Chapter 9
Leukocyte's classification, structure,
function, and assessments
Medical Laboratory Sciences
Dr. Ali Abdelfattah
Headlines
➢ Types, maturation stages, and functions of WBCs
▪ Granulocytes
▪ Monocytes
▪ Lymphoid cells
➢ Laboratory assessment of WBCs
Introduction
➢Leukocytes (also known as white blood cells, or WBCs) are so
named because they are relatively colourless compared to red blood
cells.
➢The basic function of blood leukocytes is as a defense system
against infectious foreign invaders. Leucocytes may be divided into
two broad groups:
▪ Phagocytes
Include neutrophils, eosinophils, basophils, and monocytes
and comprise the cells of the innate immune system, which
can act very quickly after an infection
▪ Lymphocytes
Mediate the adaptive immune response, which can develop
immunological memory cells
Leukocytes
Types of Leukocytes:
➢Myeloid cells ➢Lymphoid cells
•Neutrophils • T cells
•Eosinophils • B-cells
•Basophils • Natural killer (Nk) cells
•Monocytes
Granulocytes Agranulocytes
Polymorphonuclear cells (PMNs), Mononuclear cells, nucleus is not
nucleus is segmented or lobulated segmented but is round, oval, or
indented
Neutrophils
➢ Neutrophil development occurs in the bone marrow
➢ There are three pools of developing neutrophils in the BM
10-14 days
Once neutrophils are in the tissues, their life span
is variable, depending on whether or not they are 6-10 hours
responding to infectious or inflammatory agents.
In the absence of these agents, the neutrophil’s life
span is measured in hours. Spontaneous neutrophil
apoptosis is regulated by Bcl-2 family.
Neutrophil maturation
As the neutrophil
matures:
• Size decreases
• Nucleoli disappear
• Chromatin condenses,
indents, and finally
segments
• The cytoplasm goes
from an agranular
cytoplasm (blue color)
to a granular cytoplasm
(pink color)
Neutrophil maturation
➢ Myeloblast
• Is the first recognizable precursor cell
• Makes up 5% of the nucleated cells in BM
• 14 to 20 µm in diameter
• has a high N:C ratio
• Round to oval nucleus with fine
chromatin
• visible 2-5 nucleoli
• Agranular basophilic cytoplasm.
Sporadically, the cytoplasm contains
primary granules
• Not present in normal PB
Neutrophil maturation
➢Promyelocyte
• Comprises up to 5% of the nucleated cells
in the BM
• They are relatively larger than the
myeloblast and measure 16 to 25 µm in
diameter.
• The nucleus is round to oval and is often
eccentric with indistinct nucleoli
• Decreased N:C ratio as compared with
Myeloblast
• Coarse chromatin
• Primary granules are produced
• Blue cytoplasm, with a perinuclear halo
• Not present in normal PB
Neutrophil maturation
➢ Myelocyte
• The production of primary granules ceases, and the
cell begins to manufacture secondary (specific)
neutrophil granules
• 15-18 µm in diameter, smaller than promyelocytes
• Decreased N:C ratio as compared with
promyelocytes
• Eccentric oval Nucleus- (one side is flat),
• Chromatin strand – condensed, unevenly stained
• Last myeloid precursor capable of division
• Cytoplasm more pink
• Not seen in normal PB
Neutrophil maturation
➢ Metamyelocyte
• Nucleus becomes more indented (kidney bean shaped)
with Clumped chromatin
• Central or eccentric nucleus, N:C ratio = 1:1
• 14-16 µm in diameter, Smaller than myelocytes
• Synthesis of tertiary granules
• Cytoplasm: Small pinkish granules
• Cells do not divide and absent in normal PB
➢ Band Neutrophil
• Nuclear indentation is great
• Appearance of horseshoe, central or eccentric
• Nuclear chromatin is pyknotic
• 10-16 µm in diameter, smaller than
metamyelocytes
• In PB: up to 5%
Neutrophil maturation
➢ Mature neutrophils
• Neutrophils – most numerous WBC
• Protection against bacteria and fungi through granules
• Cell size- 12-15µm in diameter
• Nucleus – three to five nuclear lobes connected by a thin
filament/strand
• The granules of neutrophils react with both acidic and basic parts
of the stain, pinkish-blue colour.
Normal values
Differential:50-70%
Absolute:1700-7500/µL
Neutrophils
Alterations in the peripheral blood concentration of neutrophils are
often the first indication of an underlying pathology
A decrease in the number of blood neutrophils is called
neutropenia and when this occurs it can lead to infections
An increase in the number of blood neutrophils is called
neutrophilia and this occurs as a response to bacterial
infection, metabolic or drug intoxication, or tissue necrosis.
Presence of immature cells is termed a shift to the left
In response of neutrophils to bacterial infections,
neutrophilia and an increase in the % of bands and
even some metamyelocytes in the peripheral blood
Neutrophil granules
➢ primary granules (Azurophilic): hydrolytic enzymes (e.g.,
elastase, serine proteases) and myeloperoxidase (MPO).
The key elements of the innate immune system and provide
defence against invading pathogens
➢ Specific, or secondary, granules - contain lysozyme,
Hydrogen peroxide, and high levels of the iron-binding protein
lactoferrin (inhibiting bacterial growth by consuming iron).
Important in modulating inflammation
➢ Tertiary granules – contain gelatinase (mediates delivery of
new adhesion proteins to the plasma membrane).
Important in maintaining the cellular adhesion
Neutrophil function
Only about half of the peripheral neutrophils are freely
circulating in the blood, while the other half are attached to the
vessel walls (called the marginating pool). Upon antigenic
stimulation, the marginating neutrophils will move into
circulation and then into the tissues (diapedesis).
Next primary and specific granules
and secretory vesicles fuse with the
phagocytic vacuole, releasing their
contents (degranulation).
The enzymes and antimicrobial
peptides present in the granules may
Neutrophil
act to kill the ingested
microorganisms. In this process most
neutrophils die and are themselves
phagocytosed by macrophages
Phagocytosis
Granulopoiesis
➢ Mature neutrophils,
eosinophils, basophils
have similar patterns
GM-CSF GM-CSF of proliferation,
GM-CSF
G-CSF
G-CSF
IL-5
G-CSF
IL-3 differentiation,
division, storage in BM
and delivery to PB.
Eosinophils
Eosinophil myelocytes can be recognized but earlier stages are
indistinguishable from neutrophil precursors, containing reddish-
orange secondary granules in cytoplasm
➢ Mature eosinophils usually display a bilobed
nucleus with coarser cytoplasmic granules
➢ The granules (basic proteins) in eosinophils
have affinity for the acid (eosin) part of the
Giemsa stain and they stain reddish-orange
colour
Normal values
Differential: 1-3%
Absolute:10-300/µL
Eosinophils
➢ Growth factors: GM-CSF, G-CSF, and IL-5
➢ Granules contain very high peroxidase content, lysozymes,
Major Basic Protein (MBP), and inducing histamine release.
➢ Eosinophils have a circulating half-life of roughly 18 hours.
➢ Survival time of eosinophils in human tissues ranges from
2 to 5 days
➢ Eosinophils function in phagocytosis and killing of bacteria, but
they are more sluggish and less efficient than neutrophils.
➢ They have a special role in allergic responses, defence against
parasites and removal of fibrin formed during inflammation.
Thus, they play a role in local immunity and tissue repair
Basophils
➢ Basophils also have a similar maturation process
➢ Nucleus – usually two lobes with dark cytoplasmic granules
which overlie the nucleus and contain heparin and histamine
➢ The granules (acidic proteins) of basophils have affinity for the
basic (methylene blue) part of the stain and they stain bluish-
black.
Normal values
Differential: 0-2%
Absolute: 0-200/µL
Basophils
➢ life span of basophils is relatively longer than that of the other
granulocytes, 60 hours.
➢ They have immunoglobulin E (IgE) attachment sites, and their
degranulation is associated with histamine release, similar in
function to mast cells, mast cells are not considered to be
leukocytes. They are tissue effector cells of allergic responses
and inflammatory reactions
➢ They are mediators of the inflammatory response. The release of
granules in hypersensitivity reactions leads to vasodilation,
increased vascular permeability and smooth muscle contractions
Monocytes
➢ Monocyte development is similar to neutrophil development
because both cell types are derived from the GMP.
➢ Macrophage colony-stimulating factor (M-CSF) is the
major cytokine responsible for the growth and
differentiation of monocytes.
M-CSF
Monocyte Maturation
➢ Monoblasts
• Monoblasts in normal bone marrow are
very rare and are difficult to distinguish
• from myeloblasts based on morphology. It
can be differentiated from a myeloblast by
cytochemical stains.
➢ Promonocytes
▪ They are a little smaller than monoblasts,
12 to 18 µm in diameter
▪ Their nucleus is slightly indented or folded
and at least one nucleolus is apparent.
The cytoplasm is blue and contains scattered
azure granules that are fewer and smaller than
those seen in promyelocytes
Mature Monocytes
➢ The largest mature leukocytes. Cell size: 15-20 µm
➢ Nucleus –large oval indented nucleus with clumped chromatin
(kidney shaped)
➢ The abundant cytoplasm stains blue and contains many fine
vacuoles, giving a ground glass appearance
➢ They transform into macrophages in tissues, phagocytic cells
Normal values
Differential: 2-10%
Absolute:100-1300/µL Figure 17.4e
Monocyte Function
➢ When mature monocytes leave the blood and enter the tissues,
they mature into macrophages. This is accompanied by
progressive enlargement of the cell. The cells can live for
months in the tissues.
➢ They function in phagocytosis of microorganisms and cellular
debris
Antigen Presenting cell
They are important in the
processing of and
presentation of antigens to
lymphocytes for activation
and differentiation.
Lymphocytes
Lymphocytes are divided into
three major groups: T cells (so
named because they develop
in the thymus), B cells Pre-Pro-B cell ETP
(because they develop in the Thymu
Early-immature B
BM), and natural killer cells Pro-B cell s
(NK, which develop in both Pro-T
Mid-immature B
the BM and the thymus). T cell
Pre-B cell Pre-T
and B cells are major players Late-immature B
cell
Immatur
in adaptive immunity. NK Early-B cell
e-T cell
cells make up a small
percentage of lymphocytes
and are part of innate
immunity
Lymphocytes
For both B and T cells, development can be subdivided into
antigen-independent and antigen-dependent phases.
➢ Antigen independent lymphocyte development occurs in the
bone marrow and thymus (sometimes referred to as primary
lymphatic organs)
➢ Antigen-dependent lymphocyte development occurs in the
spleen, lymph nodes, and tonsils (sometimes referred to as
peripheral or secondary lymphatic organs)
Normal values
Differential: 18-42%
Absolute: 1000-3200/µL
B Lymphocyte development
B lymphocytes develop initially in the bone marrow and go
through three stages known as pro-B, pre-B, and early B cells
(late immature B cells) which is called naïve B cells
(immunocompetent cell). During these stages, immunoglobulin
gene rearrangement occurs so that each B cell produces a
unique immunoglobulin
➢ Naïve B cells which have not yet been exposed to antigen
leave the BM to migrate to secondary lymphatic organs,
where B cells may come in contact with antigen, which
results in cell division and the production of memory cells
as well as effector cells. Effector B cells are antibody-
producing cells known as plasma cells. Approximately 3%
to 21% of circulating lymphocytes are B cells.
T Lymphocyte development
➢ Early Lymphoid progenitors (ETP) migrate from the BM to the
thymic cortex, where they progress through stages known as pro-
T, pre-T, and immature T cells.
➢ During these phases they undergo antigen receptor gene
rearrangement to produce T cell receptors that are unique to each
T cell. Immature T cells then proceed to the thymic medulla,
where apoptosis of self-reactive T cells occurs.
➢ The remaining immature T cells (naive T cells) leave the thymus
and migrate to secondary lymphatic organs, where they come in
contact with antigens. This results in cell activation and the
production of either memory cells or effector T cells that mediate
the immune response (T helper (CD4), T suppressor, or T
cytotoxic cells (CD8)). The effector T cells are often referred to
as reactive lymphocytes. T cells comprise 51% to 88% of
circulating lymphocytes
Lymphocytes
➢ One cannot tell the difference between a B or T cell under the
microscope.
Immunocompetant Reactive
Plasma cell
Lymphocyte Lymphocyte
• Nucleus – stains dark purple The reactive lymphocytes
with high N:C ratio have increased cytoplasm
• Around 8-9 µm in diameter
with variable basophilia.
Lymphocyte kinetics
Lymphocytes are different from the other leukocytes in
several ways, including the following:
➢Although early lymphocyte progenitors such as CLP originate in
the BM, T and NK lymphocytes develop and mature outside the BM
➢The peripheral blood contains only about 5% of the total body
lymphocytes. Most of the lymphocytes reside in lymphoid organs
➢Lymphocytes are not end cells. They are resting cells, and when
stimulated, they undergo mitosis to produce memory and effector
cells.
➢Unlike other leukocytes, lymphocytes recirculate from the blood to
the tissues and back to the blood.
➢B and T lymphocytes are capable of rearranging antigen receptor
gene segments to produce a wide variety of antibodies and surface
receptors.
Leukopoiesis assessment
Leukopoiesis can be assessed clinically by:
1. Complete blood count (CBC) on peripheral blood (PB)
▪ White blood cell (WBC, Leukocyte) count - total number of
white cells (An increase : Leukocytosis or a decrease:
Leukopenia) in PB. It does not distinguish WBC types
Reference Interval: Adult, 3.5-11 x103/μL, Newborn: 9.0-30 x 103/μL
▪ WBC differential count classifies WBC types
Cells relative absolute Increase/ Decrease
Neutrophils 50-70% 1.7-7.5 x 103/μL Neutrophilia/ Neutropenia
Lymphocytes 18-42% 1.0-3.2 x 103/μL Lymphocytosis/
Lymphocytopenia
Monocytes 2-10% 0.1-1.3 x 103/μL Monocytosis/ Monocytopenia
Eosinophils 1-3% 0.0-0.3 x 103/μL Eosinophilia
Basophil 0-2% 0.0-0.2 x 103/μL Basophilia
Leukopoiesis assessment
2. Blood film examination (morphological assessment)
• Estimate total counts
• Corrected WBC count
• Differential counts
• Abnormal WBC
➢ To perform a WBC estimate, the average count of the WBCs in 10
fields per high-power field (40x) is multiplied by 2000
➢ Corrected WBC counts
The Nucleated RBCs (NRBCs) are counted as WBCs because they are
indistinguishable by automation or hemacytometer. If five or more NRBCs per
100 WBCs are observed on the differential count on a stained peripheral blood
film, the WBC count must be corrected. Report the result as the “corrected”
WBC count
Leukopoiesis assessment
3. Bone marrow (BM) examination
➢ BM Cellularity
➢ M:E ratio, Normal M:E ratio is 1.5:1 to 5:1
➢ Morphologic examinations,
▪ The maturation stages of the myeloid and lymphoid cells
▪ malignant cells ?
➢ Cytochemistry: differentiate cell types
➢ Flow cytometry analysis: cell surface antigens
