Zone of Best Morphology - region where the cells are Zone of Best Morphology (4x)

MODULE 12- PERIPHERAL BLOOD spaced far enough apart to be differentiated and counted.
The morphology is best in this zone.
Blood is a fluid connective tissue composed of formed
elements (red blood cells, white blood cells,
and platelets) circulating in a fluid called plasma. It provides Feather Edge - right side of the slide with the lowest number
a mechanism by which gases, nutrients, wastes, and cells of cells. The morphology of the cells is typically poor.
can be transported throughout the body.
This scan was obtained with a 40x dry objective. Except for
Special stains are used to differentiate the types of blood identifying the zone of best morphology, individual cells need
cells. Wright's stain, a mixture of eosin Y (acidic) and to be viewed at higher magnification to examine their
methylene blue (basic) dyes, is superior in this regard to
morphology.
hematoxylin and eosin H&E.
Blood MH 033 BLOOD SMEAR
Zone of Best Morphology (40x)
Whole Slide

Composition of human blood:

 50 to 65% plasma
 36 to 50% red blood cells (erythrocytes)
 < 1% white blood cells (leukocytes)

Composition of white blood cells:

 Granulocytes - contain azurophilic (primary)

granules and specific (secondary) granules
 60-70% neutrophils (polymorphonuclear
leukocytes, PMNs) Head of Blood Film (4x)
Feather Edge (4x)
 2 to 4% eosinophils
 <1% basophils
 Agranulocytes - contain only azurophilic
(primary) granules
 25 to 30% lymphocytes
 5% monocytes

BLOOD SMEAR

The histology of blood cells is studied in smears prepared by

spreading a drop of blood into a thin layer on a microscope
slide.
Head of Blood Film (40x)

Blood smears are made by placing a drop of blood on one

end of a slide, and using a spreader slide to disperse the
blood from left to right.

Head of Blood Film - left side of the slide with the largest
number of cells. The tight packing of cells distorts
their morphology.
Feather Edge (40x) Platelets are typically found as clusters:

EM 206 RED BLOOD CELLS Example: Platelets (60x)

Red blood cells (erythrocytes) transport oxygen to cells and Platelets are involved in the formation of blood
remove carbon dioxide from the same cells. clots. Too few platelets can cause excessive
bleeding, while too many platelets can cause
Two red blood cells within a blood clot as seen by scanning blood clot formation. Their life span is between 7
electron microscopy (SEM). to 10 days.

A cross-section of a platelet is shown in EM 159 Platelet by

transmission electron microscopy.
Red blood cells (red)

 Biconcave discs (i.e., both sides of the disc curve

inwards).
BLOOD CELLS  ~7.8 µm diameter.
 Their shape maximizes the surface area for the
The histology of blood cells is examined using a single, high-
exchange of oxygen and carbon dioxide.
resolution 60x scan acquired from the zone of best
morphology of this blood smear.  Flexible to allow passage through small capillaries.

Identify the different types of blood cells by distinguishing Fibrin strands (tan)
characteristics, such as, size, shape, nuclear morphology,
and the presence or absence of granules, and the
appearance of granules.
MH 033hr BLOOD SMEAR EM 159 PLATELET

Red blood cells (or erythrocytes) are the most abundant Platelets (thrombocytes) are small non-nucleated cell
type of blood cell (98% of all cells). fragments produced by budding from megakaryocytes under
the influence of thrombopoietin. Platelets plug damaged
 Small cells (7 to 8 µm diameter) blood vessel walls and are involved in the formation of blood
 Biconcave disc clots.
 No nucleus
 Platelets are small biconcave discs (2 to 4 µm
 Eosinophilic cytoplasm (bright pink) because of
diameter).
the high concentration of the protein hemoglobin
 Plasma Membrane (dark green) - covered by a
Red blood cells are involved in transporting oxygen and thick glycocalyx.
carbon dioxide. They circulate in blood for between 100 to MH 033hr BLOOD SMEAR  Open Canalicular System -
120 days. invaginations of the plasma membrane
Platelets are small cell fragments produced by budding from that facilitate signaling and secretion.
megakaryocytes under the influence of thrombopoietin.  Hyalomere - outer region that contains circular
Each megakaryocyte produces between 5,000 and 10,000 rings of microtubules (cross-sections; purple) and
platelets. microfilaments.
 Granulomere - central region that contains
Platelets are much smaller than red blood cells. organelles and secretion granules.
 Mitochondria (red)
 Small discs (2 to 4 µm diameter)  Dense Tubular System (cyan) - stores
 No nucleus calcium (similar to sarcoplasmic
 Central region is basophilic structure reticulum in skeletal muscle).
(granulomere)
 Outer region does not stain (hyalomere)
  Alpha Granules (purple) - large
granules that contain blood clotting
substances. Open Canalicular System Alpha Granules (purple)
 Dense (delta) Granules (yellow) -
smaller granules that contain factors
that facilitate platelet adhesion and
vasoconstriction.
 λ Granules - similar to lysosomes (not
visible).

Whole Micrograph

Mitochondria
Dense (delta) Granules

Platelet

Dense Tubular System

MH 033hr BLOOD SMEAR

Neutrophils (also known as polymorphonuclear leukocytes

[PMN]) are the most abundant type of white blood cells (60-
70%).

 12 to 15 µm diameter
Plasma membrane and microtubules  Segmented nucleus with 2 to 5 lobes
 Cytoplasm is a pale blue-pink
 Specific granules are pink-salmon
 Few azurophilic granules
 No longer capable of mitosis

Examples:

 Neutrophil #1 (60x)
 Neutrophil #2 (60x)
 Neutrophil #3 (60x)
 Neutrophils #4 (60x)
 Neutrophil 3 EM 169 NEUTROPHIL

 Neutrophil #5 (60x) Neutrophils are phagocytic cells that engulf and kill microbes
(especially bacteria). They have a multilobed nucleus and
Neutrophils phagocytose and destroy bacteria. They contain numerous granules.
circulate in blood for 8 to 16 hrs and survive in peripheral
tissues for only 1 to 2 days.  Specific Granules (purple) - smaller, round (less
electron-dense) or elongated granules.
The ultrastructure of neutrophils is shown in EM 169  Antimicrobial proteins, bactericidal
Neutrophil by transmission electron microscopy. Neutrophil enzymes, and extracellular matrix
activation and intracellular killing of microbes is also degrading enzymes.
described.  NADPH oxidase components needed to
produce toxic oxygen radicals and
Neutrophil 1 hydrogen peroxide (H2O2).
 Fuse with phagosomes or are secreted.
 Azurophilic Granules (black) - larger, round
Neutrophil 4 (electron-dense) granules. Lysosome-related
organelles that contain extra proteins.
 Antimicrobial proteins, bactericidal
enzymes, and lysosomal degradative
enzymes.
 Myeloperoxidase needed to convert
hydrogen peroxide (H2O2) into highly
toxic hypochlorous acid (HOCl; similar
to bleach).
 Fuse with phagosomes (less often are
secreted).

Neutrophil 2  Gelatinase Granules - look like specific granules.
 Adhesion receptors, chemotactic
receptors, and extracellular matrix
degrading enzymes.
Neutrophil 5  Secretion granules.

Although a neutrophil contains numerous granules, it

contains relatively few organelles.

 Plasma Membrane (dark green) - extends small

processes (pseudopodia) involved in motility and
engulf microbes during phagocytosis.
 Large number of cell surface receptors
for growth, adhesion, chemotaxis, and
cell-to-cell interactions.
 Nucleus (blue) / Nuclear Envelope (purple) - three
to five lobes connected by thin strands of
chromatin.
 Golgi Apparatus - poorly developed (not visible).
 Mitochondria (red) - small, few in number.
 Endoplasmic Reticulum (cyan) - scattered
fragments.
  Glycogen (#1 and #2; dark green) - numerous oxygen radicals and hydrogen Eosinophil 1
clusters of small granules. peroxide (H2O2).
 Cytoplasm (green)  Myeloperoxidase (from
azurophilic granules) converts
Neutrophils use anaerobic glycolysis of the abundant hydrogen peroxide to highly
glycogen as an energy source. Useful in the low oxygen toxic hypochlorous acid
environment of inflamed (or damaged) tissue. (HOCl; similar to bleach)
which is extremely
Neutrophil Activation antibacterial.

Neutrophils are among the first immune cells to arrive at a

site of infection. They leave the circulation and migrate into
tissues where they phagocytose microbes and other foreign Neutrophils primed by some cytokines or overstimulation
substances. release specific and azurophilic granules damaging host
tissues. Eosinophil 2
Neutrophil granules play a pivotal role in these processes.
MH 033hr BLOOD SMEAR
 Gelatinase granules are secreted during
movement through the walls of blood vessels Eosinophils are a small component of white blood cells (2-
(diapedesis). 4%).
 Exposes additional adhesion and
chemotactic receptors on the cell  12 to 15 µm diameter
surface  Nucleus is bilobed
 Releases extracellular matrix degrading  Cytoplasm is a pale blue-pink
enzymes to digest the basal lamina.  Eosinophilic, specific granules - oval, 1 to 1.5 µm
 Limited secretion of specific granules to aid diameter, red/orange
migration through tissues.  Few azurophilic granules
 Exposes receptors on the cell surface  No longer capable of mitosis
that recognize microbes. Eosinophil 3
 Releases extracellular matrix degrading Examples:
enzymes.
 Eosinophil #1 (60x)
Neutrophils are attracted by a variety of signaling molecules  Eosinophil #2 (60x)
that bind to cell surface receptors (chemotaxis).  Eosinophil #3 (60x)
 Eosinophil #4 (60x)
Neutrophils rapidly engage, engulf, and kill microbes.
Eosinophils kill multicellular parasites and phagocytose
 Microbes are recognized by cell surface receptors. immune complexes. They circulate in the blood for 8 to 16
 Phagocytosis of microbes into phagosomes. hrs and survive in peripheral tissues for only 2 to 5 days.
 Specific and azurophilic granules fuse with
phagosomes to form phagolysosomes. The ultrastructure of eosinophils is shown in EM 161
 Microbes inside phagolysosomes are damaged Eosinophil and EM 119 Eosinophil by transmission electron
and killed using the contents from both types of microscopy. Eosinophil activation and extracellular killing of
granules. parasites is also described.
 Antimicrobial proteins, bactericidal
enzymes, serine proteases, and acid
hydrolases.
 Generation of more effective cytotoxic
oxidants (i.e., respiratory burst).
 NADPH oxidase (from specific
granules) produces toxic
Eosinophil 4 Basophil 2 Lymphocytes are released from bone marrow into the
peripheral circulation. They exit blood into one of the
peripheral lymphatic organs and continue to divide.

The ultrastructure of lymphocytes is shown in EM 199

Lymphocyte and EM 171 Lymphocyte by transmission
electron microscopy.

SMALL LYMPHOCYTES

Lymphocyte 1

MH 033hr BLOOD SMEAR

Basophils are rare white bloods (<1%). MH 033hr BLOOD SMEAR

 Smaller cells (10 to 12 µm diameter) Lymphocytes are a large component of white blood cells
 Nucleus bilobed or S-shaped (20 to 25%).
 Cytoplasm is a pale blue-pink
 Specific granules - 1 to 1.5 µm diameter,many Small Lymphocytes
very basophilic (blue to black)
 Few azurophilic granules  Round cells of 6 to 9 µm diameter
 No longer capable of mitosis  Intense staining of the spherical nucleus of
mostly condensed chromatin Lymphocyte 2
Examples:  Cytoplasm is limited to a small rim that is pale
blue
 Basophil #1 (60x)  No specific granules
 Basophil #2 (60x)  Capable of mitosis

Basophils are found at sites of infection and release Examples:

substances mediate inflammatory responses.
 Lymphocyte #1 (60x)
The ultrastructure of basophils is shown in EM 168  Lymphocyte #2 (60x)
Basophil by transmission electron microscopy.  Lymphocyte #3 (60x)

Basophil 1 Large Lymphocytes

 Round cells of 9 to 15 µm diameter

 Blotchy staining (sometimes referred to as hill and
Lymphocyte 3
valley) of the spherical nucleus
 More cytoplasm than in small lymphocytes
 No specific granules
 Capable of mitosis

Examples:

 Lymphocyte #1 (60x)
 Lymphocyte #2 (60x)
 Lymphocyte #3 (60x)
 LARGE LYMPHOCYTES Monocytes are the largest type of white blood cell. Monocyte 2

Lymphocyte 1  Large cells (12 to 20 µm diameter)

 Large, kidney-shaped or indented nucleus often
with a "raked" chromatin pattern
 Cytoplasm is pale blue
 Few azurophilic granules

Examples:

 Monocyte #1 (60x)
 Monocyte #2 (60x)
 Monocyte #3 (60x)

Monocytes are released from the bone marrow into the

peripheral circulation. Their time span in blood is only 16
hrs. Monocytes in connective tissue differentiate into Monocyte 3
Lymphocyte 2 macrophages.

Monocytes are involved in phagocytosis and antigen

presentation.

The ultrastructure of monocytes is shown in EM 170

Monocyte by transmission electron microscopy.

Monocyte 1

Lymphocyte 3 Summary of blood cells identified on this slide.

MH 033hr BLOOD SMEAR

Summary of blood cells:

Red Blood Cells (erythrocytes)

 Light red biconcave discs (~7.5 µm diameter)

without nuclei

Platelets (thrombocytes)

 Small, basophilic discs (2 to 4 µm diameter)

White Blood Cells (leukocytes) White Blood Cells (leukocytes):

 Neutrophils (polymorphonuclear leukocytes, Neutrophils (polymorphonuclear leukocytes)

PMN) (#1, #2, #3, #4 #5) - have distinctive nuclei
with 2 to 5 lobes (polymorphic) and azure granules  Small granules: pale purple-pink
 Eosinophils (#1, #2, #3 and #4) - contain  Nuclei with 2 to 5 lobes : blue-purple
distinctive large, eosinophilic granules. Nuclei
usually have two lobes Eosinophils
 Basophils (#1 and #2) - rare cells with distinctive
large, basophilic granules.  Large granules: bright orange-red
 Lymphocytes - occur in a range of sizes.  Nuclei with 2 lobes: blue-purple
 Small Lymphocytes (#1, #2 and #3) -
small cells (6 to 9 µm diameter) with a Basophils
thin rim of cytoplasm. Nuclei are round
and densely stained heterochromatin.  Large granules: deep blue-purple
 Nuclei with 2 lobes (or S-shaped) : blue-purple
 Large Lymphocytes (#1, #2 and #3) -
larger cells (9 to 15 µm diameter) with Azurophilic granules: blue-purple
more cytoplasm than small
lymphocytes. Nuclei contain a mixture of Cytoplasm: pale blue
euchromatin and heterochromatin.

 Monocytes (#1, #2 and #3) - large cells (12 to 20

µm diameter) with "kidney-shaped" or notched
nuclei with chromatin having a "raked"
appearance. The cytoplasm is bluish-gray.

REVIEW

This diagram allows the morphology and staining

characteristics of different types of blood cells to be easily
compared. The key features used to distinguish between
them are highlighted.
Blood Cells

Examples of blood cells and the Key features used to

distinguish between them:

Red Blood Cells (erythrocytes)

 Biconcave discs: pink-red

 No nuclei

Platelets (thrombocytes)

 Small disc: lavender