HEMATOPOIESIS

 Definition – the formation/production and development of blood cells.

 Destruction and formation is balanced, therefore the number is cells is constant.
 Cell population is maintained through complex network of tissues, organs, stem cells, and
controlling factors.
 Network is responsible for the maturation and division of undifferentiated cells into
operational cell lines – WBC, RBC, and platelets.

Hematopoietic system
 Bone marrow, liver, spleen, lymph nodes, thymus where production, maturation, and
destruction occur.
 Marrow – hematopoiesis occurs in the extravascular part of the red marrow; single layer of
epithelial cells separate extravascular from intravascular compartment (venous sinuses)
 Almost mature cells –leave marrow parenchyma by squeezing through cytoplasmic
fenestrations in sinus endothelial lining cells -emerging into venous sinuses
 Infancy – early childhood – hematopoiesis in entire medullary space.
 By 4, fat cells appear in the long bones
 18 to 20 – sternum, ribs, pelvis vertebrae, skull (flat bones), proximal portion of long bones.
 > 40 – marrow has equal amounts of hematopoietic tissue and fat.
-Extra medullary hematopoiesis – active hematopiesis again found in spleen, liver, and other
tissues as a compensatory mechanism, during increased demand for blood cells (occurs
outside of the bone marrow).

Multipotential stem cell

 Produces mature circulating blood cells that have the capacity for self-renewal and
proliferation and differentiation into progenitor cells committed to one cell lineage.
 Provides cellular reserve for the progenitor stem cell.
 The committed stem cell – myeloid and lymphoid, are genetically destined to make
irreversibly differentiated cells and recognized by morphologically distinct progeny,
daughter cells.
Lymphoid stem cell
 T-lymph’s – cellular immune functions; directly cytotoxic or helping/suppressing immune
activities through interaction with other immunocompetent cells.
 -B- lymph’s – differentiate into plasmacytes, which secret Ig’s.
 Null cells – Killer cells – interact with Ab to cause destruction of Ab-coated targets.
 Natural killer – lyses target cells through direct cytotoxic activity.

CSF’S and Interleukins

- Regulate blood cell development by mediating proliferation, maturation of hematopoietic
progenitor cells.

- Also regulate survival and function of mature blood cells.

The effects of the CSF’s are mediated by specific surface receptors present on precursor
cells and mature cells.

 Interleukins – protein molecules that work in conjunction with CSFs to stimulate

particular cell lineages to proliferate and differentiate marrow.
IL-1 α and β, IL-2, IL –3, IL –4, IL –5, IL –6, IL –7

1
 Growth Factors, Other (cytokines)
 Erythropoietin – regulates growth of erythroid progenitor cells into mature erythrocytes
-Also stimulates bursts-colony formation.
-A hormonal glycoprotein, produced primarily by the kidneys.
-Primary stimulus for production is tissue hypoxia.
 Thrombopoietin – stimulates thrombopoiesis by stimulating megakaryocytic maturation
and the subsequent release of platelets.
--Combinations of two or more hematopoietic growth factors can support proliferation and
differentiation more effectively than can the individual
growth factors.

MORPHOLOGY OF HUMAN BLOOD AND MARROW CELLS

 Stem cells differentiate and become committed to production of one cell line, and
progenitor cells mature to develop into morphologically recognizable cell lines with
distinctive features.
 The stages of maturation are classified according to cellular characteristics and
staining properties, like, size, nuclear chromatin patterns, presence of nucleoli,
amount and color of cytoplasm, and ratio of nucleus to cytoplasm.
 Difference between cell lines and stages of same cell line.
 Cell maturation – slow progress from one stage to another .
 Asynchronism – When a part of the celll ( nucleus or cytoplasm ) matures at a
different rate from rest of the cell .
 Cytoplasm – RNA content decreases; granules increase. Ratio of cytoplasm/nucleus
increases.
 Nucleus - Immature forms are round /oval and large.
Mature forms take on specific shapes and get smaller.
Chromatin material (DNA) becomes more coarse (from fine, delicate)
Nucleoli become less visible as cell matures.
 Size of the cell – becomes smaller as cell matures. Mature cells sizes are as follows:
Erythrocytes……………………….....6 –8 microns
Band neutrophils …………………….9 – 15 microns
Seg neutrophils ……………………...9 -15microns
Eosinophils ………………………….9 – 15 microns
Basophils ……………………………8 – 15microns
Lymphocytes………………………...8 – 10 microns
10-12 microns
12-16microns
Monocytes ……………………….....14-20microns
Platelets (package of cytoplasm) …...1 –4 microns

ERYTHROPOIESIS
 Erythron – the total population of mature erythrocytes and their precursors in blood
and bone marrow and other sites.
 The widely distributed red blood cells function as a unit.
 Occurs in the central sinus beds of medullary marrow over 5 days.
 Apoptosis – programmed cell death; depends on the binding of receptor Fas and
FasL located on the membrane of the immature erythroid precursors (as
erythropoietin also binds “specific high-affinity receptors”)
2
  The two controlling systems achieve an equilibrium of production, usage,
destruction, homeostasis of erythroid line
Rubriblast (pronormoblast) – Prorubricyte (basophilic normoblast) – Rubricyte
(polychromatic normoblast) - Metarubricyte (orthochromatic normoblast) – Diffusely
Basophilic Erythrocyte (polychromatophilic erythrocyte) – Erythrocyte.
RUBRIBLAST
 Earliest recognizable cell of erythrocyte series.
 Round primitive nucleus with visible nucleoli and chromatin strands that are indistinct
and dispersed.
 Cytoplasm – royal blue
PRORUBRICYTE
 Daughter cells of rubriblasts; smaller.
 Difference from rubriblast – coarsening of the chromatin pattern and the nucleoli are not
visible with light microscope.
 Blue cytoplasm (RNA) with pink hue/tinge – presence of Hgb. Deeper, richer blue than
the blast; increased RNA completely masks the hgb pigmentation.
RUBRICYTE
 Relatively more cytoplasm mixed pink and blue (murky gray-blue)
 Chromatin pattern quite variable; condensed
METARUBRICYTE
 Solid blue-black degenerated nucleus, incapable of further DNA synthesis.
 Cytoplasm predominantly pinkish; bluish hue because of residual organelles –
mitochondria, ER, polyribosomes
RETICULOCYTE
 The pyknotic nucleus of a metarubricyte is extruded, leaving a diffusely basophilic or
polychromatophilic cell. Some of the bluish color remains due to the presence of RNA.
 Contains 2/3 of RBC’s total Hgb contents – predominant pigment
 Released in 2-3 days into peripheral blood, then takes 1-2 days to mature into an
erythrocyte.
 New methylene blue stain – reveal ribosomes in a granulofilamentous arrangement –
then classified as reticulocytes
 # increased with anemia or hypoxia- released early from marrow.
DISCOCYTE
 Normal, mature erythrocyte – biconcave disc.
 Central pallor – less intense stain where cell is thinnest.
 Not able to synthesize protein – no nucleus or mitochondria but can sustain self.
 Are flexible and deformable, making them capable of unusual changes in shape that are
necessary for passage through the microcirculation to transport oxygen.

GRANULOCYTOPOIESIS
 Or myelopoiesis – production of neutrophils, eosinophils, basophils
 Neutrophils, eosinophils and basophils have similar patterns of proliferation,
differentiation, and division.
 During maturation there is a reduction in nuclear volume, condensation of chromatin,
appearance and disappearance of primary granules, appearance of secondary granules,
color changes in cytoplasm from blue to pinkish red, and change in size of cells.

3
Myeloblast → promyelocyte → myelocyte → metamyelocyte → band neutrophil →
Eosinophil, basophil
segmented neutrophil, eosinophil, basophil

Myeloblast
 Earliest recognizable cell of the granulocytic series
 Has a round nucleus that stains predominantly reddish-blue and has a smooth nuclear
membrane
 Has a slight to moderate cytoplasm that is bluish and non-granular
 Is smaller and has less blue cytoplasm than a rubriblast
Promelocyte:
 First time to see granules, primary granules, that stain a dark blue or
reddish blue (azurophilic granules) and may be round or irregular in shape.
 The granules contain acid phosphatase and peroxidase, hydrolytic enzymes, elastase, beta-
glucuronidase and other basic proteins.
 The nucleus is still round and eccentric or central with a light reddish-blue colour and slight
aggregation of chromatin at the nuclear membrane.
Myelocyte
 where less dense, secondary granules are formed. Primary granules are no
longer synthesized.
 The secondary granules are specific for neutrophils and contain amino
peptidase, alkaline phosphatase, collagenase, lysozyme, lactoferrin, plasminogen activators.
 The nucleus is oval or round and slightly indented; it is commonly eccentric, but may be
central. It is reddish-blue having a fine chromatin pattern with slightly aggregated or
granular pattern.
Metamyelocyte
 identifying factor is the nucleus becomes slightly indented (bean- or
kidney-shaped)
 The indentation is less than half the width of the arbitrary round nucleus
 There is noticeable condensation of chromatin
 The cell does not divide nor does it contain nucleoli
 The cytoplasm is clear pink and moderate with many small, pinkish secondary granules that
fill the cytoplasm, and a few primary granules that may still remain.
Band neutrophil
 Nuclear indentation is greater than half the width of the nucleus. The opposite edges of the
nucleus become almost parallel
 The cytoplasm is pink and abundant.
 The secondary granules are small and evenly distributed.
Segmented neutrophil
 The nucleus is normally separated into two to five lobes with a narrow filament or strand
connecting the lobes
 Nuclear chromatin is heavily clumped or pyknotic and stains purplish red
 The cytoplasm in an ideal stain is light pink and the small, numerous, and evenly distributed
secondary granules either stain pink or take a neutral stain.
 Neutrophil secondary granules are lysosomes that contain alkaline phosphatase
 Mature neutrophils are approximately twice the size of normal erythrocytes

4
Tissue Neutrophil
 Large, narrow cells with ample cytoplasm that is irregular with blunt pseudopods and often
multipointed and may have nebulous cytoplasmic streamers
 The cells are readily indented by adjacent marrow cells or are squeezed between them
 The cytoplasm stains light blue and has a fine, lattice-like structure
 Granules vary in number and stain-varying shades of red to blue but majority has a reddish
purple stain. They are beadlike aggregates
 They occur infrequently in normal bone marrow, but are found in increased numbers in
patients having conditions in which there is a proliferation of neutrophilic cells such as
myelocytic leukemia, myelocytic-monocytic leukemia, and myelofibrosis; also found in
neutropenic states (arrest in maturation).

Eosinophils
 Eosinophils pass through the same developmental stages as neutrophils.
 The earliest eosinophil (eosinophilic myelocyte) has a few dark bluish primary granules
intermingled with the few specific, reddish-orange granules.
 During development the bluish granules become less visible and disappear, and the round,
specific, or secondary bright red eosinophilic granules fill the cytoplasm.
 Eosinophils spend 3 to 6 days in production in the marrow before appearing in the peripheral
blood. Bone marrow provides a storage area for eosinophils so that they can be rapidly
mobilized when needed.
 Eosinophils migrate from blood to tissue, such as bronchial mucosa, skin, gastrointestinal
tract, and vagina in about 12 days.
 Eosinophil granulocytes are one of the immune system components responsible for
combating multicellular parasites and certain infections in vertebrates.

 Along with mast cells, they also control mechanisms associated with allergy and asthma.

 They are granulocytes that develop during hematopoiesis in the bone marrow before
migrating into blood.

 These cells are eosinophilic or 'acid-loving' as shown by their affinity to coal and tar dyes:
Normally transparent, it is this affinity that causes them to appear brick-red after staining
with eosin, a red dye, using the Romanowsky method.

 The staining is concentrated in small granules within the cellular cytoplasm, which contain
many chemical mediators, such as histamines and proteins such as eosinophil peroxidase,
ribonuclease (RNase), deoxyribonucleases, lipase, plasminogen, and major basic protein.
These mediators are released by a process called degranulation following activation of the
eosinophil, and are toxic to both parasite and host tissues.

Basophils
 Basophils may be identified as basophilic myelocytes, metamyelocytes, bands, and
segmented cells based upon the shape of their nuclei. The shape of the nucleus is, however,
often masked by large basophilic granules.
 The specific blue granules of basophils are formed in the myelocytic stage and continue to
be produced throughout all later maturation stages.

5
  There are also some smaller granules that do not stain as darkly as the specific basophil
granules but instead tend to be more reddish-blue.
 Basophils appear in many specific kinds of inflammatory reactions, particularly those that
cause allergic symptoms. Basophils contain anticoagulant heparin, which prevents blood
from clotting too quickly. They also contain the vasodilator histamine, which promotes
blood flow to tissues.
 Basophil granulocytes, sometimes referred to as basophils, are the least common of the
granulocytes, representing about 0.01% to 0.3% of circulating white blood cells.

 The name comes from the fact that these leukocytes are basophilic, i.e., they are susceptible
to staining by basic dyes, as shown in the picture.

 Basophils contain large cytoplasmic granules which obscure the cell nucleus under the
microscope. However, when unstained, the nucleus is visible and it usually has 2 lobes. The
mast cell, a cell in tissues, has many similar characteristics. For example, both cell types
store histamine, a chemical that is secreted by the cells when stimulated in certain ways
(histamine causes some of the symptoms of an allergic reaction). Like all circulating
granulocytes, basophils can be recruited out of the blood into a tissue when needed

 Basophils appear in many specific kinds of inflammatory reactions, particularly those that
cause allergic symptoms.
 Basophils contain anticoagulant heparin, which prevents blood from clotting too quickly.
They also contain the vasodilator histamine, which promotes blood flow to tissues.
 They can be found in unusually high numbers at sites of ectoparasite infection, e.g., ticks.
 Like eosinophils, basophils play a role in both parasitic infections and allergies. They are
found in tissues where allergic reactions are occurring and probably contribute to the
severity of these reactions.
 Basophils have protein receptors on their cell surface that bind IgE, an immunoglobulin
involved in macroparasite defense and allergy. It is the bound IgE antibody that confers a
selective response of these cells to environmental substances, for example, pollen proteins or
helminth antigens.
 Recent studies in mice suggest that basophils may also regulate the behavior of T cells and
mediate the magnitude of the secondary immune response.

MONOPOEISIS
 The mononuclear-phagocyte system (MPS) is composed of monocytes, macrophages, and
their precursors
Monoblasts and promonocytes
Monoblasts divide and give rise to promonocytes.
 Promonocytes and monoblasts are not easily identifiable in bone marrow or peripheral blood
smears except in disorders in which there is marked proliferation of monocytic cells.
Monocytes and macrophages
 Monocytes and macrophages constitute the monocyte-macrophage phagocytic system.
Monocytes have abundant cytoplasm in relation to the nucleus.
 With Wright’s stain the cytoplasm turns a dull-blue in contrast to the pink cytoplasm of
neutrophils.
 Numerous fine, small, reddish – or purplish-stained, evenly distributed granules in the
cytoplasm give the cell a ground-glass appearance.

6
  Digestive vacuoles may be observed in the cytoplasm. In disease states phagocytized
erythrocytes, nuclei, cell fragments, bacteria, fungi, and pigment may be present.
 The nuclei of monocytes frequently may be kidney – shaped, deeply folded, or indented or
occasionally lobular.
 One of the distinctive features of is the appearance of brain like convolution in the nucleus.
Another characteristic is the lacy chromatin network of fine strands intermingled with small
chromatin clumps.
 Three helpful characteristic features of brain like nuclear convolutions, blunt pseudopods,
and dull gray-blue cytoplasm.
 As monocytes grow they become too large to pass readily through capillaries, and so they
move into tissue and convert into macrophages in many organs.

LYMPHOPOIESIS
 Common lymphoid progenitor cell can be differentiated into either T or B cells. T. cells
differentiate in the thymus, B cells in adult bone marrow.
 Primary lymphoid organs, thymus, marrow, secondary lymphoid organs, lymph nodes, mucosal
tissues, spleen.
LYMPHOBLASTS AND PROLYMPHOCYTES
 The earliest lymphocytes are identified as lymphoblasts and prolymphocytes. The nuclear
chromatin strands in lymphoblasts are thin, evenly stained, and not clumped.
 Prolymphocytes have an intermediate chromatin pattern that has clumps in some areas of
the nucleus but do not appear as clumped as in mature lymphocytes.
LYMPHOCYTES
 Lymphocytes are the second most numerous white blood cells in the blood.
 Most lymphocytes are small.
 Large lymphocytes may be 12 to 15 μm or more. Size varies with the thickness of the smear.
 Small lymphocytes usually are round with smooth margins.
 With Wright’s stain, color of the cytoplasm is blue, with intensity of blue varying from light
to dark in different cells.
 The intensity of the blue stain is greater at the periphery of the cell than near the golgi area
adjacent to the nucleus.
 The majority of lymphocytes do not have granules .Some large cells may have a variable
number of a few well-defined granules of large size that are unevenly distributed, and may
be easily counted.
 The diameter of the nucleus of the smallest lymphocyte in the peripheral blood is slightly
larger than or the same size as a normal erythrocyte in the same microscopic field. The
lymphocyte’s nucleus in relation to its cytoplasm is large.
 T, B, and null cells cannot be separately identified morphologically but can be distinguished
functionally and by immunologic marker studies.
 In reaction to appropriate antigenic stimuli, lymphocytes have been shown to transform
into cells that are immunologically competent. Usually these cells are large with abundant
cytoplasm.
 The increase in size is due to an increase in DNA in the nucleus and RNA in the cytoplasm.
 The cytoplasm may appear bubby and vacuoles may be seen.
 Most striking variants are observed in infectious mononucleosis; they are also present in
other viral diseases (CMV, infectious hepatitis), post–transfusion reactions, organ transplant
and serum sickness.

7
 To distinguish monocytes from large lymphocytes, nuclear chromatin structure, character
of the cytoplasm, shape of the cells is useful.
 The nucleus of a lymphocyte tends to be clumped rather than linear or lacy, as it is in a
monocyte greater tendency for the nuclear chromatin to be condensed at the periphery of
the nucleus in the lymphocyte. Brain-like convolutions present in a monocyte are not
observed in a lymphocyte.
 Lymphocytes may have distinct bluish-red granules.
 The large bluish-red granules are interspersed with numerous fine granules in the cytoplasm
and cannot be enumerated. Large granules prominent and can be counted easily because
there are no other granules. Monocyte has a ground – glass appearance whereas lymphocyte
has a relatively clear non-granular background.
 Large lymphocytes are often deeply intended by neighboring red blood cells. Monocytes
tend to project blunt pseudopods between cells or to compress cells, rather than being
indented by them.
 Plasmablasts are identified primarily in the presence of proplasmacytes and plasmacytes
but cannot be differentiated easily from other blasts.
 Plasmablast and proplasmacytes, not observed in the bone marrow, are seen in diseases
associated with abnormal immunoglobin production, e.g. multiple myeloma.
 Plasmacytes represent the end stage of B-lymphocyte lineage.
 Mature plasmacytes - size 15 to 25μm

MEGAKARYOCYTOPOIESIS
 The megakaryocyte is the largest hematopoietic cell in the bone marrow
 The mission of megakaryocytes is to proliferate and then fragment their cytoplasm into
platelets when needed in order to maintain a normal number.
 Because of their size and volume, megakaryocyte precursors increase their amount of
nuclear chromatin (or DNA doublings) without cytoplasmic division (process of
endomitosis).
 A promegakaryocyte not only increases the size of the nucleus, but it becomes lobed.
 The membrane demarcation system establishes the outer limit of each platelet which
arises as a cytoplasmic fragment.
 After maturatuion is completed, the megakaryocyte membrane ruptures, the entire
megakaryocyte cytoplasm fragments, and thrombopoiesis occurs.
 Platelets are cytoplasmic fragments and have no nucleus.
 Platelets stain light blue and contain a variable number of small reddish blue granules.
 Platelets tend to adhere to each other and may form small aggregate in a well – made,
normal smear.

OSTEOBLASTS
 Osteoblast is a large cell with ample cytoplasm and a small, round, eccentrically
p[laced nucleus .
 Osteoblasts are most often seen in marrow from young children, are responsible for
the formation, calcification, and maintenance of trabeculae bone. Osteoblasts
morphologically resemble plasmacytes.
 The relatively unstained zone of the plasmacytes is adjacent to the nucleus whereas
the chromophobic zone of the osteoblast is often distinctly separate from the nuclear
margin.

8
 OSTEOCLASTS
 Osteoclasts are giant, multinucleated, irregularly shaped marrow phagocytes that are
capable of absorbing of bone; have from 2 to 50 nuclei.
 Osteoclasts and megakaryocytes are sometimes difficult to differentiate.
 The nuclei of megakaryocytes are connected by nuclear strand, whereas nuclei of
osteoclasts are separated, uniform in size, have no visible connection to each other.
 Osteoclasts secrete enzymes that aid in dissolution of osteoid tissue and calcific
bone.

TRENDS IN THERAPEUTIC MANIPULATION OF HEMATOPOESIS

 Colony- stimulating factors have been employed to strengthen patients with
cancer and acquired immunodeficiency syndrome (AIDS) and to guard against
infection for bone marrow transplant patients.
 Also used to treat anemia due either to surgery or to kidney failure.
 Interleukins are used clinically for wound healing, activating lymphocytes, and
assisting in the growth of transplanted or damaged bone marrow.

Trabecula/e – any of the thin bars of bony tissue in spongy *bone