HEMA1: HEMATOLOGY 1 (LECTURE)

LECTURE 8: LEUKOPOIESIS, KINETICS AND FUNCTION

1st SEMESTER | S.Y. 2024-2025

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 RATI
(um) O
SHAPE CHROMATI NUCLEO STAINING GRANULES
N LI
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”
eccentric in normal ones

MYELOCYTE 15-18 Oval or Coarser none Mixture of SECONDRY 1:1 Final stage of
(6%-17% in BM) round and basophilic GRANULES Mitosis
condensed &
acidophili
c
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 STAFF 9-15 Elongated Coarse & none Beige or Faint 1:1-
(0-5% in Peripheral, 9- or band- clumped salmon 1:2
32% in BM) shaped (C staining
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- circulation
BLACK

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

CABAY, J.B. | BMLS3 | PLT COLLEGE, INC.

 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
- Mitotic pool – consist of myeloblast, promyelocyte and myelocyte
- Maturating pool – consists of metamyelocyte
 - Storage pool – consists of metamyelocyte, bands and segmented PMNs
- Blood
- Circulating pool – 50% of PMNs circulating freely
- 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)
- 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:
- 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.
- 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.
- 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:
- 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.
- Can also be activated independently of IgE,
which leads to inflammatory reactions.
- Can function as antigen-presenting cells to
induce the differentiation of TH2 cells.
- Anti-inflammatory and immunosuppressive
functions - can both enhance and suppress
features of the immune response.
- 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 Basophilic, non- 4:1-3:1
folding and granular
clefting
1-2 nucleoli
PROMONOCYTE 12-18 Oval, indented, Blue-gray 3:1-2:1
folded cytoplasm
MONOCYTE 15-20 Round, KIDNEY- Blue-gray 2:1-1:1
LARGEST CELL SHAPED or cytoplasm
IN PERIPHERAL HORSESHOE Many fine
BLOOD shaped, showing azurophilic
brain-like granules (azure
convolutions dust) appearing
 No nucleoli as “GROUND
visible 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:
- 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.
- 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.
- 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 NUCELUS CYTOPLASM
(um)
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/ 8-20 Eccentric Deeply basophilic
PLASMA CELL Exhibits Moderately abundant
“CARTWHEEL” like Large, well-defined
pattern 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.