HEMATOLOGY 1 (LECTURE)

- Blood indices / red cell indices

o Includes MCH, MCV, and MCHC
- Haemas: Blood
o Tells the morphologic feature of the RBC
- Logos: Study
- Study of the cellular elements of the blood
- Three formed elements: RBCs, WBCs, Platelets
- This study includes: BLOOD COMPOSITION
o Formation of cells
o Stages involve in the formation of cells
o Different tests associated to evaluate
cells
o Diseases associated with these cells

HISTORY
• 1657: Describe worms in the blood by
Athanasius Kircher
• 1658: Discovery of erythrocytes by
Swammerdam
• 1674: Human erythrocytes described by Anton ➢ Plasma – composed of 95% water and 5%
Van Leuwenhoek chemical constituents of the blood (proteins,
• 1842: Platelets were described carbohydrates, lipids, electrolytes, etc.)
• 1846: PMN (polymorphonuclear neutrophils) ➢ Buffy coat – contains all blood cells except RBCs
distinguished from other leukocytes by Wharton o Contains WBCs and platelets
Jones o Also contains reticulocytes at the
• 1879: First complete classification of leukocytes bottom portion of the buffy coat
by Ehrlich o May also contain immature and
• 1902: Development of Wright’s stain by James abnormal cells
Homer Wright ➢ Erythrocytes – contains RBCs
• 1920: Hematology was considered a separate
science from clinical pathology GENERAL CHARACTERISTICS OF BLOOD
➢ Blood Volume:
o Adult Male: 5 to 6 liters
o Adult Female: 4 to 5 liters
Terms
o Newborns: ≈350mL (prone to anemia)
TERMS DEFINITION
➢ Viscosity:
a Without/ absence
blast Youngest/ nucleated/ immature o 3.5 to 4.5x Thicker than water because
chromic Color of its components (liquid in nature)
Cyte Cell ➢ Color:
Dys Abnormal o Venous Blood: Dark Red (unoxygenated)
aemia In the blood o Arterial Blood: Bright Red (oxygenated)
Ferro/ ferric/ sidero Iron
➢ In vivo and in vitro appearance:
Hyper Increased
Hypo Decreased o In vivo: blood is liquid in state to easily
Iso Equal circulate within the body
Macro Large o In vitro: from liquid to solid (it forms clot)
Micro Small ➢ pH:
Myelo Marrow o Neutral level: 7.35 to 7.45 pH
Normo Normal
▪ Venous Blood: 7.35 pH
Oid Like
▪ Arterial Blood: 7.45 pH
o Lungs and Kidneys helps in maintaining
Common abbreviations
the pH level of the blood
ABBREVIATIONS MEANING
➢ Specific Gravity:
FBC Full blood count o Whole Blood: 1.045 to 1.066 (contains
Femtoliter cells, and other chemical constituents)
fL (to measure the volume of the cell, which is
directly proportional to their size)
o Plasma: 1.025 to 1.029 (+ fibrinogen)
Hb/ hgb Hemoglobin concentration o Serum: 1.024 to 1.028 (- fibrinogen)
Hct Hematocrit
Mean cell hemoglobin FUNCTIONS OF BLOOD
MCH
(color; measures the weight of hemoglobin) • Respiratory (oxygen delivery capacity of RBCs)
MCV
Mean cell volume • Nutritional (oxygen serves as nutrition)
(size)
• Excretory (removing any unwanted materials)
Mean cell hemoglobin concentration
MCHC (color; measures the actual concentration
• Buffering Action (balance pH)
of hemoglobin) • Maintenance of Constant Body Temperature
CBC Complete blood count • Transportation of hormones and other
pg Picogram endocrine secretion that regulates cell function
• Body Defense Mechanism
 HEMATOLOGY 1 (LECTURE)

Quality assurance – the sum of all activities the

➢ Complete Blood Count – Most basic and laboratory is engaged so we can ensure the results are
common test correct, accurate, and precise.
o Red Blood Cell Count (determine how
much RBCs are present to transport O2) QUALITY ASSESSMENT IN HEMATOLOGY
o White Blood Cell Count (to know if a LABORATORY
person can eliminate certain pathogen ➢ Pre-Analytical – Everything before the testing
or microorganism) (blood collection, labelling, proper use of
o Platelet Count (to know if someone is at anticoagulants, storage, transport, etc.)
risk for bleeding or not) ➢ Analytical – Actual testing of the specimen
o Differential Count (differentiate WBCs to o Quality control (just part of quality
know what type of infection you have) assurance) takes place
o Hemoglobin ➢ Post-Analytical – How you generate, interpret,
To diagnosis diseases,
o Hematocrit especially anemia and analyze the results.
o Blood Indices
➢ Reticulocyte Count – determine if the bone
MAJOR ACTIVITIES
marrow is capable of producing RBCs
➢ Erythrocyte Sedimentation Rate (ESR) – helps to ➢ Preventive – activities done in prior to the
identify if a person has inflammation or not examination of the specimen or sample that is
intended to establish system conducive accuracy
testing
➢ Assessment – activities done during testing to
determine whether test systems are performing
EDTA (Ethylene Diamine Tetraacetic Acid) correctly
- Color: Lavender, purple, or violet ➢ Corrective – done when error is detected to
- Action: Inhibits calcium correct the system
- Forms: o Recalibration of the instrument/
o Versene (disodium salts) machine
o Sequestrene (tripotassium salts)
- Optimum Concentration: 1.5mg/mL of blood
- Use:
o CBC, Blood Smear
o Preserves morphology of the cells
- Excess anticoagulant (EDTA) will affect blood
tests, particularly ESR and hematocrit (decreased
test result values)

Citrate
- Color:
o Blue: 9 parts blood, 1 part anticoagulant
(9:1)
o Black: 4 parts blood, 1 part anticoagulant
(4:1)
- Action: Inhibits Calcium
- Use: Platelet Count, ESR (for black top), and
coagulation studies (for blue top)

Heparin
- Color: Green
- Action: Inhibits thrombin
- Optimum Concentration: 15 to 20 units of QUALITY ASSURANCE PROGRAMS
heparin /mL of blood • Test request procedures
- Use: • Patient identification
o Blood gas analysis, Osmotic fragility test, • Specimen procurement
Platelet retention test • Specimen labeling
o Do not use in blood smear (background • Specimen transportation and processing
will appear bluish) procedures
• Laboratory personnel performance
Oxalate • Laboratory instrumentation, reagents, and
- Color: Gray or Black analytical (examination) test procedures
- Action: Inhibits calcium • Turnaround times
- Optimum Concentration: 1 to 2mg/mL of blood • Accuracy of the final result
- Use: ESR Testing
 HEMATOLOGY 1 (LECTURE)

NON-ANALYTICAL FACTORS IN QUALITY CBC QUALITY CONTROL

ASSESSMENT ➢ Commercial Controls
• Qualified personnel o 3 levels (low, normal, high)
• Laboratory Policies o Values stored in instrument computer
• Laboratory procedure manual o Lewey-Jennings graph generated and
• Test requisitioning stored for each parameter
• Patient identification and specimen ➢ Mode to Mode QC
procurement and labeling o Most automated hematology
• Specimen collection, transport, processing and instruments have a primary and
storage secondary mode of sample aspiration
• Preventive maintenance of equipment ▪ Primary: Automated or Closed
▪ Secondary: Manual or Open
• Appropriate methodology
➢ Delta Checks
• Accuracy in reporting results and documentation
o When the laboratory information
system (LIS) and the instrument are
interfaced (connected) delta checks are
- A system of ensuring accuracy and precision in conducted by the LIS on select
the laboratory parameters
- Involves process of monitoring the o Comparing of current results to previous
characteristics of the analytical processes and results
detects analytical errors during the test
- only a part of quality assurance (a part of FUNCTIONS OF QUALITY CONTROL PROGRAM
analytical) • Providing a guide to the functioning of
- main objective: to ensure the machine is working equipment reagents, and individual technique
well, all reagents is in good condition, and there
• Confirming the accuracy of testing when
are no technical/ medtech errors.
compared with reference values
- done to ensure the results you’ll give to px. are
• Detecting an increase in the frequency of both
accurate and precise.
high and low minimally acceptable values
- To check the stability of the machines and
(dispersion)
reagents
• Detecting any progressive drift or values to one
side of the average value for at least 3 days
BASIC TERMINOLOGIES (trends)
➢ Sensitivity – Quality of being sensitive • Demonstrating an abrupt shift or change from
➢ Specificity – Specific to analyte of interest the established average value for 3 days in a row
➢ Accuracy – Close to the target value (shift)
➢ Precision – Quality of being exact and accurate

KINDS OF QUALITY CONTROL

➢ Intralab – Involves the analyses of control
samples together with the patient specimen
o Internal quality control
o Done on a daily basis
➢ Interlab – For maintaining long-term accuracy of
the analytical methods
o External quality control involves
proficiency testing
o Done periodically

QUALITY CONTROL MATERIAL

• Resembles human sample
• Inexpensive
• No communicable disease
• No matrix effect
• Known analyte concentration
• Convenient packaging

TOOLS OF QUALITY ASSURANCE AND QUALITY

CONTROL
➢ Standard Solution – solutions of known
concentration and value
➢ Blank – use to check the quality of automated
analyzer
 HEMATOLOGY 1 (LECTURE)

Hemoglobin production
➢ Hemoglobin F or Fetal Hemogobin (HbF)
HEMATOPOIESIS o Most predominant hemoglobin starting
- A controlled, continuous, and regulated process from hepatic stage and as long as we are
of blood cell production (RBCs, WBCs, and still inside our mother’s womb
platelets) o The first 6 months after delivery, HbF is
- It deals with the following: still abundant.
o Cell renewal o Globin contents present:
o Proliferation ▪ 2 Alpha globin chains
o Differentiation ▪ 2 Gamma globin chains
o Maturation ➢ Hemoglobin A or Adult hemoglobin (HbA)
- this process results in the formation and o Little amount during pregnancy
development of a mature and functioning cells in o Beyond 6 months of delivery, HbA will be
the blood abundant
- only mature cells are allowed to circulate in our o Beta globin will start to increase its
blood production
- hematopoiesis should never stop o 2 Types of Hemoglobin A:
- In blood cell production, your body will produce ▪ HbA1 (95%)
what you need (no more, no less) • 2 Alpha globin chains
• 2 Beta globin chains
▪ HbA2 (2-3%)
MESOBLASTIC PHASE • 2 Alpha globin chains
• 2 Delta globin chains
- Chief site: Yolk sac
o There is still 1-2% of HbF in adults
- Time: As early as 19th day of gestation
o Cells from the mesoderm will then
migrate to the yolk sac MEDULLARY PHASE (MYELOID PHASE)
o The yolk sac can remain active up to 8 to - Chief site: Bone Marrow
12 weeks of gestation - Time: 5th to 6th month of gestation
- Type of cell produced: erythroblasts (immature - Type of cell produced: hematopoietic cells that
or primitive red blood cells) will give rise to the formation of a functioning
- Hemoglobin – a protein that is made up of 4 units mature RBCs, WBCs, and platelets
of heme that is paired with a globin o HSCs and mesenchymal cells migrate
- Thalassemia – an inherited blood disorder into the core of the bone
caused when the body doesn’t make enough of - Bone marrow – inner part of the bone
hemoglobin - Measurable levels of growth factors
- Occurs in most of the bones, wherein among
Embryonic hemoglobins adults, the principal source production are the
Only present during the mesoblastic stage Flat bones (e.g. Sternum, Ribs, Pelvis)
➢ Gower I Hemoglobin
o Globin contents present:
▪ 2 Epsilon globin chains
▪ 2 Zeta globin chains
➢ Gower II Hemoglobin
o Globin contents present:
▪ 2 Alpha globin chains
▪ 2 Epsilon globin chains
➢ Portland Hemoglobin
o Globin contents present:
▪ 2 Zeta globin chains
▪ 2 Gamma globin chains

HEPATIC PHASE
- Chief site: Liver
- Time: Begins at 5 to 7 gestational weeks
- Type of cell produced:
o Characterized by recognizable clusters LYMPHOID DEVELOPMENT
of developing erythroblasts, Primary lymphoid tissues
granulocytes, and monocytes
• Bone marrow
o Lymphoid cells and Megakaropoiesis
• Thymus
- Other active sites: Spleen, thymus, kidneys,
lymph nodes
Secondary lymphoid tissues
• Lymph nodes
• Spleen
• Liver
 HEMATOLOGY 1 (LECTURE)

THE BONE MARROW

- Contains hematopoietic cells (immature and
mature), stromal cells, and blood vessels
- Main function: production of blood cells
- All blood formed elements ultimately develop
from this undifferentiated and primitive
precursor called hematopoietic stem cell (HSC)
- Red at birth (cellular)
- In young adults around 20 years old, the marrow
is about 60% active
- The cell production usually happens in the flat
bones
- Retrogression – replacement from red to yellow
bone marrow (active to inactive)
- adults: equal area of red and yellow marrow

Bone Marrow Cellularity

➢ Normocellular – normal bone marrow in a
Red Bone Marrow stained smear
- Active o 30-70% hematopoietic cells (including
- Composed of the hematopoietic cells (immature the mature & immature forms)
and mature) and macrophages arranged in ➢ Hypercellular (Hyperplasia) – abnormal increase
extravascular cords in the production of cells
o hematopoietic cells will later on develop o more than 70% hematopoietic cells
and mature in specific niches within the ➢ Hypocellular (Hypoplasia) – abnormal decrease
cords in the production of cells
o once matured, it can now easily go to the o less than 30% hematopoietic cells
blood circulation ➢ Aplastic (Aplasia) – there is very few or a total
- usually at birth, the bone marrow is 100% red absence of hematopoietic cells
- Main Functions:
o Production of Blood Cells Nutrient and Periosteal Arteries
o Iron storage ➢ Nutrient Artery – supplies blood only for the
o B-cell development bone marrow
➢ Periosteal Artery – provides nutrients both for
Yellow Bone Marrow the bone and for the bone marrow
- Inactive (not capable of producing cells)
- There is an accumulation of fats
- Usually in adults
- there are some situation wherein this bone
marrow may become active again
o if there will be an increased demand of
blood cells
 HEMATOLOGY 1 (LECTURE)

HEMATOPOIETIC MICROENVIRONMENT MARROW DIFFERENTIAL

- Nurturing and protecting HSCs Cell Type Range (%)
- Balance in quiescence (inactive/dormant), self- Erythroblasts 18-24
Myeloblasts Type I 0-1
renewal, and differentiation of HSCs
Myeloblasts Type II 0-2
- Stromal cells Promyelocytes 1-4
o responsible for supplying semifluid PMN’s and precursors 53-63
matrix (stroma) Monocytes 0-2
o excretes extracellular matrix that serves Eosinophils and precursors 1-3
as an anchor for the developing Basophils and precursors 0-1
Lymphocytes 8-12
hematopoietic cells
Plasma cells 0-2
o they form an extracellular matrix on the
- It requires a doctor to collect the specimen and
niches, in order to promote cell
- It also requires local anesthesia
adhesion, to regulate HSCs, and for the
- preferably, you need to count 1000 cells, but
maintenance of proteins needed by the
routinely, they only count at least 500 cell
maturing cells
Other Normal Marrow Cells
Stromal Cells
• Macrophages
➢ Endothelial cells – they regulate the flow of
• Mast cells
particles, entering and leaving the
• Osteoblasts
hematopoietic spaces
➢ Adipocytes (Fat cells) – they are essential for the • Osteoclasts
secretion of various steroids that influence
erythropoiesis, maintain bone integrity, and MARROW SPECIMENS
regulate the volume of bone marrow • Collection site:
➢ Macrophages – is important for phagocytosis o Preferred site: Posterior Iliac crest
and the removal of unwanted materials o Occasionally preferred sites:
o is essential for they secrete cytokines, ▪ Anterior Iliac crest
which later on regulate hematopoiesis ▪ Spinal processes
➢ Osteoblasts – are referred to as the bone- ▪ Vertebral bodies
forming cells ▪ Sternum
o comet-like appearance o Among newborns: Upper end of Tibia
o are sometimes mistaken as plasma cells • Trephine Biopsy: using Jamshidi needle with a
➢ Osteoclasts – are referred to as the bone- gauge size of 11
resorbing cells or bone-destroying cells • Aspiration: using University of Illinois Sternal
➢ Reticular cells – they support the vascular Needle
sinuses and hematopoietic cells

Extracellular Matrix of the Marrow

- Blood cell production in hematopoietic tissue
• Fibronectin
other than bone marrow.
• Collagen - it occurs whenever the bone marrow cannot
• Laminin meet the physiologic needs of the body
• Thrombospondin - Produce cells from other sources
• Tenascin - Sites: liver, spleen, lymph nodes, and thymus
• Proteoglycans (secretes chondroitin sulfate,
heparin sulfate, hyaluronate, etc.)
LIVER
- Chief metabolic site of the body
MYELOID TO ERYTHROID RATIO (M:E) - Stores essential minerals and vitamins that are
- Ratio of granulocytes and their precursors to needed in the synthesis of DNA and RNA
nucleated erythroid precursors - Major site of cell production in the second
- It is a comparison or numerical expression of the trimester of fetal development
distribution of myeloid precursor (granulocytes) - Functions:
and erythroid precursor (RBCs) o protein synthesis and degradation
- Normal M:E ratio = 2:1 to 4:1 (average: 3:1) o CHO and lipid metabolism
o there is more myeloid precursor than o drug and toxin clearance
erythroid precursor o iron recycling and storage
o RBCs can survive in the body for 120 days o hemoglobin degradation
o WBCs stay in the blood for 1 to 2 days
- Abnormal M:E ratio
SPLEEN
o Infection (6:1)
o Leukemia (25:1) - Largest lymphoid organ in the body
o Myeloid hyperplasia (20:1) - Essential for lymphopoiesis and phagocytosis
o Myeloid hypoplasia - “graveyard of the body”
o Erythroid hyperplasia - Removes old and damaged blood cells
o Erythroid hypoplasia - when Spleen is enlarged, more cells may then be
stored, removed, or damaged
 HEMATOLOGY 1 (LECTURE)

- Essential for the removal of RBCs

o Culling – phagocytized; all organelles are
degraded
o Pitting – macrophages of the spleen will
remove RBCs as well as its inclusions
- Functions:
o sequesters platelets (stores a portion of
platelets in cases of emergency)
o removal of unwanted/abnormal cells in
blood

3 types of splenic tissue

➢ white pulp – follicles with germinal centers that
- The Pluripotent stem cell, gives rise to two
contains lymphocytes and macrophages
progenitor cells, the Lymphoid & Myeloid stem
➢ red pulp – primarily composed of vascular
cells
sinuses that contains specialized macrophages
o These cells need to be stimulated and
➢ marginal zone – it surrounds the white pulp and
allow them to be committed in the
contains blood vessels, macrophages, and some
production of a specific cell
of the hematopoietic cells
o They need certain stimulants known as
growth factors in order for them to be
LYMPH NODES committed in producing a specific cell
- Bean-shaped structures located along the o Colony-Forming Units (CFUs) are
lymphatic capillaries formed after progenitor cell has been
- Functions: stimulated by a growth factor
o lymphocyte proliferation - Lymphoid stem cell: this is where lymphocytes (B
o initiation of specific immune response cell or T cell) are derived
o filter unwanted substances - Myeloid stem cell: this is where other cells are
- Regions: Cortex and Medulla derived (RBCs, WBCs, platelets)

THYMUS Culture-Derived Colony-Forming Units

- Originates from endodermal and mesenchymal Abbreviation Cell line
tissue Granulocyte, Erythrocyte, Megakaryocyte, and
CFU-GEMM
Monocyte
- Populated initially by primitive lymphoid cells CFU-E Erythrocyte
from the yolk sac and the liver CFU-Meg Megakaryocyte
- T cells are capable of presenting antigens CFU-M Monocyte
- Function: development and maturation of T-cells CFU-GM Granulocyte, Monocyte
- Lobules: Cortex and Medulla CFU-BASO Myeloid to basophil
CFU-EO Myeloid to eosinophil
CFU-G Myeloid to neutrophil
CFU-pre-T T lymphocyte
CFU-pre-B B lymphocyte
- All cells are derived from a pool of stem cells that
are self-renewing
- Pluripotential & multipotential stem cells give Stem Cell Cycle Kinetics
rise to committed stem cells for each cell line - 2.5 billion erythrocytes
Per kilogram of body
- 2.5 billion platelets weight, daily
- 1 billion granulocytes
- 1:1000 nucleated blood cells

Hematopoietic Stem Cells STEM CELLS

• Self-renewing: they differentiate into one or Earliest identifiable stem cells
another type of committed cell • CD34+
• Pluripotent: multiple cells can be produced in • CD38-
one stem cell • HLA-DRlow
• Apoptosis: can reconstitute the hematopoietic • Thy1low
system of a lethally irradiated host • Lin-

TWO CELL THEORIES Clusters of differentiation

➢ Monophyletic Theory • CD38 and HLA-DR: loss of “stemness”
o all blood cells are derived from a single • CD33 and CD38: committed myeloid progenitors
progenitor stem cell called • CD10 and CD38: committed lymphoid
Pluripotential Hematopoietic Stem Cell progenitors
(PHSC) • CD7: T-lymphoid progenitor cells and natural
o It is the most widely accepted theory killer cells
➢ Polyphyletic Theory • CD19: B-lymphoid progenitors
o each blood cells is derived from its
unique stem cell
 HEMATOLOGY 1 (LECTURE)

Cytokines with Positive Influence

➢ Erythropoietin (EPO) – for RBC production
➢ Thrombopoietin (TPO) – for the production of
megakaryocyte, which will produce platelets
➢ Granulocyte CSF (G-CSF)
➢ Granulocyte-macrophage CSF (GM-CSF)
➢ Interleukins (1, 3, 6, 7, 9, 11)
➢ KIT Ligands (KITLG)
➢ Tyrosine kinase 3 (FLT3 LG)

Progenitor Cells GF/ IL/ Cytokines Mature Cells

CFU-MEG TPO, GM-CSF Platelets
CFU-GM CFU-M GM-CSF, M-CSK, IL-3 Monocytes
CFU-GM CFU-G GM-CSF, G-CSF, IL-3 PMN
BFU-E CFU-E EPO, GM-CSF, IL-3 RBC
CFU-Eo GM-CSF, IL-3, IL-5 Eosinophils
CFU-Ba IL-3, IL-4 Basophils

Cytokines with Negative Influence

➢ Transforming Growth Factor-β
➢ Tumor Necrosis Factor-α
➢ Interferons

Cytokines Functions
• prevent hematopoietic precursor cells from
dying
• stimulate stem cells to divide
• stimulate cell production
• inhibit apoptosis
• regulate cell differentiation
 HEMATOLOGY 1 (LECTURE)

- Regulated process for maintaining adequate

numbers of red blood cells in the peripheral
blood
- A process by which erythroid precursor cells
differentiates to become mature
- Production of red blood cells
- Main regulator: EPO (erythropoietin)
o A hormone released by kidneys
whenever there is hypoxia (oxygen level
in tissues is low)
▪ Particularly in renal peritubular
interstitial cells
o Liver also produces little amount of EPO RBC production
o Binds to certain receptors to stimulate
1. It starts with pluripotent hematopoietic stem cell
the progenitor to produce RBCs
2. Through stromal cells and cytokines within the
o It will recruit colony forming units (CFU)
bone marrow, it leads to CFU-GEMM
and burst forming units (BFU), and later
3. From CFU-GEMM, it gives rise to the earliest
on prevents apoptosis of erythroid
identifiable colony of RBCs called BFU-E (Burst
progenitors
Forming Unit – Erythroid)
o It can induce hemoglobin synthesis
o Usually in semi-solid media, these
- Mature red blood cells are not capable of
colonies are large
synthesizing hemoglobin
o CFUs are small in semi-solid media
o BFU-Es contain very few receptors for
Developmental Changes EPO
4. Under the influence of other stimulants such as
IL-3, CSFs, and KIT Ligands, later on, BFU will then
give rise to CFU
o It takes 1 week for the BFU to mature as
CFU
5. CFUs will now have multiple receptors with EPO
o the more EPO receptors, the more EPO
will bind to the receptor
6. Once EPO bind to the receptors, it then stimulate
the maturation, proliferation, and differentiation
of cells.
- As the cell matures, the overall diameter of the o It will then induce the synthesis of
cell becomes smaller hemoglobin
- The Nucleus Cytoplasm ratio of the maturing cell 7. It takes 1 week for the CFU to become
will also decrease pronormoblast (earliest recognizable stage in
- The nuclear chromatin becomes coarser, erythropoiesis under the light microscope)
clumped, and condensed 8. It takes 4 to 7 days for the pronormoblast to
- The nucleus is said to be pyknotic as later they become a mature RBC
mature. The nuclei inside will later on disappears o As the cell mature, they undergo division
- It will then precedes the cessation or end of o In a single pronormoblast, around 8 to
protein synthesis 32 RBCs may be produced
- As the cell mature, the cytoplasmic color
changes from blue → grayish → pinkish
(indicating that hemoglobin is already
synthesized) PRONORMOBLAST
- The eosinophilia of the RBCs correlates with the
accumulation of hemoglobin as they mature

THREE ERYTHROID PRECURSOR NOMENCLATURE SYSTEMS

Normoblastic Rubriblastic Erythroblastic
Pronormoblast Rubriblast Proerythroblast
Basophilic normoblast Prorubricyte Basophilic
erythroblast
Polychromatic Rubricyte Polychromatic
(polychromatophilic) (polychromatophilic)
normoblast erythroblast
- Earliest recognizable stage and first
Orthochromic Metarubricyte Orthochromic
normoblast erythroblast morphologically identifiable RBC precursor
Polychromatic Polychromatic Polychromatic - Other names: rubriblast or proerythroblast
(polychromatophilic) (polychromatophilic) (polychromatophilic)
erythrocyte* erythrocyte* erythrocyte*
- Size: 12 to 20 µm in diameter
Erythrocyte Erythrocyte Erythrocyte - Cytoplasm: basophilic or dark blue because of
* Polychromatic erythrocytes are called reticulocytes the concentration of ribosomes and RNA within
when observed with vital stains. the cell
 HEMATOLOGY 1 (LECTURE)

- Cell division: capable of mitosis ORTHOCHROMIC NORMOBLAST

- Cellular activity:
o The accumulation of the components
necessary for hemoglobin synthesis
o Hemoglobin is not yet synthesized
o The globin production may begin at this
stage
- Length of time: can last about > 24 hours

Globin Production - Other names: metarubricyte or orthochromic

- Immature stages contain substances needed for erythroblast
hemoglobin production - Nucleus:
- Immature cells has nucleus, which contains DNA o Last stage in erythropoiesis to contain
that will later on transcribed to become RNA the nucleus
- RNA will then translated into amino acids, which o Condensed nucleus (pyknotic)
can now produce protein globin o Pyknosis will then later on be followed
- Heme: Made up of two substances known as by karyorrhexis
Protoporphyrin IX and Iron o Eccentric nucleus
- Cellular activity:
BASOPHILIC NORMOBLAST o Later in this stage, the nucleus is ejected
from the cell
o The nucleus moves to the cell membrane
and into a pseudopod-like projection
o Loss of vimentin, a protein responsible
for holding organelles in proper location
o The nucleus containing projections
separate from the cell by having the
membrane sealed and later on pinched
- Other names: prorubricyte or basophilic
off the projection with the nucleus
erythroblast
enveloped by a cell membrane
- Nucleus:
o Once out, the nucleus will then be called
o The chromatin begins to condense
as pyrenocyte, which will later on be
o revealing clumps along the periphery of
removed by the macrophages within the
the nuclear membrane
bone marrow
o coarser chromatin
o Hemoglobin synthesis still occur, but it
o centrally located nucleus
decreases
- Cytoplasm: intensely blue, bluer than
- Length of time: can last about 48 hours
normoblast
- Cell division: capable of mitosis
- Cellular activity: There is detectable hemoglobin RETICULOCYTE
synthesis
o Hemoglobin is not yet visible under the
microscope because of the organelles
within the cell
o Can detect the presence and synthesis of
hemoglobin in certain tests
- Length of time: can last about > 24 hours
- Other name: Polychromatic erythrocyte
POLYCHROMATIC - They are immature cells but they can normally
(POLYCHROMATOPHILIC) NORMOBLAST be present in the blood
- Cytoplasm: Appears to be polychromatic
- Nucleus: No longer contains nucleus
- Cellular activity: They are still capable of
synthesizing hemoglobin because they still have
remnants of RNA
o Last stage capable of synthesizing
hemoglobin
- Other names: rubricyte or polychromatic - Length of time: can last about 1 to 2 days within
(polychromatophilic) erythroblast the bone marrow
- Cytoplasm: has multiple colors o After 1 day of staying in the blood, it
o From bluish color, there will be a then becomes a fully matured RBC
presence of gray and pink color
- Cell division: capable of mitosis (last stage) Reticulocyte count
- Cellular activity: hemoglobin is now visible - If you want to count reticulocyte and
because of the pinkish color demonstrate the remnants of RNA, supravital
o More hemoglobin are synthesized stain is used
- Length of time: can last about 30 hours o Staining the cell while they are living
 HEMATOLOGY 1 (LECTURE)

- Counting reticulocytes is an important indicator - The number of white cells varies from one
of bone marrow function person to another
o Want to know the erythropoietic activity o Can vary according to sex, age, ethnicity,
of the bone marrow and the activity of the patient
o Want to know if there is an effective
erythropoiesis ongoing in the body Myeloid Lineage
o Counting reticulocyte in cases of
hemolysis will identify whether the bone
marrow is responding or not

ERYTHROCYTE

- Give rise to granulocytes and monocytes

- Does not show changes in cell and nuclear
morphologies
- Size: approximately 6 to 8 µm in diameter - GM-CSF stimulates the proliferation and
- Structure: differentiation of neutrophil and macrophages
o 1/3 central pallor area (gives RBC an colonies from CFUs
extra surface area so that they will not
be easily lysed)
- Function: delivers oxygen - Neutrophils share a common progenitor with
- Length of time: can last about 120 days in the monocytes called Granulocyte-Monocyte
peripheral blood Progenitor (GMP)
- Main stimulant: Granulocyte Colony-Stimulating
Factor (G-CSF)

3 Pools of Developing Neutrophils

- Production of leukocytes
➢ Stem Cell Pool – Consists of HSCs that are
- WBCs are colorless
capable of self-renewal and differentiation
- Could then be differentiated into:
o Granulocytes: neutrophils, eosinophils, ➢ Proliferation (Mitotic) Pool – Stages capable of
and basophils mitosis or cell division
o Mononuclear cells: monocytes and o Consists of CMP, GMP, myeloblast,
lymphocytes promyelocyte, and myelocyte
- Lymphocytes will be derived from lymphoid o Myeloblast – earliest recognizable stage
stem cells (lymphopoiesis), then the rest will be o It takes 6 days for the myeloblast to
derived from myeloid stem cells (myelopoiesis) become myelocyte
➢ Maturation (storage) Pool – Consists of cells
undergoing nuclear maturation that form the
marrow reserve and are available for release into
the peripheral blood
o Transition time to maturation pool: 4 to
6 days

MYELOBLAST

- Earliest recognizable stage in granulopoiesis,

particularly in neutrophil development
- Nucleus: Contains large, round/oval nucleus
which occupies almost the entire cell
- Cell division: Capable of mitosis
 HEMATOLOGY 1 (LECTURE)

3 types of myeloblast METAMYELOCYTE

➢ Type I Myeloblast
o Does not contain any visible granules
under light microscope
➢ Type II Myeloblast
o visible granules under light microscope
o contains primary (azurophilic) granules
in the cytoplasm - The nucleus is indented (kidney bean shape or
➢ Type III Myeloblast
peanut shape appearance)
o visible granules under light microscope - Tertiary granules are synthesized (gelatinase
o Rare normal in bone marrows, but they granules)
can be seen in certain types of acute o Formed during metamyelocyte and band
myeloid leukemias stages
o Second to be released
PROMYELOCYTE tertiary granules
• Gelatinase
• Collagenase
• Lysozyme
• Acetyltransferase
• Β2-microglobulin

BAND CELL
- There is a paranuclear halo “hof” which is
usually observed in promyelocytic stages
- Cytoplasm: evenly basophilic
- Cell division: Capable of mitosis
- it contains primary (azurophilic) granules or
non-specific granules
o formed during the promyelocyte stage - Nucleated Marrow Cells: 9% to 32%
o last to be released (exocytosis) - Nucleated Peripheral Blood Cells: 0% to 5%
primary (azurophilic) granules - They can be present in the blood
• Myeloperoxidase - Exclusive only for neutrophils
• Acid β-glycerophosphatase - Smear examination: segmented neutrophil
• Cathepsins (according to CLSI standards)
• Defensins o Band cells should be included within the
• Elastase neutrophil count
• Proteinase-3 - Secretory granules (secretory vesicles) are
• Others formed
o Formed during band and segmented
neutrophil stages
MYELOCYTES
o First to be released (fuse to plasma
membrane)
secretory granules
• CD11b/CD18
• Alkaline phosphatase
• Vesicle-associated membrane-2
• CD10, CD13, CD14, CD16
- Cell division: last stage capable of mitosis • Cytochrome b588
- production of primary granules decreases until it • Complement 1q receptor
totally stops • Complement receptor-1
- Secondary specific granules – slowly spread to
the cell until the cytoplasm turns into lavender SEGMENTED NEUTROPHILS
pink than bluish
o Formed during myelocyte and
metamyelocyte stage
o Third to be released
secondary (specific) granules
• Β2-microglobulin
• Collagenare - Neutrophils are the most abundant cell among
• Gelatinase white blood cells
• Lactoferrin - Rose violet or pinkish colored specific granules
• Neutrophil gelatinase-associated - Nucleus: 3-5 lobes
lipocalin - Length of time: can last about 7 hours in the
• Transcobalamin I peripheral blood
• Others o After 7 hours, they leave the blood and
go to tissues
 HEMATOLOGY 1 (LECTURE)

- Basophil granules
secondary (specific) granules
- Development is similar to that described for
• Histamine
neutrophils
• Platelet-activating factor
- Same stages with added interleukins
• Leukotriene C4
- Will not undergo band cells
• Interleukin-4
- Promyelocytic stage in eosinophils
• Interleukin-13
o Can be identified cytochemically
• Vascular endothelial growth factor A
because of the presence of Charcot-
• Vascular endothelial growth factor B
Leyden crystals on their primary
• Heparan sulfate
granules
- Bilobed, reddish-orange colored granules
- Myelocyte to eosinophil: 3 to 3.5 days
- Length of time: can last about 18 hours in the - Similar to neutrophil development because both
peripheral blood cell types are derived from the GMP
o can survive up to 2 to 5 days in tissues - They share the main progenitor as the
- Secondary Granules: Formed throughout neutrophils (CMP and GMP)
remaining maturation - Main stimulant: MCSF (macrophage colony
o Major basic protein (core) stimulating factor)
o Eosinophil cationic protein (matrix) - Monoblast → Promonocyte → Monocyte
o Eosinophil-derived neurotoxin (matrix) o Monoblast: earliest recognizable stage
o Eosinophil peroxidase (matrix) o Monocyte: largest cell in the blood
o Lysozyme (matrix)
o Catalase (core and matrix) MONOCYTES
o 13-Glucuronidase (core and matrix)
o Cathepsin D (core and matrix)
o Interleukin-2, -4, and -5 (core)
o Interleukin-6 (matrix)
o Granulocyte-macrophage colony-
stimulating factor (core)
o Others
- Small Lysosomal Granules
o Acid phosphatase - Blue-gray cytoplasm with some ground glass
o Arylsulfatase B appearance
o Catalase - The same as macrophages (tissues)
o Cytochrome b588 - Kidney bean shape nucleus that could be folded,
o Elastase showing brain-like convolutions
o Eosinophil cationic protein - Presence of vacuoles
- Lipid Bodies - May produce 16 monocytes in 60 hours
o Cyclooxygenase - Monocyte destination
o 5-Lipoxygenase differentiation into macrophages
o 15-Lipoxygenase • Liver (Kupffer cells)
o Leukotriene C4 synthase • Lungs (alveolar macrophages)
o Eosinophil peroxidase • Brain (microglia)
o Esterase • Skin (Langerhans cells)
- Storage Vesicles • Spleen (splenic macrophages)
o Carry proteins from secondary granules • Intestines (intestinal macrophages)
to be released into the extracellular • Peritoneum (peritoneal macrophages)
medium • Bone (osteoclasts)
• Synovial macrophages (type A cell)
• Kidneys (renal macrophages)
• Reproductive organ macrophages
- Derived from progenitors in the bone marrow
• Lymph nodes (dendritic cells)
and spleen
- Similar to neutrophilic development
- They differentiate under the influence of
different cytokines and interleukins - Lymphoid stem cell gives rise to T-lymphocyte
- Population: and B-lymphocyte lineages
o IL-3: IgE dependent - Thymus: T-cell maturation
o TSLP (thymic stromal lymphopoietin): - Bone marrow: B-cell maturation
non-IgE dependent - Plasma cells – present in marrow, lymphatic
- Mast cells – cells with morphologic and tissue
functional similarities (basophils in tissues) - 3 major groups:
- Basophils are essential during hypersensitivity o T-cells
and allergic reactions ▪ Cell-mediated immunity
- They are usually confused as a lymphocyte ▪ Non-phagocytic
- Nucleus is not clearly seen because of the darkly ▪ T-helper cells or cytotoxic T cells
stained granules
 HEMATOLOGY 1 (LECTURE)

o B-cells
▪ Humoral immunity
- Maturation series of a hematological cell that is
▪ Non-phagocytic
committed for platelet production
▪ Will later on turn into plasma
- Production of megakaryocytes
cells that will help in antibody
- growth factor: thrombopoietin
production
- Platelets are part of the cytoplasm or
o Natural Killer cells
cytoplasmic fragments of megakaryocytes
- B cells and T cells are morphologically the same
- A little portion of megakaryocytes is seen in the
under the microscope
lungs, but majority is located at the bone marrow
o Simply record it as lymphocyte
- Megakaryocyte lineage progenitors:
o BFU-Meg and CFU-Meg
▪ Needed for proliferation
- Antigen independent – the development of ▪ Capable of undergoing mitosis
lymphocytes occurs within the bone marrow and o LD-CFU-Meg
thymus (primary lymphoid organs) ▪ Loses its ability to undergo
o Even without stimulations, b cells and t mitosis
cells can be produced ▪ It retain DNA replication in
- Antigen dependent (secondary lymphoid cytoplasmic maturation
organs) – occurs in other parts of the body such ▪ Undergo a specialized form of
as spleen, lymph nodes, tonsils, and MALT division called as endomitosis
o Antigen stimulation (only the nucleus divides)
- B-cell maturation - Endomitosis – only the nucleus divides
o Pro-B o Contains multiple nuclei inside
o Pre-B o This is the reason why megakaryocyte is
o Immature B cells considered as the largest cell in the bone
▪ Not yet exposed marrow
▪ Antigen naïve B cells (produced o Lacks telophase and cytokinesis
by the bone marrow) - As the cell matures:
▪ Mature in secondary lymphoid o Cell size increases
organs because of antigen o N:C ratio decreases
stimulation o Number of nucleus increases
- T-cell maturation – in this phase, they undergo ▪ It can go as high as 32 nucleus
antigen receptor gene rearrangement so that ▪ The more nucleus a
they can produce T cell receptors megakaryocyte has, the more
o Pro-T platelets will shed out from the
o Pre-T cytoplasm
o Immature T cells - It takes 7 days for the megakaryoblast (MK I) to
▪ Not yet exposed become megakaryocyte (MK III)
▪ Antigen naïve T cells (produced - For every 1000 bone marrow cells, only 1 to 4
by the thymus) megakaryocyte is produced
▪ Mature in secondary lymphoid - For every megakaryocyte, it can produce 2000 to
organs because of antigen 4000 platelets
stimulation o Only 70% circulates in the blood
- NK cells o 30% is sequestered in the spleen
o CD56+, CD16+, CD3-, CD7+ large - Once megakaryocyte stage is achieved, it can
granular lymphocytes now undergo thrombocytopoiesis (platelet
o Mature NK cells are large with other shedding)
resting lymphocyte because of an o Platelets start to come out from the
increase amount of cytoplasm cytoplasm
o Cytoplasm contains azurophilic granules
which are negative for peroxidases PLATELETS
- Smallest cell in the blood
LYMPHOCYTE - Contains granules that are needed in the
formation of clot
- Size: 2 to 4 µm in diameter with 8 to 10
femtoliters in volume
- It contains mitochondria but it does not contain
nucleus
- In vivo: without any injury, platelets are round
- Size: o As platelets circulate in our body, they
o Small: 8 to 10 µm in diameter are inactive
o Medium: 10 to 12 µm in diameter - In vitro: change in shape and assume irregular
o Large: 12 to 16 µm in diameter appearance when active
- Almost same size as the red blood cells o Platelets are activated when there is
- Has a very large nucleus that almost occupy he bleeding or injury
entire cell
- Can still see a little portion of the cytoplasm
 HEMATOLOGY 1 (LECTURE)

• The components interact with one another to

ensure that the RBC will become intact and
- Main function: to carry and deliver oxygen with
deformable
the help of protein hemoglobin
- Aside from hemoglobin, red cells need an intact
membrane, an energy source, and a protector COMPOSITION OF THE ERYTHROCYTE
for them to survive in the circulation Carbohydrates
- They occur only on the external surface of the
red cell
- They occur as glycolipids and glycoproteins
CHARACTERISTICS OF RBC o Glycolipids: anchor the glycocalyx of the
• Size: 6 to 8 µm in diameter RBC
• Volume: ▪ Glycocalyx prevents microbial
o Normocytic: 80 to 100 fL attack and provides protection
o Microcytic: lower than 80 fL from any unnecessary
o Macrocytic: higher than 100 fL mechanical damages
• Lifespan: 120 days o Glycoproteins: supports the
• Shape: biconcave, which then supports the carbohydrates that will later on binds
deformation of the RBC with lipids to support glycocalyx
o Need to be deformable so that they can - These carbohydrates are not used for energy
carry and release oxygen and to get - It will bear copies of carbohydrates base blood
through the tiny vessels group antigens
o 1/3 of the RBC has pale or pallor area - Examples of blood group antigens are:
which is essential to have an extra o ABO or ABH blood group system
surface area to enable the red cells to o Lewis blood group system
stretch and pass through the narrow - Carbohydrates are essential for red cell’s
capillaries and vessels antigenic activities
o The pallor area can also prevent lysis - Not all blood groups are dependent on
o As the red cell age, they lose membrane carbohydrates
surface area, making the cell susceptible
to lysis and unable to pass through tiny Lipids
vessels - Lipid components of the red cell membrane are:
• Internal components: o External: phospholipids, glycolipids,
o Do not have nucleus, ribosome, sphingomyelin, phosphatidylcholine
mitochondria, and other organelles o Internal: phosphatidylethanolamine,
o Under wright’s stain, only the reddish to phosphatidylinositol,
pinkish color cytoplasm is seen phosphatidylserine
(indicating that there is hemoglobin) - Lipids will be essential for the movement and
• Cell membrane components: carbohydrates, permeability of the RBCs
lipids, and proteins - It facilitates entry or exits of substances within
• Metabolic pathways: glycolytic activity or RBCs
pathways - Red cells are not 100% permeable
• Destruction: extravascularly or intravascularly

ERYTHROCYTE MEMBRANE
Functions
• Maintain cell shape and deformability
• Maintain osmotic balance between the plasma
and the cell’s cytoplasm
• Act as a supporting skeletal system for surface
antigens and different receptors within the RBCs
o There are different substances in their
membrane that will then serve as a
receptor or a site for antigens
• Transportation of essential cellular ions and
gases because there is a constant exchange of - The red cell membrane, with regards to the
substances inside and outside the RBC lipids, is bilayer
- It has equal parts of phospholipids and
Composition cholesterol with a bit of fatty acids and glycerides
• 52% Proteins, 40% Lipids, and 8% Carbohydrates - The concentration of lipid is used to maintain the
o Cholesterol and phospholipids would be balance on the deformability, elasticity, and
the main principal structure of the RBC strength of the RBCs
- Cholesterol
• Proteins make up majority of the membrane
o 30% are free unesterified cholesterol
structure by its mass
o 10% glycerides and fatty acids
 HEMATOLOGY 1 (LECTURE)

- Through lipids, there is a polar and non-polar o helps in maintaining the osmotic balance
ends which serves as a seal for water within the red blood cells to ensure that
- Cholesterol at the end has a constant exchange red cells will not be lysed
of cholesterol within the cell membrane and in o Sodium-Potassium ATPase – Controls
the plasma transport of sodium and potassium
o To maintain that balance, it will then be ▪ K is inside while Na is outside
facilitated by an enzyme called LCAT • Intracellularly, the ratio
(Lecithin–cholesterol acyltransferase) of Na and K is 1:12
- As the concentration of cholesterol increases, • Extracellularly, the ratio
the membrane becomes stronger. of Na and K is 25:1
- If cholesterol is too much, the red cell’s elasticity ▪ Red cell membrane is
loses impermeable to sodium,
potassium, and calcium
Proteins ▪ Red cell is permeable to water,
- There are 300 RBC membrane proteins bicarbonates, and chloride
▪ Aquaporin 1 – forms pores or
Integral or transmembrane protein channels wherein the surface
- Serves as the receptor site for different charges create inward
substances that will bind or act on the RBCs waterflow in response to
- serve as transport and adhesion sites for RBC internal osmotic changes
- Serve as signaling receptors • Sodium can enter and
- Any deficiencies or problems with integral potassium can now
proteins will then result to loss of red cells leave the RBCs
deformability, problems with adhesion, or o Calcium ATPase – Calcium pump moves
cellular activity abnormalities calcium out of the cell to plasma against
- Linked or interconnected with cytoskeletal a high concentration gradient
protein to support cell membrane and prevent o To facilitate entry or exit of ions, energy
the loss of lipids (ATP) should be consumed
- In the integral proteins, carbohydrate
determinants are needed for different blood
group systems
o Glycophorin A: MN blood group
o Glycophorin B: Ss blood group
o Glycophorin C & D: Gerbich blood group
- Glycophorins provide RBCs with sialic acid (red and D

cells membrane are negatively charged)

o all red cells are negatively charged so
that they will repel one another (zeta
potential)
- there are other proteins that will also help in
transporting different substances such as ions,
electrolytes, water, etc.
- GPI Anchor (glycosylphosphatidylinositol)
o Around 30 proteins binds to GPI
o Essential to protect the red cells from
any possible lysis caused by complement
o Example:
▪ Decay Accelerating Factor (DAF)
▪ Membrane Inhibitor of Reactive
Lysis (MIRL)
o The main activity of these two
substances is to prevent lysis of RBCs by
complement
o Phosphatidylinositol Glycan anchor
biosynthesis class A (PIGA)
▪ Gene that codes for glycosyl
transferase needed for the
formation of GPI
- Membrane proteins can be extracted using
o Paroxysmal Nocturnal Hemoglobinuria
Sodium Dodecyl Sulfate-Polyacrylamide Gel
(PNH) – red cells destruction caused by
Electrophoresis (SDS-Page) and will then be
complement
separated based on their molecular weight and
- Membrane Enzyme System – transmembrane
net charge
proteins are the ones that facilitate the
- These proteins will be stained using periodic
movement of substrate and cofactors in and out
acid-schiff (PAS) or Coomassie blue to identify
of the cell
which proteins are present or absent
 HEMATOLOGY 1 (LECTURE)

Peripheral or cytoskeletal protein - In this pathway, it provides reduced glutathione

- Provides the shape and deformability for RBCs to prevent oxidative stress
- Do not penetrate the bilayer - To form NADPH for glutathione reduction, G6PD
- Only on the outside of the cell (glucose-6-phosphate dehydrogenase) is
- Cytoskeletal proteins acts as a tough supporting needed
framework for lipid bilayer - When red cells are exposed to oxidative damage
o Essential to make sure that red cells are and stress, the hemoglobin inside will then be
intact and can be deformable denatured.
- Some skeletal proteins: - When hemoglobin is denatured, it will form
o Actin forms short filaments which is then inclusion bodies within the RBCs called as “Heinz
regulated by tropomyosin bodies”
o Adducin and tropomodulin will then o Use supravital stain to recover Heinz
seal the end portion of the actin bodies in the laboratory
o Dematin acts as a stabilizer for the actin o supravital stain can be used to
junctional complex enumerate reticulocyte and
o Ankyrin forms complex to the integral demonstrate Heinz bodies
protein to connect and support lipid
bilayer Rapoport-Luebering Pathway
- Anaerobic pathway
- Generates 2,3-DPG, which regulates hemoglobin
affinity to oxygen
o hemoglobin should let go oxygen if the
tissue needs it (2,3-DPG is present)
o hemoglobin should also conserve oxygen
if the tissue does not need it (2,3-DPG is
not present)
- affinity of oxygen to hemoglobin can also be
affected by carbon dioxide, pH, or temperature
- For the red cells to fully survive and become
efficient in delivering and transporting oxygen, Methemoglobin Reductase Pathway
they will need energy - Also known as cytochrome b5 reductase
- They will source it from carbohydrates, pathway
especially glucose, in the circulation - Methemoglobin: iron is on its oxidized form
o Carbohydrates in the cell membrane is (ferric state)
not intended for energy o In order for oxygen to bind, iron should
o Once the glucose enters the cell with the be on its reduced state (ferrous state)
help of GLUT-1, they will then undergo - Aim: ensure that the iron will be converted into
glycolysis (breakdown of glucose) its ferrous state
▪ Either anaerobic or aerobic
NUTRITIONAL REQUIREMENTS
PATHWAYS - Red cells need:
Embden-Meyerhof Pathway o Proteins
- Main glycolytic pathway o Lipids
- 90% of the glucose broken down will then be in o Carbohydrates
this pathway o iron (majority of iron is within the RBCs,
- Anaerobic metabolism which then breaks down particularly in the hemoglobin)
glucose to form lactic acid to form 2 ATPs o B vitamins
- Serves as the major energy generating pathway ▪ B6 for heme synthesis
of RBCs ▪ B12 (cobalamin) and folic acid
- For every molecule of glucose broken down, 2 (folate) for immature RBCs for
molecules of ATP are formed DNA
- Green leafy vegetables are rich in iron
Hexose Monophosphate Shunt
- Aerobic or oxidative glycolysis occurs through a ERYTHROKINETICS
diversion of glucose catabolism into the HMP, - Dynamics of RBC production and destruction
also known as Pentose Phosphate Pathway - Erythron: collection of all stages of erythrocytes
(PPP) throughout the body
- About 10% of the glucose that enter the red cell o Entirety of erythroid cells in the body
will undergo this pathway o Encompasses all stages of RBCs, mature
- Allows the red cell to be protected from lysis, and immature, in the bone marrow and
oxidation, or oxidative burst caused by different within the peripheral blood circulation,
chemicals or oxidants in the system as well as in other organs
- It extends the lifespan of the RBCs by - RBC Mass: refers to cells in the circulation
maintaining the lipids, proteins, enzymes,
hemoglobin, etc.
 HEMATOLOGY 1 (LECTURE)

ERYTHROPOIETIN - The iron content of hemoglobin will be recycled,

- Basically came from kidneys and it will then be bind to transferrin (transport
- Erythropoietin production is stimulated by protein) and goes to the bone marrow and
hypoxia (oxygen in tissues are low) stored there as ferritin or hemosiderin.
- EPO will stimulate the precursor cells in the bone - The globin component of hemoglobin will also be
marrow to produce RBCs recycled.
- Functions: - The remaining portion of the heme will later on
o Early release of reticulocytes (shift be broken down by different enzymatic activities
reticulocytes) - Heme will be converted to become biliverdin
o Inhibition of apoptosis of red cell through heme oxygenase.
progenitors - The biliverdin will be reduced by biliverdin
o Reduced marrow transit time reductase to become indirect or unconjugated
▪ EPO will increase the rate of bilirubin (B1)
cellular processes - The bilirubin is slow moving
▪ EPO will speed up the - Bilirubin will be carried by albumin, and will then
maturation process and goes to the liver
shortens the cell cycle time - In the liver, it will be conjugated by an enzyme
- Measurement: Chemiluminescence using plasma called uridine diphosphate
and other body fluids glucuronosyltransferase (UDPGT) to form direct
o If a person has renal disease or any bilirubin (B2)
kidney problems, do not use urine as - The direct bilirubin will later on released to the
specimen intestine, some are converted into urobilinogen
- EPO can be used as medication for those patients (with the action of bacterial flora), and some are
who have chronic kidney problems reabsorbed in the circulation going into the
kidneys and is excreted as urobilinogen in urine
- Some urobilinogen will be secreted in the stool
in the form of stercobilinogen
- When RBCs become old, lose their membrane
surface area, has no longer enough proteins, and INTRAVASCULAR HEMOLYSIS
has too much lipids, they become a liability in the
- 10% of the red cells are removed within the
blood
vessels
o They should be removed to protect the
- Other healthy red cells might be affected if most
young and healthy cells still circulating in
of the cells are removed intravascularly
the body
- Complement is involved (mechanical hemolysis)
o young and healthy cells may also be
- Also destroyed by the splenic macrophages
damaged if they are mixed with old and
- Measure haptoglobin and hemopexin to know
damaged RBCs
whether there is an intravascular hemolysis
- 1% of red cells leave the circulation every day
o Low number of haptoglobin and
- As RBC ages, there is a decrease on its enzymes,
hemopexin because they are
a decrease in ATP, decrease in size, and an
phagocytized
increase in density
- increased lysis of cells intravascularly (>10%) can
- All of the RBCs contents will come out if
be observed through the presence of
destroyed
hemoglobin, as well as iron, in the urine
- There could be three mechanisms with
EXTRAVASCULAR HEMOLYSIS haptoglobin, hemopexin, and metheme-albumin
- 90% of the red cells are removed within the complex within the blood vessel
macrophages in the spleen
- Old and damaged red cells will not survive in the
spleen because they have fragile cell membrane
- Young and healthy cells will survive if they pass
through the spleen since they can still deform
- Complement is not involved. Splenic
macrophages will be the one who will destroy
RBCs
 HEMATOLOGY 1 (LECTURE)

haptoglobin mechanism
- In the circulation, red cells are lysed
- Hemoglobin binds to the haptoglobin, forming
hemoglobin-haptoglobin complex
o Binds until it is saturated and depleted
- hemoglobin-haptoglobin complex will later on
be degraded by macrophages liberating out
again the globin and iron
o the remaining portion of the heme will
then be converted into biliverdin →
indirect bilirubin → conjugated to form
direct urobilinogen → urobilinogen in
urine and stercobilinogen in stool

hemopexin mechanism
- In the circulation, red cells are lysed
- Hemoglobin binds to the hemopexin
- Albumin will bind to metheme
- Bilirubin will be carried by albumin, and will then
goes to the liver
- In the liver, it will undergo conjugation to form
direct urobilinogen → urobilinogen in urine and
stercobilinogen in stool

➢ Kidneys – erythropoietin production

➢ Liver – store iron, store protein, Vit. B12 and
Folic acid, and synthesize globin
o Produce erythropoietin (10%)
➢ Bone marrow – site of RBCs formation
➢ Spleen – for RBCs destruction

1. Kidneys respond to a lower than normal oxygen

concentration in the blood by releasing the
hormone erythropoietin
2. Erythropoietin travels to the red bone marrow
and stimulates an increase in the production of
red blood cells (RBCs)
3. The red bone marrow manufactures RBCs from
stem cells that live inside the marrow
4. RBCs squeeze through blood vessel membranes
to enter the circulation
5. The heart and lungs work to supply continuous
movement and oxygenation of RBCs
6. Damaged or old RBCs are destroyed primarily by
the spleen
 HEMATOLOGY 1 (LECTURE)

- 2 important chromosomes for the production of

globins:
- a protein in your red blood cells that carries
o Chromosome 16: responsible for the
oxygen to your body's organs and tissues and
synthesis or generation of alpha and
transports carbon dioxide from your organs and
zeta globins
tissues back to your lungs.
o Chromosome 11: responsible for the
- Brief History:
synthesis or generation of beta, delta,
o in 1862, Felix Seyler identified the
epsilon, and gamma globins
respiratory protein hemoglobin
- Specific sequence of amino acids is known and is
o he discovered the characteristic color
important in the identification of abnormal
spectrum of hemoglobin and proved
hemoglobins involving substitution of specific
that this was the coloring matter of the
amino acids
blood
o hemoglobin makes the blood red Greek Greek No. of amino
Comments
designation name acids
- Each hemoglobin molecule is a mixture of heme
α Alpha 141
and globin, which comes in pair β Beta 146
- 1 heme can carry 1 mole of oxygen Differs from beta
δ Delta 146
o 1 hemoglobin = 4 hemes = 4 moles of O2 chain by 10 a.a.
o 4 heme and 4 globin ε Epsilon 146 Embryonic only
- Heme makes the red cells red Differs from beta
γ Gamma 146
chain by 39 a.a.
o a globular protein which weighs around ζ Zeta 141 Embryonic only
68,000 Daltons
o it almost occupy 1/3 of the red cells - Amino acids are organized and arranged
weight specifically
- each red cell contains millions of hemoglobin - Amino acids have their own specific location
(640 million) - Changes in position of amino acids will create
- hemoglobin also transports waste products, changes on the structure of hemoglobin, making
particularly carbon dioxide (which adds up to the them susceptible to lysis
acidity of our body, and is excreted out of the - Example: abnormal hemoglobin S
body with the help of our lungs) o The 6th amino acid of Beta globin, which
- adult at rest needs around 250mL O2/min is normally glutamic acid, is replaced
- Hemoglobin is slightly soluble in water = 3mL with valine
O2/L of blood o The replacement makes the entire red
o 0.08 mmol of O2 of blood cell susceptible to lysis
o 16 g/dL Hb = 2.41 mmol of O2 - The most abundant hemoglobin among adults is
o 8 g/dL Hb = 1.22 mmol of O2 HbA1
- Only immature cells are capable of producing o 2 alpha and 2 beta globin
hemoglobin o 2 (141) + 2 (146) = 574 amino acids
o Because they contain nucleus o These amino acids should be complete
- denatured hemoglobin due to oxidative stress and arranged specifically to ensure that
will lose its ability to carry and transport oxygen red cells are protected from lysis
- the connection of heme and globin through
chemical bonds forms the basis of hemoglobin
molecule
- hemoglobin synthesis:
o 65% of hemoglobin synthesis occurs
among nucleated red cells
(polychromatophilic normoblast and
orthochromic normoblast)
o The other 35% of hemoglobin synthesis
occurs on reticulocytes

GLOBIN
- makes the hemoglobin a protein Globin Chain Cluster
- globin determines the type of hemoglobin a ➢ Primary – specified sequence of amino acid
person has (embryonic hemoglobin, fetal residues
hemoglobin, adult hemoglobin, etc.) o Together, they form a certain type of
- Varied sequence of amino acids globin chain
o DNA → mRNA → amino acids → o Tells the identity of the globin, as well as
proteins the hemoglobin
- Difference in globin chains designation relates ➢ Secondary – dividing the chain into 8 separate
both to the sequence and number of amino acids helical segments (A to H)
o Makes the hemoglobin structure flexible
so that it can accept and release out
oxygen
 HEMATOLOGY 1 (LECTURE)

o When the individual heme groups Protoporphyrin IX (PIX)

unload oxygen in the tissues, the beta - Nitrogenous substance synthesized partly in
chains are pulled apart mitochondria and partly in the cytoplasm of a
▪ So that 2,3 DPG can now enter nucleated red cell
the hemoglobin molecule
▪ 2,3 DPG prevent the affinity of Iron
oxygen with hemoglobin
- ferrous form is required to convert
▪ Occurs during hypoxia so that
protoporphyrin to become heme
oxygen is delivered to tissues
- location: red cells, liver, bone marrow, and
o When the oxygen enters the red cell, the
macrophages
beta chains will move closer to one
- sources: diet (green leafy vegetables) and
another
destroyed or lysed RBCs
▪ Tissues are oxygenated
- transport: transferrin and ferroportin
▪ 2,3 DPG cannot enter the
- immature red cell with ferritin: sideroblast
hemoglobin molecule
- mature red cell: siderocyte
▪ More oxygen can bind to
- stain with: Prussian blue (Perl’s stain)
hemoglobin
- Most abundant transition metal in the body
- Majority of iron are present inside the red cells
- Blood has metallic taste because of iron
- Iron uptake is precisely controlled to maintain
iron balance
- Iron may be stored as ferritin in the enterocytes,
or exported into the circulation by an iron-
transport protein called ferroportin
o Ferroportin is important in the last step
in the intestinal iron absorption
o it will allow macrophages to recycle iron
HEME by moving it out from the macrophages
Heme Production in liver, spleen, and bone marrow
- Heme synthesis occurs in most body cells except
for mature RBCs
- Of all the body tissues, red bone marrow and the
liver are considered the most predominant heme
producers, particularly referring to porphyrin
- Requires the formation of protoporphyrin IX and
the availability of iron
o the body produces porphyrin
o iron is sourced out from diet and lysed
red cells
- the synthesis of heme begins in the mitochondria
with the formation of D-ALA from glycine &
succinyl coenzyme A
o D-ALA will later on form into
protoporphyrin IX
o The rate of heme formation depends on
the rate of synthesis of D-ALA
o The more D-ALA is formed, the more
heme is synthesized
- Ferrochelatase – helps the iron to stay on its
ferrous state

ta

uroporphyrinogen
decarboxylase

coproporphyrinogen
oxidase
IX + iron - hepcidin – a liver produced hormone which is
Protophyrinogen (ferrous) the master regulatory hormone of systemic iron
oxidase
metabolism
o it controls the plasma iron concentration
by inhibiting the activity of ferroportin
 HEMATOLOGY 1 (LECTURE)

o if there is a deficiency in hepcidin,

ferroportin will continuously export out The oxygen
iron into the circulation (iron overload) dissociation curve of
o too much hepdicin will result to loss of normal adult blood.
ferroportin, which will decrease the The oxygen tension
at 50% oxygen
release of iron into the circulation, saturation (P50) is
resulting to anemia approximately
- the liver and the endothelial macrophages 27 Torr.
functions as major iron storage
o 1-2 mg of iron is absorbed and loss
everyday
- The total amount of iron in the body can be 4 Factors of Oxygen Affinity with Hemoglobin
regulated by absorption Shift to Shift to
Factor
- Iron loss occurs only when there is bleeding the Left the Right
pH
(Bohr effect) High Low
2,3 DPG affecting the oxygen affinity with pH

- Additional component of hemoglobin CO2

(Haldane effect)
- Facilitate the affinity of oxygen to the Facilitates CO2 binding in the tissues; CO2 Low High
hemoglobin diffuses to red cells so that hemoglobin can
act as a buffer
2,3 DPG Low High
Temperature Low High
• Embryonic (3 months)
o Gower I
o Gower II
o Portland
CARBOXYHEMOGLOBIN
• Adult hemoglobin - Carbon monoxide will bind with oxygen
o HbF (abundant during fetal life) - Cannot bind and carry oxygen
o Hb A1 (most abundant among adults) - Gasoline motor, gas heaters, defective stoves,
o Hb A2 smoking
- Hemoglobin binds more with carbon monoxide
as compared with oxygen
- Carbon monoxide is lighter than oxygen
➢ Oxyhemoglobin – Hemoglobin with ferrous iron - The affinity of the hemoglobin molecule for
and oxygen which is seen in arterial circulation carbon monoxide is 200x greater
➢ Deoxyhemoglobin – hemoglobin with ferrous - People with carboxyhemoglobin poisoning
iron but no oxygen which is seen in venous usually will have cherry red discoloration of the
circulation blood
o Has the presence of 2,3 DPG - It is reversible (can go back to normal)

METHEMOGLOBIN
- Derivative of hemoglobin in which the ferrous
ions is oxidized to ferric state
- Chocolate brown discoloration of blood,
cyanosis, and functional anemia
- It is reversible (can go back to normal)
- Hemoglobin M may be responsible for
OXYHEMOGLOBIN DISSOCIATION CURVE methemoglobinemia at birth
- Hemoglobin has the ability to bind large
quantities of O2, however, hemoglobin must be SULFHEMOGLOBIN
willing to release O2 when needed - During oxidation of hemoglobin, sulfur is
- Shift to the left: the oxygen affinity of the incorporated into the heme rings of hemoglobin,
hemoglobin increases‚ and less oxygen is resulting to a green hemochrome
released at a given oxygen tension. - People with sulfhemoglobinemia has mauve
o Oxygen binds more with hemoglobin lavender colored blood
o High affinity of oxygen with hemoglobin - It cannot transport oxygen, but it may be
o Conserving oxygen because tissues are combined with carbon monoxide called
already oxygenated carboxysulfhemoglobin
o Hemoglobin F causes shift to the left - Mixture of oxidized, partially denatured forms of
▪ High oxygen and Hb affinity hemoglobin that form during oxidative heolysis
increases placental oxygen - Associated with sulfonamides administration,
- Shift to the right: the oxygen affinity of the severe constipation, Clostridium perfringens,
hemoglobin decreases‚ and more oxygen is enterogenous cyanosis, and oxidative stress
released at a given oxygen tension. (formation of Heinz bodies)
o Oxygen binds less with hemoglobin - Cannot be reduced back to hemoglobin
o Low affinity of oxygen with hemoglobin o It is irreversible
o Important when tissues are hypoxic o RBC should be destroyed
 HEMATOLOGY 1 (LECTURE)

Macrocytic
- Non-nucleated, biconcave disc-like cell - > 100 fL
- 6-8 microns in diameter - The cells did not get smaller as they mature
- normocytic and normochromic - Cells did not mature accordingly
- Central area of pallor (1/3): gives the cell an - Most likely associated because of nutritional
extra surface area deficiency within the RBCs
- In order for red cells to survive in the circulation, - Usually associated with liver diseases and
they must megaloblastic states
o Have a healthy cell membrane that is o Megaloblastic states do not only affect
made up of proteins, lipids, and the RBCs. It also affects cells that require
carbohydrates B12 or folic acid for DNA metabolism
o Be deformable so that they can easily let
go of oxygen and enter the vessels COLOR
o Have enough hemoglobin inside - Hemoglobin primarily gives color to the red cells
o Have a balance in between the - Associated with MCH (mean cell hemoglobin)
intracellular and extracellular and MCHC (mean cell hemoglobin
environment of the red cell concentration)
o Have enough nutrients that will supply - Anisochromia – any variation in red cell color
RBCs with energy, particularly glucose affecting its hemoglobin
that enters the red cells through
Embden-Meyerhof Pathway Normochromic
- MCH and MCHC are both normal
Nutritional Requirements o MCH: 27-32 pg
• CHON and amino acids o MCHC: 32-36%
• Vit B12, folic acid, Vit. B6 - Has normal structure of red cell
• Fe++
• Riboflavin, panthotenic acid, nicotinic acid Hypochromic or Hypochromia
- pale red blood cells
- Low hemoglobin
- Has > 1/3 pallor area
SIZE - Microcytic
- 6-8 um in diameter - Associated with IDA, thalassemia, anemia of
- Volume of the cell is directly proportional to their chronic disease, sideroblastic anemia, and
size myelodysplastic anemia
- Associated with MCV (mean cell volume)
- Anisocytosis – any variation in red cell size Hypochromasia Grading
GRADE DESCRIPTION
Normocytic 1+ Central pallor is one half of cell diameter
2+ Central pallor is two thirds of cell diameter
- 80-100 fL
3+ Central pallor is three fourths of cell diameter
- Normal in structure 4+ Thin rim of hemoglobin
- There are certain diseases that still shows normal
RBCs Hyperchromic or Hyperchromia
- Problem is more related to quantity
- Does not have pallor area
- Examples:
- Purely red cell
o aplastic anemia – low bone marrow cell
- Susceptible to hemolysis
production
- Does not mean that the red cell has excess
o hemolytic anemia – red cells that are
hemoglobin
not yet lysed; size is still normal
o bleeding – there is a decrease of red cells
because of bleeding Polychromasia
- variation in hemoglobin content showing a slight
Microcytic blue tinge (wright stain), gray-blue and larger
than normal; residual RNA
- < 80 fL
- normally present in the blood
- Smaller in size
- referring to reticulocytes
- The decrease production or content of
- associated with hemolytic anemia if there is too
hemoglobin within the RBCs
much polychromatic red cells in the blood
- Examples:
o Thalassemia – the body doesn’t make
enough hemoglobin Polychromasia Grading
GRADE % POLYCHROMATIC RED CELLS
o iron deficiency anemia
Slight 1%
o anemia of chronic disease 1+ 3%
o sideroblastic anemia – protoporphyrin 2+ 5%
IX is absent 3+ 10%
4+ > 11%
 HEMATOLOGY 1 (LECTURE)

SHAPE o Can be caused by improper drying of

- Shape: round and discoid in shape blood smears
- Poikilocytosis – any variation in red cell shape o Also called as Burr’s cells in patients with
kidney diseases
Poikilocytes secondary to Developmental
Macrocytosis
➢ Oval Macrocytes
o Elongated and the diameter is wide
o Markedly increased MCV (>125 fL)
o Red cells did not mature properly
o Megaloblastic erythropoiesis
o No central area of pallor
o Megaloblastic anemia
o There could be bipolar arrangement of
hemoglobin in cases of ovalocytosis
o Seen in: ➢ Codocytes
▪ Folic acid deficiency o Also known as target cells or Mexican
▪ Vitamin B12 deficiency hat cell
▪ Pernicious anemia o There is a peripheral rim of hemoglobin
➢ Round Hypochromic Macrocyte surrounded by a clear space in a central
o Seen in: hemoglobinized area
▪ Alcoholism o There is an excess surface membrane to
▪ Hypothyroidism volume ratio
▪ Liver disease o Can be associated with an increase
➢ Blue-tinged Macrocyte cholesterol and phospholipids in the cell
o Seen in: membrane
▪ Neonate response to anemic o There is an imbalance in the distribution
stress of hemoglobin within the RBCs
▪ Response to anemic stress o Target cells are always seen in cases of
thalassemia
Poikilocytes secondary to Membrane Abormalities o These cells are also present in cases of
➢ Acanthocytes liver diseases and iron deficiency anemia
o Also known as thorn cell or spur cell
o A spheroid with irregular spikes
o Can be form because of abnormal lipids
or abnormal ratio of lecithin and
sphingomyelin within the red cell
membrane
o Doesn’t have pallor area (susceptible to
lysis)
o Can be seen in the presence of hemolytic ➢ Spherocytes
anemia, alcoholism, hepatitis, o Hyperchromic red cells
malabsorption state, or in the case of o Ball-shaped RBCs
abetalipoproteinemia (lack of LDL) o Has low surface area to volume ratio
▪ LDL transports cholesterol from among spherocytes, making them
liver to tissues susceptible to hemolysis
o Usually seen in patients with
spherocytosis, hereditary spherocytosis,
hemolytic anemia, autoimmune
hemolytic anemia, or burned patients
o Sometimes, it is seen in a blood bag that
has been stored for quite a long period
of time
o Caused by antigen-antibody reaction
o Spherocytes are formed because of a
deficiency in the RBCs peripheral
proteins, particularly spectrin

➢ Echinocytes
o Also known as sea-urchin cells
o Crenated RBCs
o Spikes are evenly distributed and the
projections are short
o Form when RBCs is exposed to
hypertonic solution or the cells do not
have enough energy
 HEMATOLOGY 1 (LECTURE)

o Sometimes encountered when the

patient is burned
o Keratocytes – schistocyte with horn-like
projections

➢ Stomatocytes
o Also known as mouth cells or bowl-
shaped cells
o Caused by an increased permeability of ➢ Dacryocyte
cell membrane to sodium o Sometimes shortened as dacrocytes
o “crying cell”
o Seen in patients who are alcoholic, have
o Has tear-drop appearance
liver diseases, liver cirrhosis, and in cases
o Formed because of the squeezing and
of Rh null conditions (persons who don’t
fragmentation of RBCs when they pass
posses Rh antigens on their red cells)
the spleen
▪ Results when cells successfully
passed through the spleen
o Present when a person has myeloid
dysplasia (the marrow keeps on
producing abnormal cells)

➢ Elliptocytes
o Elongated with narrow diameter
o Rod or cigar-shaped cells
o The defect is in the peripheral protein of
the RBC, particularly deficient in protein
band 4.1
o Seen in cases of elliptocytosis, iron Poikilocytes secondary to Abnormal Hb Content
deficiency anemia, and thalassemia ➢ Drepanocytes
o Also known as sickle cell
o Has leaf-like or crescent-shaped cell
appearance
o Formed because of the polymerization
of abnormal hemoglobin S

Poikilocytes secondary to Trauma

➢ Schistocytes
o Red cells are split
o Fragmentations are produced, especially
if it is within the blood vessel wall
o Results when RBCs bumped into clots, Other Poikilocytes
damaged vessels, or prosthesis. ➢ Blister Cell
o The presence of these cells is associated o Also known as helmet cells
with a condition called o Also formed because of a trauma in the
microangiopathic hemolytic anemia red cells
(cells are lysed because of the problem o Precursor cells before turning to
within the blood vessel) Schistocytes, they will first be blister cell
 HEMATOLOGY 1 (LECTURE)

➢ Basophilic Stipplings – evidence of precipitation

of ribosomes and RNA due to heavy metal
poisoning within the erythropoietic cells
o D-ALA is affected because of heavy
metal poisoning
▪ D-ALA helps in the formation of
heme
▪ If D-ALA is affected, problem in
heme synthesis may occur
➢ Degmacyte
o Accumulation of ribosomes and RNA
o Also known as bite cell
within the cell
o Looks like a bitten piece of donut
▪ Ribosomes and RNA help in the
o Formed because of Heinz bodies caused
formation of globin
by oxidative stress
o Has a donut appearance with blueberry
▪ G6PD deficiency
on top
o Spleen will try to remove the Heinz
o 2 types of basophilic stipplings
bodies in the red cells, but it will leave a
▪ Fine: precipitation of too much
permanent mark on the cell
RNA that is usually associated
with polychromasia
▪ Coarse: problems with
hemoglobin synthesis,
especially in cases of lead
poisoning

➢ Cabot Ring – because of improper development

of cells, microtubules and mitotic spindle
remains in the cell
o Fragments of nuclear material

• Slight: less than 5%

• Moderate: 5-15%
• Marked: more than 15%

Particular variation:
• Occasional: <1%
• Few: 1-5%
• Frequent: 5-10%
• Many: >10% ➢ Heinz Bodies – formed because of oxidative
stress
o Golf ball-like appearance
Formed because of an abnormal development of RBCs o Defect in the pentose-phosphate
(improper or incomplete development and maturation) pathway or deficiency with G6PD,
➢ Howell-Jolly bodies – small round fragments of resulting to the increase susceptibility of
nucleus, resulting from karyorrhexis (when red cell to oxidative cell
nucleus is excluded) o Supravital stain is used to easily recover
o Staining techniques is performed to Heinz bodies
confirm the remnants of nucleus
o Feulgen stain – most specific stain and
reaction for Howell-jolly bodies
 HEMATOLOGY 1 (LECTURE)

➢ Ringed sideroblast – an immature cells, there is

an accumulation of iron deposits
o sidero means “iron”
o present in cases of lead poisoning and
porphyria (porphyrin IX synthesis is not
enough)
o Prussian blue (Perl’s stain) is used to
confirm the presence of iron

➢ Pappenheimer bodies – basophilic inclusions

that aggregates in clusters near the periphery
➢ Hemoglobin H inclusion – represents the o Formed because of the accumulation of
precipitation of abnormal hemoglobin H ribosomes, mitochondria, and unused
o Form of alpha thalassemia accompanied iron
by hemolysis
o Formed because of abnormalities within
the globin chain
o Supravital stain is used to easily recover
HbH inclusions

➢ Microorganisms
o malaria

➢ Hb CC crystals – presence of hexagonal

hemoglobin with blunt ends and stained dark
o Formed because of the precipitation of
hemoglobin C crystals

➢ Hb SC crystals – combination of abnormal

hemoglobin S and C
 HEMATOLOGY 1 (LECTURE)

MORPHOLOGY GRADE
Polychromatophilia 1+ = 1-5/field
Helmet cell, Dacrocyte 2+ = 6-10/field
Spherocyte, Acanthocyte 3+ = >10/field
Schistocyte
Poikilocytosis 1+ = 3-10/field
Codocyte, Burr cell 2+ = 11-20/field
Stomatocyte, Ovalocyte 3+= >20/field
Elliptocyte
Rouleaux 1+ = 3-4 aggregates
2+ = 5-10 aggregates
3+ = numerous aggregates
Sickle cells POSITIVE only
Basophilic stippling
Pappenheimer bodies
Howell- Jolly

o Babesia
▪ has Maltese cross formation
▪ thick-borne
▪ Sometimes mistaken as malaria

➢ Agglutination
o Can still be reported
o Because of antibodies (can be cold
agglutinins, hemolytic anemia, or
pneumonia)

➢ Rouleaux – stack of coins

o Can be an evidence of increase protein
in the blood
o Too much antibody is present
o Commonly seen in multiple myeloma
o Adding drops of NSS can separate and
disperse the RBCs
 HEMATOLOGY 1 (LECTURE)

o characterized by the presence of hyper-

segmented polymorphonucleocytes
Classifications o increased macrocytic WBCs
➢ Granulocytes ▪ failed to give them necessary
• 3-5 lobulations nutrients and vitamins
Neutrophils
• Rose pink or purplish granules
- in the circulatory system:
• Bilobed nucleus
Eosinophils
• Red-orange granules
o Marginating pool: injury or
• Cannot see the lobulations inflammation (migration)
Basophils
• Darkly stained purplish granules o Circulating pool: without inflammation
(migrates randomly)
➢ Agranulocytes
- Neutrophils are believed to survive 2-5 days after
Lymphocytes • Same size as red cells
• Largest cell with kidney bean-
entering the tissues
Monocytes shaped nucleus
• Contains vacuoles

Functions NUCLEAR ABNORMALITIES

• Phagocyte: major function of neutrophils is to - Number of segments
respond rapidly to microbial invasion to kill the - Normal lobes: 3-5 lobes
invaders (phagocytosis)
o Ex. Neutrophils, Eosinophils, Basophils, Pelger-Huet Anomaly
and Monocytes - Hyposegmentation (less than three lobes)
• Immunocyte: they participate in the body’s - an autosomal dominant disorder
immune activities by producing antibodies - There is an incomplete segmentation of the
o Ex. lymphocytes (B-cells or T-cells) nucleus
- Can be associated with taking 3rd generation
Steps in Phagocytosis chemotherapy drugs
1. Recognition - White cells still function normally
2. Migration - Nuclei are round oval or bilobed which are
o Chemotaxis – WBC move to the site of spectacle-like, pince-nez like, dumbbell or
infection peanut with intense nuclear clumping of
o Diapedesis – WBC pass through blood chromatin
vessel - Pelger-Huet anomaly is also described as a blood
3. Adherence or attachment laminopathy associated with the lamin B
o physical contact between the white cell receptor.
and the unwanted materials
o Opsonin – enhancement of attachment
that allows the WBC to further bind to
the microorganism
4. Phagocytosis or ingestion
o formation of a phagosome
5. Degranulation
o fusion with cytoplasmic granules to form
a phagolysosome
o the cell releases granules containing
enzymes
Pseudo-Pelger-Huet Anomaly
6. Oxidative metabolism and bacterial killing
7. Excretion - Acquired anomaly
- Caused by high stress conditions like burns, drug
reactions, MDS, malignancy, myeloproliferative
disorder
BAND CELLS / STAB CELLS
- Incomplete lobulation (no lobulations) Hypersegmentation
- Considered immature, but normally present in - Also called polycytes or macropolycytes
the blood - Megaloblastic anemia
- S or C shaped nucleus - Hypersegmentation is sometimes referred to as
- cell undergoing granulopoiesis, derived from a a myeloid "right shift"
metamyelocyte, and leading to a mature - Accompanied with abnormal RBCs
granulocyte
- Left shift:
o increased release of precursors from the
bone marrow
o can end up with leukemia
- Right shift:
o cells did not mature properly or
completely
 HEMATOLOGY 1 (LECTURE)

CYTOPLASMIC ABNORMALITIES Dohle bodies

- Still look normal - Single or multiple blue inclusions
- Should associate with the clinical conditions of - Aggregates of free ribosomes of rER
the patient - Confused with May-hegglin
- Increase aggregations of ER in cases of infection
Alder-Reilly Granules or toxic states
- Large purple-black coarse cytoplasmic granules
- Accumulation of degraded mucopolysaccharides
- Lack enzymes that will break down
mucopolysaccharides
- Often mistaken as toxic granules

May-Hegglin Anomaly
- Pale-blue inclusion
- Derived from RNA
- With the presence of giant platelets
SYNDROME ENZYME DEFICIENCY SUBSTANCE STORED - rare, autosomal dominant platelet disorder
Hurler’s Alpha-L-iduronidase Mucopolysaccaride 1 characterized by variable thrombocytopenia
Hunter’s Iduronidate sulfatase Mucopolysaccaride 2 (low platelet count)
Form A: Heparin N- - Caused by a mutation in the MYH9 gene,
sulfatase
Sanfilippo’s Mucopolysaccaride 3 resulting to precipitation and aggregation of RNA
Form B: N-acetyl-a-
glucosaminidase
Scheie’s Alpha-L-iduronidase Mucopolysaccaride 5

Auer Rods
- Pink or red rod shaped structures
- Fused primary granules
- Found in myeloid and monocytic series only
- Rods formed because primary granules are fused Toxic Granules
with one another - Large purple to black granules
- Positive in myeloperoxidase test - toxic granulation is clinically significant because
- Leukemia it appears to reflect a poorer prognosis.
o Acute: immature cells are released - Increased accumulations of granules
o Chronic: mature cells are released - Caused by poorly staining of smear
- Formed because it is artifactual
o Even: artifactual
o Uneven: true toxic granulation

Chediak-Higashi granules
- Giant red, blue to grayish round inclusions
- These bodies are formed by aggregation and
fusion of the primary and secondary specific
granules, resulting membrane defects
- Neutropenia and thrombocytopenia are the LE Cell
complications - Lupus Erythematosus cell
- The primary defect is in special granules present - An autoimmune disorder
in skin pigment cells and certain white blood cells - Neutrophil with large purple homogenous round
- Chédiak–Higashi syndrome is caused by inclusion
mutations in the LYST gene - Even staining
- It affects the ability of the WBCs to undergo - hallmark of systemic lupus erythematosus (SLE)
chemotaxis o characterized by increased immune
- Also affects platelets, particularly results in the activity in the body
absence of dense granules o produces a lot of immune complexes
- Classic feature is that patients exhibit albinism o results from phagocytosis of apoptotic
- Associated with mutations on LYST gene, which bodies induced by autoantibodies
results to chaotic environment for lysosomes,
enzymes, or granules
 HEMATOLOGY 1 (LECTURE)

Tart Cell Hairy Cell

- Pseudo LE cell - Lymphocyte with hair-like cytoplasmic
- Monocyte with ingested lymphocyte projections surrounding the nucleus
- Uneven staining - Hairy cell leukemia
- Increased production of B cells
- Confirmatory staining technique: positive in
TRAP (Tartrate Resistant Acid Phosphatase)

Reactive Lymphocyte
- Atypical lymphocyte
o Happens when there is ongoing immune
activity in the body Sezary Cell
- Stimulated
- Round lymph cell with nucleus that is grooved or
- Variant lymphocyte
convoluted because of unwanted increase of
lymphocytes from tissues
- Associated with sezary syndrome and mycosis
fungoides

Downey Cell
➢ Type I: Turk’s irritation cell
o with block of chromatin bodies
➢ Type II: associated with IM (infectious
mononucleosis) cells
o Most popular
o Caused by Epstein-Barr virus (EBV)
which targets B cells Flame Cell
o T cells that reacts to B cells infected by - Plasma cell with red to pink cytoplasm
the virus - There is an increase production of antibodies in
o Round mass of chromatin the body
o Ballerina skirt appearance - Multiple myeloma (increased production of Ig )
➢ Type III: vacuolated
o Swiss chief or moth eaten appearance

Grape Cell
Basket Cell / Smudge Cell
- Also known as berry cell
- Basket cell: 1 to 2 cells
- Plasma cell with vacuoles
- Smudge cell: 2 or more
- Large protein globules
o Associated with Chronic Lymphocytic
- Presence of Russel bodies (individual globes of
Leukemia (CLL)
antibody)
- Degenerated nucleus because of increase
- Multiple myeloma (increased production of Ig )
pressure when preparing smear
or even macroglobulinemia
o To prevent, add a drop of bovine
albumin into the slide before performing
the smear
 HEMATOLOGY 1 (LECTURE)

▪ Appear as double line or triple

line under microscope
CELL COUNTS ▪ Separates the squares
- Manual cell count: o Horizontal and vertical lines:
o Red Blood Cell Count ▪ Serve as a guide in counting cells
o Platelet Count
o White Blood Cell Count
- Materials Needed:
o Hemacytometer
o Thoma Pipet
o Suction device
o Thick coverslip
o Cell Counter
o Diluting fluids
- Whether it is RBC, WBC, or platelet counting,
they will have the same principles and concepts.
- The only difference is the diluting fluids used
- Most routine cell counting procedure in
hematology laboratory is with automation
- Diluting fluids:
RBC WBC
isotonic solution to hypotonic solution to
preserve RBCs lyse RBCs
• NSS • 1-3% acetic acid Calculations
• 3.8% Na citrate • 1% HCl
• Dacies • Turk’s diluting fluid 𝑎𝑣𝑒. 𝑛𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
𝑡𝑜𝑡𝑎𝑙 𝑐𝑜𝑢𝑛𝑡 =
• Hayem’s (contains distilled 𝑎𝑟𝑒𝑎 (𝑚𝑚2 )𝑥 𝑑𝑒𝑝𝑡ℎ (0.1𝑚𝑚)
• Toisson’s water, acetic acid, and
• Bethell’s gentian violet stain) - Depth factor: distance between the cover slip
• Gower’s and the counting chamber (constant)
- Cells counted: depends on the number of the
Hematocytometer cells counted (variable)
o 2 counting chambers should be counted
to check the distribution of the cells
▪ The difference should only
about 10%
▪ Get their average
- Dilution Factor: depends on the dilution of the
specimen
o RBC pipette: 1:200
o WBC pipette: 1:20
- Area Factor: refers to the area of the squares
counted
o Depends on the area of the squares in
- “Heart of manual cell count”
the Neubauer
- Make counting procedure organized and
▪ primary square: 9mm2
systematic
▪ secondary square: 1mm2
- Has 2 counting chambers (one at the top and
▪ RBC tertiary square: 0.04mm2
one at the bottom)
- The results are in cubic millimeter, so convert it
- Every counting chamber contains:
to SI unit using
o Primary square: 1 large square
o RBC: x1012/L
▪ area: 9mm2
▪ multiply the results by 0.000001
▪ contains 9 secondary squares
o WBC and Platelet: x109/L
o Secondary squares: 9 squares
▪ multiply the results by 0.001
▪ 1mm by 1mm
▪ 4 squares located at the corner
are called WBC square N-RBC
o WBC square: - Nucleated red blood cells
▪ Contains 16 tertiary squares - Falsely counted as WBC
o RBC Square: - Not lysed by WBC diluting fluids
▪ Central secondary square - Correct the WBC Count:
▪ Contains 25 tertiary squares o Adults: if there are >5 nRBC/100 cells on
▪ Each tertiary squares contains diff. count
16 quaternary squares o Newborns: if there are >10 nRBC/100
▪ Only 5 tertiary squares are cells on diff. count
counted (4 corners and central 𝑢𝑛𝑐𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 𝑥 100
square) 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑛𝑅𝐵𝐶 𝑝𝑒𝑟 100𝑊𝐵𝐶 + 100
o Dark lines:
 HEMATOLOGY 1 (LECTURE)

Indirect Platelet Count

- Counting platelet in a blood smear
- Procedure:
o Count and observe 10 OIO fields
o Get the average and multiply it by
20,000
- Example:
𝑝𝑙𝑎𝑡𝑒𝑙𝑒𝑡 𝑐𝑜𝑢𝑛𝑡 90
= 𝑥 20,000 = 180,000/𝑚𝑚2
10 𝑂𝐼𝑂 𝑓𝑖𝑒𝑙𝑑𝑠 10
180,000/𝑚𝑚2 𝑥 0.001 = 𝟏𝟖𝟎𝒙𝟏𝟎𝟗 /𝑳

- The result will always be considered as estimate

because the distribution of cells in a blood smear
will vary
o Thick smear: high platelet count
o Thin smear: low platelet count
- If platelet is counted in a hemacytometer, it is
then called as direct platelet counting
Absolute Count
Platelet estimate of Report platelet estimate as
- More accurate than the relative count
0 – 49,000/uL Markedly decreased - Actual number of specific WBC in a liter of blood
50,000 – 99,000/uL Moderately decreased - It represents the number of white blood cells for
100,000 – 149,000/uL Slightly decreased every liter of blood
150,000 – 199,000/uL Low normal - The sum of the values should and must be equal
200,000 – 400,000/uL Normal to the value of the WBC count
401,000 – 599,000/uL Slightly increased - Absolute count = percentage x WBC Count
600,000 – 800,000/uL Moderately increased - Example: WBC = 10 x 109/L
Above 800,000/uL Markedly increased o 50% N x 10 = 5 x 109/L
o 30% L x 10 = 3 x 109/L
Disposable Blood Cell Count Dilution Systems o 20% M x 10 = 2 x 109/L
- Capillary pipette and diluent reservoir systems
o WBC count and platelet count Eosinophil Count
- Available in some laboratories - To assess the adrenocortical function
- 1.98mL of 1% ammonium oxalate - Diluting Fluids:
- Blood: 20uL o Pilot’s
- Automatically set the dilution at 1:100 o Manner’s
o Randolphs’s
Differential Count o Hinkleman
- One hundred WBCs are counted and classified o Tannen’s
through the use of push-down button counters - Diluting Fluids Composition:
(relative count) o Phloxine/eosin/neutral red: stain
o It only represents the 100 cells o Propylene glycol
- Results are reported as percentages o Na2CO3
- Principle: A stained smear is examined to o Heparin
determine the percentage of each type of
leukocyte present and assess the erythrocyte HEMOGLOBIN
and platelet morphology. - Gives red color to the RBCs
- adjust the number of counted white cell to have - Carries oxygen from lungs to tissues, and later on
a better accuracy of manual differential counting takes carbon dioxide going back to the lungs
o especially when WBC count is high or - It is important that the red cells are lysed before
low measuring hemoglobin
- this is the time where we can further determine - Higher in the morning and lower in the evening
or identify the presence of nucleated red cells - For screening of anemia
- Reporting: - Higher in high altitudes (oxygen level is low)
o Reactive Lymphocytes: - Increase in strenuous muscular activity
▪ separate %, as a % of total - Colorimetric methods:
lymphocytes o Acid hematin
▪ Semi-quantitatively: occasional o Alkali hematin
to many ▪ not used among newborn
o Toxic granulation: patients because Hemoglobin F
▪ Present resist alkaline solution
▪ Semi-quantitatively: slight to o Cyanmethemoglobin
marked / 1+ to 3+ ▪ Standard method
- Specimen: ▪ read spectrophotometrically at
o Peripheral blood 540 nm wavelength
o Bone marrow ▪ Reagent: Drabkin
o Body fluid sediments
 HEMATOLOGY 1 (LECTURE)

Potassium hemoglobin is converted to Mean Cell Hemoglobin (MCH)

ferricyanide become methemoglobin
Potassium cyanide methemoglobin is converted to HGB (g/dL)
become cyanmethemoglobin 𝐌𝐂𝐇 = 𝑥 10
RBC count (x 1012 /L)
Non-ionic detergent for lysing RBCs
Dihydrogen allows the solution to be read - Average weight of hemoglobin in a red blood cell
potassium phosphate spectrophotometrically expressed in picogram (pg)
- Other techniques: - Not usually used in classifying anemia
o Specific gravity technique using copper - Normal value: 26-32 pg
sulfate - Example: If the hemoglobin = 16 g/dL and the
o Chemical analysis by measuring iron RBC count = 5 x 1012/L, what is the MCH?
o Gasometric by measuring oxygen 16
𝐌𝐂𝐇 = 𝑥 10 = 𝟑𝟐 𝐩𝐠
5
HEMATOCRIT
Mean Cell Hemoglobin Concentration (MCHC)
- “Packed Cell Volume”
- Volume of packed RBCs that occupies a given HGB (g/dL)
𝐌𝐂𝐇𝐂 = 𝑥 100
volume of whole blood HCT (%)
- Used in diagnosing, monitoring, and assessing
- Average concentration of hemoglobin in each
cases of anemia
individual red blood cell expressed in grams per
- Reported: percentage(%) or L/L
deciliter (g/dL) or percentage
- Two methods:
- Much better in classifying anemia
o Macromethod: wintrobe tube
- Normal value: 32-36 g/dL
▪ centrifuged for 30 minutes at
o RBCs will not contain more than 36 g/dL
2000-3000rpm
of hemoglobin
o Micromethod: capillary tube
o Hyperchromic: red cells do not have
▪ centrifuged for 5 to 10 minutes
pallor area; it does not mean that the red
at 10000-15000rpm
cell has an elevated hemoglobin
concentration
Rule of Three - Causes of elevated hemoglobin concentration:
- Applicable if red cells are normocytic- o Computation is not correct
normochromic o Presence of spherocytes (proceed with
- The value of the hematocrit should be three OFT to check for spherocytes)
times the value of the hemoglobin + or – 3 o Conditions like cold agglutinins (collect
- Example: another blood and warm it before
o HGB = 12 g/dL testing)
o HCT = 36% (0.36 L/L) - Causes of decreased hemoglobin concentration:
o According to the rule of three o Abnormal hemoglobins such as
▪ HGB(12)x 3 = HCT (36) hemoglobin S or hemoglobin C
o An acceptable range for the hematocrit o Specimen is lipemic
would be 33% to 39%. These values - Example: If the hemoglobin = 16 g/dL and the
conform to the rule of three. HCT = 48%, what is the MCHC?
16
RED CELL INDICES 𝐌𝐂𝐇 = 𝑥 100 = 𝟑𝟑. 𝟑 𝐠/𝐝𝐋
48
- Used to define the size and hemoglobin content
of the red blood cell
- Aids in diagnosing and differentiating anemia
- Serves as a quality control check to assess the
parameters in CBC

Mean Cell Volume (MCV)

HCT (%)
𝐌𝐂𝐕 = 𝑥 10 RED CELL DISTRIBUTION WIDTH (RDW)
RBC count (x 1012 /L)
S. D.
- Average volume of red blood cells expressed in 𝐑𝐃𝐖 = 𝑥 100
Mean MCV
femtoliters (fL)
- Volume directly reflects the size of the RBC - It reflects the degree of red cell variation in size
- Volume: - Reflects the degree of anisocytosis
o Normocytic: 80 to 100 fL - Normal Value: 9.0-14.5
o Microcytic: lower than 80 fL - Other results:
o Macrocytic: higher than 100 fL o Microcytic/macrocytic: normal RDW +
- Example: If the HCT = 45% and the RBC count = 5 decreased or increased MCV
x 1012/L, what is the MCV? o Anisocyte with size within the reference
range: increased RDW + Normal MCV
45
𝐌𝐂𝐕 = 𝑥 10 = 𝟗𝟎 𝐟𝐋 o Anisocyte with size below or above the
5
normal range: abnormal MCV and RDW
 HEMATOLOGY 1 (LECTURE)

REPRESENTATIVE CRITICAL VALUES Absolute Reticulocyte Count (ARC)

Hemoglobin less than 5.0 g/dL
Hematocrit less than 15% reticulocytes (%) x RBC Count (x 1012 /L)
𝐀𝐑𝐂 =
Platelet count less than 30,000 per microliter 100
less than 2,500 per microliter and
WBC count or
greater than 30,000 per microliter
reticulocytes (%) x RBC Count
𝐀𝐑𝐂 = 𝑥 1000 = (𝑣𝑎𝑙𝑢𝑒) x 109/L
100
- The actual number of reticulocytes in 1 liter of
RETICULOCYTE COUNT blood
- An indicator of the rate of erythrocyte - It will reflect the true number or true value of
production reticulocytes for every liter of blood
- To assess the erythropoietic activity of the bone - Values between 20 x 109/L and 115 x 109/L are
marrow, especially in cases of hemolysis within the reference interval for most
- Use of supravital stains populations
- The count is expressed as a percentage of total
erythrocytes Example:
- Reference range: If a patient’s reticulocyte count is 2% and the RBC count
o Adult: 0.5-1.5% is 2.20 x 1012/L, the ARC is calculated as follows (note that
o Newborn: 2.0-6.0% the calculated result has to be converted from 1012/L to
109/L):
Relative Reticulocyte Count 2 x (2.20 x 1012 /L)
𝐀𝐑𝐂 = = 𝟒𝟒 𝐱 𝟏𝟎𝟗 𝐋
number of reticulocytes 100
𝐑𝐞𝐭𝐢𝐜𝐮𝐥𝐨𝐜𝐲𝐭𝐞𝐬 (%) = 𝑥 100
1,000 (RBCs counted) or
2 x 2.20
Example: 𝐀𝐑𝐂 = 𝑥 1000 = 𝟒𝟒 𝐱 𝟏𝟎𝟗 𝐋
100
There are 15 reticulocytes counted in 8 months old
infant. Calculate the relative reticulocyte count. Corrected Reticulocyte Count (CRC)
15
𝐑𝐞𝐭𝐢𝐜𝐮𝐥𝐨𝐜𝐲𝐭𝐞𝐬 (%) = 𝑥 100 = 𝟏. 𝟓% Patient ′ s Hematocrit
1,000 𝐂𝐑𝐂 = reticulocytes (%) x
Standard Hematocrit
Interpretation: NORMAL
- In specimens with low hematocrit, percentage of
reticulocytes may be falsely elevated as the
Miller Disc whole blood contains fewer RBCs
- Design to reduce the labor-intensive process of - Usually, reticulocyte is expressed as the
counting reticulocytes percentage of the total number of circulating
- Disc is inserted into the eyepiece of the RBCs.
microscope o But that percentage may then be
incorrect because there may be changes
in the percentage caused by the changes
in the total number of RBCs in the blood
o Because of these changes, we can then
correct the reticulocyte count
- In CRC, we correct the reticulocyte count if the
hematocrit of the patient is affected
- A correction factor is used, with the average
normal hematocrit considered to be 45% or 0.45

- Red cells are counted in the smaller square (B) Example:

- Retics are counted in the larger square (A) If an adult male has a hematocrit of 30% (0.30 L/L) and a
- Minimum of 112 red cells should be counted in reticulocyte count of 3%, the corrected reticulocyte
the small square count would be
o 112 red cells in the small square are 0.30
roughly equivalent to 1008 red cells 𝐂𝐑𝐂 = 3% x = 0.02 x 100 = 𝟐%
0.45
- The area of the smaller is 1/9 of the area of the
larger square (reciprocal: 9) Reticulocyte Production Index (RPI)
no. of reticulocytes in a square 𝐀
(large square) x 100 corrected reticulocytes count in %
𝐑𝐞𝐭𝐢𝐜𝐮𝐥𝐨𝐜𝐲𝐭𝐞𝐬 (%) = 𝐑𝐏𝐈 =
no. of RBCs in square 𝐁 (small square) x 9 maturation time in days
- Measures erythropoietic activity when stress
Example: reticulocytes are present
If 15 reticulocytes are counted in the large square and
- shift or stress reticulocytes – early release of
112 red blood cells are counted in the small square, reticulocytes (< 1 day)
15 x 100 o they are released into the blood
𝐑𝐞𝐭𝐢𝐜𝐮𝐥𝐨𝐜𝐲𝐭𝐞𝐬 (%) = = 𝟏. 𝟓%
112 x 9 prematurely to compensate for anemia
o they will stay for about 2-3 days more
before they totally mature
 HEMATOLOGY 1 (LECTURE)

- if reticulocyte is counted in cases of hemolytic - Used in the laboratory to detect spherocytosis

anemia, and when the bone marrow is (spherocytes: red cells that do not have pallor
responding well, expect that the reticulocyte area)
count is high and elevated - Test on how capable the red cells in resisting lysis
- Results:
Patient’s Hematocrit Correction Factor o Increased OFT (decrease resistance):
Value (%) (Maturation Time, Days) found in autoimmune hemolytic
40-45 1
anemias and hereditary spherocytosis
35-39 1.5
25-34 2 and whenever spherocytes are found
15-24 2.5 ▪ perform Coomb’s testing or
<15 3 Direct Antiglobulin Testing
(DAT) for the confirmation of
Example 5: hemolytic anemia (positive)
If the corrected reticulocyte count is 2.0% and the o Decreased OFT (increase resistance):
patient’s hematocrit is 0.30 L/L, the RPI is occurs following splenectomy and in
corrected reticulocytes count in % 2.0 liver disease, sickle cell anemia, IDA, and
𝐑𝐏𝐈 = = = 𝟏. 𝟎 thalassemia
maturation time in days 2.0
▪ Pallor area is high
Interpretation of Results ▪ Surface area to volume ratio is
• RPI > 3: there is an adequate erythropoietic high
response from the bone marrow response
• RPI < 2: there is an inadequate erythropoietic
response from the bone marrow Hematocrit
- Includes:
Automated Reticulocyte Count o Centrifuge-based device
- Analyzers evaluate reticulocytes using optical o Conductivity Method: Plasma conducts
scatter or fluorescence after the RBCs are electrical current, whereas WBC acts as
treated with fluorescent dyes or nucleic acid insulators
stains to stain residual RNA in the reticulocytes - Factors that may affect hematocrit values:
- The percentage and absolute counts are further o Decreased total protein will result to
provided by automated analyzers falsely decreased hematocrit
o Increased WBC will result to falsely
ERYTHROCYTE SEDIMENTATION RATE (ESR) increased hematocrit
- Sometimes called as “sed rate”
- Non-specific measurement used to detect and Hemoglobin
monitor an inflammatory response to tissue - measured by modified hemoglobinometers or
injury by oximeters integrated with a blood gas
- Distance in millimeters at which the RBCs fall in analyzer
1 hour
- One of the most important factors affecting ESR Cell Counts
is plasma components of patient’s blood - Traditional cell counting methods
o Because it is where the acute phase - employs a buffy coat analysis method
reactants are found (increased proteins - can be used in platelet, granulocyte, and other
during inflammation) cells
o Albumin is not an acute phase reactant
o Proteins
▪ Positively charged heavy
proteins will push down the - automation particularly in CBC
RBCs a lot faster - with our current hematologic analyzer, it uses a
▪ The more red cells are pushed combination of different principles
down, the higher the ESR - Not all parameters are measured. Some
▪ The higher the ESR, the more it parameters are just computed values
correlated with inflammation - Two General Principles:
o Electronic resistance (impedance):
Stages of ESR measures cell’s volume
1. Initial rouleaux formation (10 minutes) o Optical Scatter: focuses on cell’s size and
2. Rapid settling of RBCs (40 minutes) other cell’s characteristics
3. Final sedimentation of RBCs (10 minutes) - Other Principles:
o Fluorescence: combines with light
scattering to analyze specialized
OSMOTIC FRAGILITY TEST (OFT) components of the cells such as RNA
- A measure of the ability of red cells to take up o Electrical conductivity: goes with the
fluid without lysing internal structure of the cell
- Employed to help diagnose different types of o Cytochemistry: uses enzymes and
anemia, in which the physical properties of red employ it with light scattering
cells are altered.
 HEMATOLOGY 1 (LECTURE)

- Types of automated analyzers for CBC: Factors Affecting Volume Measurement

o 3 part differential machine – • Aperture diameter
differentiating WBCs into lymphocytes, o Red cell/platelet
monocytes, and granulocytes o White cell
o 5 part differential machine – o Protein build-up: decreases diameter,
differentiating WBCs into lymphocytes, which results to falsely elevated volume,
monocytes, and neutrophils, making the cell count falsely lower
eosinophils, and basophils • Cell carryover
- 6 part differential machine: additional of • Coincident passage loss (passage of > 1 cell)
nucleated RBCs in differentiating WBCs • Orientation of the cell in the center of the
aperture
Electronic Resistance (Impedance) • Deformability of the RBC
- Widely known as coulter principle • Recirculation of cells back into the sensing zone
- Most commonly used methodology in CBC
- 3 part differential machine Optical or Light Scattering
- Utilizes non-conductive properties of blood cells - A hydrodynamically focused sample stream is
o as blood cell passes through orifice of directed through a quartz flow cell past a focused
aperture it displaces its own volume light source
o RBCs and Platelets counted together, - 5 part differential machine
separated by pulse heights - Uses both laser and non-laser light in measuring
o Hydrodynamic focusing forces cells to different parameters for CBC
pass single file through sensing zone - Can also measure other cell characteristics,
- There will be an increased resistance between particularly its internal components
electrodes, which later on results to electrical - Cells counted as passed through focused beam
pulse of light (LASER)
- The pulses reflect the size and volume of the cell - Sum of diffraction, refraction, and reflection
- Based on the detection and measurement of o Diffraction: bending of light around
changes in the electrical resistance produced by corners
the cell as they pass through the electrical o Refraction: bending of light due to a
current change in speed
- WBCs and hemoglobin are counted together o Reflection: the light rays turn back by
because they both need to lyse RBCs first obstruction
- Results: - It uses low angled light and high angled light in
o Platelets: 2-20 fL measuring cells
▪ MPV: 6.8-10.2 fL o Low angled light: measures the volume
o Red Cells: 36 fL or size of the cell
o Lymphocytes: 35-90 fL o 90° light or side scatter light: measures
o Mononuclear cells: 90-160 fL the internal complexity of the cell
o Granulocytes: 160-450 fL ▪ Structures such as nucleus and
cytoplasmic granularity
- Multi angle polarized scatter separation
(M.A.P.S.S.)
o Forward-angle light scatter (0°)
o Forward low-angle light scatter (2° to 3°)
o Forward high-angle (5° to 15°)
o Orthogonal light scatter (90°)
 HEMATOLOGY 1 (LECTURE)

Radiofrequency UNIT OF COMMON METHOD OF

PARAMETER
REPORTING DETERMINATION
- Used in conjunction with electrical impedance Impedance count x calibration
- Cell interior density is proportional to pulse WBC x 109/L
(cal) factor
height or change in the RF signal Impedance count x calibration
RBC x 1012/L
- Can help in analyzing the internal complexity of (cal) factor
the cell, which also convert the electrical Colorimetric absorbance in
HGB g/dL
proportion to hemoglobin
impedance into a 5 part differential machine
From RBC histogram, # of RBCs x
MCV fL size of RBCs x cal constant or
𝐻𝐶𝑇 𝑥 10
Calculated:
𝑅𝐵𝐶
𝑅𝐵𝐶 𝑥 𝑀𝐶𝑉
HCT % Calculated:
10
𝐻𝐺𝐵 𝑥 10
MCH pg Calculated:
𝑅𝐵𝐶
𝐻𝐺𝐵 𝑥 100
MCHC g/dL or % Calculated:
𝐻𝐶𝑇
RDW % Impedance (from histogram)
Impedance count x calibration
Platelet c 109/L
(cal) factor
Absolute:
x 109/L
WBC Diff Light Scatter, flow cytometry
Percent of
WBC: %

CBC QUALITY CONTROL

➢ Commercial Controls
o 3 levels (low, normal, high)
o Values stored in instrument computer
o Levey-Jennings graph generated and
stored for each parameter
➢ Mode to Mode QC
o Most automated hematology
instruments have a primary and
secondary mode of sample aspiration.
o Controls must be run on BOTH and
correlate.
▪ Primary: Automated or Closed
▪ Secondary: Manual or Open
➢ Delta Checks
o When the Laboratory Information
System (LIS) and the instrument are
interfaced (connected) delta checks are
conducted by the LIS on select
parameters.
o Comparing of current results to previous
results

ADVANTAGES AND DISADVANTAGES

OF THE AUTOMATED ANALYZER
Advantages
• rapid, objective, statistically significant
• not subject to the distributional bias of the
manual count.
• more efficient and cost effective
• The precision of the automated differential
makes the absolute leukocyte counts reliable
and reproducible.
• Flagging system

Disadvantages
• produce cell counts which are falsely increased
or decreased.
• Some analyzers check only the volume and
number of particles .
• Platelet clumps may be misclassified as
leukocytes or erythrocytes, and nucleated red
blood cells can be misclassified as leukocytes or,
specifically, lymphocytes.
 HEMATOLOGY 1 (LECTURE)

- Reduction of red cell mass - Small and pale red cells because hemoglobin is
- Decreased concentration of hemoglobin not enough
- There is a decreased ability of RBCs to transport - Any deficiency problem with hemoglobin
out oxygen components may result in decrease production
- Hemoglobin level is the first one to check to of hemoglobin
evaluate anemia - Causes:
o Male: < 12 g/dL o Iron deficiency anemia
o Female: < 11 g/dL o Chronic disease
- Relative anemia: normal RBC mass, low volume o Sideroblastic anemia
o Transient or temporary o Thalassemia
o normal retics
o Example: dehydrated IRON DEFICIENCY ANEMIA (IDA)
- Absolute anemia: low RBC mass, normal volume - Most common cause of anemia
o low RBC delivery to circulation (bone
- Usually smaller in size
marrow is not producing enough RBCs) - Due to dietary inadequacy, malabsorption,
o there is an impaired production of RBCs
increase iron loss, & increased iron requirement
o loss of RBC from circulation
- Do not diagnose IDA by measuring iron alone
▪ destruction of RBCs because of - Low ferritin confirms IDA
antibodies or certain external
factors such as malaria
Lab Findings
▪ bleeding because of accidents,
acute or chronic bleeding, etc. ✓ CBC: decreased hemoglobin
✓ Retics: low to normal
✓ OFT: decreased
Signs and Symptoms
✓ Bone marrow: erythroid hyperplasia
• easy fatigability ✓ Others:
• dyspnea on exertion o Decreased: serum iron, % saturation,
• bounding pulse serum ferritin (stored iron)
• palpitations o Increased: Total Iron Binding Capacity
• systolic murmur (TIBC – ability of the iron to be stored as
• headache, faintness, and vertigo ferritin) and Free Erythrocyte
• pallor Protoporphyrin (FEP)
• low BP
• spoon nails ANEMIA OF CHRONIC DISEASE
- Due to chronic infections, inflammatory process,
Evaluation of Anemia and malignant neoplasms
• red cell count - Blockage in deliver of iron to the developing red
• red cell indices cells
• hemoglobin and hematocrit - Low serum iron and TIBC; high serum ferritin
• red cell distribution width (RDW)
• Peripheral Blood Smear (PBS): to evaluate the SIDEROBLASTIC ANEMIA
morphological features of RBCs - Abnormalities in heme metabolism
• Reticulocyte count: index of normal - Adequate iron stores but unable to incorporate it
erythropoiesis into hemoglobin because there is lack of
o Especially in cases of hemolytic anemia protoporphyrin IX
to determine whether the bone marrow - Presence of nucleated RBC with iron granules
is responding or not - Primary: idiopathic (cause is unknown)
• Osmotic Fragility Test (OFT): evaluates the - Secondary: side effect (other diseases that
relationship of RBCs surface area to its volume results to sideroblastic anemia such as B12
ratio deficiency, porphyria, and leukemia)
o Especially in cases of spherocytosis - RDW increased
• Bone marrow examination - Hypercellular BM
- Normal retics
CLASSIFICATION OF ANEMIA - High serum iron, serum ferritin, and LDH
Physical Characteristics - (+) stain: Prussian blue (Perl’s stain)
- Pappenheimer bodies on Giemsa
• Degree of hemoglobinization: diameter of
central pallor
Evaluation of Anemia
• Size of red cells
Serum Serum
TIBC FEP RDW
Iron Ferritin
Pathogenesis IDA Low High Low High High
• Disorders of red cell formation Chronic
Low Low High High Normal
Disease
• Excessive loss of red cell
Sideroblastic High Normal High Low High
• Abnormal distribution of red cells
 HEMATOLOGY 1 (LECTURE)

Intrinsic Hemolytic Anemia

- Normal in size and color Hereditary defects
Abnormalities of red cell membranes
- The number of circulating red cells are low
Hereditary Spherocytosis
- Quality/Quantity - Autosomal dominant trait in whites
- Delivery of oxygen is depleted - Causes:
- Causes: o Increased temperature
o Aplastic anemia o Certain antibodies
o Deficiency in spectrin
o Hemolytic anemia
- Loss of red cell membrane resulting in decreased
o Renal disease surface area
o Acute blood loss - Cells are less deformable
- Clinical features: jaundice, splenomegaly, skeletal
abnormalities, chronic leg ulcers, gall stones,
APLASTIC ANEMIA spherocytes, and stomatocytes in PBS
- Absence of hematopoietic cells in the BM - Lab findings:
- Usually associated with pancytopenia (all cell o Increased: OFT, B1, LDH, urobilinogen
▪ Since OFT is also increased in cases of AIHA,
counts are low) perform Direct Antiglobulin Test (DAT) to
- Associated with: detect the presence of antibodies
o Drug exposure, such as chloramphenicol o Decreased: haptoglobin
and sulfonamides Hereditary Elliptocytosis
o Viral infections such as parvo virus B19 - Inherited disorder characterized by deficiencies in
protein band 4.1
(one of the common causes of aplastic - Defective membrane protein skeleton structure,
anemia), measles, cytomegalovirus, and elongated elliptical cells
Epstein-Barr virus - Variant: hereditary pyropoikilocytosis
o Exposure to toxins such as benzene, Inherited RBC enzyme defects
G6PD Deficiency
pesticides, or radiation
- X-linked disorder
o Tuberculosis - Deficiency in G6PD will result to the loss of NADPH
o Chemicals - Inability to neutralize oxidation stress
- Fanconis anemia: congenitally acquired anemia - Hemoglobin molecule is unstable, susceptible to
- Severe if 3 of the 4 criteria are present: hemolysis
- When RBCs are oxidized, hemoglobin is denatured
o Neutrophil count: < 500/uL
- Rapid intravascular destruction
o Platelet count: < 20,000/uL - Resistance to malaria
o Reticulocyte count: < 10,000/uL - Clinical patterns:
o Markedly hypocellular marrow o Neonatal jaundice (increased bilirubin)
o Congenital hemolytic anemia
- Common in people 50 years old and above
o Drug-induced hemolysis (caused by primaquine)
- 50% of people with aplastic anemia has a six o Favism (after consuming fava beans)
months mortality rate - Classes of G6PD deficiency
- Treatment: bone marrow transplantation o Class I: severe deficiency
▪ Hemolysis is chronic
▪ < 10% activity of G6PD
Pure Red Cell Aplasia o Class II: severe deficiency
- Hypoplasia of erythrocyte precursors only ▪ Hemolysis is intermittent
- Other cell precursors are still normal ▪ < 10% activity of G6PD
o Class III: mild deficiency
- Severe anemia with reticulocytopenia ▪ 10-60%
- If the causes of pure red cell aplasia is not ▪ Exposure to stress
addressed, it can progress to aplastic anemia - Lab diagnosis
- Associated with: o Inclusion bodies: Heinz bodies (recovered using
o Hemolytic anemia supravital stain)
o Increased retics, bilirubin, serum LDH
o Parvovirus infection o Hemoglobinuria
o Drugs o Decreased haptoglobin
o Thymoma o Negative Coomb’s test
- Diamond-blackfan syndrome: congenitally - G6PD testing is part of the newborn screening
- Confirmatory: G6PD assay or G6PD fluorescence
acquired testing
- Other tests:
HEMOLYTIC ANEMIA o Ascorbate cyanide test (non-specific)
▪ Red cells are exposed to sodium cyanide
- Shortened red cell survival and sodium ascorbate
- Destruction of RBCs ▪ Normally, RBCs Should remain intact
- Intracorpuscular or extracorpuscular defects ▪ Solution should remain red
- Classification: ▪ If there is G6PD deficiency, red cells are
lysed resulting to brownish color solution
o Intrinsic: red cell itself has a problem
Pyruvate Kinase Deficiency
o Extrinsic: red cells are normal but there - Problem in E-M pathway
are external factors that destroy RBCs - Pyruvate kinase is an enzyme needed in the production
- Intravascular: RBC is lysed within the vessel of energy
o The hemoglobin binds with haptoglobin o Lack of this enzyme will result to lack of energy
o Sodium and calcium can now easily enter the
- Extravascular: RBC is lysed outside the vessel RBC, resulting to lysis
(particularly by macrophages in the spleen) - Failure to generate sufficient ATP results in defective
control ions
- Jaundice and splenomegaly
- Confirmatory test: PK assay
 HEMATOLOGY 1 (LECTURE)

Disorders of Hgb production - Laboratory findings:

Hemoglobinopathy o Elevated reticulocyte count
Thalassemia o Increased concentration of indirect bilirubin
o Hemoglobinuria
o Positive Donath-Landsteiner of Rosenbach or
Acquired defects Ehrlich or Sanford test
Paroxysmal Nocturnal Hemoglobinuria o Positive for methemalbumin
- Red cells are lysed because of complement Alloantibody
- It is manifested by hemosiderin in the urine Transfusion Reactions
- Deficiency in DAF - Blood incompatibility
- Decay Accelerating Factor (DAF) regulates complement - Laboratory findings:
- Increased susceptibility to complement mediated red cell o Direct antiglobulin test (DAT) positive
lysis o High hemoglobin (even in the urine)
o EM: protuberances on the red cell surface
- Chemical abnormalities
o Deficient acetylcholinesterase activity and abnormally Non-Immune Hemolytic Anemia
constituted glycoprotein - Antibodies are not involved
- Lab diagnosis - Transient forceful contact of the body with hard surfaces
o Screening: Sugar of sucrose lysis test o Associate with athletes, boxers, triathletes, marathon
▪ Excessive hemolysis when exposed to low ionic runners, tennis players
strength solution - May be caused by mechanical trauma, microangiopathic
▪ Px’s RBC + serum + sucrose hemolytic anemia, chemical and toxic agents, infections,
▪ Make sure that the serum is compatible to the hypersplenism, and systemic disease
patient - Mechanical trauma: chemicals, drugs, snake venom,
o Confirmatory: Ham’s acid hemolysis test prosthetic heart valves
▪ Excessive hemolysis when exposed to o Defective prosthetic heart valves has been associated
complement containing serum at low pH with Waring Blender Syndrome
▪ Has three different set-up - Thermal burns: red cells are exposed to increased
• Tube 1: Px’s RBC + serum + acid (0.2 N HCl) temperatures
o Lysis is present - Infections: damages red cell membranes
• Tube 2: Px’s RBC + own serum o Microbial infections such as Plasmodium falciparum or
o Lysis is present Clostridium perfringens
• Tube 3: Px’s RBC + inactivated serum (56°C) - Associated with microangiopathic hemolytic anemia (cells
o Lysis is not present because are lysed because of the problem within the blood vessel):
complement is destroyed o Disseminated Intravascular Coagulation (DIC): fibrin is
deposited in small vessels
▪ Unwanted clots is formed
Extrinsic Hemolytic Anemia o Hemolytic Uremic Syndrome: renal damage
Immune Hemolytic Anemia ▪ Kidney problems resulting to bleeding forming a
Autoantibody clot
o Thrombotic Thrombocytopenic Purpura: deficiency of
Cold Autoimmune Hemolytic Anemia (cAIHA)
enzyme ADAMST13
- Due to IgM cold reactive antibody
▪ The body keep on forming unwanted clots
- Occurs in association with infection, malignancies, or
autoimmune disorders
- The cold reacting antibodies binds to the RBCs resulting HEMORRHAGIC ANEMIA (BLEEDING)
to destruction
- Laboratory findings: - Acute bleeding: sudden loss of blood resulting
o Direct antiglobulin test (DAT) is positive from injury or trauma
▪ To check for complement coated RBCs - Chronic bleeding: long-term and gradual
o Cold agglutinins titer is increased o Example: gastrointestinal bleeding
▪ Cold agglutinin syndrome
- Cold agglutinins:
- Red cells are normocytic and normochromic
o Anti-I: infected with mycoplasma pneumoniae - Body adjusts, increased heart rate
o Anti-i: infected with Epstein-Barr virus that cause - Expanding circulatory volume
infectious mononucleosis o Hematologic response to acute blood
- Peripheral smears
loss
o Polychromatophilia
o Spherocytosis - Increase platelet count, circulating granulocytes
o Agglutination of red cells - Increased EPO levels in 6 hours
Warm Autoimmune Hemolytic Anemia (wAIHA) - Reticulocytosis in 24 hours
- RBCs react with IgG and/or complement
- Idiopathic cases or secondary
- Laboratory findings: RENAL DISEASE
o High: OFT, bilirubin, retics - Decreased release and production of
o Direct antiglobulin test (DAT) positive
erythropoietin
▪ To differentiate it between spherocytosis
- Autoimmune hemolysis is positive because of the - Decreased in RBC count
antibody - Red cells are normocytic and normochromic

Example: Paroxysmal Cold Hemoglobinuria

- Associated with biphasic IgG antibodies
o When red cells are exposed to cold environment, MEGALOBLASTIC ANEMIA
complement binds to the RBCs but there is no
lysis - Deficiency in:
▪ IgG antibody allows complement to bind o Vitamin B12 (Cobalamin)
on red cells o Folic acid (folate)
o When red cells are exposed to warm
- Defective DNA production
environment (37°C), RBCs are destroyed
- This is a rare state in which hemolysis occurs when - Earliest signs: hypersegmented PMNs, oval
blood is warmed after previous exposure to chilling macrocytes
- Associated with Donath-Landsteiner antibody or Anti-
P antibody
 HEMATOLOGY 1 (LECTURE)

- Megaloblastic state affecting the RBCs

- Megaloblastic: it does not only affect RBCs. It
- Excess production of cells
also affects other cells such as WBCs and
- Primary erythrocytosis: uncontrolled growth of
platelets
cells for no apparent purpose
o All cells contain nucleus which need the
- Associated with Polycythemia vera
same set of nutrients for their
maturation such as B12 and folic acid
- Lab diagnosis: Differentials of erythrocytosis
o Pancytopenia (all cells are low) - Primary polycythemia: PV
o Macroovalocytes - Secondary polycythemia: high altitude, CPD,
o WBCs and platelets are low cyanotic congenital heart disease, low cardiac
o Hypersegmented PMNs output, hypoventilation syndrome, high affinity
o Low retics Hgb variants, neoplasms-renal artery stenosis,
o BM: marked erythroid hyperplasia renal transplant
o Blood chem: increased B1, serum iron
and LDH
o Inclusions: Howell-Jowell bodies, as well
- Abnormal cell production, unresponsive to
as pappenheimer bodies, cabot rings, treatment
and basophilic stipplings - Hallmark is ineffective erythropoiesis, with
markedly hypercellular marrow and abundant
Vitamin B12 (Cobalmine) Deficiency abnormal erythroid precursors
- Intrinsic factor: needed to absorb Vit. B12 in - Precursors are megaloblastic
terminal ileum - It will later on lead to leukemia if it is not
o intrinsic factor is synthesized by parietal managed immediately
cells - A person has anemia (there is insufficient
- Pernicious anemia: most common cause, amount of RBCs) because the bone marrow
characterized by a deficiency in intrinsic factor produces and releases immature cells
(presence of antibody that acts against the
intrinsic factor or parietal cells)
o Gastrectomy, atophic gastrisis
o Veganism - Usually a genetically acquired inborn error of
o Diphyllobothrium latum infection metabolism
- Features: weakness, jaundice, sore tongue, - Deficiencies of enzymes involved in porphyrin-
numbness, and other CNS disease heme biosynthesis pathway
- Laboratory findings: - Diagnosis: spectroscopy and biochemical
o Low vitamin B12 analysis of blood, urine, and stool
o Folic acid may be decreased - Urine porphobilinogen is mostly elevated
o Elevated metabolites because it is not - Associated with sideroblast cells (accumulation
absorbed of iron in RBCs)
- Schilling test: for megaloblastic state - Patients are photosensitive
o Abnormal in cases of vit. B12 deficiency
Genetic Porphyrias
Vitamin B9 (Folate or Folic Acid) Deficiency - Manifestation may be neurologic (excruciating
- It does not affect the CNS pain and other neurologic symptoms),
- Due to: cutaneous
o Dietary (most common) - Acute intermittent porphyria (AIP)
o Intestinal malabsorption
o Increased demand Acquired Porphyrias
o Excess loss - Associated with lead poisoning
o Defective synthesis - Lead will block the protoporphyrin pathway,
which will affect the heme synthesis
NON-MEGALOBLASTIC ANEMIA o D-ALA will not be converted into
porphyrins
- Anemia of liver disease
- Adults (occupational) and children (PICA)
- Membrane abnormalities of RBCs
- Enzymes depressed:
- Liver provides vital components for the
o Delta amino levulinic dehydratase
maturation of RBCs
o Heme synthetase
o Problems with liver will result to
- Features: abdominal pain, weakness
incomplete development of RBCs at well
- Lab diagnosis:
as its membrane
o Anemia (micro/normo)
o Increased reticulocytes
Liver Disease
o Decreased OFT
- Non-megaloblastic anemia o Inclusions: basophilic stipplings
- Decreased cholesterol synthesis
- Spur cells, acanthocytes
 HEMATOLOGY 1 (LECTURE)

2. Beta Thalassemia
➢ Hemochromatosis - Production of B chain will occur only at 3-6
o Increased GIT absorption and systemic months after birth
iron overload - Homozygous beta thalassemia: (major, cooley’s
o iron deposits in liver anemia, Mediterranean anemia) severe lifelong
➢ Hemosiderosis anemia
o secondary iron accumulation. - Heterozygous beta thalassemia: one normal and
o Iron deposits in parenchymal cells and one abnormal beta chain (minor)
Kupffer cells in the portal tract Other Names Description
Minor • Heterozygous - results when one of the 2
• Cooley’s trait genes that produce beta
• Rietti-Greppi- globin is defective
Micheli disease - usually presents a mild,
- characterized by decreased rate of production or asymptomatic anemia
total absence of globin chains Intermediate • Thalassemia - more severe but do not
Intermedia require regular transfusion
- less globin = less hemoglobin produced = less - occasional transfusions
delivery of oxygen to tissues Major • Homozygous - decrease or complete lack
- 2 important chromosomes for the production of • Cooley’s Anemia of beta chains
• Mediterranean - most severe form
globins: Anemia - TRANSFUSION dependent
o Chromosome 16: responsible for the • Target Cell Anemia anemia
synthesis or generation of alpha and
zeta globins 3. Hereditary Persistence of Hb F (HPHF)
o Chromosome 11: responsible for the - thalassemia with increased levels of fetal
synthesis or generation of beta, delta, hemoglobin
epsilon, and gamma globins - partial or total suppression of beta and delta
- gene for synthesis is located in chromosome 16 chains and Hb F increased to compensate
and chromosome 11 - less delivery of oxygen to tissues
o Alpha Thalassemia – decreased - test for Hb F:
production of alpha chains o alkali denaturation test
o Beta Thalassemia – decreased ▪ Singer test
production of beta chains ▪ Betke test
- demographics: o Acid elution test: citric acid phosphate
o Southeast Asia and Mediterranean buffer is used (high amount of Hb F
region yields to pinkish to reddish solution)
- Clinical presentation:
o Minor: mild anemia (confused with IDA)
4. Hemoglobin Lepore
o Intermedia: moderate anemia
- a rare class of thalassemia caused by crossing
o Major: severe anemia (Hydrops Fetalis)
over of beta and delta genes

TYPES OF THALASSEMIA 5. Hemoglobinopathy + Thalassemia

1. Alpha Thalassemia ➢ Hemoglobin S-Thalassemia
- Each individual has 4 genes for hemoglobin o is a double heterozygous abnormality
- Severity of disease depends on the number of o the abnormal genes for Hb S and
genes/globin chains defective or missing thalassemia are coinherited
o If 2 = alpha trait (minor) o Types:
o If 3 = H hemoglobin (intermedia) ▪ Hb S-α-thalassemia
▪ Can be observed using ▪ Hb SS-α-thalassemia
supravital stain ▪ Hb S-β-thalassemia
o If 4 = Barts hemoglobin (major) ➢ Hemoglobin C-Thalassemia
Hemoglobin o β thalassemia with inherited Hb C
Description
Present ➢ Hemoglobin E-Thalassemia
Silent Carrier (- α / α α) deletion of one α birth: 1%-2% Hb
globin gene, leaving 3 Bart’s
o co-inherited of Hemoglobin E and β
functional α globin adult: normal Hb A, thalassemia that results to a marked
genes Hb Bart reduction of β chain production.
α Thalassemia Trait deletion of two α birth: 2%-10% Hb
Homozygous (- α / - α) globin gene Bart’s
Heterozygous (- - / α α) adult: normal Hb A LABORATORY FINDINGS OF THALASSEMIA
Hemoglobin H Disease caused by the birth: 10%-40% Hb
(- - / - α) presence of only one Bart’s replaced by • CBC
gene producing α Hb H 30-50% o ↓Hb and Hct
chains. remainder: Hb F,
HbA₂, Hb Bart’s o ↑RBC count
and Hb A o ↓RBC indices (MCV and MCHC)
adult: 70% Hb A
o ↑RDW
Hydrops Fetalis (- - / - -) results in the absence birth: 80%-90% Hb
of all α chains Bart’s 5%-20% Hb • Peripheral Smear
synthesis and Portland, trace of o microcytic hypochromic
incompatible with life Hb H
o exhibits anisocytosis and poikilocytosis
(target cells and elliptocytes)
o presence of NRBC
o polychromasia and basophilic stippling
 HEMATOLOGY 1 (LECTURE)

• Increased Reticulocyte count ▪ protected from Plasmodium

• Bone marrow examination falciparum infection
o shows hypercellular with extreme ▪ normal PBS
erythroid hyperplasia ▪ positive sickle cell solubility test
• Decreased OFT
• Supravital stain Laboratory Diagnosis
o shows Hb H inclusions • Peripheral Blood Smear:
• Electrophoresis (migration pattern) o marked poikilocytosis, inclusion bodies,
o differentiates hemoglobin variants sickle cells (6-18%)
• Mass Spectrophotometry o increased WBCs, platelets, retics
o assess the difference in mass of the • Bone Marrow:
globin chains o marked erythroid hyperplasia
o detects single amino acid substitutions o high iron storage
in the globin chains • Blood Chemistry:
• DNA analysis o increased B1, serum iron
o identify globin gene mutations
▪ PCR HEMOGLOBIN SC (Hgb SC) DISEASE
▪ Signal Amplification System
- Hgb SC disease is a double heterozygous
• Increased Indirect Bilirubin condition where an abnormal sickle gene from
one parent and an abnormal C gene from the
other parent inherited.
- The problem is more on the amino acids of o One beta is replaces by valine and the
globins other beta is replaced with lysine
- These are a group of inherited disorders causing - Laboratory:
structurally abnormal globin chain synthesis due o Moderate to severe normocytic/
to amino acid substitutions (qualitative defect) normochromic anemia with target cells
- changes in RBC deformability and o characterized by SC crystals
electrophoretic mobility can occur. o may see rare sickle cells or C crystals
- Homozygous/ disease conditions (both globin o positive haemoglobin solubility
chains affected) are more serious than screening test.
heterozygous/trait conditions (only one globin
chain affected). HEMOGLOBIN C (Hgb C) DISEASE
- resembles Hgb S but instead of valine, lysine is
SICKLE CELL DISEASE seen on the 6th position of B chain
- Hgb S – most common abnormal hemoglobin - mild hemolytic anemia
- normal glutamic acid at 6th position in the B chain - Hgb C crystals and clam shaped cells
is replaced by valine - Laboratory:
- Results in: altered solubility, altered ability to o Normochromic/ normocytic anemia
withstand oxidation, instability, increased with target cells
propensity for methemoglobin production, o characterized by intracellular rodlike C
increased or decreased oxygen affinity crystals
- Hgb A is lacking, Hgb S is present - Hgb C migrates with hemoglobins A2, E, and O on
- Sickling is increased alkaline hemoglobin electrophoresis; can
o low oxygen tension differentiate hemoglobins using acid
o low pH electrophoresis.
o increased body temp
- confers protection against falciparum malaria HEMOGLOBIN E DISEASE
- laboratory tests: - Caused when lysine replaces glutamic acid at
o Sodium metabisulfite test position 26 on the beta chain.
▪ Positive: sickling - Homozygous condition results in mild anemia
o Solubility test with microcytes and target cells; heterozygotes
▪ Reagent: sodium dithionate are asymptomatic.
▪ Positive: turbidity - Hgb E migrates with hemoglobin A2, C, and O an
- Clinical considerations: alkaline hemoglobin electrophoresis.
o Homozygous S (SS) – lifelong, severe,
hemolytic anemia
HEMOGLOBIN D (PUNJAB) DISEASE
▪ hemolytic crisis, vaso-occlusive
episodes, prone to infection - Caused when glycine replaces glutamic acid at
(pneumococcus), gallstone is position 121 on the beta chain.
common, bone pain and - Hgb D migrates with Hgb S and Hgb G on alkaline
tenderness hemoglobin electrophoresis.
o Heterozygotes – one Hgb S gene, one
Hgb A
▪ Sickle cell trait (Hgb AS)
▪ usually with no apparent disease
HEMATOLOGY 1 (LECTURE)
 HEMATOLOGY 1 (LECTURE)

o People with the disease are always at

risk in acquiring infections with catalase
➢ Quantitative disorders – abnormalities affecting
positive organisms
the number of WBCs
o Laboratory test: Nitroblue tetrazolium
o Proliferative: Increase in WBCs
test (NBT test)
▪ reactive leukocytes
▪ The buffy coat is mixed with the
▪ neoplasms (cancer)
bacterial suspension
o Leukopenias: Decrease in WBCs
▪ Formation of blue precipitate
➢ Qualitative disorders – abnormalities affecting
the phagocytic activities of WBCs ABNORMAL IN MACROPHAGES/MONOCYTES
Lipid storage diseases – abnormalities affecting the
function of monocyte/macrophages caused by enzyme
LEUKOPENIA deficiency
➢ Decreased production: irradiation, drugs, viral ➢ Gaucher’s disease
infection, congenital o accumulation of glucocerebroside
o The bone marrow is not synthesizing because of the deficiency in enzyme
glucocerebrosidase
enough WBCs
o Associated with aplastic anemia o Wrinkled/crumpled cytoplasm
➢ Niemann-Pick disease
➢ Ineffective production: megaloblastic anemia,
myelodysplastic syndromes o Accumulation of sphingomyelin because
o The bone marrow produces abnormal of the deficiency in enzyme
cells, making it ineffective sphingomyelinase
o Foamy cytoplasm
o If one cell production is ineffective, the
other cell production will also be ➢ Tay-Sach’s disease
ineffective o Accumulation of glycolipids and
➢ Increased destruction: isoimmune neonatal, ganglioside because of the deficiency in
enzyme hexosaminidase A
autoimmune, complement-activation-induced
hemolysis o Vacuolated cytoplasm
o The bone marrow can produce cells but ➢ Sandhoff’s disease
the WBC count is low o Accumulation of glycolipids and
o There may be certain chemicals or ganglioside because of the deficiency in
substances that destroys WBCs enzyme hexosaminidase A and B
o Vacuolated cytoplasm
o Can be immune or non-immune
➢ Splenic sequestration: makes the WBC count ➢ Sea Blue Histiocytes
o Accumulation of lipids
low
o Spleen is enlarged because more WBCs o Specific enzyme deficiency is unknown
are sequestered o Blue-green cytoplasm
➢ Increased margination: has a lot white cell Disease Defect
precursor than red cell precursor Leukocyte Adhesion Def. 1 B chain of CD11/CD18 integrins
Sialylated oligosaccharide
o Low WBC count because more white Leukocyte Adhesion Def. 1
selectin
cells leave the circulation PMN Specific Granulocyte Defective chemotaxis
CGD Decrease oxidative burst
Acquired X linked membrane component
Acquired cytoplasmic component
Has leukopenia; decreased WBC count
MPO Deficiency Absent MPO-H2O2 system
• Thermal injury Chediak Higashi Multiple defects
• Diabetes mellitus
• Malignancy
• Sepsis - It affects all of the formed elements
• Malnutrition - Abnormal, uncontrolled proliferation,
unregulated, and accumulation of one or more
of the hematopoietic cells
➢ Job’s syndrome - Symptoms: fever, weight loss, increased
o normal random activity sweating
o Abnormal chemotactic activity o There could be bone pain from a very
➢ Lazy leukocyte syndrome large leukemic mass especially in cases
o Abnormal random activity of acute leukemia
o Abnormal chemotactic activity - Classifying leukemia based on the origin of the
➢ Chronic Granulomatous Disease (CGD) cell
o Inability to phagocytized
microorganisms Incidence
o Impaired NADPH oxidase - Dominant cause of cancer death in children
o Problem in respiratory burst under 15 years old
o Cannot fully kill or phagocytized the - ALL: most common in children
microorganism - AML: adults under 60
- CLL: adults over 60
 HEMATOLOGY 1 (LECTURE)

Classification - Features: bone pain and tenderness, organ

• Duration infiltration, symptoms related to depressions of
o Acute: lasts for days up to 6 months normal marrow function
o Subacute: 2 to 6 months - Usually encountered with organ failure or
o Chronic: variable (minimum of 1-2 years) organomegaly
• Number of WBC in the peripheral blood - Laboratory Findings:
o Leukemic: > 15k/uL o WBC count: varying blast
o Subleukemic: < 15k/uL o Platelets: usually decreased
▪ White cell count is low o Anemia: present
▪ Abnormal and immature cells
are present in the peripheral Acute Lymphoblastic Leukemia (ALL)
blood - immature form of the lymphoid lineage
o Aleukemic: < 15k/uL predominates
▪ No abnormal and immature cells - Primarily a disease of the children and young
in the peripheral blood adults
▪ Abnormal cells are in the bone - According to morphology of lymphoblasts
marrow o Pre-B cell ALL: t(9;22)
• Types of WBC involved o B cell ALL: t(4;11)
o Acute: last only for days to 6 months o T cell ALL: t(7;11)
▪ Accumulation of immature cells - Prognosis
▪ Immature cells go to the o More than 90% of children with all can
peripheral blood be cured
▪ Immature cells starts to o All in children between 2-10 years old
infiltrate other organs once the with early pre-B phenotype and
spleen does not longer filter hypodiploidy in the range of 51-60
white cells chromosomes: most favorable
▪ Associated with organ failure
▪ Hepatosplenomegaly Types of Acute Lymphoblastic Anemia
o Chronic: minimum of 1 to 2 years ➢ L1
▪ Abundant mature cells o Childhood ALL
▪ Can turn into acute once the o Lymphoblasts: small and homogenous
bone marrow is saturated

Agencies that classify the disease

➢ FAB Classification
o It divides acute leukemias into
lymphoblastic and myeloblastic
o Subdivided according to cellular
morphology, cytochemical staining ➢ L2
results, cytogenetic studies, and T and B o Adult ALL
lymphocytes markers o Lymphoblasts: large and heterogenous
o Acute leukemia: ≥ 30% blasts in the bone o They vary in size
marrow
➢ WHO Classification
o Based on cell morphology, cytochemical
stains, immunologic probes of cell
markers, cytogenetics, molecular
aspects, and clinical manifestations
o Standard in classifying acute leukemia
o More strict and has wholistic approach
o Acute leukemia: ≥ 20% blasts in the bone ➢ L3 (Burkitt type)
marrow o Rare
o Lymphoblasts: large and homogenous
Treatment o t(8;14) with a rearrangement on MYC
• Bone Marrow Transplantation oncogene
• Radiation o it appears to be vacuolated
• Chemotherapy o it has a leukemic manifestation of
• Supportive treatment (Blood transfusion) Burkitt’s disease

ACUTE LEUKEMIA
- Accumulation of blasts due to:
o clonal expansion of transformed stem
cells
o failure of maturation
- there is a prolonged generation time
- depression of the hematopoietic cycle
 HEMATOLOGY 1 (LECTURE)

Acute Myeloblastic Leukemia (AML)

- immature form of the myeloid lineage
predominates
- primarily in adults between 15-39 years old
- Morphology: more abundant cytoplasm, Auer
rods, MPO granules, delicate nuclear chromatin
➢ M5a – Acute monoblastic leukemia without
Chromosomal abnormalities maturation
• 90% of AML o 20 to >80% monocytic cells
• t(9;22)(9q34;q11) Ph’ 10-15% of M1 Poor Prog o Monoblasts: > 80%
• t(8;21)(q22;q22) 20-25% of M2 favorable prog
• t(15;17)(q22;q21) 70-80% M3 RAR-PML fusion
gene
• Absence of Ch.16 20-25% M4 favorable prog
• Absence of Ch.11 30-40% M5 poor prog
• Absence or deletions of Ch.5 or 7 poor prog

Types of Acute Myeloblastic Leukemia

➢ M0 – Accumulation of undifferentiated blasts ➢ M5b – Acute monoblastic leukemia with
➢ M1 – Acute myeloblastic leukemia without maturation
maturation o 20 to >80% monocytic cells
o > 30% blast in the bone marrow o Monoblasts: < 80%
o < 10% granulocytic cells

➢ M6
o Also known as Erythroleukemia or Di
➢ M2 – Acute myeloblastic leukemia with
Guglielmo syndrome
maturation
o 30 % blasts
o > 30% blast in the bone marrow
o > 50% erythroblastic precursors
o > 10% granulocytic cells
o It affects the RBC precursors

➢ M7 – Acute megakaryocytic leukemia

➢ M3 – Acute promyelocytic leukemia o 30% blasts
o > 30% blast in the bone marrow o > 30% megakaryocytes
o > 10% granulocytic cells
o > 50% promyelocytes
o Associated with disseminated
intravascular coagulopathy (DIC)
o Associated with translocations in
chromosomes 15 and 17
o Fusion of primary granules (Auer rods)
▪ Faggot cells: cells that have
bundles of Auer rods

CHRONIC LEUKEMIA
- Predominance of mature cells in the peripheral
blood
- Two types:
o Lymphoproliferative
➢ M4 – Acute myelomonocytic leukemia o Myeloproliferative
o 30% blasts in the bone marrow - Laboratory Findings:
o 20 to < 80% monocytic cells o WBC count: high
o Also known as Naegeli o Platelet count: normal to increase
o M4e: M4 accompanied by eosinophilia o Anemia: normal to mild
 HEMATOLOGY 1 (LECTURE)

Chronic Myelogenous Leukemia (CML) o Common thrombosis, infarction gastric

- mature form of the myeloid lineage ulcer, high BP, stroke, heart attack
predominates o Also associated with JAK genes
- Stem cell disorder affecting the granulocytic, o There will be pancytosis (all cell counts
erythrocytic, and megakaryocytic cell lines in the blood are elevated) and
- It is associated with Philadelphia chromosome panmyelosis (increase number of cell in
o One arm of chromosome 22 is the bone marrow)
translocated to chromosome 9 Absolute
o Negative: poor prognosis (they do not - True polycythemia (the problem is in the
respond well to therapy) production of cells)
o Positive: good prognosis (they respond - Increase hematocrit = increase BM
well to therapy) production
- Associated with Chronic Myeloproliferative - Primary: panmyelosis
Disorders o Increase bone marrow production of
o Clonal neoplasia of multipotent stem cell RBC, WBC, and platelets
o With differentiation and maturation o EPO: low
o Bone marrow fibrosis: reactive process - Secondary with appropriate EPO production:
response to hypoxia
Clinical features o Patients with pulmonary or cardiac
➢ Chronic phase (3 years) diseases
o Expansion of the granulocytic mass o Increase BM production of RBC, WBC,
o Marked increase cellularity and platelets
o Basophilia o EPO: high
o Predominantly myeloid hyperplasia - Secondary with inappropriate EPO
o Splenomegaly production:
o Total absence of LAP o No hypoxia
➢ Accelerated phase (50%) o Tumor of kidneys, liver, brain, arenals
o Gradual failure of response to treatment o Increase RBC, normal WBC, and
(poor prognosis) platelets
o Increasing anemia and o EPO: high
thrombocytopenia Relative
o Additional cytogenetic abnormalities - temporary and transient
➢ Blastic crisis - Increase hematocrit, decrease plasma
o AML (70%), ALL (30%) volume
o Usually ineffective to treatment - Seen when the patient is dehydrated,
stressed, and anxious
Examples of Chronic Myelogenous Leukemia - Spurious polycythemia
➢ Essential thrombocythemia
o Dominant proliferation of Additional information:
megakaryocytic cell line Myelodysplastic syndromes
o No Philadelphia chromosome - Pre-leukemic state
o Platelet count > 1000 x 109/L - Only abnormal or immature cells are produced
o With spontaneous aggregation of - Clonal stem cell disorders characterized by:
abnormal platelets o Maturation defects
o Increase susceptibility of forming o Ineffective erythropoiesis
unwanted and unnecessary clots o Peripheral pancytopenia in spite of
o Formation of clots in the cardiovascular marrow cellularity
and central nervous system - This may result to anemia
o It is still not clear what causes Blasts in Blasts in
Ringed
thrombocythemia, but some literature MDS peripheral Bone
sideroblasts
blood (%) Marrow (%)
said that it is associated with genetic Refractory Anemia/
abnormalities, particularly in Janus Refractory Cytopenia <1% <5% +/-
Kinase (JAK) genes (RA/RC)
Refractory Anemia with
➢ Myelofibrosis with Myeloid Metaplasia ringed sideroblasts <1% <5% >15%
o Fibrosis and granulocytic hyperplasia of (RARS)
the bone marrow with granulocytic and Refractory Anemia with
<5% 5-20% +/-
excess blasts (RAEB)
megakaryocytic proliferation in the liver Refractory Anemia with
and spleen excess blasts in >5% 20-30% +/-
transformation (RAEBIT)
o The patient may suffer from bleeding
Chronic myelomonocytic
o Giant platelets and occasional circulating leukemia (CMML)
<5% 5-20% -

megakaryocytic fragments
➢ Polycythemia Vera Leukemoid reaction
o Exact opposite of aplastic anemia - Not related to leukemia
o All cell counts are high - Not a disease (only a description)
o Characterized by an absolute increase in - Excessive leukocytic response in the blood
red blood cells, white blood cells, and - WBC count > 50x109/L
platelets - Exaggerated response to infection
 HEMATOLOGY 1 (LECTURE)

- Blood picture resembles leukemia

- Increase WBC count with shift to the left
- Sometimes mistaken as CML
o Leukocyte Alkaline Phosphatase (LAP) –
special stain to differentiate CML from
Leukemoid reaction ➢ Mycosis fungoides
o Leukemoid reaction: high LAP score o Positive: PAS
o CML: low LAP score o Incidence: elderly men
o Cutaneous T cell lymphoma + circulating
Chronic Lymphocytic Leukemia (CLL) lymphoma cells
- Most indolent form of all leukemias ▪ Starts with skin itching
- Silent type of leukemia o Splenomegaly and LAD
- 25% of all cases of leukemia in Western countries o Associated with Sezary cells
- Median age: 60 years o Types: pre-mycotic and mycotic
➢ Lymphoma – proliferation of malignant cells in
Chromosomal abnormalities solid lymphatic tissues
- 50% abnormal karyotype o malignancy of lymphoid tissues
- Most common among adults o it can later on spread into the bone
- Associated with deletions in chromosomes 11, marrow and blood
13, and 17, also in Trisomy 12 in some studies o Non-Hodgkin’s
- Require early treatment and have significantly ▪ Classification: Rappaport
shorter survival ▪ Proliferation of neoplastic
lymphocytes (cancer cells)
Clinical features ▪ Associated with Burkitt’s
- Often asymptomatic lymphoma
- Non-specific symptoms o Hodgkin’s
- There could be lymphadenopathy (LAD) with ▪ Reed-Sternberg cell is present
hepatosplenomegaly (50-60%) because of the • Has an owl’s eye
increased accumulation of lymphocytes appearance
• Can be associated with
Laboratory features Epstein-Barr virus
- Absolute lymphocytosis ▪ Proliferation of cells reacting to
- Often hypogammaglobulinemia increase cancer cells
bacterial infection o Diagnosis: definitive-lymph node
- Abnormal antibodies may be present, biopsy
particularly autoantibodies (10-15%) that act ▪ Rye: appearance on histologic
against platelets and RBCs lymph node biopsy
- Present of smudge cells in blood smear ▪ Ann Arbor: staging based on
- 5% monoclonal serum IgG tissue involvement
- CD5+ cells

Prognosis Myeloperoxidase (MPO) (+) AML

- Median survival: 4-6 years - Specimen should be fresh (–) ALL
- Not altered by therapy Sudan Black B (+) AML
- Depend primarily on stage - Staining of fats (–) ALL
- Transformation to acute leukemia with blast Terminal Deoxyribonucleotidyl (+) ALL
Transferase (TdT) (–) AML
crisis is rare Periodic Acid Schiff (PAS) (+) M6 or erythroleukemia
- Superimposed large cell lymphoma Napthol AS-D Chloroacetate (+) granulocytic cells
- Transformation to prolymphocytic leukemia Esterase (Specific Esterase) (–) monocytic cells
α-Naphthyl Acetate Esterase (Non- (+) monocytic cells
specific Esterase) (–) granulocytic cells
Examples of Chronic Lymphocytic Leukemia
α-Naphthyl Butyrate Esterase (+) monocytic cells
➢ Prolymphocytic leukemia (Non-specific Esterase) (–) granulocytic cells
o Problem affecting B cells and T cells Acid Phosphatase – Cannot be (+) Hairy cell leukemia
o Rare, common among aged men stored but it can be treated by
o Massive hepatosplenomegaly with tartrate to become TRAP
Leukocyte Alkaline Phosphatase 0: no red/brown
minimal LAD
(LAP) precipitate
o Poor prognosis - Count 100 neutrophils 1+: slightly diffused red
o Absolute lymphocytosis - Presence of red or brown precipitate
➢ Hairy cell leukemia precipitate 2+: moderately diffused
o “leukemic reticuloendotheliosis” - Normal LAP score: 30-185 3+: heavily diffused
- Increase in: Leukemoid 4+: very heavily diffused
o Positive: Tartrate resistant acid reaction, PV, 3rd trimester in
phosphatase (TRAP) pregnancy
o B cell (CD19 or CD20) with fine hair-like - Decrease in: CML, PNH
cytoplasmic membrane projections Toluidine Blue – binds with acid (+) mast cells and
mucopolysaccharides in blood cells basophils
o Prominent splenomegaly
o Treatment: splenectomy
 HEMATOLOGY 1 (LABORATORY)

• Biological Hazard
• Sharp Hazard
• Chemical Hazard
• Radiation Hazard
• Electrical Hazard
• Fire Hazard
• Physical Hazard
• Other Hazards

FIRE HAZARD
- Enforcement of a non-smoking policy
- Placement of fire extinguishers every 75 feet,
checked monthly and maintained annually
- Placement of fire detection system and manual
fire alarm near exit doors which is less than 200
ft away and should be tested every three months
SAFETY PRECAUTIONS - Written fire prevention and response
- Exposure to blood and body fluids is the most procedures and fire drills
common risk associated in hematology
laboratory
- Bloodborne pathogens are pathogenic
microorganisms present in blood causing
infection or diseases

OSHA STANDARDS
- OSHA provides standards to maintain safe work
environment
- The following practices are enforced inside the
laboratory:
o Handwashing
o Food, drink and medications not allowed
o Applying cosmetics are prohibited
o Fomites or any surfaces must be kept away
from mouth and all mucous membranes
o Contaminated sharps must be disposed
properly
o Personal Protective Equipment must be
worn at all times following the proper
donning
o Equipment should be check and
maintained

• Wash your hands BEFORE: entering workplace,

handling equipment, before filling up napkin
dispensers, eating
• Wash your hands AFTER: going to toilet, meal,
smoking, cleaning, handling wastes, removing
gloves, touching parts of the body, every patient
interaction, handling chemicals
• HOW TO WASH YOUR HANDS? Turn on tap, wet
CHEMICAL HAZARD
hands with warm water then apply liquid soap,
lather and rub at least for 20 seconds. Clean each - Labelling of all chemicals properly
nail, between each finger, front and back of the - Follow handling, storage requirements
hands up to the wrist then rinse off soap using - Use adequate ventilation
water pointing downwards. Dry hands using - Spill response procedures should be included in
disposable paper towel. Turn off the water tap the safety procedures
using another disposable paper towel. - MSDS should be available and reviewed by
laboratory personnel
 HEMATOLOGY 1 (LABORATORY)

Standard Waste Protocol (PHIL)

Disposal
• Flushing Down the Drain to the Sewer System
• Incineration
• Landfill Burial
• Recycling

- Public Law 11058

- ”An Act Strengthening Compliance with
ELECTRICAL HAZARD Occupational Safety and Health Standards and
- Use of adapters, gang plugs and extension cords Providing Penalties for Violations Thereof”
are prohibited - Goal: Provide all employees (clinical laboratory
- Stepping on cords, rolling heavy equipment over personnel included) with a safe work
cords should be prohibited environment
- Before repair or adjustment of electrical
equipment, unplug first the equipment making GOVERNMENT REGULATORY AGENCIES
sure that the hand is dry and no jewelry should ➢ Department of Labor: 29 Code of Federal
be present Regulations Parts 1900-1910
o Hazard Communication Standard
NEEDLE PUNCTURE o Hazardous Waste Operations
- Containers should be puncture proof o Occupational Exposure to bloodborne
- Improper disposal is the major cause of needle pathogens Standards
prick incident ➢ Department of the Interior, Environmental
- Replaced once the container is ¾ full Protection Agency: 40 Code of Federal
Regulations Parts 200-399
o Clean Air Act and Clean Water Act
PHYSICAL HAZARD
o Toxic Substances Control Act
- An agent, factor, or circumstance that can cause o Comprehensive Environmental
harm without contact. Response, Compensation and Liability
- It includes: Act (CERCLA)
o Ergonomic Hazard ➢ Voluntary Agencies/Accrediting Agencies
o Vibration Hazard o The Joint Commission
o Noise Hazard o College of American Pathologist
o Centers for Disease Control and
Prevention (CDC)
Segregation o Clinical and Laboratory Standards
Institutes
• Biodegradable
• Non-biodegradable
• Hazardous waste
o Special Waste
o Biological Waste
o Chemical Waste
o Radioactive Waste

WASTE DISPOSAL STANDARD

Blood Containing Waste
- Objects contaminated with blood should be
autoclaved before disposal
- Blood should be treated before disposal;
treatment involves the use of aldehyde, chlorine
compounds, phenolic compounds or thru
autoclaving before pouring down the sink with
running water
 HEMATOLOGY 1 (LABORATORY)

- Has 2 counting chambers (one at the top and

one at the bottom)
RBC COUNT - Every counting chamber contains:
- The number of RBCs in 1 liter or 1 microliter of o Primary square: 1 large square
blood ▪ area: 9mm2
- Manual RBC counts are rarely performed ▪ contains 9 secondary squares
because of the inaccuracy of the count and o Secondary squares: 9 squares
questionable necessity ▪ 1mm by 1mm
- Red blood cells are important for the delivery of ▪ 4 squares located at the corner
oxygen are called WBC square
o WBC square:
WBC COUNT ▪ Contains 16 tertiary squares
- The number of WBCs in 1 liter or 1 microliter of o RBC Square:
blood ▪ Central secondary square
- Utilized to indicate infection ▪ Contains 25 tertiary squares
- Important terms: ▪ Each tertiary squares contains
o Leukocytosis 16 quaternary squares
o Leukopenia ▪ Only 5 tertiary squares are
counted (4 corners and central
square)
o Dark lines:
• Hemacytometer ▪ Appear as double line or triple
• Thoma Pipet (for diluting blood) line under microscope
o Red cell pipette ▪ Separates the squares
▪ Contains red bead o Horizontal and vertical lines:
▪ Up to 101 units of volume ▪ Serve as a guide in counting cells
▪ 10 graduations (each graduation
is equivalent to 0.1)
▪ The stem of the pipette can
- Manual cell counting of WBCs and RBCs has the
contain a total volume of 1
same procedure, except in diluting fluids.
o White cell pipette
- Isotonic solution is used in counting RBCs to
▪ Contains white bead
avoid RBC lysis
▪ Up to 11 units of volume
- hypotonic solution is used in counting WBCs to
• Suction device
lyse RBCs and to easily count the WBCs
• Thick coverslip (glass)
- in automated machines, there are two different
• Cell Counter
pathways: RBC counting and WBC counting
• Diluting fluids o Note: you cannot count both of them in
the same pathway
HEMACYTOMETER
- “Heart of manual cell count” RBC DILUTING FLUIDS
- Make counting procedure organized and
• NSS
systematic
• 3.8% Na citrate
- Different Types
• Dacies
o Improved Neubauer (most commonly
• Hayem’s
used)
o Neubauer • Toisson’s
o Fuchs – Rosenthal • Bethell’s
o Speirs – Levy • Gower’s
o Tuerk’s
o Bass – Jones WBC DILUTING FLUIDS
• 1-3% acetic acid
Improved Neubauer • 1% HCl
• Turk’s diluting fluid (contains distilled water,
acetic acid, and gentian violet stain)

PROCEDURE FOR RBC COUNT

1. Draw blood up to 0.5 mark using the RBC Pipette.
o Note: if the blood was drawn too far
above the 0.5 mark, the procedure
should be repeated using a new pipette,
excess blood causes an inaccurate result.
2. Wipe the outside walls of the pipette with clean
gauze.
 HEMATOLOGY 1 (LABORATORY)

o Note: do not allow capillary attraction to 2. Wipe the outer walls of the pipette
draw fluid from the tip onto the gauze. o Note: do not allow capillary attraction to
Gauze tends to absorb the liquid portion draw fluid from the tip onto the gauze.
of the blood, causing an inaccurate Gauze tends to absorb the liquid portion
result. of the blood, causing an inaccurate
3. Dip the pipette into diluting fluid, and then draw result.
the diluting fluid into the pipette, slowly, until 3. Dip the pipette into diluting fluid, and then draw
the mixture reaches the 101 mark. Gently rotate the diluting fluid into the pipette, slowly, until
the pipette to ensure a proper amount of mixing. the mixture reaches the 11 mark. Gently rotate
The dilution is 1:200 the pipette to ensure a proper amount of mixing.
4. Remove the tubing from the pipette, and then The dilution is 1:20
mix it on a horizontal axis for 5 minutes. 4. Remove the tubing from the pipette, and then
5. Discard the first 3-4 drops of the diluted sample. mix it on a horizontal axis for 5 minutes.
6. Charge both sides of the hemocytometer o Note: to control the flow of the drops
counting chamber with a drop of the diluted place the index finger on the bore to
sample and allow to stand for few minutes. where the rubber tubing is fitted
7. While keeping the hemacytometer in a 5. Discard the first 3-4 drops of the diluted sample.
horizontal position, place it on the microscope 6. Charge both sides of the hemocytometer
stage. counting chamber with a drop of the diluted
8. Using HPO, count the red cells in the 5R’s squares sample and allow to stand for few minutes.
of the central secondary square. 7. While keeping the hemacytometer in a
9. Calculate the number of RBC per liter of each of horizontal position, place it on the microscope
the hemocytometer. Average the results and stage.
report this number. 8. Count the white blood cells in the 4 ‘W’ squares.
Using the L.P.O., the cells appear as round bodies
Simplified procedure with a definite outline. It doubtful whether an
1. Collect blood and place it in an EDTA tube object is a leukocyte or artefact, keep on moving
2. Using thoma pipette, aspirate the blood up to 0.5 the fine adjustment and take note of the well
mark of the stem defined outline or better still examine under the
3. Wipe the tip of the pipette to make sure that H.P.O. to verify.
there will be no excess blood
4. Aspirate the diluting fluid until the solution reach
its total volume
5. Mix or shake the pipette for 5-10 minutes
6. Discard the first 3-5 drops of the solution to get
rid of the diluting fluid
7. Place a cover slip on top of the counting chamber
8. Charge the solution on both counting chambers
of the hemacytometer
9. Incubate the hemacytometer in a petri dish with
wet tissue paper inside for 5-10 minutes so that
the solution will remain moist
10. Count the cells under microscope
a. Scanner: primary square
b. LPO: secondary square
c. HPO: tertiary square

PROCEDURE FOR WBC COUNT

1. Using the WBC Pipette, aspirate blood up to 0.5
mark of the capillary stem of the pipette • All cells inside the squares are included
o Note: if the blood was drawn too far • Cells touching the upper and left lines are
above the 0.5 mark, the procedure included
should be repeated using a new pipette, • Cells touching the lower and right lines are not
excess blood causes an inaccurate result. included
 HEMATOLOGY 1 (LABORATORY)

Example:
Formula for RBC and WBC count 𝑢𝑛𝑐𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 𝑥 100
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑛𝑅𝐵𝐶 𝑝𝑒𝑟 100𝑊𝐵𝐶 + 100
𝑎𝑣𝑒. 𝑛𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
𝑡𝑜𝑡𝑎𝑙 𝑐𝑜𝑢𝑛𝑡 =
𝑎𝑟𝑒𝑎 (𝑚𝑚2 )𝑥 𝑑𝑒𝑝𝑡ℎ (0.1𝑚𝑚) 13 𝑥 100 1300
= = = 𝟏𝟏. 𝟔𝟏𝒙𝟏𝟎𝟗 /𝑳
12 + 100 112
EXAMPLES FOR RBC COUNT
Example 1:
𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 NORMAL VALUES FOR RBC COUNT
RBC count =
𝑎𝑟𝑒𝑎 (𝑚𝑚2 ) 𝑥 𝑑𝑒𝑝𝑡ℎ (0.1) • Female: 3.6 – 5.6 x 1012/L
=
400 𝑥 200
= 𝟒, 𝟎𝟎𝟎, 𝟎𝟎𝟎/µ𝑳 𝑜𝑟 𝟒𝒙𝟏𝟎𝟏𝟐 /𝑳 • Male: 4.2 – 6.0 x 1012/L
0.2𝑚𝑚2 𝑥 0.1𝑚𝑚 • At Birth: 5.0 – 6.5 x 1012/L

EXAMPLES FOR WBC COUNT

NORMAL VALUES FOR WBC COUNT
Example 1:
• Male and Female: 4.0 -11.0 x 109/L
WBC count =
𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 • At birth: 10.0 – 30.0 x 109/L
𝑎𝑟𝑒𝑎 (𝑚𝑚2 ) 𝑥 𝑑𝑒𝑝𝑡ℎ (0.1)
96 𝑥 20
= = 𝟒𝟖𝟎𝟎/𝒎𝒎𝟑 𝑜𝑟 𝟒𝟖𝟎𝟎/µ𝑳
4𝑚𝑚2 𝑥 0.1𝑚𝑚
𝑜𝑟 𝟒. 𝟖𝒙𝟏𝟎𝟑 /µ𝑳 𝑜𝑟 𝟒. 𝟖𝒙𝟏𝟎𝟗 /𝑳 RBC COUNT
• Highest in the morning and at its lowest in the
Example 2: evening
𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 • Increased: myeloproliferative disorder such as
WBC count = polycythemia
𝑎𝑟𝑒𝑎 (𝑚𝑚2 ) 𝑥 𝑑𝑒𝑝𝑡ℎ (0.1)
120 𝑥 20 • Decreased: anemic cases, bleeding
= = 𝟔𝟎𝟎𝟎/𝒎𝒎𝟑 𝑜𝑟 𝟔𝟎𝟎𝟎/µ𝑳
4𝑚𝑚2 𝑥 0.1𝑚𝑚
𝑜𝑟 𝟔𝒙𝟏𝟎𝟑 /µ𝑳 𝑜𝑟 𝟔𝒙𝟏𝟎𝟗 /𝑳 WBC COUNT
➢ Leukocytosis
o Bacterial Infections
o Appendicitis
• Dust and fingerprints may cause difficulty in
o Leukemia
distinguishing the cells
o Pregnancy
• Diluting fluid should be free of contaminants
o Uremia
• If the count is low, a greater area may be counted
o Ulcers
• Chamber must be charged properly to ensure
➢ Leukopenia
accurate count o Viral infection
• Allow cells to settle for 10mins before counting o Brucellosis
o Typhoid fever
OTHER SOURCES OF ERROR FOR RBC COUNT o Rheumatoid arthritis
• Use of other, more accurate manual RBC o cirrhosis
procedures, such as the microhematocrit and
hemoglobin concentration, is desirable when
automation is not available

OTHER SOURCES OF ERROR FOR WBC COUNT

• N-RBC causes false elevation of WBC count
• Note: The accuracy of the manual WBC count can
be assessed by performing a WBC estimate on a
Wright-stained peripheral blood film made from
the same specimen

N-RBCs
- Nucleated red blood cells
- Falsely counted as WBC
- Not lysed by WBC diluting fluids
- Correct the WBC count:
o Adults: if there are >5 nRBC/100 cells on
diff. count
o Newborns: if there are >10 nRBC/100
cells on diff. count
𝑢𝑛𝑐𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 𝑥 100
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑛𝑅𝐵𝐶 𝑝𝑒𝑟 100𝑊𝐵𝐶 + 100
 HEMATOLOGY 1 (LABORATORY)

Procedures
- Hemoglobin is one of the most important 1. Introduce 0.1 N HCl solution up to 2 mark of the
component of the RBC Sahli graduated tube
- It is responsible for carrying and binding oxygen 2. Using the Sahli pipette, draw blood up to 0.02 cc
among RBCs mark. Observe the same technique as in the use
- Without hemoglobin, red cells are nonfunctional of WBC or RBC pipette
- It is a globular protein that weighs about 68,000 3. Dispense the measured volume of the blood into
Daltons, which accounts for about 1/3 of the the graduated tube. Gently draw up and down to
RBCs total weight rinse the pipette and also to mix the resulting
- Hemoglobin is made up of heme and globin colloidal suspension
o Heme is a mixture of protoporphyrin IX o Once the red blood cell comes into
and iron contact with the HCL, it will be lysed and
o For every heme, it carries 1 mole of it makes the solution brown in color.
oxygen 4. Thoroughly mix with the use of the stirrer and
- Each hemoglobin molecule is made up of 4 allow to stand for 5 minutes
subunits 5. Place the Sahli graduated tube in a standard
o Every subunits is made up of heme and comparator block
globin
o Therefore, there are 4 subunits of heme
and 4 subunits of globin for every
hemoglobin molecule
o Heme and globin should always come in
pair
- Hemoglobin can also carry and transport carbon
dioxide 6. Add distilled water drop by drop, stirring after
o By binding carbon dioxide, hemoglobin each addition and compare with the comparator
can now act as a buffer (help in block until the color of the solution is the same
balancing out pH level) as with the comparator block
7. When the resulting solution compares with the
color standard in the comparator block, get the
- Also referred to as measurement of hemoglobin reading either from the grams or percentage
- Methodologies of measuring hemoglobin scale based on the markings of the Sahli
o Colorimetric methods such as acid graduated tube
Hematin and cyanmethemoglobin 8. Write your result (expressed in g/dL)
o There will be changes in the color of the
solution CYANMETHEMOGLOBIN
o Additional method: alkali hematin - Also known as hemiglobincyanide
- It is important that the red cells are lysed before - Standard approved by the Clinical and
measuring hemoglobin, whether it is manual or Laboratory Standards Institute
automated, so that hemoglobin will come out o Widely accepted internationally
from the cells o Manual or automated method
o In automated method, RBC counting and - An aliquot of blood is mixed with drabkin so that
hemoglobin measurement cannot be the hemoglobin will be converted to
performed on the same chamber cyanmethemoglobin, which results to changes in
color
ACID HEMATIN o It will be read spectrophotometrically at
Materials and Instruments 540 nm wavelength
o The color intensity is compared with a
• Specimen: Capillary or unclotted venous blood
known standard
• Hemoglobinometer or Ohmmeter set consisting o It is mathematically converted
of: (computation is based on the value of
o Comparator block or color standard the drabkin reagent)
o Sahli pipette: - Reagent: Drabkin (an alkaline solution)
▪ Straight pipette - Beer’s Law: the concentration of the unknown is
▪ Has single mark (20 [Link]. or directly proportional to its absorbance and
0.02 cc.) inversely proportional to the transmittance of
o Sahli graduated tube: calibrated grams light
on one side of the scale and percentage - It is the method of choice because
on the opposite scale o it is stable
o Stirrer: used to mix instead of shaking to o standards and solutions are readily
minimize bubble on the surface of the available
solution o it can measure all forms or derivatives of
o Dropper hemoglobin except for sulfhemoglobin
o Distilled water o it could be easily read using a
o 0.1 N HCl solution spectrophotometer at 540 nm
 HEMATOLOGY 1 (LABORATORY)

Components of Drabkin’s reagent

• Potassium ferricyanide – hemoglobin is - Higher in the morning and lower in the evening
converted to become methemoglobin - Carries oxygen from the lungs to the tissues and
• Potassium cyanide – methemoglobin is takes carbon dioxide
converted to become cyanmethemoglobin - Screen for anemia and may detect RBC
• Non-ionic detergent – for lysing RBCs breakdown/hemolytic anemia
• Dihydrogen potassium phosphate – allows the - Higher in high altitudes (oxygen level is low)
solution to be read spectrophotometrically - Increase in strenuous muscular activity

Principle LOW HEMOGLOBIN

Potassium ferricyanide potassium cyanide • Anemia
Hb (Fe+2) → methemoglobin (Fe+3) → cyanmethemoglobin • blood loss
- Absorbance of cyanmethemoglobin at 540 nm is • Iron, folate, and vitamin B6, B12 deficiencies
directly proportional to the Hb concentration
- Uses a standard curve HIGH HEMOGLOBIN
- Sodium Lauryl Sulphate (SLS) – it does not • Sickle cell anemia
generate toxic products when converting
• Thalassemia
hemoglobin to SLS-methemoglobin
• Transfusion reaction
• Hemolysis
Materials and Instruments
• Dehydration
• Anticoagulated blood (venous blood) • Polycythemia vera
• Test tubes • High altitude
• Pipette
• Spectrophotometer
NORMAL HEMOGLOBIN VALUES
• Drabkin’s reagent
• Parafilm • At birth: 15-20 g/dL
• Women: 12-16 g/dL
Procedures • Men: 13-18 g/dL
1. Place 5 cc of Drabkin’s reagent into a test tube
2. Using the Sahli pipette, draw blood to 0.02 cc SOURCES OF ERROR
mark. Make sure to wipe the outer wall of the 1. Cyanmethemoglobin reagent is sensitive to light.
pipette, and then dispense the blood to the test It should be stored in a brown bottle or in a dark
tube place.
3. Gently draw up and down to rinse the pipette 2. A high WBC count (greater than 20 x 109/L) or a
and also to mix the resulting colloidal high platelet count (greater than 700 x 109/L) can
suspension. cause turbidity and a falsely high result. In this
o Note: the pipette should be rinsed case the reagent-sample solution can be
thoroughly with the reagent to ensure centrifuged and the supernatant measured.
that no blood remains 3. Lipemia also can cause turbidity and a falsely
4. Cover and mix well by inversion. Let it stand for high result. It can be corrected by adding 0.01 mL
5 minutes of the patient’s plasma to 5 mL of the
5. Transfer the mixture to a cuvette cyanmethemoglobin reagent and using this
6. Set the spectrophotometer to 100% solution as the reagent blank.
transmittance at the wavelength of 540 nm, 4. Cells containing Hb S and Hb C may be resistant
using a cyanmethemoglobin reagent as a blank to hemolysis, causing turbidity; this can be
7. Continue reading the patient’s sample, and corrected by making a 1:2 dilution with distilled
record the percentage transmittance. water (1 part diluted sample plus 1 part water)
8. The hemoglobin concentration is obtained from and multiplying the results from the standard
a calibration curve prepared with the use of curve by 2.
standards (stable standard cyanmethemoglobin 5. Abnormal globulins, such as those found in
solution of concentration representing 1:250 patients with plasma cell myeloma or
dilution of whole blood containing 5, 10, and 15 Waldenström macroglobulinemia, may
grams of hemoglobin/100mL are available precipitate in the reagent. If this occurs, add 0.1g
commercially for calibration purposes and for of potassium carbonate to the
periodic check-up on the accuracy of the cyanmethemoglobin reagent. Commercially
colorimeter) available cyanmethemoglobin reagent has been
modified to contain KH2PO4 salt, so this problem
ALKALI HEMATIN is not likely to occur.
6. Carboxyhemoglobin takes 1 hour to convert to
- additional or other method in measuring
cyanmethemoglobin and theoretically could
hemoglobin
cause erroneous results in samples from heavy
- Same principle and procedure with acid hematin,
smokers. The degree of error is probably not
but different solution is used (0.1 N NaOH)
clinically significant, however.
- It is not used among newborn patients
7. Because the hemoglobin reagent contains
o Hemoglobin F resist alkaline solution
cyanide, it is highly toxic and must be used
cautiously. Consult the safety data sheet
 HEMATOLOGY 1 (LABORATORY)

supplied by the manufacturer. Acidification of

cyanide in the reagent releases highly toxic
hydrogen cyanide gas. A licensed waste disposal
service should be contracted to discard the
reagent; reagent-sample solutions should not be
discarded into sinks.
8. Commercial absorbance standards kits are
available to calibrate spectrophotometers.
9. Handheld systems are commercially available to
measure the hemoglobin concentration. An
example is the HemoCue (HemoCue, Inc., Brea,
CA) in which hemoglobin is converted to
azidemethemoglobin and is read
photometrically at two wavelengths (570 nm
and 880 nm).

OTHER METHODS
➢ Gasometric (Van Slyke oxygen capacity) – based
on the concentration of oxygen for every gram of
hemoglobin
o 1gm Hb = 1.34 mL O2
o Lower than 1.34 may indicate that the
hemoglobin concentration of the patient
is low
➢ Specific Gravity method (Copper sulfate) –
usually used in mobile blood donation
o [Link] = 1.053
o approximation of the patient’s
hemoglobin
o a drop of donor’s blood is added to the
copper sulfate solution in a 30mL
container
o when the drop of blood reaches the
bottom of the beaker within 15 seconds,
it means that the blood has enough
hemoglobin concentration
o when the drop of blood does not reach
the bottom of the beaker within 15
seconds, it means that the blood has low
hemoglobin concentration
▪ do not accept the blood donor
o in a 30mL container, 25 specimens can
be used
o copper sulfate should be changed every
single day
➢ Chemical (Kennedy’s, Wong’s) – based on the
concentration of iron
o 1gm Hb = 3.47 mg Iron
 HEMATOLOGY 1 (LABORATORY)

o Chance of having trapped plasma in

between RBCs
- Ratio of the volume of packed red blood cells to
▪ Amount of plasma that still
the volume of whole blood
remains in RBC portion
- “Packed Cell Volume” or PCV
▪ The amount or level of
o Referring to RBCs alone
centrifugation is lower
- Reported as percentage or L/L (Ex. 35% or 0.35)
▪ Results to higher hematocrit
- Deals with the amount of space occupied by the
value
RBCs in a given blood volume
- Useful in evaluating cases of anemia
Wintrobe & Landsberg: double oxalate
- 115mm in length
- The bore is 3mm in diameter
- Marks:
o 0 to 10 upward mark is used for
measuring hematocrit
o 0 to 10 downward mark is used for
measuring ESR
- Do not include the slanted portion of the slanted
packed cells when measuring hematocrit

Principle
- Whole blood is centrifuged to determine packing
of red blood cells
- After centrifugation of blood, it forms different
layers according to their densities

Materials and Instruments

• Wintrobe tube with rubber caps
• Long stem pipette
• Centrifuge
• Anticoagulated blood (this procedure needs
about 3.0 mL of whole blood)
• Cotton
Normal Values
• At birth: 45-60% Procedure
• Female: 36-48% 1. Mix the blood
• Male: 40-55% 2. With the use of a long stem pipette, fill the
Wintrobe tube to the 10 mark.
o Note: No air bubbles should be present
on the surface of the blood, it may be
removed by touching it with a tissue
- In manual method, the actual value of the paper or with a pipette. If the bubbles
hematocrit can be measured are present in the middle of the tube,
- In automated analyzers, hematocrit values are aspirate the blood and proceed to step
just computed numbers or values no. 2 again.
o Automated machines do not have any 3. Centrifuge the blood for 30 minutes
centrifuge inside 4. Read the height of the packed red blood cells on
the scale at the right side of the tube, which is
MACROHEMATOCRIT METHOD graduated from 0-10 cm from the bottom to top,
- Whole blood is transferred to a Wintrobe tube read upward.
and centrifuged for 30 minutes at 2000-3000rpm
- Perform venipuncture to obtain large volume of Other tubes used
blood • Van Allen: sodium oxalate
- Disadvantages: • Haden: sodium oxalate
o More time consuming • Sanford-Magath: sodium oxalate
o Larger volume of blood is needed • Bray: heparin
 HEMATOLOGY 1 (LABORATORY)

MICROHEMATOCRIT METHOD
- Perform skin puncture to obtain peripheral ➢ Increased
blood (lesser amount of blood) o Insufficient centrifugation
- Specimen: EDTA blood, capillary blood o Inclusion of buffy coat
o If EDTA blood is used, transfer the blood o Disorders such as sickle cell anemia,
in a capillary tube with blue band macrocytic anemias, hypochromic
o Red band capillary tube has heparin anemias
anticoagulant o Dehydration
o Blue band capillary tube has no o Hemoconcentration during specimen
anticoagulant collection
- Sample is transferred to a capillary tube and ➢ Decreased
centrifuged to a microhematocrit centrifuge o Improper sealing of capillet
- centrifuged for 5 to 10 minutes at 10000- o Increased conc. of anticoagulants
15000rpm o Prolonged centrifugation
- has better packing of RBCs o Acute blood loss
- trapped plasma: amount of plasma that still o Difficulty in collecting blood, which may
remains in RBC portion after microhematocrit introduce interstitial fluid in the blood
has been spun
o 1-3% higher
o Increased: macrocytic anemia,
spherocytosis, thalassemia,
hypochromic anemia, sickle cell anemia

Materials and Instruments

• Heparinized capillary tube
• Microhematocrit centrifuge
• Microhematocrit reader
• Lancet
• Cotton
• Alcohol

Procedure
1. After necessary preparation, make a capillary
puncture and produce a rounded drop of blood
2. In a horizontal position, put one end of capillary
rube in the drop of blood and fill the tube about
2/3 full
o Note: the tube will be filled by capillary
action. If using tubes with a colored ring
at one end, fill from opposite end
3. Seal one end of the capillary tube with the clay
by placing the dry end of the tube into the clay in
a vertical position
4. After sealing with wax, seal it again with wax
5. Assemble the tube in microhematocrit
centrifuge in such a way that the unsealed end is
nearest the center of the centrifuge
6. Spin at 10000 to 15000 rpm for 5 minutes
7. Using the microhematocrit reading device,
determine the HCT
o Note: buffy coat should not be included
in the reading
8. The reading on the window of the hematocrit
reader corresponds to the hematocrit value
 HEMATOLOGY 1 (LABORATORY)

- 0 to 200 downward markings

- Modified Westergren
- Also called as “sed rate”
o EDTA anticoagulant is used.
- Basically measures the rate of settling of RBCs
o It must be diluted to the ratio of 4
o It is the distance travelled by the RBCs in
volume of blood to 1 volume of 0.9%
millimeters (mm) per hour
sodium chloride
- Does not involve centrifugation
- Non-specific measurement used to detect and
monitor an inflammatory response to tissue
injury
- Low specificity and sensitivity
- Only used as a screening procedure in detecting
inflammation
- Distance in millimeters at which the RBCs fall in
1 hour
- It should be incorporated with other laboratory
procedures, such as CRP, CBC, etc., for suspected Procedure
inflammation 1. Mix the blood citrate or blood-EDTA-saline
- It is also useful in the diagnosis of polymyalgia mixture thoroughly.
rheumatica (affects the muscles) and temporal 2. Aspirate a bubble-free specimen into a clean and
arteritis dry Westergren pipette (dispet). Fill to the zero
mark. Do not pipette by mouth.
Principle 3. Place the pipette into the vertical rack at 20°C to
- When anticoagulated blood is allowed to stand 25°C in an area free from vibrations, drafts, and
at room temperature undisturbed for a period of direct sunlight.
time (1 hour), the red blood cells settle toward 4. After 60 minutes, read the distance in
the bottom of the tube millimeters from the bottom of the plasma
- The phenomenon depends on an meniscus to the top of the sedimented
interrelationship of variables, such as the plasma erythrocytes.
protein composition, the concentration of 5. Record the value as millimeters in 1 hour.
erythrocytes, and the shape of the erythrocytes.
- Red cells repel one other because they are WINTROBE
negatively charged - Oxalate-anticoagulated whole blood
o The repulsive forces are then partially or
totally counteracted if there are
increased quantities of positively
charged plasma proteins in the
specimen
o Acute phase reactants – increased
proteins during inflammation
o Albumin is not an acute phase reactant
- One of the important factors to be considered in
ESR is the chemical constituents of the plasma, Procedure
particularly proteins 1. Use fresh blood collected in EDTA anticoagulant.
o Positively charged heavy proteins will A minimum of 2 mL of whole blood is needed.
push down the RBCs a lot faster 2. After mixing the blood thoroughly, fill a Pasteur
o The more red cells are pushed down, the pipette using a rubber pipette bulb.
higher the ESR 3. Place the filled pipette into the Wintrobe tube
o The higher the ESR, the more it until the tip reaches the bottom of the tube.
correlated with inflammation 4. Carefully squeeze the bulb and expel the blood
into the Wintrobe tube while pulling the Pasteur
STAGES OF ESR pipette up from the bottom of the tube. There
must be steady, even pressure on the bulb to
➢ Initial rouleaux formation: 10 minutes
expel blood into the tube as well as continuous
o The more proteins in the plasma, the
movement of the pipette up the tube to prevent
more red cells will form rouleaux, and
the introduction of air bubbles into the column
the more red cells will settle down
of blood.
➢ Rapid settling of RBCs: 40 minutes
5. Fill the Wintrobe tube to the 0 mark.
➢ Final sedimentation of RBCs: 10 minutes
6. Place the tube into a Wintrobe rack (tube holder)
and allow to stand undisturbed for 1 hour at
room temperature. The rack must be perfectly
WESTERGREN METHOD level and placed in a draft-free room.
7. Record the number of millimeters the red blood
- Ratio is 4 volume of blood to 1 volume of sodium
cells have fallen. Read the tube from the bottom
citrate (black top citrated tubes)
of the plasma meniscus
- Most commonly used
- Taller tubes (300mm in length)
 HEMATOLOGY 1 (LABORATORY)

o Some RBCs may change their shape

when kept at room temperature for
WESTERGREN TUBE WINTROBE TUBE
more than 4 hours
Length 300 mm +/- 0.5 mm 115 mm or 11.5 cm
Internal Bore 2.65 mm +/- 0.5 mm 3 mm • Bubbles in the column of blood
External Bore 5.5 mm +/- 0.5 mm • Clotted specimen
Graduation/
200 100 • Tubes must not be subjected to vibrations
Markings

This is not yet widely acceptable since it cannot

completely finish the stages of sedimentation. The only
advantage is smaller blood sample is needed and uses
shorter time.
➢ Ves-Matic System (Diesse, Inc., Hialeah, FL) –
optoelectronic sensor which measures the
change in opacity of a column of blood as
sedimentation of blood progresses.
➢ Sedimat 15 (Polymedco, Cortlandt Manor, NY) –
uses the principle of infrared measurement
➢ ESRSTATPLUS System (Hema Technologies,
Lebanon, NJ) – based on centrifugation

• Increased anticoagulant (low ESR, inhibits Technically, there is no such thing as low ESR because
rouleaux formation) normal values start at zero
• Use of sodium/potassium oxalate and heparin ➢ Westergren
o Shrink the RBCs, making the ESR falsely o M <50yo = 0-15 mm
elevated o M >50yo = 0-20 mm
• Significant change in the temperature of the o F <50yo = 0-20 mm
room o F >50yo = 0-30 mm
• Slight tilt of the pipette o Children = 0-10 mm
o If the tube is tilted 3°, it will result to 30% ➢ Wintrobe
increase or error in the ESR o Male = 0-9 mm
• Blood specimens must be analyzed within 4 o Female = 0-20 mm
hours of collection if kept at room temperature o Children = 0-13 mm
 HEMATOLOGY 1 (LABORATORY)

- Complete hemolysis will be observed in tube

numbers 16 and 17 (solution is totally red in
- A measure of the ability of the RBC to take up
color)
fluid without lysing
- In cases of spherocytes
- Employed to help diagnose different types of
o initial hemolysis is observed in tube
anemia, in which the physical properties of red
numbers 23 or 24
cells are altered.
o complete hemolysis is observed in tube
- Used in the laboratory to detect spherocytosis
numbers 20 or 21
(spherocytes: red cells that do not have pallor
- in some cases that the OFT is high, it could
area)
indicate spherocytosis or autoimmune
- Test on how capable the red cells in resisting lysis
hemolytic anemia (AIHA)
o there is hemolysis
Principle o perform Coomb’s testing or Direct
- Red cells suspended in hypotonic solution of Antiglobulin Testing (DAT) – tests for
sodium chloride take up water, swell, become antibody activity
spheroidal, and after reaching the critical o positive result
volume, eventually burst.
- Cells are exposed to varying concentrations of Data and Results
hypotonic solutions
• Check for initial and complete hemolysis
- Cells that are thicker than normal have a
• Initial hemolysis: tinge of pinkness in the
decreased surface/volume ratio
supernatant and some cells at the bottom of the
o They are easily susceptible to lysis
tube
- Thin or flat cells have an increased
• Complete hemolysis: clear, red supernatant with
surface/volume ratio
no microscopic cells at the bottom of the tube
o Has more surface area
o The more the red cell can resist lysis
o Red cells do not have enough
hemoglobin inside

Materials
• 12 Kahn tubes or ordinary test tubes
• Test tube rack
• 0.5% NaCI
• Distilled water
• Capillary pipette or Sahli pipette
• Anticoagulated blood (Heparinized blood)

Procedures (Sanford Method)

1. Arrange 12 tubes in a rack. Label it from number
25 up to number 14, starting from the left.
2. With the capillary or Sahli pipette, place in each
tube the number of drops of stock 0.5% NaCI
indicated by the number of the test tube.
o Note: Hold the pipette at the same angle
to ensure uniformity of drops
3. With the same pipette, add into each tube the
number of drops of distilled water required to
bring the value in each tube to 25 drops.
4. Mix by lateral tapping
5. Draw blood into a Sahli-Hellige pipette up to 20 • Hemolysis – tube number with initial and
ul or 0.02 mL and dispense in each test tube complete hemolysis x 0.02 mL (amount of blood
6. Mix by inverting the tube once or twice in each tube)
7. Stand for two hours • Example:
o Initial hemolysis:
tube number 21 x 0.02 = 0.42%
• Increased OFT (decrease resistance): found in
hemolytic anemias and hereditary spherocytosis
and whenever spherocytes are found
• Decreased OFT (increase resistance): occurs
following splenectomy and in liver disease, sickle
- Normal expected lysis of the RBCs will then be
cell anemia, Iron Deficiency Anemia, and
seen in tube numbers 21 and 22 (recorded as
thalassemia
initial hemolysis)
o Occurs when red cells are hypochromic
- In initial hemolysis, red cells are not totally lysed
(has more than 1/3 pallor area), usually
yet
observed in IDA
 HEMATOLOGY 1 (LABORATORY)

Procedures
- Also known as polychromatic erythrocytes 1. Place three drops of reticulocytes stain in a small
- Last immature RBC stage that are normally test tube.
present in the peripheral blood 2. Add three drops of well mixed whole blood to
- Last stage in erythropoiesis capable of the tube containing the stain.
synthesizing hemoglobin 3. Mix the tube and allow to stand at room
- They stay in the bone marrow for about 1-2 days temperature or incubate at 37°C for 15 minutes
before they go to the blood 4. After 15 minutes, mix the contents of the tube
o They will stay in the blood for 1 day well and prepare smears and allow it to dry.
before they totally mature as red cells 5. Examine the smear under the oil
- Contains remnant cytoplasmic RNA and 6. Count 1000 RBCs and enumerate, out of those
organelles such as mitochondria and ribosomes 1000 RBCs, how many of those cells showed
o Perform supravital staining to see the bluish filaments, granules, or dots inside the cells
remnants o Approximately, in every oil immersion
field, there are about 200 RBCs
o Simply count and observe five oil
RETICULOCYTE COUNT immersion fields then multiply it by 5
- An indicator of the rate of erythrocyte
production
- Use of supravital stains using methylene blue or
brilliant cresyl blue
o Staining the cells while they are living
o Before preparing the smear, allow first
the blood to contact with the stain, so
that the cell can take up the stain
o In Wright’s stain, reticulocytes appear
polychromatic (pink, gray and blue)
- The count is expressed as a percentage of total
erythrocytes
- To determine the bone marrow’s activity Reference Range
whether a person has an effective erythropoiesis
or not • Adult: 0.5-1.5%
- Indicate the ability of the bone marrow to • Newborn: 2.0-6.0%
increase RBC production in anemia due to blood
loss or excessive RBC destruction Sources of errors
- It can also help in determining whether the bone • Blood and stain not mixed before films are made.
marrow is responding in cases of hemolytic o The specific gravity of the reticulocyte is
anemia lower than the matured RBCs
• Very anemic or polycythemic patient
Principle o The proportion of the stain and of the
- Whole blood, anticoagulated with EDTA, is blood should be adjusted accordingly
stained with a supravital stain • Moisture in air and poor drying of slide
- Any non-nucleated red blood cell that contains • Other RBC inclusions that stain supravitally
two or more particles of blue-stained include Heinz, Howell-Jolly, and Pappenheimer
granulofilamentous material after new bodies
methylene blue staining is defined/counted as a
reticulocyte
RELATIVE RETICULOCYTE COUNT
number of reticulocytes
𝐑𝐞𝐭𝐢𝐜𝐮𝐥𝐨𝐜𝐲𝐭𝐞𝐬 (%) = 𝑥 100
1,000 (RBCs counted)

Example 1:
There are 15 reticulocytes counted in 8 months old
infant. Calculate the relative reticulocyte count.
15
𝐑𝐞𝐭𝐢𝐜𝐮𝐥𝐨𝐜𝐲𝐭𝐞𝐬 (%) = 𝑥 100 = 𝟏. 𝟓%
Materials 1,000
• Slides Interpretation: NORMAL
• Anticoagulated blood
• Supravital stains: Miller Disc
o Brilliant cresyl blue - Design to reduce the labor-intensive process of
o New methylene blue counting reticulocytes
• Pipette - Disc is inserted into the eyepiece of the
• Microscope microscope
• Cedarwood oil
 HEMATOLOGY 1 (LABORATORY)

o But that percentage may then be

incorrect because there may be changes
in the percentage caused by the changes
in the total number of RBCs in the blood
o Because of these changes, we can then
correct the reticulocyte count
- In CRC, we correct the reticulocyte count if the
hematocrit of the patient is affected
- A correction factor is used, with the average
normal hematocrit considered to be 45% or 0.45
- Red cells are counted in the smaller square (B)
- Retics are counted in the larger square (A) Example 4:
- Minimum of 112 red cells should be counted in If an adult male has a hematocrit of 30% (0.30 L/L) and a
the small square reticulocyte count of 3%, the corrected reticulocyte
o 112 red cells in the small square are count would be
roughly equivalent to 1008 red cells 0.30
- The area of the smaller is 1/9 of the area of the 𝐂𝐑𝐂 = 3% x = 0.02 x 100 = 𝟐%
0.45
larger square (reciprocal: 9)
no. of reticulocytes in a square 𝐀 RETICULOCYTE PRODUCTION INDEX (RPI)
(large square) x 100
𝐑𝐞𝐭𝐢𝐜𝐮𝐥𝐨𝐜𝐲𝐭𝐞𝐬 (%) = corrected reticulocytes count in %
no. of RBCs in square 𝐁 (small square) x 9 𝐑𝐏𝐈 =
maturation time in days
Example 2: - Measures erythropoietic activity when stress
If 15 reticulocytes are counted in the large square and reticulocytes are present
112 red blood cells are counted in the small square, - shift or stress reticulocytes – early release of
15 x 100 reticulocytes (< 1 day)
𝐑𝐞𝐭𝐢𝐜𝐮𝐥𝐨𝐜𝐲𝐭𝐞𝐬 (%) = = 𝟏. 𝟓% o they are released into the blood
112 x 9
prematurely to compensate for anemia
ABSOLUTE RETICULOCYTE COUNT (ARC) o they will stay for about 2-3 days more
before they totally mature
reticulocytes (%) x RBC Count (x 1012 /L) - if reticulocyte is counted in cases of hemolytic
𝐀𝐑𝐂 =
100 anemia, and when the bone marrow is
or responding well, expect that the reticulocyte
reticulocytes (%) x RBC Count
count is high and elevated
𝐀𝐑𝐂 = 𝑥 1000 = (𝑣𝑎𝑙𝑢𝑒) x 109/L
100
Patient’s Hematocrit Correction Factor
- The actual number of reticulocytes in 1 liter of Value (%) (Maturation Time, Days)
blood 40-45 1
- It will reflect the true number or true value of 35-39 1.5
reticulocytes for every liter of blood 25-34 2
15-24 2.5
- Values between 20 x 109/L and 115 x 109/L are <15 3
within the reference interval for most
populations
Example 5:
If the corrected reticulocyte count is 2.0% and the
Example 3: patient’s hematocrit is 0.30 L/L, the RPI is
If a patient’s reticulocyte count is 2% and the RBC count
is 2.20 x 1012/L, the ARC is calculated as follows (note that corrected reticulocytes count in % 2.0
𝐑𝐏𝐈 = = = 𝟏. 𝟎
the calculated result has to be converted from 1012/L to maturation time in days 2.0
109/L):
Interpretation of Results
2 x (2.20 x 1012 /L)
𝐀𝐑𝐂 = = 𝟒𝟒 𝐱 𝟏𝟎𝟗 𝐋 • RPI > 3: there is an adequate erythropoietic
100
response from the bone marrow response
or • RPI < 2: there is an inadequate erythropoietic
2 x 2.20 response from the bone marrow
𝐀𝐑𝐂 = 𝑥 1000 = 𝟒𝟒 𝐱 𝟏𝟎𝟗 𝐋
100

CORRECTED RETICULOCYTE COUNT (CRC)

- Analyzers evaluate reticulocytes using optical
Patient ′ s Hematocrit scatter or fluorescence after the RBCs are
𝐂𝐑𝐂 = reticulocytes (%) x
Standard Hematocrit treated with fluorescent dyes or nucleic acid
- In specimens with low hematocrit, percentage of stains to stain residual RNA in the reticulocytes
reticulocytes may be falsely elevated as the - The percentage and absolute counts are further
whole blood contains fewer RBCs provided by automated analyzers
- Usually, reticulocyte is expressed as the
percentage of the total number of circulating
RBCs.
 HEMATOLOGY 1 (LABORATORY)

Example 3:
If the hemoglobin = 16 g/dL and the HCT = 48%, what is
- Still included in complete blood count (both
the MCHC?
manual and automated)
- Help in picturing out the morphological 16
𝐌𝐂𝐇 = 𝑥 100 = 𝟑𝟑. 𝟑 𝐠/𝐝𝐋
characteristics of the RBCs 48
- The values obtained are used in classifying
different types of anemia

MEAN CELL VOLUME (MCV)

HCT (%)
𝐌𝐂𝐕 = 𝑥 10
RBC count (x 1012 /L)
- Average volume of red blood cells expressed in
femtoliters (fL)
- Volume directly reflects the size of the RBC
- Volume:
o Normocytic: 80 to 100 fL
o Microcytic: lower than 80 fL
o Macrocytic: higher than 100 fL

Example 1:
If the HCT = 45% and the RBC count = 5 x 1012/L, what is
the MCV?
45
𝐌𝐂𝐕 = 𝑥 10 = 𝟗𝟎 𝐟𝐋
5

MEAN CELL HEMOGLOBIN (MCH)

HGB (g/dL)
𝐌𝐂𝐇 = 𝑥 10
RBC count (x 1012 /L)
- Average weight of hemoglobin in a red blood cell
expressed in picogram (pg)
- Not usually used in classifying anemia
- Normal value: 26-32 pg

Example 2:
If the hemoglobin = 16 g/dL and the RBC count = 5 x
1012/L, what is the MCH?
16
𝐌𝐂𝐇 = 𝑥 10 = 𝟑𝟐 𝐩𝐠
5

MEAN CELL HEMOGLOBIN

CONCENTRATION (MCHC)
HGB (g/dL)
𝐌𝐂𝐇𝐂 = 𝑥 100
HCT (%)
- Average concentration of hemoglobin in each
individual red blood cell expressed in grams per
deciliter (g/dL) or percentage
- Much better in classifying anemia
- Normal value: 32-36 g/dL
o RBCs will not contain more than 36 g/dL
of hemoglobin
o Hyperchromic: red cells do not have
pallor area; it does not mean that the red
cell has an elevated hemoglobin
concentration
- Causes of elevated hemoglobin concentration:
o Computation is not correct
o Presence of spherocytes (proceed with
OFT to check for spherocytes)
o Conditions like cold agglutinins (collect
another blood and warm it before
testing)
 HEMATOLOGY 1 (LABORATORY)

• Automated slide making (Spunner’s method)

o Automatically prepares a smear after
- Also called as blood films
the machine measures CBC
- Gives a picture of the true condition of the blood
o The machine will check the patient’s
- Whatever size, shape, and appearance of the
hematocrit to know how much blood is
cells in the blood, we can identify it by examining
to be used in preparing the smear
a smear
o Some machines will also stain the smear
- Automated machines cannot identify which
after it is prepared
specific abnormality is/are present
o There are also machines that are
- Examine a blood smear when CBC results in an
equipped with microscopic pictures (has
automated machines is abnormal
objectives) that will capture photos that
will appear on the monitor of the
USES OF BLOOD SMEAR EXAMINATION machine
• WBC differential count
• Platelet estimation Wedge Technique
• WBC relative count - The most convenient and commonly used
• Morphologic evaluation of formed elements method for making peripheral blood films
- One slide serves as the film slide, and the other
SPECIMEN FOR SMEAR EXAMINATION is the pusher or spreader slide
- Most ideal specimen: EDTA Blood (versene or - Blood drop: 2-3 mm
sequestrene) o Diff-safe dispenser: it makes the size of
o Preferably liquid tripotassium EDTA the drop of blood standard
blood because it easily mixed with blood - 0.25 inch away from the end of the slide
- Prepare the smear within 2-3 hours after - Hematocrit:
collecting blood o High hematocrit count: lower the angle
o Should have stained and examined o Low hematocrit count: higher the angle
- Blood films in RT for more than 5 hours often
have unacceptable artifacts
- It is better than using capillary blood
- Advantages of EDTA:
o Multiple slides can be made if necessary
o Generally prevents platelets from
clumping on the glass slide
- Disadvantage of EDTA:
o If patient has certain antibodies, it may
result to platelet satellitism (platelets
aggregate at the wall of the neutrophils)
o It occurs because of certain antibodies
that reacts with EDTA anticoagulant
o Collect another blood using citrate (it
will only be used for platelet counting)

Factors affecting blood smear preparation

Thick Thin
Pressure Low High
- Capillary blood sample: Angle High Low
o Finger and heel punctures Speed Fast Slow
o Used when anticoagulant is not available Size Large Small
o Limitations:
▪ Some platelet clumping must be Features of a well-made wedge
expected (will not interfere the peripheral blood film
results) 1. The film is two thirds to three fourths the length
▪ Only a few films can be made of the slide
directly from blood from a skin 2. The film is finger shaped, very slightly rounded at
puncture the feather edge, not bullet shaped; this provides
the widest area for examination.
PERIPHERAL BLOOD SMEAR PREPARATION 3. The lateral edges of the film are visible.
Types of blood smear techniques: 4. The film is smooth without irregularities, holes,
• Wedge technique (double slide / spreader slide or streaks.
/ push smear) 5. When the slide is held up to the light, the thin
• Coverslip technique (Ehrlich’s method) portion (feather edge) of the film has a
• Coverslip and slide technique (Beacom’s "rainbow" appearance.
method) 6. The whole drop of blood is picked up and spread.
 HEMATOLOGY 1 (LABORATORY)

Well-stained Blood Smear

➢ Macroscopically:
o Reddish brown: Wright's stain (pink)
➢ Microscopically: Minimum precipitate with no
artifacts
o RBC: pink
o Nuclei of leukocytes: purple
o Eosinophilic granules: red orange
o Basophilic granules: dark purple
o Platelets: dark lilac
o Monocytes: gray ground glass
appearance

Staining Problems
➢ Too Acidic Stain:
o Thin blood smear
o insufficient staining time
o prolonged buffering or washing
o old stain
o Correction:
▪ lengthen staining time
▪ check stain and buffer pH
▪ shorten buffering or wash time
➢ Too Alkaline Stain:
o thick blood smear
o prolonged staining
o insufficient washing
Automated Slide Making and Staining o alkaline pH of stain components
o Correction:
▪ check pH
▪ shorten stain time
▪ prolong buffering time

Troubleshooting Poorly Stained Blood Films

Problems
• RBCs appear gray
• WBCs are too dark
• Eosinophil granules are gray, not orange
First
STAINING OF SMEARS Scenario
Causes
• Stain or buffer too alkaline (most common)
- Polychromatic stains are used because cells
• Inadequate rinsing
have different characteristics • Prolonged staining
o Contains both eosin and methylene blue • Heparinized blood sample
- Pure Wright stain Problems
- Wright-Giemsa stain • RBCs are too pale or are red
- Purpose: make the cells more visible and to allow • WBCs are barely visible
Second
Causes
their morphology to be evaluated Scenario
• Stain or buffer too acidic (most common)
- Fixative: Methanol • Underbuffering (too short)
- pH: 6.4 to 6.8 • Over-rinsing
o 0.05M sodium phosphate buffer (6.4 pH)
o Aged distilled water (6.4-6.8 pH) MACROSCOPIC EXAMINATION
- Free methylene blue is basic and stains acidic
(and basophilic) cellular components, such as • Bluer than normal: increased blood proteins and
ribonucleic acid (RNA) possible rouleaux
- Free eosin is acidic and stains basic (and o Especially in cases of multiple myeloma
eosinophilic) components, such as hemoglobin • Grainy: RBC agglutination (usually seen in cold
and eosinophilic granules agglutinin disease)
- Some laboratories use coplin jars to hold the • Holes all over the smear: increased lipid levels
slides while submerging it in a staining solution • Blue specks at the feathery edge: markedly
and leave it in a couple of minutes increased WBC and platelet counts

MICROSCOPIC EXAMINATION
Low Power Objective (LPO/10X)
- Check if the sample is suitable for examination
- Cannot identify cells
- not allowed to count cells
- overall film quality
- color
 HEMATOLOGY 1 (LABORATORY)

- distribution of cells WBC abnormalities are also reported

- possible to check for the presence of fibrin Reporting:
strands ➢ Reactive Lymphocytes:
- RBC distribution o separate % as a % of total lymphocytes
- scanned quickly for any large abnormal cells o Semi-quantitatively: occasional to many
- feather edge and lateral edges should be ➢ Toxic granulation:
checked quickly for WBC distribution o Present
o Semi-quantitatively: slight to marked /
High Power Objective (HPO/40X) 1+ to 3+
- select the correct area of the film in which to Specimen:
begin the differential count • Peripheral blood
- evaluate cellular morphology • Bone marrow
- WBC estimate • Body fluid sediments
o Count 10 HPO field, get the average, and
multiply it by 2,000

Oil Immersion Objective (OIO/100X)

- provides the highest magnification
- WBC differential count
- RBC, WBC, and platelet morphology evaluation
- platelet estimate
- segmented neutrophils can be readily
differentiated from bands
- RBC and WBC inclusions
- Reactive or abnormal cells enumeration
- n-RBC counting (if present)

OPTIMAL ASSESSMENT AREA

- Heel: between the thick area and the very thin
feather edge (transition)
- Ideal: Procedure
o RBCs are uniformly and singly 1. Begin the slide examination with a correctly prepared and stained
distributed, with few touching or smear
overlapping, and have their normal 2. Focus the microscope on the 10× objective (low power). Scan the
smear to check for cell distribution, clumping, and abnormal cells.
biconcave appearance
Add a drop of immersion oil and switch to the 100× (oil immersion)
o 200 to 250 RBCs per oil immersion field objective. Begin the count by determining a suitable area. Extend
the examination from the area where approximately half of the
erythrocytes are barely overlapping to an area where the
erythrocytes touch each other. It is important to examine cellular
➢ Cross-sectional or crenellation: From side to side morphology and to count leukocytes in areas that are neither too
thick nor too thin. In areas that are too thick, cellular details such as
➢ Longitudinal: From the tail toward the head of nuclear chromatin patterns are difficult to examine. In areas that
the smear are too thin, distortion of cells makes it risky to identify a cell type.
➢ Battlement: uses a pattern of consecutive fields 3. Count the leukocytes using a tracking pattern. Each cell identified
should be immediately tallied as a neutrophil (band), neutrophil
beginning near the tail on a horizontal edge (segmented), or polymorphonuclear neutrophil (PMN);
o Most ideal lymphocyte; monocyte; eosinophil; or basophil.
4. Abnormalities of leukocytes, erythrocytes, and platelets should be
noted. Normally, 8 to 20 platelets are present in an oil immersion fi
eld in a properly prepared smear (where the RBCs barely touch each
other). After examining at least 10 different fi elds, the average
number of platelets can be multiplied by a factor of 20,000 to arrive
at an approximate total circulating platelet concentration.
Nucleated erythrocytes are not included in the total count but are
noted per 100 white blood cells (WBCs). A total of at least 100
leukocytes should be counted. Express the results as a percentage
of total leukocytes counted.

- One hundred WBCs are counted and classified Absolute count

through the use of push-down button counters - More accurate than the relative count
- Results are reported as percentages - Actual number of specific WBC in a liter of blood
- Principle: A stained smear is examined to - Absolute count = percentage x WBC Count
determine the percentage of each type of - Out of 100 cells counted, the results will be
leukocyte present and assess the erythrocyte referred to as WBC relative differential count
and platelet morphology.
Example:
WBC = 10 x 109/L
Differential Count (100 WBC)
• Neutrophils: 60% x 10 = 6 x 109/L
• Lymphocytes: 30% x 10 = 3 x 109/L
• Monocytes: 10% x 10 = 1 x 109/L