CHAPTER 4: LEUKOPOIESIS, KINETICS AND FUNCTION

I. INTRODUCTION
- Leukocytes also known as WBC are relatively colorless. Number in circulation varies with age, sex, activity, time of
day and ethnicity.
- Typical reference interval is 4.5x10^9/L – 11.5x10^9/L
- Granulocytes – cytoplasm filled with granules with differing staining characteristics and whose nuclei are
segmented or lobulated (Eosinophils, Basophils, and Neutrophils)
- Mononuclear cells – have nuclei that are non-segmented but are round, oval, indented, or folded
- Overall function of Leukocytes is in mediating immunity

II. GRANULOPOIESIS
- Orderly production of mature granulocytes (Neutrophil, Eosinophil, Basophil)
- Maturation sequence from the blast stage to the release of mature granulocytes into the peripheral blood takes
about 14 days
A. STAGES OF GRANULOCYTE MATURATION:

STAGE CELL NUCELUS CYTOPLASM N/C ADDT’L

SIZE RATIO
(um) SHAPE CHROMATIN NUCLE STAINING GRANULES
OLI
MYELOBLAST 14-20 Round or Fine 2-5 Basophilic None 4:1 3 TYPES:
(0%-3% in BM) slightly I: 8:-4:1 N/C ratio,
oval no visible granules
II: presence of
primary granules,
<20/cell
III: rare, seen in
certain types of AML
PROMELOCYTE 16-25 Oval or Slightly 1-3 Basophilic PRIMARY 3:1- Presence of halo,
(1%-5% in BM) round, coarse GRANULES 2:1 also called “Hof” in
eccentric normal ones
MYELOCYTE 15-18 Oval or Coarser and none Mixture of SECONDRY 1:1 Final stage of
(6%-17% in BM) round condensed basophilic GRANULES Mitosis
&
acidophilic
METAMYELOCYTE/ 14-16 KIDNEY Coarse & none Beige or Tertiary 1:1
JUVENILE SHAPED or clumped salmon granules
(3%-20% in BM) INDENTED staining (gelatinase)
BAND or STAB or 9-15 Elongated Coarse & none Beige or Faint 1:1-
STAFF (0-5% in or band- clumped salmon 1:2
Peripheral, 9-32% in shaped (C staining
BM) or S)
MATURE FORMS
NEUTROPHIL 9-15 2-5 LOBES Highly none Beige or PINK to 1:2 7-30% in BM
condensed salmon ROSE 50-70% of WBCs in
staining VIOLET circulation
EOSINOPHIL 9-15 2 lobes Condensed none Beige or REDDISH- 1:2 1-3% in BM
salmon ORANGE 1-3% of WBCs in
staining circulation
BASOPHIL 10-16 Unsegment Condensed none Beige or DARK 1:2 <1% in BM
ed or salmon PURPLE or 0-2% of WBCs in
bilobed staining BLUE-BLACK circulation
B. GRANULOCYTIC STAGES IN SUMMARY:
MYELOBLAST - 1st recognizable stage in granulocytic series
PROMYELOCYTE – 1st appearance of Primary granules
MYELOCYTE – 1st appearance of Secondary granules, last stage capable of mitosis
METAMYELOCYTE/JUVENILE – youngest stage not capable of mitosis
BAND/STAFF/STAB – youngest cell to appear normally in peripheral blood

C. GRANULES
III. GRANULOCYTES

A. NEUTROPHIL/POLYMORPHONUCLEAR CELL (PMNs) – responds to BACTERIAL INFECTION.

 Granules: pink to rose-violet specific granules with neutral affinity for stains
- In mature neutrophils, ratio of primary to secondary granules is 2-3:1
- Myeloperoxidase (MPO) together with H2O2 & halide aids in killing bacteria (Respiratory burst or
Oxidative burst)
- Lysozyme or muramidase: present in 1° and 2° granules and degrades glycopeptides and
hydrolyzes carbohydrates which compose the bacterial cell wall.
- Lactofferin: iron-binding glycoprotein that competes with bacteria for iron possibly inhibiting
growth and may also promote PMN adherence to endothelial cells.
 Life span: 9-10 days (from myeloblast to death)
 Neutrophil pools in the body:
- Bone marrow – where the production and maturation occur
o Mitotic pool – consist of myeloblast, promyelocyte and myelocyte
o Maturating pool – consists of metamyelocyte
o Storage pool – consists of metamyelocyte, bands and segmented PMNs
- Blood
o Circulating pool – 50% of PMNs circulating freely
o Marginating pool – 50% of PMNs adhere to the vessel walls or are sequestered in the
capillaries (not included in the WBC count and differential count)
o Increase in WBC count can be seen in a fearful person, in the presence of stress or in
epinephrine administration caused by shift of PMNs from marginating pool to the circulation
 Major function: Phagocytosis & killing of foreign material or infectious agents.
  Secondary functions:
o Generation of neutrophil extracellular traps (NETs). These structures have enzymes from
neutrophil granules attached to them and have been shown to be able to trap and kill gram-
positive and gram-negative bacteria as well as fungi. NETs are generated at the time that
neutrophils die as a result of antibacterial activity. The term NETosis has been used to
describe this unique form of neutrophil cell death that results in the release of NETs.
o Secretory function. Neutrophils are a source of transcobalamin I or R binder protein, which
is necessary for the proper absorption of vitamin B12.

B. EOSINOPHIL - responds to PARASITIC & HELMINTHIC INFECTION and ALLERGY

 The time from the last myelocyte mitotic division to the emergence of mature eosinophils from the
marrow is about 3.5 days.
 Once in the circulation, eosinophils have a circulating half-life of roughly 18 hours. However, the
half-life of eosinophils is prolonged when eosinophilia occurs.
 Tissue destinations of eosinophils under normal circumstances appear to be underlying columnar
epithelial surfaces in the respiratory, gastrointestinal, and genitourinary tracts. Survival time of
eosinophils in human tissues ranges from 2 to 5 days.
 Concentration is normally high at night.
 Granules: reddish-orange specific granules with affinity for eosin which is acidic part of the stain
- Major Basic Protein – arginine-rich protein that plays a major role in killing parasites
 Charcot-Leyden crystals – formed from the disintegration of eosinophils; made up of
lysophospholipase found in the cytoplasm of eosinophils. Can be seen in:
o Allergic asthma (nasal mucus)
o Pulmonary eosinophilic infiltrates (pleural fluid)
o Parasitic infections (stool)
 Major functions:
o Defense against helminthic parasites
o Has a role in allergic reactions by lessening hypersensitivity reactions through the release of
amine oxidase, which neutralizes histamine
o Eosinophil production is increased in infection by parasitic helminthes, and in vitro studies
have found that the eosinophil is capable of destroying tissue-invading helminths through
the secretion of MBP and eosinophil cationic protein as well as the production of reactive
oxygen species. There is also a suggestion that eosinophils play a role in preventing
reinfection.
 Other functions:
 o Transmigrate into the thymus of the newborn and are believed to be involved in the deletion
of double-positive thymocytes
o Capable of acting as antigen-presenting cells and promoting the proliferation of effector T
cells.
o They are also implicated in the initiation of either type 1 or type 2 immune responses
because of their ability to rapidly secrete preformed cytokines in a stimulus-specific manner.
They are also important factors in acute and chronic allograft rejection.
o Regulate mast cell function through the release of major basic protein (MBP), which causes
mast cell degranulation as well as cytokine production, and they also produce nerve growth
factor that promotes mast cell survival and activation.
 Eosinophilia is a hallmark of allergic disorders, of which asthma has been the best studied. The
number of eosinophils in blood and sputum correlates with disease severity. This has led to the
suggestion that the eosinophil is one of the causes of airway inflammation and mucosal cell
damage through secretion or production of a combination of basic proteins, lipid mediators, reactive
oxygen species, and cytokines such as IL-5.
 Eosinophil accumulation in the gastrointestinal tract occurs in allergic disorders such as food allergy,
allergic colitis, and inflammatory bowel disease such as Crohn’s disease and ulcerative colitis.

C. BASOPHIL - responds to ALLERGIC or HYPERSENSITIVITY REACTIONS

 Life span: 60 hours, relatively longer than that of the other granulocytes.
 Granules: water-soluble blue-black specific granules with affinity to Methylene blue which is the BASIC part
of the stain
- Histamine – mediates some hypersensitivity reactions
 Functions:
o Basophils are capable of releasing large quantities of subtype 2 helper T cell (TH2) cytokines such as
IL-4 and IL-13 that regulate the TH2 immune response
o Induce B cells to synthesize IgE.
o Initiators of the allergic inflammation through the release of preformed cytokines.
o Play a role in angiogenesis through the expression of vascular endothelial growth factor (VEGF) and
its receptors
 o Along with eosinophils, basophils are involved in the control of Helminth infections by enclosing
toxic egg products with granulomas and preventing tissue damage. They promote eosinophilia, are
associated with hindering migration of larvae to other organs, and contribute to efficient worm
expulsion.
o Play a nonredundant role in mediating acquired immunity against ticks.

D. MAST CELL
 Not considered to be leukocytes. They are tissue effector cells of allergic responses and inflammatory
reactions.
o Included here because (1) their precursors circulate in the peripheral blood for a brief period on
their way to their tissue destinations, and (2) mast cells have several phenotypic and functional
similarities with both basophils and eosinophils.
 Mast cell progenitors (MCPs) originate from the bone marrow and spleen. The progenitors are then released
to the blood before finally reaching tissues such as the intestine and lung, where they mediate their actions.
 The major cytokine responsible for mast cell maturation and differentiation is KIT ligand (stem cell factor)
 Once the MCP reaches its tissue destination, complete maturation into mature mast cells occurs.
 Functions:
o Effector cells in allergic reactions through the release of a wide variety of lipid mediators, proteases,
proteoglycans, and cytokines as a result of cross-linking of IgE on the mast cell surface by specific
allergens.
o Can also be activated independently of IgE, which leads to inflammatory reactions.
o Can function as antigen-presenting cells to induce the differentiation of TH2 cells.
o Anti-inflammatory and immunosuppressive functions - can both enhance and suppress features of
the immune response.
o May act as immunologic “gatekeepers” because of their location in mucosal surfaces and their role
in barrier function.
IV. MONONUCLEAR CELLS

A. MONOCYTES
STAGE CELL SIZE (um) NUCLEUS CYTOPLASM N/C RATIO
MONOBLAST 12-20 Round with folding Basophilic, non- 4:1-3:1
and clefting granular
1-2 nucleoli
PROMONOCYTE 12-18 Oval, indented, Blue-gray cytoplasm 3:1-2:1
folded
MONOCYTE 15-20 Round, KIDNEY- Blue-gray cytoplasm 2:1-1:1
LARGEST CELL IN SHAPED or Many fine
PERIPHERAL BLOOD HORSESHOE shaped, azurophilic granules
showing brain-like (azure dust)
convolutions appearing as
No nucleoli visible “GROUND GLASS”
(frosted)
 2% - 11% of circulating leukocytes
 Under normal circumstances, promonocytes undergo two mitotic divisions in 60 hours to produce a total of
four monocytes. Under conditions of increased demand for monocytes, promonocytes undergo four
divisions to yield a total of 16 monocytes in 60 hours.
 No storage pool of mature monocytes in the bone marrow. Unlike neutrophils, monocytes are released
immediately into the circulation upon maturation. Relatively large reservoir of immature monocytes resides
in the subcapsular red pulp of the spleen. Monocytes in this splenic reservoir appear to respond to tissue
injury such as myocardial infarction by migrating to the site of tissue injury to participate in wound healing.
 Monocytes remain in the circulation approximately 3 days.
 Once in the tissues, monocytes differentiate into macrophages, osteoclasts, or dendritic cells, depending on
the microenvironment of the local tissues.
 Macrophages can be as large as 40 to 50 mm in diameter, having an oval nucleus with a net-like (reticulated)
chromatin pattern. Their cytoplasm is pale, often vacuolated, and often filled with debris of phagocytized
cells or organisms.
 The life span of macrophages in the tissues depends on whether they are responding to inflammation or
infection, or they are “resident” macrophages such as Kupffer cells or alveolar macrophages. Resident
macrophages survive far longer than tissue neutrophils. For example, Kupffer cells have a life span of
approximately 21 days. Inflammatory macrophages, on the other hand, have a life span measured in hours.
 Monocyte/Macrophage Functions:
o Innate immunity: Monocytes/macrophages recognize a wide range of bacterial pathogens by means
of pattern recognition receptors (Toll-like receptors) that stimulate inflammatory cytokine
production and phagocytosis. Macrophages can synthesize nitric oxide, which is cytotoxic against
viruses, bacteria, fungi, protozoa, helminths, and tumor cells. Monocytes and macrophages also
have Fc receptors and complement receptors. Hence, they can phagocytize foreign organisms or
materials that have been coated with antibodies or complement components.
o Adaptive immunity: Both macrophages and dendritic cells degrade antigen and present antigen
fragments on their surfaces (antigen-presenting cells). Because of this, they interact with and
activate both T lymphocytes and B lymphocytes to initiate the adaptive immune response. Dendritic
cells are the most efficient and potent of the antigen-presenting cells.
o Housekeeping functions: These include removal of debris and dead cells at sites of infection or
tissue damage, destruction of senescent red blood cells and maintenance of a storage pool of iron
for erythropoiesis, and synthesis of a wide variety of proteins, including coagulation factors,
complement components, interleukins, growth factors, and enzymes.
B. LYMPHOCYTE
 Divided into three major groups: T cells, B cells, and natural killer (NK) cells.
o T and B cells are major players in adaptive immunity.
 3 CHARACTERISTICS OF ADAPTIVE IMMUNITY:
- It relies on an enormous number of distinct lymphocytes, each having surface receptors for
a different specific molecular structure on a foreign antigen
- After an encounter with a particular antigen, memory cells are produced that will react
faster and more vigorously to that same antigen on re-exposure.
- Self-antigens are “ignored” under normal circumstances (referred to as tolerance).
o NK cells make up a small percentage of lymphocytes and are part of innate immunity
o B lymphocytes or B cells - Antibody-producing lymphocytes; develop in the bone marrow.
o Cellular immunity is accomplished by two types of lymphocytes: T cells, so named because they
develop in the thymus, and NK cells, which develop in both the bone marrow and the thymus
 Lymphocytes:
o T cells: 51%-88% of circulating lymphocytes
o B cells: 3%-21% of circulating lymphocytes.
o NK cells: 4%-29% of circulating lymphocytes.
 Lymphocytes are different from the other leukocytes in several ways:
o Lymphocytes are not end cells. They are resting cells, and when stimulated, they undergo mitosis to
produce both memory and effector cells.
o Unlike other leukocytes, lymphocytes recirculate from the blood to the tissues and back to the
blood.
o B and T lymphocytes are capable of rearranging antigen receptor gene segments to produce a wide
variety of antibodies and surface receptors.
o Although early lymphocyte progenitors such as the common lymphoid progenitor originate in the
bone marrow, T and NK lymphocytes develop and mature outside the bone marrow
 Lymphocytes make up between 18% - 42% of circulating leukocytes
 LYMPHOCYTE PRODUCTION:
STAGE CELL SIZE (um) NUCELUS CYTOPLASM
 LYMPHOBLAST 10-18 Coarse chromatin No granules present
Round or oval in shape Appears smooth
1-2 nucleoli Moderate to dark blue
PROLYMPHOCYTE Same as More clumped chromatin Usually nongranular
lymphoblast or Round or oval in shape Moderate to dark blue
smaller 1-2 nucleoli
MATURE 8-10 Dense chromatin Usually forms a thin rim
SMALL LYMPHOCYTE Round or oval in shape around the nucleus
No nucleoli ROBIN’S EGG BLUE
MEDIUM LYMPHOCYTE 10-12 Chromatin not as dense as small More abundant that in
lymphocyte small lymphocyte
Round or oval in shape Pale to moderate blue
No nucleoli
LARGE LYMPHOCYTE 12-16 Round or oval in shape Abundant
No nucleoli Clear, very pale blue

 PLASMA CELLS

STAGE CELL SIZE (um) NUCELUS CYTOPLASM

PLASMABLAST 18-25 Eccentric Basophilic
Abundant
Nongranular
PROPLASMACYTE 15-25 Eccentric Intensely basophilic
Non granular
PLASMACYTE/ PLASMA 8-20 Eccentric Deeply basophilic
CELL Exhibits “CARTWHEEL” Moderately abundant
like pattern Large, well-defined
hof/perinuclear halo
Non granular
 LYMPHOCYTE FUNCTIONS:
 B cells:
o Essential for antibody production.
o Have a role in antigen presentation to T cells and may be necessary for optimal CD4
activation.
o Produce cytokines that regulate a variety of T cell and antigen-presenting cell
functions.
 T cells: can be divided into CD4+ T cells and CD8+ T cells.
o CD4+ effector lymphocytes are further subdivided into:
- Th1: mediate immune responses against intracellular pathogens.
- Th2: mediate host defense against extracellular parasites, including
helminths; also important in the induction of asthma and other allergic
diseases.
- Th17: involved in the immune responses against extracellular bacteria and
fungi.
- T reg (CD4+ & CD25+ regulatory T): play a role in maintaining self-tolerance
by regulating immune responses.
o CD8+ (cytotoxic T lymphocytes) effector lymphocytes are capable of killing target
cells by secreting granules containing granzyme and perforin or by activating
apoptotic pathways in the target cell.
 NK cells:
o Part of innate immunity and are capable of killing certain tumor cells and virus-
infected cells without prior sensitization.
o Modulate the functions of other cells, including macrophages and T cells.