogeneic transplant after the
complete remission.
Indications for the allotransplant are: young people, high-risk profile at the baseline and high level of minimal
residual disease after treatment. If you have persistence with minimal residual disease you have to
consolidate the response with allotransplant.

Globally, the difference between allotransplant and chemotherapy alone is quite small, but if we consider
high-risk patients, the allotransplant is better than the chemotherapy alone.
When examining a patient with ALL consider age, prognostic profile, and dynamic responses in terms of
minimal residual disease. So, a patient with high-risk profile, low response, high level of minimal residual
disease can be considered for allotransplant. You have to consider that with allotransplant we have a related
mortality of ¼ of patients.
The limitation for the allotransplant is the age: we transplant patients with acute leukemia until 60 years of
age because transplant toxicity and risk of death is very high. In the young people with Philadelphia+ ALL the
approach is to treat with inhibitors and then consolidate the response with a familial or an unrelated donor,
but in the very elderly the outcome was around 1 year and now we have patients with 3-4-5 years with a
quite good quality life and a quite good disease control.

8 Plasma cells neoplasms

Monoclonal Gammopathies
Monoclonal gammopathy is a synonym for paraproteinemia. These are usually found in association with
hematologic neoplasms, especially multiple myeloma. They also occur in other benign and malignant
conditions.
Gammopathies are diseases of the antibodies. Routine examination of some patients can find monoclonal
component in the gamma component of the electrophoresis. Conditions related to polyclonal
hypergammalbuminia are hepatitis infection, chronic inflammation and so on.
After this detection we need to identify the types of monoclonal component.
After these 2 assessments, we need to detect the dosage of the monoclonal component/paraprotein.
Detection of the amount of the different classes is important because sometimes there is an increase of IgG,
with low levels of IgA and IgM, especially in clear disorder like multiple myeloma.
Then we need to detect the component in urine. In the urine we can find only the light chain (Bence Jones
proteins). This is done by fixation of the urines.
Last but not least is the free light chain in serum. The free light
chains in serum are quite high in pathological situations such as
multiple myeloma, amyloidosis and so on. The ratio is also
important, weather is normal or in favour of kappa or lambda.

Half of patients present Monoclonal Gammopathy of

undetermined significant- MGUS, 20% of the patients present
multiple myeloma (MM). MGUS is a condition, and MM is a
disease.
But there are other conditions of monoclonal gammopathy like
lymphoproliferative disorders, i.e. lymphomas. Remember that
chronic lymphocytic leukemia and especially indolent lymphomas
have monoclonal gammopathy component of small amount.

8.1 MGUS
Monoclonal gammopathy of undetermined significance (MGUS), is a condition in which a paraprotein is
found in the blood during standard laboratory tests. It resembles multiple myeloma and similar diseases, but
the levels of antibody are lower, the number of plasma cells (white blood cells that secrete antibodies) in the
bone marrow is lower, it has no symptoms or problems, and no treatment is indicated. However, multiple
myeloma develops at the rate of about 1–2% a year, so doctors recommend monitoring it yearly.
This condition can transform into a real disorder, and risk of transformation exists in all patients.
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MGUS is a common, age-related medical condition characterized by an accumulation of bone marrow
plasma cells derived from a single abnormal clone. Patients may be diagnosed with MGUS if they fulfil the
following four criteria:
1. A monoclonal paraprotein band lesser than 30 g/L (< 3g/dL);
2. Plasma cells less than 10% on bone marrow examination;
3. Patient should be asymptomatic, thus there should be no evidence of bone lesions, anemia,
hypercalcemia, or renal insufficiency related to the paraprotein. Some aspecific symptoms such as
fatigue could be present.
4. No evidence of another B-cell proliferative disorder.

The distribution of the heavy chain is the distribution or the amount of the normal classes of
immunoglobulins. IgG is the most represented class. The second ones with the same distribution are IgM
and IgA. In the US IgF are more frequent.

MGUS prevalence increases with age. People with more than 50 years have prevalence of 2%, but in people
older than 70 years, the prevalence is 3%. The median age of diagnosis is around 70 years. Males are more
affected than females.

Observation showed that 8% of the patients with MGUS progress to multiple myeloma, WM, amyloidosis or
lymphoproliferative disorders. Hence overall the rate of progression is 1% per year.
Type  of  transforma4on  depends  on  the  monoclonal  component.    
IgG and IgA can progress to symptomatic smouldering multiple myeloma, and multiple myeloma.
IgM rarely progress to multiple myeloma of IgM type, but to a neoplastic disorder with IgM component called
WM.
Are all multiple myelomas preceded by MGUS? It is hard to know, because many patients are diagnosed
with multiple myeloma but have no history of diagnosed MGUS. Virtually all cases of multiple myeloma are
preceded by MGUS.

It is important to know whether the patient is at high or low risk to transform. The risk is 1% per year but
patients with IgA and IgM present higher risk of transformation and progression. The amount of monoclonal
component is related to the rate of progression, if there is small amount of the component the risk of
progression and transformation is less pronounced.
Low risk patient have small amount of monoclonal component. With IgG and no IgM and IgA. Free light
chain is normal.
High risk patient have higher amount of monoclonal gammopathy with no IgG, only IgA and IgM
and altered ratio of the free light chain.
The predictive model is based on the type of the heavy chain and the ratio between heavy and light chains.
3/4 of cases progress to multiple myeloma. Patients with IgA and IgM MGUS present higher risk of
progression in respect to IgG patients. IgA MGUS may progress to IgA multiple myeloma. IgM MGUS may
progress to WM or other lymphomas with monoclonal component (IgM multiple myeloma is not so common).
Rate of progression is higher compared to IgG monoclonal gammopathies.  

Patients with MGUS should not be treated, and there only is the need of performing follow up. Different
follow up examinations should be performed according to the risk of progression. If MGUS is diagnosed,
follow up is done every 6 month, if stable every 2 years at the family doctor.

8.2 Smouldering Multiple Myeloma

Smouldering myeloma is a very slow-growing type of myeloma which is characterised by a proliferation of
malignant plasma cells and a subsequent overabundance of monoclonal paraprotein (M protein).
Smouldering myeloma is a very early phase of myeloma and is devoid of symptoms but a bone marrow
biopsy shows definite evidence of myeloma.
Between MGUS and multiple myeloma we have this intermediate condition in which there are more plasma
cells, higher amount of monoclonal component but with no clinical manifestation, as in MGUS. The difference
between smouldering multiple myeloma and multiple myeloma is the presence of clinical manifestation.
Smouldering multiple myeloma is a real disorder and not a condition like MGUS.
Smouldering myeloma is characterised by:
1. Serum paraprotein >30 g/L
2. Clonal plasma cells >10% on bone marrow biopsy
3. No myeloma-related organ or tissue impairment
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Smouldering multiple myeloma is a stable condition that may last years, the progression to the symptomatic
multiple myeloma is 10% per year, in comparison to the 1% per year from MGUS to multiple myeloma.
Progression is an increase in the disease, while transformation is histological. At diagnosis cytology and
histology are performed.

The median time of progression in a patient with 1 risk factor is 9 years.

The median time of progression of a patient with 3 of these factors is 2 years.
If the condition is stable the test will be repeated every 4-6 month for one year and if stable then repeated
every 6 month.
At the moment there is no indication for therapy outside clinical trials.
8.3 Multiple Myeloma
Multiple myeloma is a cancer of plasma cells, a type of white blood cell normally responsible for producing
antibodies. In multiple myeloma, collections of abnormal plasma cells accumulate in the bone marrow, where
they interfere with the production of normal blood cells. Multiple myeloma is a quite rare disorder, median
age of the patients is 70 years old; multiple myeloma is a disease of elderly people.
Most cases of multiple myeloma also feature the production of a paraprotein, an abnormal antibody which
can cause kidney problems. Bone lesions and hypercalcemia (high blood calcium levels) are also often
encountered.
Multiple myeloma is diagnosed with blood tests (serum protein electrophoresis, serum free kappa/lambda
light chain assay), bone marrow examination, urine protein electrophoresis, and X-rays of commonly
involved bones.
All cases of multiple myeloma are preceded by MGUS. The biological explanation is the continuum of the
disease, similar to other conditions in hematology, like monoclonal B cells lymphocytosis and CLL, and many
other preneoplastic conditions and neoplastic conditions. Some biological events, such as translocation or
chromosome trisomy allow the progression from MGUS to multiple myeloma.
Multiple myeloma is considered to be incurable but treatable. Remissions may be induced with steroids,
chemotherapy, proteasome inhibitors, immunomodulatory drugs such as thalidomide or lenalidomide, and
stem cell transplants.
It is more common in men and, for unknown reasons, is twice as common in African-Americans as it is in
European-Americans. The five year survival rate is 45%. Multiple myeloma is not a curable disorder, patients
with good prognosis have a median overall survival of 62 months (very short survival) while patients with
advanced stage have median survival of around 3 years.

Symptomatic myeloma:
1. Clonal plasma cells >10% on bone marrow biopsy or (in any quantity) in a biopsy from other tissues
(plasmacytoma)
2. A monoclonal protein (paraprotein) in either serum or urine (except in cases of true non-secretory
myeloma)
3. Evidence of end-organ damage related to the plasma cell disorder (related organ or tissue impairment,
ROTI, commonly referred to by the acronym "CRAB"):
• HyperCalcemia (corrected calcium >2.75 mmol/L)
• Renal insufficiency attributable to myeloma
• Anemia (hemoglobin <10 g/dL)
• Bone lesions (lytic lesions or osteoporosis with compression fractures)
At diagnosis more than 50 % of patients present bone pain, 1/3 present fatigue, and some patients present
alterations such as anemia. Common sites of skeletal bone involvement are spine, pelvis, long bones and
there can be compression from extra medullary masses.
When making the diagnosis of multiple myeloma, we also need to conduct further investigations: data about
biological lesions, cytogenetical lesion (related to bad prognosis), investigation of the skeleton.

Most of the patients are IgG type, 1/4 IgA type.

10% are light chain multiple myeloma, and in this condition there is no spike in the electrophoresis but just
low levels of Ig in the gamma components, hence not all multiple myeloma are with high levels of Ig and
spike.
Kappa light chains are more frequent found than lambda, while it is the opposite in AL amyloidosis: if you see
a lambda patient, think about amyloidosis.

In multiple myeloma there are 2 different problems: over proliferation of plasma cells and production of
paraproteins.

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• Plasma cells over proliferation causes infection, osteoblastic activation and the development of osteolytic
bone lesion, as well as hypercalcemia (typical feature of multiple myeloma and solid cancers rather than
hematological disorders). There is then bone marrow suppression and failure, cytopenia, anemia,
thrombocytopenia and leucopenia. Renal complication is another typical feature of multiple myeloma.
• The presence of monoclonal component is related to hyper viscosity syndrome.

Radiological examination in patient with multiple myeloma allows identification of lytic lesions and
identification of localization outside bone marrow and bones. These alterations can have prognostic
significance in some patients. Moreover the imaging instead of bone marrow examination can define bone
marrow involvement. The standard 10 years ago was X-ray of skeleton, however X-ray is positive only when
more than 30% of the bone is lost.
Besides X-ray, another option is low dose CT scan which identifies more lesions, has an higher sensitivity,
allows 3D evaluation, and is able to detect non skeletal masses arising from the bone.
The best approach for skeletal examination however is MRI. It is essential for displaying the spinal cord
compression, thus patients with neurological symptoms and multiple myeloma need MRI. The pattern of MRI
is also of prognostic significance: normal MRI implies a better survival than focal or diffused MRI
involvement.
PET is quite important in the assessment of the patients. It is less sensitive than MRI on the spine, but is
able to specify if the lesions are metabolically active or necrotic.
The diagnosis of multiple myeloma is based not only on symptoms, but also on biomarkers.

Nowadays, in order to assess the stages of the multiple myeloma we use the international staging system,
which is based on beta 2 microglobulin and albumin. The combination of these 2 criteria allows us to
differentiate 3 clinical stages with different median overall survival.
I. β2 microglobulin (β2M) < 3.5 mg/L, albumin ≥ 3.5 g/dL
II. β2M < 3.5 mg/L and albumin < 3.5 g/dL; or β2M 3.5–5.5 mg/L irrespective of the serum albumin
III. β2M ≥ 5.5 mg/L

In patients with multiple myeloma we have to distinguish between young and elderly.
Young patients are eligible for autologous transplantation. Autologous transplantation age limit is 60-65 but
this limit is pushed more and more because elderly patients today can be in a very good condition without
co-morbidities and can thus be fitted for auto-transplant.
One drug that is used in the treatment of MM is Melphalan which is used in the elderly patients and in the
condition of auto-transplant.
Autologous transplantation is not a real transplant, but a high dose treatment, and the hematopoietic
recovery is made by infusion of progenitor cells into the patient. The treatment is the chemotherapy, and not
the cells.
Other drugs are angiogenesis and immunomodulatory drugs: Thalidomide and Lenalidomide. Thalidomide is
known for its effect on children, it was found effective on multiple myeloma quite recently, and is used as
angiogenetic and immunomodulatory drug. The evolution of Thalidomide is Lenalidomide that is not only able
to induce the tumor cell death but also to modulate the immune effectors cells against the tumor, used also in
non Hodgkin lymphoma.
Moreover there are the proteasome inhibitors drugs like Bortezomibe. The proteasome is responsible for the
degradation of abnormal proteins, that are abundant in cancer cells, antigene processing and recycling of
proteins. It is very active in the MM cells and the drug is a reversible inhibitor of this pathway.

8.4 AL Amyloidosis
Amyloid Light-chain (AL) amyloidosis is the most common form of systemic amyloidosis in the US. The
disease arises when a person's antibody-producing cells do not function properly and produce abnormal
protein fibers made of components of antibodies called light chains. These light chains come together to form
amyloid deposits in different organs which can cause serious damage to these organs. Abnormal light chains
in blood and urine are sometimes referred to as "Bence Jones proteins”.
AL amyloidosis can occur spontaneously, however, it is often associated with other blood disorders, such as
multiple myeloma and Waldenström's macroglobulinemia. About 10% to 15% of patients with multiple
myeloma may develop overt AL amyloidosis.
What is the difference between MM and primary AL amyloidosis? There is a typical distinction: in patients
with MM the disease is due to the accumulation of the cells and the monoclonal component: a big amount of
neoplastic cells and a big amount of monoclonal component for a protein.
The situation is different in amyloidosis, in which we have a small clone but with a very bad monoclonal
component. Small monoclonal component but badly reacting, because the biochemical properties of light

70
chain make possible the formation of amyloid that eventually leads to organ damage. We can also have the
two conditions together, but this is a different situation.

It is a disease deriving from protein misfolding, which causes aggregation of proteins in beta sheet fibrils that
can target the cells in the tissue. These proteins are related to progressive organ dysfunction. AL amyloidosis
can affect a wide range of organs, and consequently present with a range of symptoms.
The kidneys are the most commonly affected organ in AL amyloidosis. Symptoms of kidney disease and
renal failure can include fluid retention, swelling, and shortness of breath. In addition to kidneys, AL
amyloidosis may affect heart, peripheral nervous system, gastrointestinal tract, blood, lungs and skin. Heart
complications, which affect more than a third of AL patients, include heart failure and irregular heart beat.
Other symptoms can include stroke, gastrointestinal disorders, enlarged liver, diminished spleen function,
diminished function of the adrenal and other endocrine glands, skin color change or growths, lung problems,
bleeding and bruising problems, fatigue and weight loss.

Lab signs and symptoms in a patient with suspected amyloidosis could be increased levels in biomarkers of
cardiac function (Nt-pro BNP). Another biomarker that needs to be evaluated n these patients is albumin in
urine. We must also check for increased size of liver and, among liver function tests, for alkaline
phosphatase elevation.

An other important issue in patients with monoclonal gammopathy is the presence of peripheral neuropathy.
This is not only important in amyloidosis, due to localization of the amyloid in the peripheral nervous system,
but it is important also for patients with monoclonal gammopathy, especially of the IgM type.
Orthostatic hypotension, aspecific symptoms, fatigue and weight loss may be due to localization of the
disease in the bowel. All patients refer fatigue, so it is difficult to distinguish a patient from another, but these
aspecific symptoms such as fatigue can be the first signs of amyloidosis. We can have also hemorrhagic
manifestations in the orbit due to localization of amyloid.

In order to diagnose amyloidosis, we need to assess the presence of a monoclonal component and free light
chains. There is also the need for a demonstration of amyloids in tissue, and the first site of testing for this is
the abdominal fat, which is analyzed with congo red stain. If negative, when still with great suspicion of al
amyloidosis, biopsy of minor salivary glands should be performed. If this biopsy results negative as well,
biopsy of the involved organ should be performed. Sometimes, colleagues suggest biopsy of rectal mucosa.

Treatments of AL amyloidosis is performed through chemotherapy, autologous transplant, and biological

agents. The most effective treatment is autologous bone marrow transplantation. However many patients are
too weak to tolerate this approach.
Other treatments can involve an application of chemotherapy similar to that used in multiple myeloma.
Why is it important to make early diagnosis? Early intervention with chemotherapy, autologous transplant,
and so on, is recommended to achieve optimal response. A patient with advanced cardiac situation will
hardly respond well.

———Clinical case.——————————————————————————————————————
Young male patient with cardiac failure and with involvement of left ventricle, with increase in size of organs
and monoclonal component in serum and urine, with the ratio in favour of kappa chains.
What to do in such a patient to rule out MM? You perform an X-ray and an MRI. In this case they were
negative, and infiltration was around 10%, so it was not a clear situation of MM. The diagnosis of amyloidosis
was done through fat biopsy.

Survival of patients with al amyloidosis is not so good. However there are different survivals according to the
time of diagnosis. The outcome is not so good, but it is improving with progression of the treatment.
Most of the time patients affected by amyloidosis die because of the typical heart involvement. Very few
patients die instead because of liver or renal failure.
So, the treatment of cardiac involvements is the most important step in amyloidosis treatment.
This is explained by the different survivals according to the cardiac markers, such as BNP and troponin. If I
have a patient with low levels of these 2 biomarkers, the patient will have a good prognosis. But if I have a
patient with elevated BNP and elevated troponin, survival will be shorter than the one for the first patient.

The survival determinants are: cardiac involvement and response to treatment, so the possibility of having
low free light chain levels after treatment.

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If after treatment there is no presence of monoclonal component, and normal ratio of free light chain, this is a
complete remission. On the contrary I have a very good partial remission when the difference between free
light chain is less than 40 mg/l. If the decrease is more than 50% this is partial remission.

8.5 Waldenstrom’s Macroglobulinemia

It is cancer affecting B cells. The main attributing antibody is immunoglobulin M (IgM). WM is an "indolent
lymphoma," (i.e., one that tends to grow and spread slowly). It is a type of lymphoproliferative disease, which
shares clinical characteristics with the indolent non-Hodgkin lymphomas. Low grade indolent lymphomas are
for instance follicular lymphoma, and other types like marginal zone lymphoma, and lymphoplasmocytic
lymphoma (can be called lymphoplasmacytoid lymphoma or immunocytoma).
The disease is characterized by an uncontrolled increase of B-cells, i.e., white blood cells formed in the bone
marrow and lymph nodes. The proliferation of B-cells interferes with the production of red blood cells,
resulting in anemia. A unique characteristic of the disease is that the B-cells produce excess amounts of
immunoglobulin protein (IgM), thickening the blood, and requiring additional treatment. WM is a rare disease
and while incurable, it is treatable.
Signs and symptoms of WM include weakness, fatigue, weight loss and chronic oozing of blood from the
nose and gums. Peripheral neuropathy can occur in 10% of patients. Lymphadenopathy, enlargement of the
spleen, and/or liver are present in 30–40% of cases. Other possible signs and symptoms include blurring or
loss of vision, headache, and (rarely) stroke or coma.

Lymphoplasmocytic lymphoma is a histopathological definition, so to make a diagnosis we have to detect

typical BM and lymph node picture. A typical double population of cells can be identified: small cells and
greater cells similar to plasma cells. Small cells are typical of chronic lymphocytic leukemia (CLL), marginal
zone lymphoma, lymphocytic lymphoma and so on; plasma cells are typical of MM. Also histologically we
have features of lymphoma and of MM and monoclonal gammopathies.
This is the same for the bone marrow, where there is infiltration of small lymphocytes and plasma cells. Mast
cells infiltration is typical as well.
The phenotype is quite typical, there is expression of CD20 and CD 19, so it is a B cell neoplasm. CD20 is a
marker for small lymphocytes, B cell lymphocytes. The expression of CD5 is instead lacking (difference from
mantle cell lymphoma). Expression of CD10 is lacking as well (difference from follicular lymphoma) is
CD10+.
This lymphoma is typically connected with presence of the IgM type monoclonal component, but not always.
There can also be an other type or no monoclonal component.

So the diagnostic criteria for WM is diagnosis of lymphoplasmocytic lymphoma and concurrent presence of
IgM monoclonal gammopathy.
There can be patients with diagnosis of lymphoplasmocytic lymphoma without having WM because there is
either the presence of another monoclonal component, or no monoclonal component.
We can also have low grade lymphomas, that are not lymphoplasmocytic, but carry IgM monoclonal
components (such as marginal zone lymphoma). However this is more rare.
A characteristic of WM is that there is bone marrow involvement, which is instead absent in related disorders
and monoclonal gammopathy of undetermined significance.
Symptoms are present in symptomatic WM, in IgM related disorders and not in monoclonal gammopathies.
Only in WM there is the presence of infiltration symptoms, which are systemic symptoms such as fever,
sweats, organomegalies, adenopathies and so on. These are typical lymphoma features.
There can also be symptoms due to the monoclonal component.
In MM there are monoclonal symptoms and infiltration symptoms.
In amyloidosis there only are monoclonal component symptoms because of their deposition.
In the IgM related disorders there are no infiltration symptoms, no organomegaly, but the monoclonal
component is causing PNS neuropathy, amyloidosis, immune thrombocytopenia, crioagglutinins and so on.

Crioagglutinins are cold antibodies that can be responsible for autoimmune hemolytic anemia. Autoimmune
hemolytic anemia due to cold antibodies is formally an IgM related disorder and in most cases we can detect
an IgM component. So crioagglutinins are a manifestation of an IgM related disorder.
Crioglobulinemia instead is a medical condition in which the blood contains large amounts of cryoglobulins
that become insoluble at reduced temperatures. This should be contrasted with cold agglutinins, which cause
agglutination of red blood cells. Cryoglobulins typically precipitate at temperatures below normal body
temperature (37°C) and will dissolve again if the blood is heated. Cryoglobulinemia can be associated with
various diseases such as multiple myeloma and hepatitis C infection. Cryoglobulins usually consist of IgM
directed against the Fc region of IgG.

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In a patient with suspected WM we should perform bone marrow biopsy, because there is the need of
demonstrating histologically the lymphoplasmacytic lymphoma. There is also the need for a test for the cold
autoantibodies and imaging. There are then some lymphoma features and here are patients with
adenopathies and organomegalies, this is why we need imaging. When you have an IgM component you
need to look at spleen, liver and lymph nodes. If you don't want to do the CT scan, perform a US scan of the
abdomen at least.

For asymptomatic patients with WM, the outcome is better than for patients with symptomatic disease.

Current medical treatments result in survival of some patients longer than 10 years; in part this is because of
better diagnostic testing which results in early diagnosis and administration of treatment.
In rare instances, WM progresses to multiple myeloma.
The International Prognostic Scoring System for Waldenström’s Macroglobulinemia (IPSSWM) is a predictive
model to characterise long-term outcome. According to the model, factors predicting survival are:
• Age >65 years
• Hemoglobin ≤11.5 g/dL
• Platelet count ≤100×109/L
• B2-microglobulin >3 mg/L
• Serum monoclonal protein concentration >70 g/L
An additional predictive factor is elevated serum lactate dehydrogenase (LDH).

The risk categories are:

• Low: ≤1 adverse variable except age. Five-year survival rate is 87%.
• Intermediate: 2 adverse characteristics or age >65 years. Five-year survival rate is 68%.
• High: >2 adverse characteristics. Five-year survival rate is 36%.

Waldenström's macroglobulinemia is characterized by an uncontrolled clonal proliferation of terminally

differentiated B lymphocytes. The most common causes are a somatic mutation in MYD88 (90% of patients)
and a somatic mutation in CXCR4 (27% of patients). There has been an association demonstrated with the
locus 6p21.3 on chromosome 6. There is a 2- to 3-fold risk increase of developing WM in people with a
personal history of autoimmune diseases with autoantibodies and particularly elevated risks associated with
liver inflammation, human immunodeficiency virus, and rickettsiosis.
The effect of this mutation is in favour of the NF-κB complex, protein complex controlling lymphocyte
development. Its regulation leads to activation in the nucleus of pro-survival genes, blocking apoptosis and
promoting tumor development. The MYD88 mutation was also shown to induce activation of Bruton's
tyrosine kinase which plays a crucial role in B cell maturation as well as mast cell activation through the high-
affinity IgE receptor.

It can be useful to use an inhibitor of BTK in treatment of WM, and this drug is called ibrutinin.  

Some infections can be related to lymphoma development. There are several methods to establish if an
infection is related to a neoplasia: one of these is the epidemiological evidence. A patient with HCV presents
2,5 more risk of lymphoma development.
In our country 10% of cases of lymphoma can be related to HCV.
However epidemiological evidence alone cannot be used in order to determine the role of the virus. It has
been shown however that crioglobulinemia was present in 100 % of patients.
For prognostic evaluation, many parameters have been used. Age and albumin were related to worst
prognosis. Patients treated with antiviral drugs responded also with lymphoma regression (remission in
44%).

IFN is active against lymphoma so maybe it's not the clearance of virus but the anti-proliferative IFN activity.
Sofosbuvir in the future can be used to cure hvc and maybe cure lymphoma.  

9 Hemostasis and Hemostatic diseases

Hemostasis is the process aimed at stopping bleeding, that means to keep blood within the vessels.
Hemostasis (from the Ancient Greek: αἱμόστασις "styptic (drug)") is a process which causes bleeding to
stop (the opposite of hemostasis is hemorrhage).

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It is the first stage of wound healing and it involves blood changing from a liquid to a gel. Intact blood vessels
are central to moderating blood's tendency to clot. The endothelial cells of intact vessels in fact prevent blood
clotting with a heparin-like molecule and thrombomodulin, and prevent platelet aggregation with nitric oxide
and prostacyclin. When endothelial injury occurs, the endothelial cells stop secretion of coagulation and
aggregation inhibitors and instead secrete von Willebrand factor which initiate the maintenance of
hemostasis after injury.
Hemostasis has three major steps which seal the hole until tissues are repaired:
1) vasoconstriction,
2) temporary blockage of a break by a platelet plug,
3) blood coagulation, or formation of a fibrin clot.
Three major components are involved: blood vessels, platelets and coagulation plasma proteins.
We identify 2 major steps of hemostasis:
- Primary hemostasis, which includes the reaction of blood vessels to a damage (spasm) and the action of
platelets, consisting in platelet adhesion and then in platelet activation and aggregation.
- Secondary hemostasis consists instead in the activation of the coagulation cascade, that results in the
generation of fibrin threads.

Primary hemostasis
When the endothelium is damaged, the normally isolated, underlying collagen is exposed to circulating
platelets, which bind directly to collagen with collagen-specific glycoprotein Ia/IIa surface receptors. This
adhesion is strengthened further by von Willebrand factor (vWF), which is released from the endothelium
and from platelets; vWF forms additional links between the platelets' glycoprotein Ib/IX/V and the collagen
fibrils. Abnormalities of VWF will result in a defective platelet adhesion and consequent development of
diseases characterized by high risk of bleeding.
This localization of platelets to the extracellular matrix promotes collagen interaction with platelet
glycoprotein VI. Binding of collagen to glycoprotein VI triggers a signalling cascade that results in activation
of platelet integrins. Activated integrins mediate tight binding of platelets to the extracellular matrix. This
process adheres platelets to the site of injury.
Activated platelets release the contents of stored granules into the blood plasma. There are two types of
granules: dense (delta) and alpha. The granules include ADP, serotonin, platelet-activating factor (PAF),
vWF, platelet factor 4, and thromboxane A2 (TXA2), which, in turn, activate additional platelets.
The granules' contents activate a Gq-linked protein receptor cascade, resulting in increased calcium
concentration in the platelets' cytosol. The calcium activates protein kinase C, which, in turn, activates
phospholipase A2 (PLA2). PLA2 then modifies the integrin membrane glycoprotein IIb/IIIa, increasing its
affinity to bind fibrinogen.
The activated platelets change shape from spherical to stellate, and the fibrinogen cross-links with
glycoprotein IIb/IIIa aid in aggregation of adjacent platelets (completing primary hemostasis). Collagen-
mediated GPVI signalling increases the platelet production of thromboxane A2 (TXA2) and decreases the
production of prostacyclin. Platelets thus secrete thromboxane A2, which acts on the platelet's own
thromboxane receptors on the platelet surface (hence the so-called "out-in" mechanism), and those of other
platelets. These receptors trigger intraplatelet signalling, which converts GPIIb/IIIa receptors to their active
form to initiate aggregation. The process of aggregation is reversible at the beginning but then it becomes
irreversible. The activation of platelets is then resulting in the synthesis of 2 biochemical mediators,
synthesised by the same enzyme (Cyclooxygenase, COX):
- In platelets, COX transforms the arachidonic acid in thromboxane, which is, an enhancer of platelet
aggregation, enhancing platelet aggregation.
- In endothelial COX transforms the arachidonic acid in prostacyclin, which is an inhibitor of hemostasis. By
doing this, healthy endothelial cells limit the site of the reaction to the damaged area alone.

Low dose aspirin is able to inhibit COX in platelets (which are parts of megakaryocytes thus are not
nucleated and they can’t synthesise enzymes), but it’s not enough to inhibit it in endothelial cells (which are
nucleated and can therefore regulate enzyme production), because the latter can replace the inhibited
enzyme with a new one.
If we use aspirin at high dose (used as anti inflammatory) we again inhibit COX in platelets, but this time also
in endothelial cells. Therefore we inhibit both aggregation and inhibition of it (in the long run we do not act
effectively on hemostasis).

There are a dozen proteins that travel along the blood plasma in an inactive state and are known as clotting
factors. Once the platelet plug has been formed by the platelets, the clotting factors begin creating the clot.
When this occurs the clotting factors begin to form a collagen fiber called fibrin. Fibrin mesh is then produced

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all around the platelet plug, which helps hold the plug in place. Once this begins, red and white blood cells
become caught up in the fibrin mesh which causes the clot to become even stronger.


Secondary hemostasis
Clots form upon the conversion of fibrinogen to fibrin, and its addition to the platelet plug.
Coagulation is the third and final step in this response. As the fibrin mesh begins to form the blood is also
transformed from a liquid to a gel like substance through involvement of clotting factors and pro-coagulants.
The coagulation process is useful in closing up and maintaining the platelet plug on larger wounds. The
release of prothrombin also plays an essential part in the coagulation process because it allows for the
formation of a thrombus, or clot, to form. This final step forces blood cells and platelets to stay trapped in the
wounded area. Though this is often a good step for wound healing, it has the ability to cause severe health
problems if the thrombus becomes detached from the vessel wall and travels through the circulatory system;
If it reaches the brain, heart or lungs it could lead to stroke, heart attack, or pulmonary embolism
respectively.

In vivo, the only active coagulation pathway is the extrinsic one, which is activated by vessel damage. The
intrinsic pathway in vivo enhances the activation of factor X. That means it enhances the extrinsic pathway.

Secondary hemostasis also includes fibrinolysis, that is aimed to contrast the excessive production of fibrin
from coagulation. Fibrinolysis does this by increasing the degradation of fibrinogen and fibrin, and it is mainly
done through plasminogen, that is converted to plasmin through the process mediated by urokinase or tissue
plasminogen activator. As all the physiologic processes, it has physiological inhibitors: plasminogen activator
inhibitor and the alpha2 antiplasmin and alpha2 macroglobulins (inhibit plasmin itself). This reaction of
degradation of fibrin and fibrinogen, results in some products that we are able to measure in the plasma of
patients and may be helpful when approaching hemostasis defects in patients. A series of fragment
(fibrinogen degradation product, FDP) result from degradation of fibrinogen, whereas the degradation of
fibrin results in D-dimers formation.
Coagulation is controlled by inhibitory mechanisms that are extremely relevant. The complex of activated
protein c and protein s is the major system of inhibition of coagulation, deactivating factor V and VIII. In
addition, thrombin is partly inhibited by antithrombin. These two mechanisms are very important, we know
inherited diseases characterized by deficiency of antithrombin characterized by enhanced risk of thrombosis.

9.1 Hemostatic diseases

We can distinguish between two major groups of disorders:
1) Those resulting in an increased risk of thrombosis (thrombophilia). They may increase the risk of arterial
and/or venous thrombosis. In arterial thrombosis, the primary hemostasis is mainly involved (so we inhibit
platelets to treat it) whereas in venous thrombosis we have mainly an activation of secondary hemostasis
(coagulation must be inhibited).
2) Those resulting in an increased risk of bleeding, involving a defect in hemostasis in either primary or
secondary phase.

9.1.1 Thrombotic diseases

Thrombosis (θρόμβωσις) is the formation of a blood clot (θρόμβος) inside a blood vessel, obstructing the
flow of blood through the circulatory system.
When a blood vessel is injured, the body uses platelets (thrombocytes) and fibrin to form a blood clot to
prevent blood loss. Even when a blood vessel is not injured, blood clots may form in the body under certain
conditions. A clot that breaks free and begins to travel around the body is known as an embolus.
When a thrombus is significantly large enough to reduce the blood flow to a tissue, hypoxia (oxygen
deprivation) can occur and metabolic products such as lactic acid can accumulate. A larger thrombus
causing a much greater obstruction to the blood flow may result in anoxia, the complete deprivation of
oxygen and infarction, tissue death. There are also a number of other conditions that can arise according to
the location of the thrombus and the organs affected.
This is a rather common disease, more common in the elderly patients. There are two categories of risk
factors for this disease: inherited and acquired risk factors.
- Inherited: we define as a inherited thrombophilia, genetically determined tendency to venous
thromboembolism. It is related to the presence of a family history or by the frequent recurrence of venous
thrombosis in the same subjects, suggesting that the problem remains active even after therapy.
- Acquired: age (incidence is a function of age), obesity, folate and b12 deficiency, drugs, chemical agents
(smoking), surgery (long lasting immobilisation), cancer (solid tumors but also hematologic tumors, such
as myeloproliferative diseases), and some autoimmune diseases.

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The main causes of thrombosis are given in Virchow's triad:
I. which lists hypercoagulability, caused for example by genetic deficiencies or autoimmune disorders
II. endothelial cell injury, caused for example by trauma, surgery, infection or turbulent flow at bifurcations.
III. disturbed blood flow, caused for example by stagnation of blood flow past the point of injury, or venous
stasis which may occur in heart failure, in or after long periods of sedentary behaviour, such as sitting on
a long airplane flight. Also, atrial fibrillation, causes stagnant blood in the left atrium (LA) and can lead to
a thromboembolism.

Signs and symptoms of venous thromboembolism

Stasis is a major factor in venous thromboembolism and this explains why usually the deep vein districts
(iliac, femoral, popliteal) are usually involved. Less frequently, and usually by intervention of local risk factors,
it may involve also upper limbs, liver, mesenteric veins, retina and cerebral sinuses.
Edema of involved limb can be observed, usually associated with redness and tenderness.
Most of the times the process is self limiting and there is no extension of thrombosis to proximal districts,
however this may happen in 1/4 of cases. The clinical picture and the possible complications thus become
significantly worse.
To confirm the diagnosis, we use clinical signs, then we look for D-Dimer and we perform doppler
ultrasonography (thanks to which it is possible to identify the thrombus within the vessel).
The D-dimer is a fibrin degradation product (or FDP) which is present in blood after a blood clot is degraded
by fibrinolysis. It is so named because it contains two cross linked D fragments of the fibrin protein. D-dimer
concentration may be determined by a blood test to help diagnose thrombosis. While a negative result
practically rules out thrombosis, a positive result can indicate thrombosis but does not rule out other potential
causes. It is also used in the diagnosis of the blood disorder disseminated intravascular coagulation.
Nowadays, all patients with a first episode of thromboembolism should be screened. Then, also people
related with individuals with proven genetic defects should be screened. Also, women with pregnancy
complications and history of recurrent miscarriage. Then also asymptomatic females with family history who
are starting venous contraception should undergo screening.

Therapy
We need first to interfere with the coagulation cascade, and this can be done in several ways. The simplest
way is to use Heparin (low molecular weight) and the oral anticoagulants (family of agents being vitamin K
antagonist, like Coumadin).
The problem with vitamin K antagonists is that not only coagulation factors but also anticoagulation factors
are vitamin K dependent (mainly protein c and s). In the first few days of therapy, the effect of the drug hits
more the anticoagulant factors, we may therefore obtain the opposite effect. The current schedule is thus to
start with oral anticoagulant together with low dose heparin. Then, after 4-5 days, when the oral
anticoagulant has reached the INR between 2 and 3, we can eliminate the low heparin and go on with oral
anticoagulant. Usually the treatment should be taken for at least 3 months and then it should be modulated
according to medical history and presence of inherited risk factors.
There are now novel anticoagulants being tested and they do not require any monitoring of the therapeutic
effect. 

In the case of major thrombosis we also take in consideration thrombolytic therapy, but only in selected
cases.

Anticoagulants and antiplatelet drugs are a type of medication that is used to eliminate or reduce the risk of
blood clots.
Heparin and Coumadin (warfarin) are two common anticoagulant medications.
Aspirin is a common antiplatelet medication.
The greatest benefit of these medications is that they prevent blood clots and the serious complications that
can occur from them. Aspirin in particular can be a lifesaver. Aspirin has been known to help reduce the risk
of a transient ischemic attack (TIA), stroke, and heart attack.
There are however serious risks we encounter when taking these drugs. Sometimes the medication can
cause bleeding problems, headaches, dizziness, pain, and discomfort.
Coumadin should not be prescribed to a patient that is at risk for bleeding, pregnant, or breastfeeding.
Coumadin can pass through the placental barrier and may cause bleeding in the foetus. Coumadin is also
commonly associated with spontaneous abortion, stillbirth, neonatal death, and preterm birth. Coumarins
(such as warfarin) are also teratogens, that is, they cause birth defects. Coumadin may also worsen
diabetes, high blood pressure, problems with falls, congestive heart failure, or liver or kidney problems.

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Complications
The major complication of venous thrombosis is pulmonary thromboembolism. The patient will suffer of acute
chest pain, thus there will be the need of making a differential diagnosis with MI. The cause could be
arrhythmia, tachycardia and, if the embolism is large, there might be a defect in blood oxygenation due to the
obstruction of a major pulmonary vessel. In this case we should have high resolution CT or scintigraphy with
double contrast medium and we should also look for deep vein thrombosis (which is the main cause of this
complication). Therapy is the same as deep vein thrombosis, but if the embolism is massive and impairing
oxygen supply, we may try thrombolytic therapy (urokinase and streptokinase) which induce lysis of the
thrombus. Pulmonary thromboembolism is a very severe condition with high mortality rate.

Factor V Leiden
Factor V Leiden thrombophilia is a genetically inherited disorder of blood clotting. Factor V Leiden is a
variant (mutated form) of human factor V that causes an increase in blood clotting (hypercoagulability). In
this disorder, the Leiden variant of factor V cannot be inactivated by the anticoagulant protein activated
protein C, so clotting is encouraged. Factor V Leiden is the most common hereditary hypercoagulability
disorder amongst European Caucasians.
In a normal person, factor V functions as a cofactor to allow factor Xa to activate an enzyme called thrombin.
Thrombin in turn cleaves fibrinogen to form fibrin, which polymerizes to form the dense meshwork that
makes up the majority of a clot. Activated protein C (aPC) is a natural anticoagulant that acts to limit the
extent of clotting by cleaving and degrading factor V.

Factor V Leiden is an autosomal dominant genetic condition that exhibits incomplete penetrance, i.e. many
people carrying the mutation do not suffer any consequences. Given that this disease displays incomplete
dominance, those who are homozygous for the mutated allele are at a heightened risk than those that are
heterozygous for the mutation.

The condition results in a factor V variant that cannot be as easily degraded by aPC (activated Protein C).
The gene that codes the protein is referred to as F5. A missense mutation prevents efficient inactivation of
factor V. When factor V remains active, it facilitates overproduction of thrombin leading to generation of
excess fibrin and excess clotting.
The excessive clotting that occurs in this disorder is almost always restricted to the veins, where the clotting
may cause a deep vein thrombosis (DVT). If the venous clots break off, these clots can travel through the
right side of the heart to the lung where they block a pulmonary blood vessel and cause a pulmonary
embolism.
Some environmental factors may also interact with genetic factors: in particular smoking, immobilization, long
flights. 

Suspicion of factor V Leiden being the cause for any thrombotic event should be considered in any
Caucasian patient below the age of 45, or in any person with a family history of venous thrombosis.
There are a few different methods by which this condition can be diagnosed. Diagnosis is done by testing the
ability of activated c protein to inhibit factor V and therefore to modulate the APPT time. During laboratory
tests, the time it takes for blood to clot is decreased in the presence of the factor V Leiden mutation.
There is also a genetic test that can be done for this disorder.

Mild hyperhomocysteinemia
Hyperhomocysteinemia is a medical condition characterized by an abnormally high level of homocysteine in
the blood, conventionally described as above 15 µmol/L.
The increase of homocysteine in blood may be sustained by both genetic defects and environmental or
acquired factors. We may have this condition as a consequence of folate or B12 deficiency (quite prevalent)
and we may have it as a genetic defect, involving the gene variant of the methyltetrahydrofolate reductase
(an enzyme involved in the conversion of cysteine).
The disease increases the risk of arterial and venous thrombosis.
Hyperhomocysteinemia is typically managed with vitamin B6, vitamin B9 and vitamin B12 supplementation.

Antiphospholipid syndrome
Antiphospholipid syndrome is an autoimmune, hypercoagulable state caused by antiphospholipid antibodies.
It is an example of purely acquired condition. APS provokes blood clots (thrombosis) in both arteries and
veins as well as pregnancy-related complications such as miscarriage, stillbirth, preterm delivery, and severe
preeclampsia.
It is an autoimmune disease characterized by autoantibodies directed against phospholipids, which are also
cofactors of the hemostatic system (anti cardiolipin, anti b2 glycoprotein and lupus anticoagulant).
Lupus anticoagulant antibodies bind to prothrombin, thus increasing its cleavage to thrombin, its active form.

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Therefore, in vivo they activate the hemostatic system. However they have a different behaviour in vitro
where they act as anticoagulants. This causes thrombocytopenia and prolonged APPT in vitro, but then this
is not what occurs in vivo.
The diagnostic criteria require one clinical event, i.e. thrombosis or pregnancy complication, and two
antibody blood tests spaced at least three months apart that confirm the presence of either lupus
anticoagulant, or anti-β2-glycoprotein-I.
Antiphospholipid syndrome can be primary or secondary.
Primary antiphospholipid syndrome occurs in the absence of any other related disease.
Secondary antiphospholipid syndrome occurs with other autoimmune diseases, such as systemic lupus
erythematosus (SLE).
In rare cases, APS leads to rapid organ failure due to generalised thrombosis and this is termed
"catastrophic antiphospholipid syndrome" (CAPS) and is associated with a high risk of death.
Antiphospholipid syndrome often requires treatment with anticoagulant medication such as heparin to reduce
the risk of further episodes of thrombosis and improve the prognosis of pregnancy. Warfarin/Coumadin is not
used during pregnancy because it can cross the placenta, unlike heparin, and is teratogenic.

9.1.2 Diseases with an increased bleeding risk (coagulopathies)

Coagulopathy (also called clotting disorder and bleeding disorder) is a condition in which the blood’s ability to
clot (coagulate) is impaired. This condition can cause prolonged or excessive bleeding, which may occur
spontaneously or following an injury or medical and dental procedures.
The normal clotting process depends on the interplay of various proteins in the blood. Coagulopathy may be
caused by reduced levels or absence of blood-clotting proteins (clotting factors or coagulation factors).
Genetic disorders, such as hemophilia and Von Willebrand's disease, can cause a reduction in clotting
factors.
Anticoagulants such as warfarin will also prevent clots from forming properly. Coagulopathy may also occur
as a result of dysfunction or reduced levels of platelets.
Bleeding disorders are characterized by defect of hemostatic system resulting in excessive bleeding. We are
able to distinguish them depending on wether they involve primary or secondary hemostasis.

Disorders of primary hemostasis

It can be due to:
• a quantitative abnormality of platelets (thrombocytopenia)
• a functional abnormality (platelets normal in number but with a functional defect, resulting as well in a
defect of primary hemostasis). Functional abnormalities of platelets are rare.
• a defect in the Von Willebrand’s factor, causing defective platelet adhesion and giving wrong hemostasis
with normal platelet count
Primary hemostasis defects result in immediate or very early bleeding after a trauma, usually in an external
bleeding site (skin or mucosa).
The lab tests to be performed in order to diagnose a primary hemostasis defect are a CBC (to identify
quantitative defects) and a test for assessing the function of platelets (Bleeding time, consisting in actively
making or inducing bleeding on one arm by standardized conditions and measuring how long it takes for the
platelets to stop the bleeding).


Thrombocytopenia
Thrombocytopenia is defined as platelet count < 100*10^9 per liter.
When focusing on defective platelet count we first have to investigate and exclude a condition of
thrombocytopenia that is not due to actual biological or clinical condition but just to analytical bias. This is an
analytical problem (due to EDTA which causes agglutination and adhesion) that may result in apparently
decreased platelet count. Platelet count should be performed using citrate as anticoagulant in the presence
of reduced platelet count. If a difference is noticed between the two results, we may conclude it was just
pseudo-thrombocytopenia
Why don’t we always use citrate? Because in order to obtain proper anticoagulation with citrate you have to
use 10% dilution concentration, and this may result in dilution of all counts including hemoglobin, WBC and
so on, so this is less reliable than using EDTA that requires less than 1% dilution. We also need to consider
that in a normal subject, if you have normal count using citrate, you are expected to obtain lower values of
platelets because of dilution: so if a subject has 150*10^9 per liter platelet count using EDTA, using citrate in
same subject you should expect 135*10^9 per liter. But if you are facing a subject having a count of 80*10^9/
L and you perform blood count using citrate and find 150*10^9/L than you may conclude that analytical
problem due to platelet agglutination in the tube is responsible for the low platelet count.

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Once we have excluded this analytical issue, than we may conclude that we are indeed facing a true
condition of thrombocytopenia.
Two different types of thrombocytopenia can be distinguished:

1) thrombocytopenia due to decreased bone marrow production. It can be due to the following reasons:
- Dehydration, Vitamin B12 or folic acid deficiency
- Leukemia or myelodysplastic syndrome or aplastic anemia
- Decreased production of thrombopoietin by the liver in liver failure
- Sepsis, systemic viral or bacterial infection
- Dengue fever
- Hereditary syndromes

2) thrombocytopenia due to increased platelet destruction. It can be due to immune or non-immune

reasons.
- Idiopathic thrombocytopenic purpura
- Thrombotic thrombocytopenic purpura
- Hemolytic-uremic syndrome
- Disseminated intravascular coagulation
- Paroxysmal nocturnal hemoglobinuria
- Antiphospholipid syndrome
- Systemic lupus erythematosus
- Post-transfusion purpura
- Neonatal alloimmune thrombocytopenia
- Hypersplenism
- Dengue fever


Immunocytopenia, is a clinical condition characterized by immune mediated platelet destruction. It is

essentially due to an Ab dependent cytotoxicity mechanism, where auto-Ab bind to Ag on the platelets’
surface, which then are uptaken by macrophages through Fc receptor.
We may identify 2 different subsets of this thrombocytopenia:
- one is very frequent in childhood, it is an acute form that most of the times occurs post infection, it usually
is a benign conditions and in 90% of the cases of its spontaneously resolved in the following 3-6 month.
- the second is the chronic form that is typical or more typical to adulthood with not negligible prevalence in
the general population.
Usually there is no evidence for infectious disease in the medical history of the subjects. As most of the
autoimmune conditions the prevalence in females is higher than in man 3:1. Usually auto Ab are directed to
several Ag on the platelet surface, in particular Glycoprotein 2b/2a and 1b/9. The clinical signs may be
extremely variable, in principle as a function of platelet count. Usually in platelet counts higher than
20-30*10^9/L we shouldn’t expect major clinical bleeding signs. The clinical signs we may observe in
subjects with immune thrombocytopenia are those we are expecting in some patients with primary
hemostatic defect, which means petechiae, bruises usually involving skin or mucosa.
Facing a patient with isolated thrombocytopenia, first we have to exclude analytical thrombocytopenia.
Second we should take a look at the peripheral blood smear. Than we have to exclude other conditions
characterized by thrombocytopenia:
- splenomegaly conditions, in particular related to chronic liver disorders, that are characterized by
sequestration of platelets in spleen and a condition of hypersplenism;
- we have to exclude that immune thrombocytopenia is not related to other systemic immune diseases, as
in this case we shouldn't focus on thrombocytopenia but on main systemic diseases, in particular lupus
erythematosus;
- we have to exclude infections, in particular HIV and HCV. A not negligible number of patients with HIV are
actually referred to the haematologist and not to infectivologist due to the infection that reduce the blood
counts. HCV thrombocytopenia is not only related to the liver but it also has a direct effect on
megakaryocytes and platelet production. In southern Europe HCV infection is highly prevalent and more
than 10% of persons older than 60 had previous contact with the virus. On average the virus infection
becomes chronic in more than 90% of the subjects so this results in a very high prevalence of the
infection. In younger persons this prevalence is significantly lower.
Once excluded all these clinical conditions we have to preform bone marrow aspirate, that may confirm
megakaryocyte hyperplasia which is clearly evidence for peripheral platelets destruction and not reduced
bone marrow production. In the meantime we can also exclude other hematologic malignancies in particular

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myeloid disorders that may result in reduced platelet production and do not present megakaryocyte
hyperplasia.
This diagnostic workup is mainly based on an exclusion process. Unfortunately the test for platelet Ab is
available but with not diagnostic occurrence, that means it characterized by moderate sensitivity: there is a
number of patients with true immune thrombocytopenia which is apparently negative to the test, it may be
due to the fact that not only single Ab are involved in the disease and we able to capture just few of them.
There is also a second test which is goal specific: a number of subjects that do not have immune
thrombocytopenia (including subjects with normal platelet count) may have detectable platelet Ab in their
blood. So basically this results have poor diagnostic accuracy.

In disorders characterized by an increased platelet destruction, there is an attempt of megakaryocytes to
compensate thrombocytopenia which ends up with the production of abnormal platelets. This abnormally
large platelets are not properly counted by standard analysis, because one of the criteria of the analyzer are
the physical parameters of the cells. We will thus have underestimation of the true platelet count.
Bone marrow morphology is characterized by a high number of megakaryocytes (megakaryocyte
hyperplasia), which is a major criteria to confirm the diagnosis.

Facing patients with primary hemostatic defects we should first focus on the platelet count: if its low, then it is
necessary to confirm this low number of platelet by performing blood count using citrate to exclude analytical
problems and if low count is confirmed and we are facing a patient with isolated thrombocytopenia, we have
to consider different conditions.
The only way to have a final confirmation is to submit the patient to bone marrow investigation clearly
showing megakaryocyte hyperplasia which is the landmark of increased platelet destruction compared to
decreased bone marrow production.
How do we treat immune thrombocytopenia? Platelet counta up to 30*10^9/L are usually asymptomatic. So
in this case we may wait and watch. Of course we must pay attention if the patient undergoes surgery, major
or minor. For minor surgery a platelet count of more than 50*10^9/L is required, and for major surgery like
abdominal or thoracic no less than 80*10^9/L. We can obtain these values through 2 different approaches:
- platelet transfusion, if we have to reach this values in short time (surgical intervention in ER). We may
expect high rate of destruction of the transfused platelets but this may still help.
- use high dose of IV Ig. The administration of high dose of IV Ig is going to saturate all the Fc receptors of
the macrophages and this will lead to an increase of platelets. The effect however is very short because
the infused Ig are destructed. There is not a negligible difference between short term and long lasting
responses. The reason is that chronic therapies with corticosteroids are significantly affected by severe or
clinically relevant side effects, so we unable to maintain this dose for long term therapy. So after 4 weeks
we have to progressively reduce the dose but this may result in loss of response in fraction of patients. Up
to 70% of patients maintain the response even with low dose or after complete withdrawal of
corticosteroids.
- Currently second line therapy is splenectomy. By removing the main site of platelet destruction we obtain a
significant increase of platelet count. Just for a practical point of view consider that we are going to admit
patients to major surgery (splenectomy) having low platelet count. This is critical clinical problem so we
transfuse the patient before the surgery or administer IV Ig.
Than if patient is not adequately respond to 2 line therapy (splenectomy) we may have additional
approaches.
Another approach is the administration of thrombopoietin mimetic agents that are significantly increasing the
production of platelets partly overcoming the excessive destruction of platelets. 


Von Willebrand’s disease

If we are facing patient with bleeding signs related to primary hemostatic defects and the platelet count is
normal we will have to focus on functional defects of primary hemostasis. It has a semidominant pattern of
inheritance and this means that one abnormal allele may result the expression of disease but for full
expression we need 2 abnormal alleles.
Von Willebrand disease (vWD) is the most common hereditary coagulation abnormality described in humans,
although it can also be acquired as a result of other medical conditions. It arises from a qualitative or
quantitative deficiency of von Willebrand factor (vWF), a multimeric protein that is required for platelet
adhesion. Von willebrand factor is the primary mediator of platelet adhesion to subendothelium.
There are three forms of vWD:

- hereditary
- vWD Type I, the most common type of the disorder and patients are typically asymptomatic or may experience
mild symptoms such as nosebleeds although there may be severe symptoms in some cases. Type 1 vWD
(60-80% of all vWD cases) is a quantitative defect which is heterozygous for the defective gene. The production of
von Willebrand factor vWF is decreased. Often the discovery of vWD occurs incidentally to other medical
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 procedures requiring a blood work-up where there is a normal platelet count, but increased bleeding time, reduced
FvW antigen test and reduced activity with ristocetin cofactor. Trouble may however arise in some patients in the
form of bleeding following surgery (including dental procedures), noticeable easy bruising, or menorrhagia (heavy
menstrual periods). There are also a minority of cases of Type 1 which may present with severe hemorrhagic
symptoms.
- vWD Type II (20-30%) is a qualitative defect and the bleeding tendency can vary between individuals. There are
normal levels of vWF, but the multimers are structurally abnormal, or subgroups of large or small multimers are
absent. Four subtypes exist:
- 2A: characterized by qualitatively defective von Willebrand factor with decreased ability to bind to platelet glycoprotein1(GP1)
and decreased capability at multimerization. The vWF is quantitatively normal but qualitatively defective. Its ability to bind to
the glycoprotein1 (GP1) receptor on the platelet membrane is diminished, resulting in decreased platelet adhesiveness and
aggregation. The ability of the defective von Willebrand factors to coalesce and form large vWF multimers is also impaired,
resulting in decreased quantity of large vWF multimers. Only small multimer units are detected in the [Link]
Willebrand factor antigen assay is normal.
- 2B: characterized by a "gain of function" defect in which the ability of the qualitatively defective von Willebrand factor to bind
to glycoprotein1 (GP1) receptor on the platelet membrane is abnormally enhanced, leading to its spontaneous binding to
platelets and subsequent rapid clearance of the bound platelets and of the large vWF multimers. Large vWF multimers are
reduced or absent from the circulation.
- 2M: characterized by a qualitative defect of von Willebrand factor which has decreased ability to bind to glycoprotein1 (GP1)
receptor on the platelet membrane and normal capability at multimerization. The vWF antigen levels are normal. High
molecular weight large vWF multimers are present in the circulation.
- 2N: deficiency of the binding of vWF to coagulation factor VIII. The vWF antigen test is normal, indicating normal quantity of
vWF. Assay for coagulation factor VIII reveals marked quantitative decrease equivalent to levels seen in hemophilia A.
- vWD Type III. Type 3 is the most severe form of von Willebrand disease (homozygous for the defective gene) and
is characterized by complete absence of production of vWF. The von Willebrand factor is undetectable in the vWF
antigen assay. Since the von Willebrand factor protects coagulation Factor VIII from proteolytic degradation, total
absence of vWF leads to extremely low Factor VIII level, equivalent to that seen in severe hemophilia A with its
clinical manifestations of life-threatening external and internal hemorrhages. The inheritance pattern of vWD type
3 is autosomal recessive while the inheritance pattern of hemophilia A is x-linked recessive.

- acquired, can occur in patients with autoantibodies. In this case the function of vWF is not inhibited but
the vWF-antibody complex is rapidly cleared from the circulation.

- pseudo or platelet type, is an autosomal dominant genetic defect of the platelets. The von Willebrand
factor is qualitatively normal and genetic testing of the von Willebrand gene and vWF protein reveals no
mutational alteration. The defect lies in the qualitatively altered glycoprotein1 (GP1) receptor on the
platelet membrane which increases its affinity to bind to the von Willebrand factor. Large platelet
aggregates and high molecular weight vWF multimers are removed from the circulation resulting in
thrombocytopenia and diminished or absent large vWF multimers.

There are various factors that affect the presentation and severity of symptoms of vWD such as blood type.

In order to diagnose this disorder we may need to perform bleeding time tests. We may also have to directly
measure vWF and this may be either quantitative or a functional.
Very useful to catch functional defect in case the protein normally detected with reduced function. We can
also measure the FvW as a carrier of factor VIII. In the presence of a defect in the carrier- an increase
catabolism of factor VIII occurs thus its levels will be reduced.

Examining the patient we will observe a prolonged bleeding time with a normal platelet count. In such
condition we should first focus our attention on VWd. We may then be able to demonstrate the partial
reduction in the Ag and of course also a partial reduction in its function as a cofactor and carrier of factor VIII.
Ristocetin is an antibiotic, previously used to treat staphylococcal infections. It is no longer used clinically
because it caused thrombocytopenia and platelet agglutination. It is now used solely to assay those
functions in vitro in the diagnosis of conditions such as von Willebrand disease (vWD). Platelet agglutination
caused by ristocetin can occur only in the presence of von Willebrand factor multimers, so if ristocetin is
added to blood lacking the factor (or its receptor), the platelets will not clump. In some types of vWD (types
2B and platelet-type), even very small amounts of ristocetin cause platelet aggregation when the patient's
platelet-rich plasma is used. This paradox is explained by these types having gain-of-function mutations
which cause the vWD high molecular-weight multimers to bind more tightly to their receptors on platelets (the
alpha chains of glycoprotein Ib (GPIb) receptors). In the case of type 2B vWD, the gain-of-function mutation
involves von Willebrand's factor (VWF gene), and in platelet-type vWD, the receptor is the object of the
mutation (GPIb).

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An high proportion of patients do not need any treatment. Some of them are actually diagnosed at adulthood.
So the most frequent scenario we have to face is when they need to undergo surgery. We have different 2
approaches:
- use cryoprecipitate including FvW and nowadays we have also recombinant FvW,
- use DDAVP which is a derivative of vasopressin that stimulates endothelial cells to release FvW. It may be
effective for a short time.

Disseminated Intravascular Coagulation (DIC)

Disseminated intravascular coagulation (DIC), also known as disseminated intravascular coagulopathy or
less commonly as consumptive coagulopathy, is a pathological process characterized by the widespread
activation of the clotting cascade that results in the formation of blood clots in the small blood vessels
throughout the body. This leads to a compromising of the blood flow to the tissues and can ultimately lead to
multiple organ damage. In addition, as the coagulation process consumes clotting factors and platelets,
normal clotting is disrupted and severe bleeding can occur from various sites.
DIC does not occur by itself but only as a complicating factor from another underlying condition. The
combination of widespread loss of tissue blood flow and simultaneous bleeding leads to an increased risk of
death in addition to that posed by the underlying disease. DIC can be overt and severe in some cases, but
milder and insidious in others.
It's a systemic process due to inappropriate activation of secondary hemostasis.
Most of these conditions can be immediately or clearly proven but sometimes they cannot. Sometimes DIC is
the first sign for obstetric complications, the same applies to aneurysm or tumors.

Under homeostatic conditions, the body is maintained in a finely tuned balance of coagulation and
fibrinolysis. The activation of the coagulation cascade yields thrombin that converts fibrinogen to fibrin; the
stable fibrin clot being the final product of hemostasis.
The fibrinolytic system then functions to break down fibrinogen and fibrin. Activation of the fibrinolytic system
generates plasmin (in the presence of thrombin), which is responsible for the lysis of fibrin clots. The
breakdown of fibrinogen and fibrin results in polypeptides called fibrin degradation products (FDPs) or fibrin
split products (FSPs). In DIC, the processes of coagulation and fibrinolysis are dysregulated, and the result
is widespread clotting with resultant bleeding. Regardless of the triggering event of DIC, once initiated, the
pathophysiology of DIC is similar in all conditions.

There are 2 major mechanisms that may lead to DIC (activation of secondary coagulation):
• systemic inflammation. Cytokines may result in coagulation activation. Tissue factor, which is the first
activator of the external coagulation pathway, is released in response to exposure to cytokines (particularly
interleukin 1). Cytokines may also result in the inhibition of fibrinolysis, or inhibition of anticoagulant
mechanisms in particular of protein c, either by reduction of thrombomodulin, that is a cofactor in activating
protein C, or by reducing the expression of protein C receptors on endothelial cells. During inflammation, a
vicious circle arises: an increase in activation of coagulation may in turn increase or stimulate inflammation,
and this may in turn increase the excretion of pro inflammatory cytokines.

• direct release of procoagulant molecules. One critical mediator of DIC is the release of a transmembrane
glycoprotein called tissue factor (TF). TF is present on the surface of many cell types (including endothelial
cells, macrophages, and monocytes) and is not normally in contact with the general circulation, but is
exposed to the circulation after vascular damage. TF is released in response to exposure to cytokines
(particularly interleukin 1), tumor necrosis factor, and endotoxin. This plays a major role in the development
of DIC in septic conditions. TF is also abundant in tissues of the lungs, brain, and placenta. This helps to
explain why DIC readily develops in patients with extensive trauma. Upon exposure to blood and platelets,
TF binds with activated factor VIIa, forming a complex which further activates factor IX and X to IXa and Xa
respectively, leading to the common coagulation pathway and the subsequent formation of thrombin and
fibrin. The release of endotoxin is the mechanism by which Gram-negative sepsis provokes DIC.

In acute promyelocytic leukemia, treatment causes the destruction of leukemic granulocyte precursors,
resulting in the release of large amounts of proteolytic enzymes from their storage granules, causing
microvascular damage. Other malignancies may enhance the expression of various oncogenes that result in
the release of TF and plasminogen activator inhibitor-1 (PAI-1), which prevents fibrinolysis.

Excessive circulating thrombin results from the excess activation of the coagulation cascade. The excess
thrombin cleaves fibrinogen, which ultimately leaves behind multiple fibrin clots in the circulation. These

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excess clots trap platelets to become larger clots, which leads to microvascular and macrovascular
thrombosis.
Coagulation inhibitors are also consumed in this process. Decreased inhibitor levels will permit more clotting
so that a positive feedback loop develops, in which increased clotting leads to more clotting. At the same
time, thrombocytopenia occurs and this has been attributed to the entrapment and consumption of platelets.
Clotting factors are consumed in the development of multiple clots, which contributes to the bleeding seen
with DIC.
Simultaneously, excess circulating thrombin assists in the conversion of plasminogen to plasmin, resulting in
fibrinolysis. The breakdown of clots results in an excess of FDPs, which have powerful anticoagulant
properties, contributing to hemorrhage. The excess plasmin also activates the complement and kinin
systems.

First symptoms and signs may be the ones of the underlined disease. Then clinical signs directly related to
disseminated intravascular coagulation. Activation of these systems in fact leads to many of the clinical
symptoms that patients experiencing DIC exhibit, such as shock, hypotension, and increased vascular
permeability. The acute form of DIC is considered an extreme expression of the intravascular coagulation
process with a complete breakdown of the normal homeostatic boundaries. DIC is associated with a poor
prognosis and a high mortality rate.
The most frequent clinical manifestation of this process is organ failure (because of ischemic damage),
which is even more frequent and relevant than external bleeding or thrombosis that is actually is quite rare.
The inappropriate activation of secondary hemostasis is generating thrombi in the small vessels (micro
circulation). And this is inducing thrombocytopenia because of disruption of platelets in the micro circulation.
There also is the consumption of coagulation factors due to inappropriate activation.


When performing lab tests we should first focus on secondary hemostasis test. We may observe a reduction
in nearly all the coagulation factors, this is in fact a consumption coagulopathy. Significant reduction in
fibrinogen may be noted as well. Prolonged PTT may be observed.
Increased fibrinolysis causes an increase of D-DIMER or FDP. There is also a reduction in the anticoagulant
proteins (thrombin 3 or protein C).

Thrombocytopenia is due to platelets disruption and consumption.
We can also observe hemolytic anemia due to increased erythrocyte fragmentation. Anemia is therefore
microcytic characterized by an increased reticulocyte count, increase in hemolytic markers, unconjugated
bilirubin and increased LDH. Haptoglobin is reduced. Finally there is also evidence of erythrocyte
fragmentation (schistocytes) that can be clearly demonstrated in a peripheral blood smear.

When approaching treatment, we first need to treat the underlining disease. If you don’t do this you will be
unable to control this process. We then need to treat disseminated intravascular coagulation and basically
we have to try to limit the coagulopathy on one hand and thrombocytopenia on the other hand. So
transfusion of platelets and of fresh frozen plasma which contains all the coagulation factors are usually the
2 products that are indicated in this condition.
Usually there is no need and no benefit from the introduction of an anticoagulant like heparin (the only case
in which it could be useful is when we are facing an overt major venus thrombus).
In selected conditions, in particular sepsis, we also use recombinant human activator protein C, that has
been proven to be effective with patients with sepsis and has been approved by the FDA and EMA.

Moschcowitz disease
It is a rare but clinically extremely relevant condition that has to be differentiated by disseminated
intravascular coagulation. Both can be classified as thrombotic microangiopathies, that are clinical conditions
mainly characterized by the formation of thrombi in the microcirculation. This disease is characterized by a
micro angiopathic hemolytic anemia as we observed in disseminated intravascular coagulation, but in this
case with a very selective organ targets that include kidney, CNS. Nearly all the cases the disease are
characterize by fever, thus this is a systemic disease.
Thrombotic thrombocytopenic purpura (TTP or Moschcowitz syndrome) is a rare disorder of the blood-
coagulation system, causing extensive microscopic clots to form in the small blood vessels throughout the
body. These small blood clots, called thrombi, can damage many organs including the kidneys, heart and
brain. With plasma exchange, mortality rate has dropped to 10% at six months.
Most cases of TTP arise from inhibition of the enzyme ADAMTS13, a metalloprotease responsible for
cleaving large multimers of von Willebrand factor (vWF) into smaller units. The increase in circulating
multimers of vWF increase platelet adhesion to areas of endothelial injury, particularly at arteriole-capillary
junctions.

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A rarer form of TTP, called Upshaw–Schulman syndrome, is genetically inherited as a dysfunction of
ADAMTS13. If large vWF multimers persist, a tendency for increased coagulation exists.
Red blood cells passing the microscopic clots are subjected to shear stress which damages their
membranes, leading to rupture of red blood cells within blood vessels, which in turn leads to anaemia and
schistocyte formation. Reduced blood flow due to thrombosis and cellular injury results in end organ
damage.
Current therapy is based on support and plasmapheresis to reduce circulating antibodies against
ADAMTS13 and replenish blood levels of the enzyme.
TTP, as with other microangiopathic hemolytic anemias (MAHAs), is caused by spontaneous aggregation of
platelets and activation of coagulation in the small blood vessels. Platelets are consumed in the aggregation
process, and bind vWF. These platelet-vWF complexes form small blood clots which circulate in the blood
vessels and cause shearing of red blood cells, resulting in their rupture.
Roughly, the two forms of TTP are idiopathic and secondary TTP. A special case is the inherited deficiency of
ADAMTS13, known as the Upshaw-Schülman syndrome.

The process is thus triggered by defects in primary hemostasis, and in fact if we perform a coagulation test in
this patients it will be normal. This is how you can differentiate this disease from DIC.

Patients present with intravascular hemolytic anemia, fever, thrombocytopenia (because of the consumption
of platelets in the thrombi) and milder organ failure. In most cases this is a very acute disease, the patient
will be developing relevant neurologic signs.
This is the one of few emergencies in haematology.
The most common hematologic malignancy associated with DIC is acute promyelocytic leukemia.

The therapy is different from DIC, and also because of this it is very important to distinguish between these 2
conditions. In this case the principle is to remove HMW FvW multimers and this may be accomplished by
plasmapheresis, that is removing patient's plasma and in the same time administering other components. So
no transfusion of plasma. This disease, when not recognized, is associated with high mortality.

Heparin-induced thrombocytopenia (HIT)

It is the development of thrombocytopenia (a low platelet count), due to the administration of various forms of
heparin, an anticoagulant. HIT predisposes to thrombosis (the abnormal formation of blood clots inside a
blood vessel), and when thrombosis is identified the condition is called heparin-induced thrombocytopenia
and thrombosis (HITT).
HIT is caused by the formation of abnormal antibodies that activate platelets. If someone receiving heparin
develops new or worsening thrombosis, or if the platelet count falls, HIT can be confirmed with specific blood
tests.
Heparin occurs naturally in the human body, but the development of HIT antibodies suggests heparin may
act as a hapten, and thus be targeted by the immune system. In HIT, the immune system forms antibodies
against heparin when it is bound to a protein called platelet factor 4 (PF4). These antibodies are usually of
the IgG class and their development usually takes about five days. However, those who have been exposed
to heparin in the last few months may still have circulating IgG, as IgG-type antibodies generally continue to
be produced even when their precipitant has been removed. This is similar to immunity against certain
microorganisms, with the difference that the HIT antibody does not persist more than three months.
The IgG antibodies form a complex with heparin and PF4 in the bloodstream. The tail of the antibody then
binds to the FcγIIa receptor, a protein on the surface of the platelet. This results in platelet activation and the
formation of platelet microparticles, which initiate the formation of blood clots; the platelet count falls as a
result, leading to thrombocytopenia.
Formation of PF4-heparin antibodies is common in people receiving heparin, but only a proportion of these
develop thrombocytopenia or thrombosis. This has been referred to as an "iceberg phenomenon".
The treatment of HIT requires both protection from thrombosis and choice of an agent that will not further
reduce the platelet count.

Disorders of secondary hemostasis

It is mainly due to coagulation factors deficiency. This is divided into:
• inherited (haemophilia and rarer disorder)
• acquired (liver, vitamin k deficiency, disseminated intravascular coagulation).
All these diseases result in coagulation deficiencies, increasing the risk of bleeding.
Secondary hemostatic defects result in delayed bleeding after trauma is usually delayed, since at the
beginning platelets are properly activated and aggregated, but then there is no stabilization by coagulation

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factors. The bleeding is usually not external, because the primary hemostasis mostly acts externally.
Bleeding therefore involves joints, muscles and internal cavities.
The lab tests to be performed in order to diagnose a secondary hemostasis defect are the coagulation tests:
APPT (for the intrinsic pathway) and prothrombin test (PT or INR, for the extrinsic pathway). Then we can
directly measure fibrinogen and we can also have a quick look at the fibrinolytic phase, dosing FDP and D-
Dimer.

Hemophilia A
It is a clinical disorder characterized by partial or complete deficiency of factor VIII, resulting from a genetic
lesion. The gene responsible for this disease is located on the X chromosome, and the disease is therefore
x-linked (it thus occurs in males and in homozygous females). The prevalence is significantly different among
males and females, with basically 1 male over 5-10 thousands females.
Factor VIII is a cofactor for factor IXa which, in the presence of Ca2+ and phospholipids forms a complex
that converts factor X to the activated form Xa.
Hemophilia is a secondary hemostatic deficiency, therefore bleeding is delayed and it involves joints,
muscles, soft tissues and visceral cavities. In newborns there can be cephalic haematoma, in case of natural
delivery. Delayed bleeding is peculiar.
Complications include symptoms and signs of pseudo-tumors due to compression of tissues, vessels and
nerves surrounding the tumor (ischemic damage of peripheral nerves) and joint damage.
The first sign of Hemophilia A is a prolonged APPT. We can then measure the concentration of factor VIII or
we can measure its activity measuring the activity of the plasma of the patient compared to a control plasma.
The severity of bleeding is related to the factor VIII activity. We define as “severe” the disease in which the
residual factor VIII activity is less than 2% of normal, “moderate” when the residual activity is between 2 and
5% and “mild” when the activity is 5-30%. About half of the patients have severe disease.
The genetic defect is a point mutation and overall there are more than 300 genetic defects described.
The prevalence would have been expected to decrease because the patients should die before giving birth.
However, the incidence is increasing because there is a relevant proportion of cases that are “de novo” and
not inherited. Most of these de novo cases are characterized by the inversion of intron 22 of factor VIII gene.
This results in a non-functional truncated protein, causing severe hemophilia.
How do we treat patients with hemophilia A? Prevention is really important: we have in fact the tools for
preventing the bleeding and therefore its chronic complications (joint damages, pseudo-tumor…).
In case of an unexpected event we may then early-treat the patient, with several products available. The
most common during the 90s was the “Plasma cryoprecipitate” but the problem is that it cannot be properly
treated for Hepatitis C and B and HIV. In particular, during 80s-90s, when HBV and HCV were unknown, a
good number were infected through transfusion.
Lyophilised factor VIII was introduced after few years, and now we have recombinant factor VIII. The aim of
the therapy is to bring patients with the severe hemophilia to levels that are associated with mild or no
symptoms (25-30% of VIII activity) and basically one unit of factor VIII (quantified as VIII present in one ml of
normal plasma) is able to increase VIII activity by 2% per kg of body weight. So an adult may require
700-1000 unit of this factor in order to have a safe residual activity. However, a fraction of patients may
develop inhibitors of factor VIII (antibodies against it) and this is relevant because they'll loose the most
powerful tool to prevent their bleeding.
Nowadays the disease in pregnancy is treatable and the choice to continue pregnancy is individual.
Finally, talking about gene therapy, hemophilia was one of the first diseases in which clinical trials and gene
therapy were carried on. This approach was able to induce an increase in synthesis of plasma factor VIII, but
for a very transient period.9 Hemostasis and Hemostatic diseases

10 Transfusion therapy

A blood transfusion is when you receive blood via an intravenous (IV) line. Blood transfusions are sometimes
necessary after an injury or surgery causes a high amount of blood loss. Some people need regular
transfusions due to medical conditions such as hemophilia or cancer. This is called transfusion therapy. In
some cases, a permanent IV can be put in place. 
The rational of transfusion therapy is that, in definite conditions, there is the need for only peculiar blood
components, or for a limited number of blood components, thus the transfusion therapy should be targeted
and limited to the component required, in order not to give too much volume to the patient.
Whole blood units (WBU) transfusions are also only limited to specific various peculiar conditions.
The components are obtained after centrifugation and separation in a close-loop system of whole blood units
collected in a plastic bag enriched in anticoagulants.

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Blood apheresis
Another possibility is blood apheresis, in which the blood taken from a healthy donor can be separated into
its component parts during blood donation. The needed component is then collected and the "unused"
components are returned to the donor. Fluid replacement is usually not needed in this type of collection.

There are large categories of component collections:

• Plasmapheresis: blood plasma. It is useful in collecting FFP (fresh frozen plasma) of a particular ABO
group. Commercial uses aside from FFP for this procedure include immunoglobulin products, plasma
derivatives, and collection of rare WBC and RBC antibodies.

• Erythrocytapheresis: red blood cells. It is the separation of erythrocytes from whole blood. It is most
commonly accomplished using the method of centrifugal sedimentation. This process is used for red blood
cell diseases such as sickle cell crises or severe malaria.

• Plateletpheresis (thrombapheresis, thrombocytapheresis): blood platelets. Plateletpheresis is the collection

of platelets by apheresis while returning the RBCs, WBCs, and component plasma. The yield is normally
the equivalent of between six and ten random platelet concentrates. Quality control demands the platelets
from apheresis be equal to or greater than 3.0 × 1011 in number and have a pH of equal to or greater than
6.2 in 90% of the products tested and must be used within five days.

• Leukapheresis: leukocytes (white blood cells). Leukopheresis is the removal of PMNs, basophils,
eosinophils for transfusion into patients whose PMNs are ineffective or where traditional therapy has failed.
The complications of this procedure are the difficulty in collection and short shelf life (24 hours at 20 to
24 °C). Quality control demands the resultant concentrate be 1.0 × 1010 granulocytes in 75% of the units
tested and that the product be irradiated to avoid graft-versus-host disease (inactivate lymphocytes).
Irradiation does not affect PMN function. Since there is usually a small amount of RBCs collected, ABO
compatibility should be employed when feasible.

• Stem cell harvesting: circulating bone marrow cells are harvested to use in bone marrow transplantation.

The advantages of targeted transfusion therapy are: better therapeutic results, reduced transfusion risk,
reduced incidence of side effects of the transfusion. There is also sparing of the blood components obtained
from the single blood donor, so that this additional components can be used for another patient.
These components are tested with immuno-hematological tests: these tests are aimed at assessing the
compatibility between the donor and the recipient. There is the need of checking the AB0 and the Rh(D)
blood groups of the donor and the recipient. For the donor the complete Rh phenotype has to be
ascertained, whereas for the recipient the blood group should be checked on two separated samples: there
is a first sample that is the first check, but, as in allogenic bone marrow transplantation, you have to assess
the HLA of the recipient and you have to make a double check. Specific transfusion compatibility tests are
performed in peculiar situations, such as in a patient with a positive Coomb's test.

Blood components can also be manipulated.

• Leukodepletion: leukocyte number varies among the distinct units collected. WBCs can cause a
sensitization of the recipient. This can cause an immunological reaction and may induce a Graft versus
Host reaction in the recipient. GvHD in the recipient after the transfusion of a blood component is very
dangerous because it is always lethal. Transfusion related GvHD occurs within 10-15 days after RBCs
infusion, and it is characterized by fever, skin rash, diarrhea, liver necrosis, an pancytopenia; these are the
same complications that occur when a patient submitted to an allogeneic bone marrow transplant
develops the GvHD. Thus it is very important to prevent this reaction. The platelets unit and the plasma
unit usually contain more WBCs than a RBCs unit, and thus it is more frequent that a reaction presents
with transfusion of a platelets unit. It is theorized that transfusions that contain white blood cells may cause
adverse effects through multiple mechanisms. White blood cells may themselves harbour infectious
diseases and some pathogens will be more concentrated in white blood cells than in the rest of the blood
product. It is also theorized that the donor white blood cells may suppress the recipient's immune system
by interacting with it. Possible side effects due to the presence of leukocytes are:
- Alloimmunity, an immune response to foreign antigens (alloantigens) from members of the same species.
Alloimmune response results in graft rejection, which is manifested as deterioration or complete loss of graft
function. Alloimmunity is caused by the difference between products of highly polymorphic genes, primarily genes
of MHC complex, of the donor and graft recipient. These products are recognized by T-lymphocytes and other
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 mononuclear leukocytes which infiltrate the graft and damage it. The consequence in this case is a refractoriness
to the platelets transfusion: when you have had this reaction, and you administer platelets to your patient, there is
no increase in platelets count, because the patient has developed antibodies against the HLA class I antigens; and
this alloimmunization can cause an high incidence of febrile non hemolytic reaction. Moreover, if later on the patient
needs to undergo an allogeneic bone marrow transplant, the presence of these antibodies against the leukocytes
antigens can cause a difficulty in identifying a suitable HSC donor, and can adversely influence the possibility of a
hemopoietic reconstitution after having performed the transplant
- Immunomodulatory effect with higher incidence of infection and relapse with solid cancers.
- The development of a transfusion late GvHD
- The transmission of viral and bacterial agents
- The reactivation of various viruses: the most frequent viruses are CMV, EBV, HIV1, and also an increased
incidence of infection due to prions (such as Creutzfeld-Jacob disease, which is very rare, but there is a possibility
of transmission of this disease)
- Development of a non cardiogenic lung edema
- Cytokine storm, due to the release of cytokines, during the reaction of leukocytes against this alloimmunization.

There are new devices used to prevent alloimmunization, and these devices are usually used to perform
leukocytes depletion: polyester microfibers (leukodepletion is caused by the direct and indirect adhesion of
leukocytes to microfibers), or polyurethane microfibers (leukodepletion is due to mechanical sifting). Filtering
can also be performed at the time of collection of the unit, or just before transfusion. Usually depletion is
performed at the time of transfusion of the blood component, even if the pre-storage filtering is preferred.
Leukodepletion is indicated in patients candidates to long term transfusion support, in patients who are
immunodeficient, and thus it is desirable to reduce the incidence of transmission of infection (especially viral
infections) and in peculiar patients such as pregnant women (candidate to hematopoietic stem cells
transplant) and neonates; these are peculiar situations in which it is desirable to perform leukodepletion.

The potential complications of leukodepletion are:

- hypotension in patients treated with ACE inhibitors and submitted to bedside leukodepletion, achieved
through polyester microfibers
- anaphylaxis
- strong pain
- pain at the site of infusion
- erythema of the conjunctiva.

• Washed blood components: especially RBCs are washed using isotonic solutions until the protein content
of the unit that is used is under 0.3 g. This is very important because our patient can have had some
reaction caused by the proteins contained within the serum of the donor. Since the washing procedure
increases the risk of microbial contamination, washed RBC concentrates can be stored at +4°C for less
than 24 hours. Moreover, patients that receive these units receive less RBCs in comparison to those that
receive standard RBCs unit, this because a certain volume of RBCs is lost during the washing. It is thus
possible that in order to achieve the standard dose of Hb, we have to give to our patient not just one RBCs
unit, but two. Anyway, if your patient have had a reaction to proteins, such as the development of pruritus
during the transfusion, it is mandatory to use washed blood component, especially of RBCs. It is important
the removal of plasma proteins in order to avoid severe allergic reactions that are mediated by the
recipient antibodies, especially IgG antibodies against plasma proteins. The washing is mandatory when
these reactions are recurrent and remain severe even after the administration of antihistaminic.

———Clinical case—————————————————————————————————— —————

In IgA deficient patients there is also another possibility. The recipient was an IgA deficient patient, had
antibodies against IgA and developed a very severe reaction to a blood component that ended in an
anaphylactic reaction. In this case it is recommended to use blood components provided by a donor with the
same deficiency, in order to avoid the reaction against IgA. In neonates, moreover, it is recommended to use
washed products, in order to reduce the administration of anti-clotting agent, to avoid the administration of
extracellular K+ and other cellular metabolic products.

• Irradiated blood components: in some special cases, it is also important to use irradiated blood
components. Irradiation is carried out with γ or X-rays, and the X-rays used are those of 25-50 Gray, in
order to prevent lymphocytes replication without affecting the cellular function. This is usually carried out in
patients who are candidates to allogeneic bone marrow transplantation, or in patients who have already
received a transplant; not only an allogeneic but also autologous stem cells transplant in order to avoid

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 any potential reaction which could be caused by the lymphocytes contained in the donor units. Irradiated
RBC units lose K+ and units obtained within 14 days shall be infused within 28 days; RBCs units used for
intra-uterine support shall be infused within 48 hours. Instead, the irradiation of platelets does not change
their expiry date, so this is a problem occurs only with RBCs. The first aim of providing irradiated blood
components is to avoid the frequently fatal risk of transfusional related GvHD which can occur after the
transfusion of immunologically competent donor lymphocytes to an immuno-incompetent recipient. Other
indications to use irradiated blood components are Hodgkin and non Hodgkins lymphomas, patients
submitted to intensive chemotherapies (including fludarabine, for example, in patients with acute myeloid
or lymphoplastic leukemia, who are submitted to a very intensive chemotherapy, and thus heavily
compromised), patients with congenital immunodeficiency and also pre-term neonates: all situations in
which the recipient is immuno-incompetent, and the donor is immunocompetent. In this case, the use of
irradiated blood components is required.

Whole blood units (WBU) transfusion

These are very rarely used, but they are used in two peculiar situations. Usually the volume of a whole blood
unit is 450 mL plus 10%, and the hematocrit is usually comprised between 46% and 44%. Depending on the
anticoagulant used, the WBU can be stored for a different time: it can be stored for 35 days if CPDA (citrate-
phosphate-dextrose-adenine) is used as the anti-clotting factor, but if you use CPD (citrate-phosphate-
dextrose), which is the anticoagulant more frequently used, the WBU can be stored for a short period of time,
which is 21 days. This is due to the fact that the storage causes a progressive reduction of RBCs levels of
2.3-BPG and ATP, an increase in intracellular K+, a pH decrease and a 80-90% reduction of clotting factors
V, VIII and XI.
When one has to decide which unit to use for the transfusion in a patient with the DIC, since you have the
need to add clotting factors, obviously, you do not use WBU, because the activity of the clotting factors
contained in the WBUs is very low. Instead you have to use fresh frozen plasma, since it contains the clotting
factors, which are perfectly functioning. After RBCs infusion, 2,3-BPG and ATP levels return in the normal
range within 24 hours.
Indications for whole blood units:
1. whole blood units can be used to correct blood volume and to improve the ability of the blood to carry O2,
the capacity of blood to carry O2. Whole blood units are usually given after a trauma or in case of
surgeries involving extensive blood losses, that occur when within 3 hours more than 50% of the
circulating mass is lost. In these cases, whole blood unit has two advantages: first it improves the colloid
osmotic pressure and you also give to your patient some coagulation factor that are not given when you
infuse into your patient crystalloid solutions; second, you do not expose the recipient to RBCs and
plasma from different donors, but you have a component that is given from one single donor, and thus a
problem of sensitization does not occur (sensitization occurs instead when you give components from
different donors)
2. whole blood units can be used for replacement of the newborn blood in case of the hemolytic disorder of
the newborn (HDN). In this case RBCs collected no more than 7 days before and fresh frozen plasma
are usually preferred. In this case you also have to consider the AB0 and Rh(D) antigens: the donor AB0
blood group must be identical to that of the recipient, whereas for the Rh(D) group the identity is not so
strict. However RhD- blood can be given to both positive and negative recipient, while the positive one
can be given only to positive patients. When you use whole blood unit for the HDN the group of the blood
unit must be the same of that of the newborn and the blood unit must not contain the antigens targeted
by maternal antibodies, in order to avoid any hemolytic reaction, so usually blood units compatible with
both the mother and the newborn are used, and usually are used negative units compatible for both.

Side effects of WBU infusion:

- transmission of infectious agents,
- circulation overload (especially in patient with an monoclonal component, especially having a
hemodilution, so if you give a blood unit you can determine a circulation overload as the Immunoglobulin
value is not the right one, because the presence of the monoclonal component determines the arrival of
liquids from tissues in the circulation and therefore there is a hemodilution),
- alloimmunization,
- hemosiderosis,
- hemolytic or allergic febrile reaction,
- GhVD,
- immunomodulation,
- lung edema,
- citrate toxicity, with paresthesia due to a eduction of Calcium,

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- post-transfusion purpura.

Immunohematological tests
Immunohematological tests must be performed every time the blood component contains more than 2 mL of
RBCs, and they can exclude or confirm the presence of clinically relevant recipient's antibodies against
RBCs, which can be:
- IgM or IgG. If active at 37 degrees they could cause intravascular hemolysis, due to complement
activation, or extravascular hemolysis due to the removal of RBCs sensed by IgG.
- Regular or Irregular: regular if constantly present in the serum of subjects who usually lack the
corresponding antigen (antibodies against AB0 antigens), and irregular if only occasionally present,
generated due to the fact that the patient has been previously sensitized toward peculiar blood groups
antigens (antibodies against other blood groups antigens). Thus, regular antibodies are the ones against
AB0 antigens. If an irregular antibody is discovered, compatibility tests are mandatory, and you have to
select the RBCs unit that does not contain that peculiar antigen. If no irregular antibody is discovered and
compatibility tests are omitted, at the time of blood component delivery the AB0 compatibility between the
donor and the recipient must be assessed by determining their blood group or by performing a cross-
match.
- Natural or Isoimmune: natural antibodies are produced despite the absence of any apparent stimulus
(anti-A antibodies in a group B patient for example), and isoimmune are produced after immunological
reaction.
These immunohematological tests include optionally a compatibility test between donor RBCs and recipients
serum.

Red Blood Cells Units

RBCs can be obtained by the whole blood after separating 200-250 mL of plasma: these units do not contain
platelets and granulocytes, and the advantage is that if the RBCs mass is the same, they have an ability to
deliver O2 as full blood, but this capability is provided by a smaller volume, so by infusing this RBCs unit we
can avoid the problems related to the use of WBU (overload), as you give to the patient a blood component
at the right volume.
If plasma elimination is achieved through a close loop device, and the hematocrit is higher than 80%, then
RBCs can be stored at 2 centigrades for up to 35 days; instead, if the plasma has been removed and the
RBCs had been suspending in a peculiar solution, their resistance is increased depending on the solution in
which they are infused.
RBC units can be stored for ten years if, after being cryopreserved in glycerol at 40% or at 20%, they are
frozen in a mechanical freezer at -85°C and stored at -65°C. In this case however glycerol must be removed
after various washes and the unit is centrifuged. They can also be frozen in liquid nitrogen at -196°C and
stored at -120°C respectively.
RBCs units can also be cryopreserved for at least 10 years. Then red cells must be resuspended in a saline
solution and stored at +2°C and infused within 24 hours, in order to avoid any bacterial or microbial
contamination.
Poly-transfused patients should receive units of RBCs stored for short periods of time in order to reduce iron
overload, that is one of the major complications in these patients; in order to avoid an iron overload you can
use iron-chelators that can be administered subcutaneously or intravenously (nowadays it is possible also to
give iron-chelators per mouth, as platelets). In every case, if the RBCs have been collected without using a
sterile close-loop device, they have to be infused within a short time, lower than 24 hours, in order to avoid
microbial contamination.

RBCs units are used in patients with anemias that do not respond to pharmacological treatments:
- anemias occurring in patients submitted to intensive chemotherapy, like those anemia occurring in the
period after chemotherapy in the patient with acute myeloid leukemia, treated with chemotherapy agents;
- in patients with chronic anemias in order to correct an isolated defect in O2 carrying capacity;
- in emergency, in patients who have had an intensive trauma, such as after a big crash: in this case, if the
patient is at risk of dying in 30 minutes and you are obliged to transfuse the patient very soon, you can not
perform the compatibility test, then you have to transfuse only the 0 unit, which is the universal donor
blood. You can give to these patients this RBCs blood component, without performing the compatibility
test.

The principal parameters for the use of RBCs concentrates are:

- patient clinical condition (obviously, if it is a patient with anemia or with a cardiopathy he requires a
transfusion for example)

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- Hb values and hematocrit values of the patients, which are the guidelines to the use of RBCs units. In
surgical patients you perform a RBCs infusion when the Hb value is lower than 8 g/dL. Instead, in
oncological patients you ask for a RBCs unit when the Hb value is under 7 g/dL. In neonates, if the Hb is
lower than 13.0 g/dL on the first day and lower than 10 g/dL on subsequent days in males. RBCs units can
be considered in case of heart or lung diseases causing a reduction in the basal O2 values, when the Hb
values is between 8 and 10 g/dL. For example, in some occasion our cardiologist ask to perform a RBCs
infusion when cardiopathic patient has an Hb value of 8 or 8.5, which is usually not a recommended value
to perform a RBCs unit transfusion in patients. An Hb value between 9 or 9.5 and 10 g/dL, a RBCs
infusion can be performed in peculiar patients with immunoglobinopathies, in order to dilute, for example in
patients heterozygous for sickle cell anemia; in this case the RBCs unit infusion is used in order to dilute
sickle cells. An Hb value above 10 g/dL is usually a condition in which the RBCs infusion is absolutely not
required, unless there is a cardiopathy.
RBCs support in poly-transfused patient: in poly-transfused patients the target Hb value should be the one
that is able to maintain a good quality of life; thus, since the patients have acquired a quality of life strictly
linked to this Hb value, the most convenient interval for RBCs support must be based on patient's clinical
history. At first, in order to establish a correct RBCs support, a correct blood counts evaluation should be
carried out periodically, in order to perform a good transfusion.

How to calculate RBCs volume to be infused:

RBCs volume = weight of the patient (kg) x 70 x desired Hb increase (g/dL) / Hb of the unit (g/dL)
Usually, in a patient of 50 Kg, the use of one unit increases the Hb value of 1 g/dL (from 6.5 g/L to 7.5 g/L, or
even up to 8 g/dL, increasing of 1.5 g/dL). This also occurs in patients whose weight is 70 kg. The very
interesting thing is that the patient feels better within a very short time from the transfusion, within 15 minutes
from the transfusion, and this beneficial effect persist over the next 24 hours if factors able to reduce RBCs
survival are absent. Thus, we can transfuse the patient, and he feels well within 15 minutes, and this
beneficial effect remains for 24 hours, if there is no factor able to reduce the survival of RBCs.

Platelets Units
Also platelets can be collected with apheresis. Donors must have not received aspirin or other drugs that
alter platelets functions in the 72 hours before donation.
Platelets must be stored under appropriate conditions, in order to have the best survival and the best effect
of the platelets unit. They should be maintained at 20-24 degrees, for 5 days, if they have been collected
through close loop system, or for 24 hours if they have been collected with an open device (open-loop
system).
Another requirement is to perform agitation in order to avoid any membrane damage that may be
responsible for reduced post-transfusional survival. In addition, the continuous agitation prevents platelets
sedimentation which causes a reduction in O2 consumption.
A storage period of less than 3 days is very useful, since it prevents both microbial contamination and pH
lowering, both are responsible for microbial contamination and reduction of platelets survival. If the bag in
which they are contained is not sealed, platelets should be infused within 4 hours.
Platelets, like RBCs that are stored in glycerol, can also be stored in cryopreserved DMSO, at a temperature
of -120 centigrade or -65 centigrades, and they can be stored for 1 year.
This type of platelets can be employed for:
- patients who have been heavily alloimmunized,
- candidates for autologous HSCT (in both of them, in order to avoid problems in patients who undergo a
bone marrow transplant, like in the hemopoietic reconstitution)
- in cases of rare phenotypes.

There is no platelets value that surely leads to hemorrhage; some patients who have 5.000 of platelets can
not present an hemorrhage, and platelets infusions are indicated in cases of thrombocytopenia or in cases of
functional platelets defect.
• Usually, you have to perform platelets infusion, when the platelets count is under 5.000/mmc, or under
10.000/mmc if the patient has fever, because fever can reduce very quickly the platelets count and thus, in
order to avoid any hemorrhage, you have to perform the platelets infusion.
• In case of bleeding disorder, for example in a patient with a cerebral hemorrhage, or another an intestinal
hemorrhage, then you have to perform the infusion if the platelets count is under 20.000/mmc.
• In case of functional platelets defects, platelets infusions are required only when the bleeding time is twice
the normal value.

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• In case of fever, clotting defects, infections, marrow aplasia, neoplastic bone marrow invasion and
hypoplasia after chemotherapy, platelets infusions are required when platelets count is comprised between
10.000/mmc and 20.000/mmc.
• In patients who experienced bleeding and in patients having to undergo a surgical procedure, when the
patients count is under 50.000/mmc. A controversial situation occurs when the patients have a DIC and the
platelets count is progressively lowering under 50.000 (this is controversial due to consumption of clotting
factors and platelets in this case, so it is better to give them in the units in order to increase the platelets
count).

Platelets concentrates must be AB0 identical with the recipient; however, platelets concentrates containing
anti A or anti B antibodies incompatible with the recipient erythrocytes can be employed if the plasma has
been completely removed and substituted with other solutions, such as the crystalloid solutions, able to
prevent hemolytic reactions or platelets destruction due to antibodies against the A and B antigens. However
these incompatible platelets units will not be so effective as the compatible ones.
Moreover, when these units contains more than 2 mL of donors RBCs, the unit of donor's blood should be
tested against recipient's serum: this is a situation in which the test must be performed.
RhD+ platelets concentrates should not be given to RhD- recipients, especially if they are females, unless
prophylaxis with anti-D-immunoglobulins has been performed; this prophylaxis uses 10 μg for every platelets
concentrates containing more than 0.5 mL of RBCs, and 50-100 μg for every platelets apheresis unit
depending on its volume, considering that 20 μg of anti-D-immunoglobulins are needed to nullify the effect of
1 mL of Rh(D)+ blood. Thus, you have always to give Rh(D)+ platelets concentrates to Rh(D)+ recipients and
never to Rh(D)- recipients.

Recipient's plasma can contain antibodies either against donor's HLA-A/B/C antigens, or more rarely against
donor platelets specific antigens. In this case, platelets concentrates HLA-A/B compatible, also submitted to
the cross-match with recipients serum, must be infused, and if they are not available, platelets concentrates
can be treated with citric acid, which removes HLA-1 antigens.
Today's procedures allow to obtain a platelet number above 2.5-3x1011: the number of platelets to be
transfused depends on the recipient body surface.

Platelets concentrates dose

Usually, 0.1 Units/kg of platelets from WBU every 48-72 hours for prophylaxis or every 24 hours for
therapeutic infusions. One platelets concentrated unit obtained through apheresis corresponds to 5 platelets
concentrates unit from WBU; thus apheresis provides an increased blood content in the platelets units.
Optimal platelets dose is calculated through this formula:

(Pltsx1011)= desired plts increase x patient’s volemia x 1.5/100

You can check the increase of platelets (effectiveness check) at 1-2 hours and 18-24 hours after the platelets
infusion: since usually 40% of platelets are removed from the circulation shortly after the infusion, the
infusion of 3.2x1011 platelets in a patient whose body surface (BS) is 2 m2 should increase platelets to
20.000/mmc.
A correct count increase is calculated with this formula:
Observed increase (109/l) x BS (m2)/ number of infused plts (x1011)
The expected platelets value is 7500/mmc after 1hr, and 5000/mmc after 18-24hrs; if platelets increase is
under 7500 after 1 hr, we an think that there is a refractoriness to platelets infusion, but this should be
confirmed by a second test.

The side effects of platelets infusion are the same of RBCs infusion:
-Transmission of infective agents
-Circulation overload
-Alloimmunization
-Hemosiderosis
-Allergic, non hemolytic febrile reactions
-GvHD
-Lung oedema
-Post-transfusion purpura
In order to prevent these drawbacks platelets concentrates can be submitted to pre-storage leukodepletion,
and to treatment with psoralen, which prevents infective contamination.

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Another possibility of using platelets is the platelet gel. It is obtained by combining fibrin glue, which has
hemostatic, adhesive and repairing properties, with hyper-concentrated platelets with a sterile device. The
gel is formed by various factors contained within the alpha granules of platelets (these are PDGF-Platelets
Derived Growth Factor, TGF-Tumor Growth Factor beta 1-2, IGF-Insulin Growth Factor), and these cytokines
have important repairing activities. It is for example used for skin ulcers and defect in skeleton regeneration,
and it is applied to the lesion for a 3 days period. The repair of the lesion occurs after few applications and
depends on lesion extension, and on its pathogenesis. It is a very effective tool to repair ocular ulcers, or
also ulcers of the mouth in our patients.

Granulocytes concentrates
Granulocytes concentrates are very rarely used: they should be quickly infused, as their function rapidly
alters. They can be stored at 22 centigrades, without agitation. Once, they were used in patients with
important infections, but there was a LETHAL effect, as these granulocytes arrive rapidly at the area of
infection, and therefore, for example, if the infection is localized in the lung, they arrive very rapidly in the
lungs, and within few hours our patient can develop an ARDS syndrome, an Acute Respiratory Distress
Syndrome. Once, they were used especially in surgical patients. Donors granulocytes should be AB0
compatible with the recipient plasma, and the daily dose that was given was 2x1010 granulocytes/m2 in two
administrations. There was a check of efficacy, by evaluating any clinical improvement. The side effects were
similar to those determined by all the other blood components.

Fresh frozen plasma

Infusion of fresh frozen plasma: if you want to provide your patient with fully functioning clotting factors you
use fresh frozen plasma, since it contains these clotting factors perfectly functioning in contrary to WBUs,
containing clotting factors but which are very ineffective. Usually fresh frozen plasma is obtained from WBU
or through apheresis, and the volume is 450 over 650 mL from one unit. When you submit only the donor to
the collection of plasma, you can obtain a unit whose volume is 400mL.
The plasma must be frozen within 6 hours from collection, in order to avoid any degradation of the clotting
factors (especially factors V, VIII and XI), and it can be stored at a temperature between -18 and -25°C for 3
months. When this plasma is defrosted, it should be quickly infused and maintained at +4°C±2, for no more
than 24 hrs.
80% of the plasma is used in the industry to produce hemo-derivatives, and only 20% is used in the clinics.
Plasma is very effective to provide clotting factors to our patients.

Indications for the use of fresh frozen plasma:

- it should NOT be employed for the correction of volemia or to correct clotting factors deficiencies not
associated with bleeding episodes;
- it can be used in patients who underwent acute bleedings caused by massive transfusion supports, for
example in patients who have had a terrible crash or accidents, when the ratio between the prothrombin
time and normal value, or thromboplastin time and normal value is more than 1.5;
- it can be used in patients with hepatitis, who have a defect in the synthesis of various clotting factors
(highly indicated);
- in bleedings secondary to anticoagulants or Vitamin K deficiencies;
- in DIC;
- coagulopathies;
- thrombotic thrombocytopenic purpura (TTP);
- uremic hemolytic syndrome and hemolysis, elevated liver enzymes, low platelets (HELL);
- cardiac surgery
- patients who have deficiency in peculiar clotting factors for which there are no hemo-derivative available.

The dose is 12-15ml/kg, and you can check the effect of fresh frozen plasma by measuring the level of PT
and PTT values. Fresh frozen plasma should be compatible for the AB0 group with he recipient's RBCs.
Usually for RBCs the 0 is the universal donor, while for plasma the AB is the universal donor, because it has
no anti-A and anti-B. An AB patient can receive plasma only from an AB donor, while an RhD compatibility is
not required: in fact, erythrocytes stroma present in Rh(D) positive plasma is insufficient to cause
alloimmunization.
The side effects are the same as those of other blood components.
In addition, it is better to avoid plasma donations from multiparous women, and poly-transfused patients,
since they may have developed antibodies against peculiar antigens.

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We have said that plasma is produced by the industries, and the unit of AB0 specific FFP (fresh frozen
plasma) frequently produced by the industries from units of 500-1600 mL volume are treated with a solvent/
detergent (S/D) able to neutralise viruses such as HIV, HBV, HCV, CMV, HLTV 1 and 2, as it destroys the
lipid envelope. However, using this unit there is an increased risk of infection from viruses with no lipid
envelope, like the parvovirus B19, or the HAV.
Since it is deficient in factor von Willebrand multimers, this unit is also less effective in DIC, and septic shock.
Another possibility is to eliminate the virus through the methylene blu treatment, which eliminates the DNA
and RNA viruses through a photodynamic reaction; the only drawback is that the activity of some clotting
factors is reduced, so these units are not so effective as the standard ones.
Another possibility is to obtain from fresh frozen plasma cryoprecipitate, when it is defrosted at +4/+6
centigrades; it is then centrifuged, so that the insoluble fraction is concentrated, and thus a volume of 40 mL
is obtained. This volume of the unit contains at least 80-120 U of FVIII, 150-250 mL of fibrinogen and
VonWillebrand factor, factor XIII and fibronectin.
The cryoprecipitate must be infused very quickly, within 6 hours; this indicated in the deficiency of factor VIII,
of fibrinogen, VWF, factor XIII and fibronectin, and the plasma obtained employed for the preparation of this
cryoprecipitate can be stored at 4 or 2 centigrades for 14 days if it has been collected in a close-loop system,
and it is indicated for the treatment of TTP.

Albumin is another component of the plasma, and it is used to increase the oncotic pressure and the plasma
volume. There are 5% solutions that are used as plasma-expander in patients with hypovolemic shock.
Albumin is also used in plasma exchange, on the second day of an extensive burn event involving more than
50% of body surface, if crystalloid solution does not improve hypovolemia, and in neonatal jaundice. There
are also solutions (20-25%) that are used only temporarily in order to increase the oncotic pressure, as in
patients with hepatitis with a very important anasarca or acute hypotension. It can be used also in patients
with cirrhosis, submitted to paracentesis, when the paracentesis volume is above 4 L, and in this case
albumin should be given at a dosage of 4 g/L, in order to avoid death.
Albumin infusion should be employed when albumin concentration (measured by electrophoresis) is under
20g/L, and this concentration is associated with an oncotic pressure of 20 mmHg, and oedemas of the legs
produced by the passage of fluids from the intravascular to the extravascular spaces. Thus in patients with
nephrosis, severe hypovolemia, pulmonary edema
The dosage of albumin can be calculated with this formula:

albumin (g) = desired increase (g/l) x patient’s weight (Kg) X 0.08

The side effects of albumin very rarely occur; it can cause pyrogen reactions, allergic reactions, and
circulation overload.

11 Hematopoietic stem cell transplantation.

Hematopoietic stem cell transplantation (HSCT) is the transplantation of multipotent hematopoietic stem
cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood. It may be autologous (the
patient's own stem cells are used) or allogeneic (the stem cells come from a donor). It is a medical procedure
in the field of hematology, most often performed for patients with certain cancers of the blood or bone
marrow, such as multiple myeloma or leukemia. In these cases, the recipient's immune system is usually
destroyed with radiation or chemotherapy before the transplantation.
Infection and graft-versus-host disease are major complications of allogeneic HSCT.
Hematopoietic stem cell transplantation remains a dangerous procedure with many possible complications; it
is reserved for patients with life-threatening diseases. As the survival of the procedure increases, its use has
expanded beyond cancer, such as autoimmune diseases.
Hematopoietic stem cell transplantation can be distinguished between:

- Autologous: the patient is both the donor and recipient. Autologous HSCT requires the extraction
(apheresis) of haematopoietic stem cells (HSC) from the patient and storage of the harvested cells in a
freezer. The patient is then treated with high-dose chemotherapy with or without radiotherapy with the
intention of eradicating the patient's malignant cell population at the cost of partial or complete bone
marrow ablation (destruction of patient's bone marrow function to grow new blood cells). The patient's own
stored stem cells are then transfused into his/her bloodstream, where they replace destroyed tissue and
resume the patient's normal blood cell production. Autologous transplants have the advantage of lower
risk of infection during the immune-compromised portion of the treatment since the recovery of immune
function is rapid. Also, the incidence of patients experiencing GVHD is very rare due to the donor and

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 recipient being the same individual. These advantages have established autologous HSCT as one of the
standard second-line treatments for such diseases as lymphoma. However, for others cancers such as
acute myeloid leukemia, the reduced mortality of the autogenous relative to allogeneic HSCT may be
outweighed by an increased likelihood of cancer relapse and related mortality, and therefore the allogeneic
treatment may be preferred for those conditions.
Principles for autologous transplantation:
1) the tumor must be chemo-radio-sensitive. With the increase of the dose of anti-neoplastic agents 

such as chemotherapy or radiotherapy, there must be an increase in tumor response, meaning that there 

is a reduction in the volume of the tumor. 

2) the suppression of hematopoiesis is the only limiting factor for carrying out this therapeutic 

procedure. That’s why you have to give the patient stem cells collected previously in order to make the
patient recover from pancytopenia caused by chemo-radio-therapy. 


- Allogenic: the donor is different from the recipient. A particular case is the Syngeneic transplant in which
recipient and donor have the same genetic code (monozygotic twins), and thus are genetically identical.
Allogeneic HSCT involves two people: the (healthy) donor and the (patient) recipient. Allogeneic HSC
donors must have a tissue (HLA) type that matches the recipient. Matching is performed on the basis of
variability at three or more loci of the HLA gene, and a perfect match at these loci is preferred. Even if
there is a good match at these critical alleles, the recipient will require immunosuppressive medications to
mitigate GVHD. Allogeneic transplant donors may be related (usually a closely HLA matched sibling),
syngeneic (a monozygotic or 'identical' twin of the patient - necessarily extremely rare since few patients
have an identical twin, but offering a source of perfectly HLA matched stem cells) or unrelated (donor who
is not related and found to have very close degree of HLA matching). Unrelated donors may be found
through a registry of bone marrow donors such as the National Marrow Donor Program. Allogeneic
transplants are also performed using umbilical cord blood as the source of stem cells. In general, by
transfusing healthy stem cells to the recipient's bloodstream to reform a healthy immune system,
allogeneic HSCTs appear to improve chances for cure or long-term remission once the immediate
transplant-related complications are resolved. A compatible donor is found by doing additional HLA-testing
from the blood of potential donors. The HLA genes fall in two categories (Type I and Type II). In general,
mismatches of the Type-I genes (i.e. HLA-A, HLA-B, or HLA-C) increase the risk of graft rejection. A
mismatch of an HLA Type II gene (i.e. HLA-DR, or HLA-DQB1) increases the risk of graft-versus-host
disease. In addition a genetic mismatch as small as a single DNA base pair is significant.
-
11.1 Autologous HSC transplant

Sources of HSC

To limit the risks of transplanted stem cell rejection or of severe GVHD in allogeneic HSCT, the donor should
preferably have the same human leukocyte antigens (HLA) as the recipient. About 25 to 30 percent of
allogeneic HSCT recipients have an HLA-identical sibling. However, even so-called "perfect matches" may
have mismatched minor alleles that contribute to graft-versus-host disease.
I. Bone marrow: In the case of a bone marrow transplant, the HSC are removed from a large bone of the
donor, typically the pelvis, through a large needle that reaches the center of the bone. If the material from
the posterior iliac crest is insufficient, bone marrow can be obtained from the sternum. The technique is
referred to as a bone marrow harvest and is performed under general anesthesia. Generally 800-1000ml
are obtained. This volume is determined by the weight: no more than 15ml/kg of patient weight can be
obtained. In the BM HSC constitute 1-3% of all nucleated cells.
II. Peripheral blood stem cells: are now the most common source of stem cells for allogeneic HSCT. They
are collected from the blood through a process known as apheresis. The donor's blood is withdrawn
through a sterile needle in one arm and passed through a machine that removes white blood cells. The
red blood cells are returned to the donor. The peripheral stem cell yield is boosted with daily
subcutaneous injections of Granulocyte-colony stimulating factor, serving to mobilize stem cells from the
donor's bone marrow into the peripheral circulation. In normal conditions, CD34+ number in peripheral
blood is about 0,1-0,4 % but this percentage dramatically increases in 2 situations:

1) during the recovery phase following cytotoxic therapy. Chemotherapy causes a reduction of WBC and
this remains low for 10 days from the end of treatment and then increases. In this circumstance also the
count of CD34+ increases.

2) after a 5 days of treatment with the granulocyte-colony stimulating factor (G-CSF). There are patients
who are unresponsive to this treatment and in these patients you can use a specific antibody directed
against an adhesion molecule called CXR4 expressed on the surface of HSC and causing their adhesion
to the bone marrow stroma; so using this anti -CXCR4 monoclonal antibody you prevent HSC adhesion

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 to stroma and so they can move to the periphery.

CD34 positive cells are obtained through a cellular separator through a leukapheretic procedure that
keeps mononucleated cells and returns to the patient the platelets and other cells.
III. Amniotic fluid: It is also possible to extract stem cells from amniotic fluid for both autologous or
heterologous use at the time of childbirth.
IV. Umbilical cord blood: is obtained when a mother donates her infant's umbilical cord and placenta after
birth. Cord blood has a higher concentration of HSC than is normally found in adult blood. However, the
small quantity of blood obtained from an Umbilical Cord (typically about 50 mL) makes it more suitable
for transplantation into small children than into adults. Newer techniques using ex-vivo expansion of cord
blood units or the use of two cord blood units from different donors allow cord blood transplants to be
used in adults. Cord blood can be harvested from the Umbilical Cord of a child being born after
preimplantation genetic diagnosis (PGD) for human leucocyte antigen (HLA) matching (see PGD for HLA
matching) in order to donate to an ill sibling requiring HSCT.

G-CSF

Granulocyte-colony stimulating factor (G-CSF or GCSF), also known as colony-stimulating factor 3 (CSF 3),
is a glycoprotein that stimulates the bone marrow to produce granulocytes and stem cells and release them
into the bloodstream. Functionally, it is a cytokine and hormone, a type of colony-stimulating factor, and is
produced by a number of different tissues.
Generally it is started 48 hours from the end of chemotherapy. The dose used is usually 10 microgram/ kg/
day administered subcutaneously for five consecutive days.

The total CD34 positive cell number is checked every day and when the absolute CD34+ cell number is 20
per micrometers the patient is ready to be submitted to the leukapheresis procedure. Usually we do not
perform the procedure the same day because the leukocytes are still low, we prefer rather to wait for the next
day so that we will have an higher number of CD34+ cells in peripheral blood; this way we can perform 1 and
not 2 collections and we can avoid some problems for the patient.

G-CSF then is stopped when the WBC count is > 3x109/l or when the procedure is ended.

This is usually the standard procedure.

Nowadays we can have special forms of G-CSF which has a longer half life so that we can have fewer
administrations of the drug compared to the standard procedure.

CXCR4

It is a chemokine receptor expressed not only by normal HSC but also by leukemic steam cells which needs
this receptor for their homing in the bone marrow stroma as the stem cells produce a ligand that is stomal
derived factor 1 (SDF-1), the CXCR4 ligand. Thanks to this binding HSC adhere to bone marrow stroma.

After a single dose of the anti-CXCR4 monoclonal ANTIBODY the HSC are quickly released in the circulation
and you can collect them even if the G-CSF administration has not been effective.

It is an alternative when G-CSF does not function.

In order to perform an AHSCT:

1) you have to collect a number of nucleated stem cell that has to be above 2-3x108 of patient 

weight. Problems arises if you collect too many or too less cells. If you collect less HSC the problem is
that the patient may remain dependent of cell transfusion for at least 6 months.
2) number of CD34 positive cell/ kG of patient weight= 3x106. 


There are also some combinations of treatments that you can use to modulate HSC and they are based on
the disease of the patient.

If the patient has a multiple myeloma you can use DCEP:


- Desamethasone 40mg/day for four days orally

- Ciclophosphamide 700mg/mq/day for two consecutive days ev

- Etoposide 100mg/mq/day for two days ev

- cisPlatinum 25mg/mq/day for two days ev

If the patient has non hodgkin lymphoma you can use IPAD:

- Idarubicine 12mg/mq ev the first day

- CisPlatinum 50mg/mq ev (Cont. Inf) day 1 and 2

- Arabinoside-cytosine 2g/mq/12hrs ev (3hrs inf) on the third day
- Desamethasone 20mg/day ev days 1, 2, 3

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If the patient has acute myeloid leukemia:
Ara-C 4g/mq/12hrs in three hrs inf, for a total of six administrations.

Nowadays we do not perform anymore AHSC in patient with acute myeloid leukemia because the disease is
in the bone marrow and you risk to harvest diseased HSC together with normal HSC.
Mini-ICE:

Idarubicine 6mg/mq/day ev for three days

Ara-C 600mg/mq/day (two hrs inf) ev for three days

Etoposide 150mg/mq/day (one hour inf) for three days

Storage of HSCs

Unlike other organs, bone marrow cells can be frozen (cryopreserved) for prolonged periods without
damaging too many cells. This is a necessity with autologous HSC because the cells must be harvested from
the recipient months in advance of the transplant treatment. In the case of allogeneic transplants, fresh HSC
are preferred in order to avoid cell loss that might occur during the freezing and thawing process. Allogeneic
cord blood is stored frozen at a cord blood bank because it is only obtainable at the time of childbirth. To
cryopreserve HSC, a preservative, DMSO, must be added, and the cells must be cooled very slowly in a
controlled-rate freezer to prevent osmotic cellular injury during ice crystal formation. HSC may be stored for
years in a cryofreezer, which typically uses liquid nitrogen.
HSC can be maintained at 4°celsius and quickly infused within 24-40 hrs. Generally, this storage is possible
only when radiotherapy has been the last agent included within the conditioning regiment schedules.

In contrast, if the last agent included cytotoxic drugs a one day wash-out period is required to avoid any
damage on HSCs, because the organism has to eliminate all the catabolites.

Cryopreservation: HSC are collected from the bone marrow then are resuspended within a cryoprotective
mixture formed by equal volumes of patients’ plasma and DMSO (dimethylsulfoxyde which prevents the
formation of crystals inside the cells and prevents any damage within the cell) and stored in liquid nitrogen at
-195° celsius even for 2-3 years. This allows to perform any type of chemotherapy based conditioning
regimen.

An interval between HSC harvest and AHSCT is possible.


Summary of the steps the patient has to follow:


[Link] can collect HSC form bone marrow or peripheral blood. These cells can be stored (cryopreserved
usually) and to avoid neoplastic cells presence you can do the purging procedure that can be done in the
bag or during treatment. Between the collection and the transplant there is a variable interval and the patient
can receive chemotherapy alone, radiotherapy or radio and chemo together. Then when you use high dose
chemotherapy there is usually 1 day wash-out and then the patient receives her autologous hemopoietic
stem cells the following day.

2 days after the transplant the WBC count of the patient is progressively dropping. Median duration of the
pancytopenia period is 12-18 days. This is the period in which the patient is at most risk for infections and
requires infusion of blood components. The lowest WBC count is reached after 9 days and during this period
the patient is at risk of infections and receives RBC and platelets, which should be irradiated in order to avoid
any reaction of the unit against the patient. Usually the patient remains in hospital for at least 20-30 days and
is isolated, in a positive air flow room. At first before the admission to the ward, a central line is positioned in
local anaesthesia and the administration of the conditioning regiments depends on the type of the onco-
ematological disorder. If the patient is affected by multiple myeloma, you can usually use just one drug in the
conditioning regiment. Instead if the patient has a NHL the conditioning regiment is called BEAM.

AHSCT indications:
- Onco-hematological disorders:
1)  non hodgkin / Hodgkin lymphomas with a high relapse risk or in relapse 

2)  Myelomas as first line treatment.
- Solid tumors: are not anymore candidates for hematopoietic steam cell transplantations (breast cancers or
other cancers).

For solid tumors only in pediatric tumors require sometimes an autologous transplant.

Conditioning regimens should be ablative, aimed at introducing an aplastic state which can be overcome
only thanks to the infusions of autologous HSC.

Employs a combination of non cross resistant drugs with distinct organ toxicity.
Which patient you can treat?
age <70 and without any other disorder.

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Mortality and side effects.

Mortality rate is 5% but it depends also on additional disorders es. arrhytmias dcc

The complications are exclusively related to the drugs employed and mortality depends also on fungal and
viral infections.

Advantages and Disadvantaged of Autologous HSCT vs. Allogeneic HSCT.

Advantages.

1) the cells are of the patient so there is no GVHD
2) lack of non take.

Drawbacks:
1)  Graft can be contaminated by neoplastic cells especially if you perform the AHSCT in acute 

leukemia
2)  Lack of the GvL effect mediated in the allogeneic setting by donor’s lymphocytes. Thus the results of
Autologous HSCT will thus be inferior to those of Allogenic HSCT because this reaction is lacking.


Purging is aimed at eliminating potential neoplastic cells (minimal residual disease) contained within the
HSCs harvest. Its clinical effectiveness is proved by the lower incidence of relapse in patient whose
leukapheretic collections lacked clonogenic cells with cytogenetic/molecular markers. If the patient receives
collections of cells lacking clonogenic cells he/she will have a lower risk of relapse; instead if neoplastic cells
are collected inside the leukapheretic cells the risk of relapse is higher. However if you perform purging with
chemotherapy agents, this determines a slow hemopoietic reconstitution.
How can you do purging?
1) in vitro through chemical methods (incubation with cyclophosphamide derivatives) or immunological
methods.
There are several immunological methods, among which:
- incubation with monoclonal antibodies targeting antigens expressed by tumor cells (antiCD20 in follicular
lymphoma). Cellular lysis occurs due to complement activation.
- incubation with immunotoxins: the monoclonal antibody targeting an antigen expressed by neoplastic cells
is conjugated with agents blocking protein synthesis (ricin).
- immunoselection: allows to eliminate cells marked by a specific antigen.
2) in vivo through peculiar immuno-chemotherapy schedules which combine chemotherapy protocols with
specific monoclonal antibodies targeting specific neoplastic cells antigenes. The antibody is used at specific
intervals and the disease is submitted to accurate molecular monitoring.

11.2 Allogoeneic HSC transplant

This is the most exciting procedure of transplant (between autologous and allogenic). This is intended at
completely eradicating all the abnormal cells. The aim is to eliminate the recipient marrow in a onco-
ematological setting. If the patient is affected by thalassemia or whatever but which is not onco-ematological,
you can leave a certain amount of the recipient cells so that the cells from donor and recipient will function
together. If instead a patient has a malignancy you are obliged to destroy his bone marrow and replace his
cells.

Therapeutic procedure is aimed at:
1)  Eradicating, or at least reducing, the number of abnormal defective cells below detectable levels. 

2)  Suppressing allosensitization to prevent rejection of donor cells. 


There is good evidence that donor cells are able to create the space they requires for their engraftment and
expansion.

Donor
At first, you have to search the donor, and you have to remember that all of us have our own HLA typed on
the cells and the genes that define them are responsible for the immunological identity of the individual.
These genes are 6 and are located on chromosome 6. Their expression defines the haplotype. HLA genes
are very close to each other and are inherited as a block (one block from the father, one block from the
mother) like a single gene. Each block is called haplotype.
As the chromosome 6 are 2, there are 2 haplotypes and 12 genes: an haplotype will be inherited from the
mother and the other from the father.
Each gene may have a lot of variants (>100) within the population and be highly polymorphic. Thus, the
potential combination of HLA gene variants are highly specific and different among distinct individuals. 


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As the sons of a couple can inherit 4 haplotype combinations, the probability of having a HLA identical sibling
is 25%.
The problem of the donors led to the development of unrelated donor registries which contain about twenty-
three millions of unrelated donors who have been typed for HLA-A and -B antigens, many also for -C, DRB1
and DQB1 antigens and an increasing number by high resolution typing, i.e. by DNA sequencing .
As HLA genes vary within the general population, the probability finding an unrelated HLA-identical donor is
between 2:20000-1:100000. This has been another reason to create an International Registry, the IBMTR
which includes all National HSCs banks.

Nowadays, the probability of finding a donor within 6 months from the donor search is 70% and this type of
transplant constitutes 50-70% of all allogeneic transplants.


Class 2 MHC genes are encoded near the centromere while class 1 MHC genes are more close to the
telomere.

Class I and class II antigens are codominantly expressed and differ in their structure, tissue distribution and
characteristics in peptide presentation to T cells

The biological function of HLA molecules is to present peptides antigens to T-cells thereby playing a central
role in T-cell-mediated adaptive immunity
Class I MHC molecules.
- Are expressed on most nucleated cells.
- Are encoded by A,B,C genes present in 522 allelic forms.
- Are composed of an α-chain encoded in the MHC and non covalently associated with a beta-2-
microglobulin, encoded on chromosome 15.
- Are recognized by CD8+ cytotoxic lymphocytes.
Class II molecules.
- Are expressed on dendritic cells, B-cells, T lymphocytes, and macrophages (collectively referred to as
antigen presenting cells).
- Are heterodimers composed of two membrane-bound α- and β-chains that are encoded by two genes that
co-localized in the MHC.
- The peptide-binding pocket is formed by the most distal domains of the two chains. Extracellular antigens
are internalized and degraded in an endocytic compartment into peptides of 10-30 amino acids that bind
class II molecules.
- Class II-peptides complex expressed on the membrane are recognized by CD4+ T-helper cells.
Peptide-HLA complexes are the ligands of clonally distributed T-cell receptors (TCRs). TCRs are also able to
recognize allogeneic HLA molecules at high frequency so that 1-10% of peripheral blood lymphocytes of a
donor can respond to a given allo-MHC antigen
Immune response against incompatible HLA antigen may be extreme as in the case of GvHD mediated by
alloreactive cytotoxic t-lymphocytes (CTL) and thus represent a major barrier to HSC

HLA typing can be done through:

- Serology, still the method of choice for low resolution typing at least for HLA-AB, due to its simplicity and
low cost
- DNA typing techniques, based on the nucleotide sequence information of the polymorphic DNA segments
using PCR technology. Mainly PCR-SSP and or reverse PCR-SSOP are used.
There also are different levels of resolution:
- Low resolution also referred to as generic or two digit typing corresponds to the identification of broad
families of alleles that cluster into serotypes (A*02)
- High-resolution or four digit typing allows the discrimination of the individual alleles within each serotypes.

Potentially HSC donors can be:

- HLA genotypically identical donor.
- HLA phenotypically identical donors (parents share a haplotype or the patient is ABDR homozygous)
- Related
- Unrelated
They differ from HLA identical donor because they differ on minor antigens that usually are not checked. Related donors
can have some difference in minor histocompatibility antigens that is higher if you use unrelated donors.
- HLA non-identical donor
- Related
- Unrelated
The difference in minor antigens is in every instances higher when you use unrelated than related donors anyway.

98
 Criteria to establish donor’s fitness:
•Age should be between 18 and 60 years .
•General blood assessment including blood group
and viral screening (CMV, EBV, Hepatitis B and C, 

HIV status), thrombophilic screening and
autoimmunity in order to identify any
rheumatological 

disorder
•ECG
•Chest X-rays
•Abdomen CT to esclude splenomegaly.

Conditions that exclude the patient from possible donation of PBSC:

1) be under treatment with acetyl-salicylic acid or platelets antiaggregants, anticoagulants, ACE- inhibitors,
lithium;

2) have splenomegaly;

3) have personal and family history of coagulation disorders in particular arterial or venous thrombosis;
4) have family history of iritis or episcleritis;

5) be a sickle cells trait carrier;

6) have difficult peripheral venous access, inappropriate for the aphaeresis procedure which is of
considerable effort and duration.

Target dose of HSC in the HSCT

Number of nucleated cells/kg of recipient’s weight = 2x10^8 2.
Number of CD34+ cell/kg of recipient’s weight = 4x10^6

When you collect blood, you obtain different percentages of the different cells.

From Bone marrow:
Median number of nucleated cells: 2x10^8/kg

Median number of CD34+ cells: 2.8x10^6/kg

Median number of T-cells: 2.2x10^7/kg

From Peripheral blood

Median number of nucleated cells: 9x10^8/kg

Median number of CD34+ cells: 7x10^6/kg

Median number of T-cells: 27x10^//kg.

T cells are responsible for the GVHD but also GV-leukemia effect.
The patient and the donor are admitted together and the donor can donate either the peripheral stem cell or
bone marrow. If he donates the peripheral blood cells, he receives double dose of stimulating factor and
every day he undergoes a physical examination to see if there is an increase in spleen size. In the same day
the recipient is admitted and he receives high dose chemotherapy with or without total body radiotherapy.

The duration of the conditioning regiment varies and then there is one day of wash out and on the same day
the donor has donated his cells the recipient gets them through a cell central line infusion.

It is possible to eliminate T cells from the cell suspension by making an immune selection with antibodies
positive for CD3 that are antigens presents on T cells.

The patient receives donor HSC and lymphocytes and there is then a period in which the WBC decrease and
reach the lowest value in 9 days and in this period the patient is at higher risk for infectious episodes but the
risk is higher than those patient submitted to the autologous transplant because our patient has received
immunosuppressant drugs and the conditioning regiment is like that if the donor is an unrelated donor.

Antiglobulins are immunoglobulins directed against lymphocytes which are administered in order to prevent
the immune cells of the recipient to react against donor HSC but also to block the reaction of donor
lymphocytes against the recipient. After having received the HSC the patient receives immunosuppressive
drugs such as cyclosporines, steroids and methotrexate in some peculiar days this further reduces the
GVHD.

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Other HSC sources

• Cord blood
Cord blood banks are grouped in the Netcord an International foundation promoting high quality cord blood
banking. This allows rapid allocation of cord blood units according to histocompatibility and number of
nucleated cells within an average time of 48 hours.
Cord blood features:
- it contains a large number of hematopoietic progenitors, more immature than those contained within the
bone marrow.
- Haematopoietic reconstitution is better after cord blood transplant than after bone marrow transplant.
- Preclinical studies have revealed that cord blood progenitors have an engraftment potential superior than
those contained within the bone marrow.

Advantages:
- cord blood transplant is a curative procedure for patients who lack a related donor and do not find a MUD
(match unrelated donor)
- It is quickly available and is commonly employed in pediatric patients, whereas in adult patients its use is
limited because of the low number of stem cells
- there is no risk for the mother and newborn
- there is a low risk of infections transmission, especially of CMV infections
- there is a reduced risk of GVHD as graft lymphocytes are more immature
- a less stringent HLA compatibility between donor and recipient is required

Disadvantages:
- There is an engraftment probability in hight weight recipients due to the limited number of HSC.
- High risk of infections due to prolonged cytosine.
- The Graft versus Leukemia effect (GvL) is reduced or lacking as graft lymphocytes are immature
- No transfer of adoptive immunity is possible.

The number of collected CB cells is the most relevant parameter and must be >2.5-4x10^7 nucleated cells/
kg of recipient.


The second most relevant factor is HLA compatibility which must be higher than or equal to 4/6 considering
HLA-A and -B by serology techniques or low resolution DNA and DRB1 by high resolution DNA techniques.
More than two mismatches are not considered to be associated with a higher transplant related mortality
(TRM).
If two cord blood units will be infused, the number of total nucleated cell (TNC) is less important than the HLA
match. Quality may vary from unit to unit and bank of origin.

Median CB doses is really small compared to the normal routine blood transfusion bag.
Nucleated cells: 0.3x10^8/kg

CD34+: 0.2x10^6/kg

T-cells: 0.4x10^6/kg

• Familiar HLA haploidentical donor

This became very relevant in the last years and it has overcome the blood cord transplant.

The patient has only 50% probability of sharing one haplotype with a relative (parents, sons, daughters, first
cousins...).

There could be an high risk of GVHD due to HLA disparity. Therefore, in order to perform this type of
transplant, T cells have to be removed from a graft.
It recently has been performed an aploidentical T cell transplant in which T cells were not removed from the
suspension and 3 days after the patient received cyclophosphamide that destroys T cells of the donor.

Indications to allogenic HSCT

1)  Onco-hematological disorders: AML, MDS, ALL, Myeloproliferative disorders, CLL, Non- Hodgkin’s
Lymphomas, Multiple Myeloma, Resistant CML.

2)  Congenital hemopathies: Thalassemia, SCID, Chediak-Higashi disease, CGD 

3)  Acquired non neoplastic hemopathies: Aplastic Anemia unresponsive to conventional 

treatment. 


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Aims of the transplant:
1. Eradication of the disease and total or partial substitution of recipient’s hematopoiesis with donor
haematopoiesis (Complete or partial chimera) 

2. Overcoming the toxicity of the conditioning regimen 

3. Overcoming the double immunological barrier with induction of immunological tolerance. 


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