HEMATOPOIESIS

PART 2: ERYTHROPOIESIS AND LEUKOPOIESIS

NORMOBLASTIC MATURATION

• Erythrocyte has one true function – to carry oxygen from the lungs to tissues.

• The major cytoplasmic component of a mature RBC is hemoglobin.

• Erythrocyte’s secondary function is to return carbon dioxide to the lungs and

buffering the pH of the blood
NORMOBLASTIC MATURATION

• RELATED TERMS:
• Erythron – total mass of RBCs circulating in the peripheral blood AND the bone
marrow RBC precursor
• RBC mass – erythrocytes in circulation
• Erythrokinetics – a term that describes the dynamics of RBC creation and destruction
• Ineffective erythropoiesis – production of defective erythroid precursor cells
• Insufficient erythropoiesis – decrease in the number of RBC precursors in the bone
marrow
NORMOBLASTIC MATURATION

Example of hormones related to erythropoiesis:

• Erythropoietin (EPO)
• Chief stimulatory cytokine for RBCs
• Major hormone that stimulates
production of erythrocytes
• Thermostable, nondializable,
glycoprotein hormone
• Normally released from the kidneys
into the blood in response to hypoxia
• 3 major effects
• Allowing early release of reticulocytes
from the bone marrow

• Primarily from the peritubular • Reducing the time needed for cells to
mature in the bone marrow
interstitial cells of the kidneys
• Preventing apoptotic cell death
• Primary target cells: BFU-E and CFU- (apoptosis rescue is the major way in
E which EPO increases RBC mass)
NORMOBLASTIC MATURATION

Example of hormones related to erythropoiesis – con’t

• Growth hormone • Prolactin
• Produced by anterior pituitary gland • Produced by the anterior pituitary gland
• Directly stimulates erythropoiesis • Directly stimulates erythropoiesis
• Testosterone • Estrogen
• Produced by the leydig cells of the • Produced by the ovaries
testes • Indirectly inhibits erythropoiesis
• Indirectly stimulates erythropoiesis
NORMOBLASTIC MATURATION

• TERMINOLOGY
NORMOBLASTIC MATURATION

• PROGENITOR CELLS vs PRECURSOR CELLS

• Progenitor cells are immature hematopoietic cells that are committed to a cell line but
are morphologically unidentifiable.

• Precursor cells are immature hematopoietic cells that are morphologically identifiable
as belonging to a given cell line
NORMOBLASTIC MATURATION

• ERYTHROID PROGENITOR
• The burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E),
both committed to the erythroid cell line, are progenitor cells that give rise to
morphologically identifiable precursor cells.
• It takes about 1 week for BFU-E to mature into CFU-E and another week for CFU-E to
become a precursor cell.
• At the CFU-E stage, the cell completes at least 3 – 5 divisions before maturing further.
NORMOBLASTIC MATURATION

• ERYTHROID PRECURSOR
• Pronormoblast – the earliest morphologically recognizable erythroid precursor
• From a single pronormoblast, 8-32 mature RBCs usually result.
NORMOBLASTIC MATURATION

• Changes that happen to RBCs during maturation:

• Overall diameter of the cell decreases
• N:C ratio decreases
• Nuclear chromatin pattern becomes condensed
• Nucleoli disappears
• Cytoplasm loses its basophilia
NORMOBLASTIC MATURATION SEQUENCE

• PRONORMOBLAST
• Size: 12-20 m in diameter
• Cytoplasm: small in amount, dark blue,
non-granular, homogenous
• Nucleus: round to oval, thin nuclear
membrane, central or slightly eccentric
(occupies 80%)
• N:C ratio: 8:1
• Nucleoli: 1-2
• Chromatin pattern: fine
NORMOBLASTIC MATURATION SEQUENCE

• BASOPHILIC NORMOBLAST
• Size: 10-15 m in diameter
• Cytoplasm: appears more abundant than in
pronormoblast, deeper-rich blue, non-granular
• Nucleus: round/slightly oval and smaller, thin
nuclear membrane, eccentric (occupies 75%)
• N:C ratio: 6:1
• Nucleoli: 0-1
• Chromatin pattern: slightly coarser than
previous
NORMOBLASTIC MATURATION SEQUENCE

• BASOPHILIC NORMOBLAST
• NOTE:
• Last stage with nucleolus
• Detectable hemoglobin synthesis occurs
(hemoglobinization)
NORMOBLASTIC MATURATION SEQUENCE

• POLYCHROMATIC NORMOBLAST
• Size: 10-12 m in diameter
• Cytoplasm: basophilic to diffusely lilac in
color (muddy or gray/murky gray-blue),
non-granular
• Nucleus: round and smaller, thick nuclear
membrane, eccentric nucleus
• N:C ratio: 4:1
• Nucleoli: 0
• Chromatin pattern: more condensed
NORMOBLASTIC MATURATION SEQUENCE

• POLYCHROMATIC NORMOBLAST
• NOTE
• Last stage capable of mitosis
• First stage where the cytoplasm becomes pink
• May be confused with lymphocyte
• Lymphocyte
• Nucleus: crushed velvet
• Cytoplasm: robin egg blue
• Rubricyte
• Nucleus: checkerboard
• Cytoplasm: muddy/gray
NORMOBLASTIC MATURATION SEQUENCE

• ORTHOCHROMIC NORMOBLAST
• Size: 8-10 m in diameter
• Cytoplasm: salmon-pink
• Nucleus: pyknotic (dense mass of
degenerated chromatin)
• N:C ratio: 1:2
• Nucleoli: 0
NORMOBLASTIC MATURATION SEQUENCE

• ORTHOCHROMIC NORMOBLAST
• NOTE:
• Last stage with nucleus
• Pyrenocyte – enveloped extruded nucleus
which is later engulfed by macrophages
• Frequently, small fragments of the nucleus
is left behind if the projection is pinched off
before the entire nucleus is enveloped (the
fragments are called Howell-Jolly bodies
when seen in circulating RBCs.
NORMOBLASTIC MATURATION SEQUENCE

• POLYCHROMATIC
ERYTHROCYTE
• Size: 8-10 m in diameter
• Cytoplasm: with small amounts of RNA
(polychromasia), with golgi apparatus
remnants and residual mitochondria that
allows continued aerobic metabolism and
hgb production
• Nucleus: none
NORMOBLASTIC MATURATION SEQUENCE

• POLYCHROMATIC
ERYTHROCYTE
• NOTE:
• Last immature erythrocyte stage
• Last stage of hgb synthesis
• Shift cells
• Polychromatophilic macrocytes
• Seen in cases of increased RBC production
• Stress reticulocytes
• Macroreticulocytes
• Seen in more severe conditions such as
hemolytic anemia
NORMOBLASTIC MATURATION SEQUENCE

• MATURE ERYTHROCYTE
• Size: 7-8 m in diameter
• Cytoplasm: salmon-pink with central pallor
occupying 1/3 of the cell diameter
• Nucleus: none
NORMOBLASTIC MATURATION SEQUENCE

• MATURE ERYTHROCYTE
• NOTE:
• Shape: biconcave
• Thickness: 1.5-2.5 m
• Average life span: 120 days
• Adult RBCs has no mitochondria (no
protein or hgb synthesis)
LEUKOCYTE DEVELOPMENT, KINETICS, AND
FUNCTION
• LEUKOCYTES
• Are named white blood cells because they are relatively colorless as compared to RBCs
• The number of different types of leukocyte varies depending on whether they are being viewed
with a light microscope after staining with a Romanowsky stain (5-6 types) or are identified
according to their surface antigens using flow cytometry (at least 10 different types).
• The number of circulating leukocytes varies with sex, age, activity, time of day, and ethnicity;
it also differs according to whether or not the leukocytes are reacting to stress, being consumed,
or being destroyed, and whether or not they are being produced by the bone marrow in
sufficient numbers.
• The overall function of leukocytes is in mediating immunity, either innate (nonspecific), as in
phagocytosis by neutrophils, or specific (adaptive), as in production of antibodies by
lymphocytes and plasma cells.
LEUKOCYTE DEVELOPMENT, KINETICS, AND
FUNCTION
• GRANULOCYTES/POLYMORPHONUCLEARS
• Group of leukocytes whose cytoplasm is filled with granules with differing staining characteristics
and whose nuclei are segmented or lobulated
• Eosinophils – have granules containing basic proteins that stain with acid stain such as eosin
• Basophils – with granules that are acidic and stain with basic stain such as methylene blue
• Neutrophils – with granules that react to both acid and basic stains; aka polymorphonuclears
• AGRANULOCYTES/MONONUCLEARS
• Cells that have nuclei that are not segmented but are round, oval, undented, or folded.
• Categorized into monocytes and lymphocytes
GRANULOCYTES/POLYMORPHONUCLEARS

• NEUTROPHILS
• Present in peripheral blood as to having segmented nucleus or still in a band shape
• Segmented neutrophils make up the vast majority of circulating leukocytes
• Predominant WBC in an adult
• phagocyte

• EOSINOPHILS
• Make up 1% to 3% of nucleated cells in the bone marrow
• account for 1% to 3% of peripheral blood leukocyte
• phagocyte
• BASOPHILS
• Are true leukocytes
• The least numerous of the WBCs, making up between 0% to 2% of circulating leukocytes and less than 1% of nucleated cells
in the BM
• phagocyte
MONONUCLEAR CELLS

• MONOCYTES
• Make up between 2% and 11% of circulating leukocytes
• phagocyte

• LYMPHOCYTES
• Predominant WBC in children <4 years of age
• immunocyte
• Divided into three major groups
• T cells and B cells which play a major role in adaptive immunity
• NK cells that make up a small percentage of lymphocytes and are part of innate immunity
MONONUCLEAR CELLS

• LYMPHOCYTES
• Differ from other leukocytes in the following ways:
• Lymphocytes are not end cells (resting cells). When stimulated, they undergo mitosis to
produce both memory and effector cells.
• Leukocytes recirculate from the blood to the tissue 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.
• T and NK lymphocyte develop and mature outside the bone marrow.
GRANULOPOIESIS

• THERE ARE THREE POOLS OF DEVELOPING GRANULOCYTE IN THE BM:

• Stem cell pool
• Consist of HSCs that are capable of self-renewal and differentiation
• Proliferation (mitotic) pool
• Consists of cells that are dividing and includes CMPs, also known as CFU-GEMMs, GMPs, myeloblasts,
promyelocyte, and myelocytes
• Maturation (storage) pool
• Consists of cells undergoing nuclear maturation that form that marrow reserve and are available for release:
metamyelocytes, band cells and mature granulocytes
GRANULOPOIESIS

• MYELOBLAST
• Size: 14-20 m in diameter
• Cytoplasm: dark blue
• Are frequently subdivided into Type I, Type II
and Type III
GRANULOPOIESIS

• MYELOBLAST
• Type I myeloblasts
• Nucleus: occupies most of the cells
• Cytoplasm: very little , slightly
basophilic
• N:C ratio: 8:1 to 4:1
• Chromatin pattern: fine
• Nucleoli: 2-4
• Granules: no visible granules when
viewed with light microscopy with
Romanowsky stain
GRANULOPOIESIS

• MYELOBLAST
• Type II myeloblasts
• Granules: presence of dispersed
primary (azurophilic) granules in the
cytoplasm
• Primary granules are also known as
nonspecific granules
• Number of granules does not exceed 20
per cell
GRANULOPOIESIS

• MYELOBLAST
• Type III myeloblasts
• Cytoplasm: more purple
• Chromatin: darker
• Granules: more than 20 granules that do
not obscure the nucleus
• Rare in normal bone marrows but can be
observed in certain types of acute
myeloid leukemias
GRANULOPOIESIS

• PROMYELOCYTE
• Size: 16-25 m in diameter
• Cytoplasm: evenly basophilic and full of primary
(azurophilic) granules
• Nucleus: round to oval, often eccentric
• Nucleoli: 1-3 but can be obscured by the granules
• Chromatin pattern: clumping (heterochromatin)
may be visible especially around the edges of the
nucleus
• A paranuclear halo “hof” is usually seen in
normal promyelocytes but not in malignant
promyelocytes of acute promyelocytic leukemia
GRANULOPOIESIS

• MYELOCYTE
• Last stage capable of mitosis
• Cessation of primary granule production
• Secondary (specific) granules starts to be
produced
• Sometimes divided in:
• Early myelocytes
• Late myelocyte
GRANULOPOIESIS

• MYELOCYTE
• Early myelocyte
• May look very similar to promyelocyte in
size and nuclear characteristic except that
patches of grainy pale pink cytoplasm
representing secondary granules begin to be
evident in the area of the Golgi apparatus
• “Dawn of neutrophilia”
GRANULOPOIESIS

• MYELOCYTE
• Late myelocyte
• Somewhat smaller than promyelocytes (15-
18 m in diameter)
• nucleus has considerably more
heterochromatin
• Nucleoli are difficult to see by light
microscopy
GRANULOPOIESIS

• METAMYELOCYTE
• Size: 14-16 m in diameter
• Cytoplasm: contains vey little residual
RNA; little or ni basophilia
• Nucleus: indented (kidney bean shaped or
peanut shaped)
• Nucleoli: absent
• Chromatin pattern: increasingly clumped
• Synthesis of tertiary granules (gelatinase
granules) may begin in this stage
GRANULOPOIESIS

• METAMYELOCYTE
• NOTE:
• Also known as juvenile cells
• First stage of nuclear indentation
GRANULOPOIESIS

• BAND
• Size: 9-15 m in diameter
• Nucleus: highly clumped; indentation that began
in the metamyelocyte stage now exceeds half
the diameter of the nucleus but segmentation has
not yet occurred; elongated, curved, sausage-
shape with round ends
• Chromatin pattern: increasingly clumped
• Tertiary granules continue to be formed in this
stage
• Secretory granules (secretory vesicle) may begin
to be formed in this stage
GRANULOPOIESIS

• BAND
• NOTE:
• aka stab cell or staff cell
• Youngest to normally appear in PB
• CLSI recommends that bands should be
included within the neutrophil count and
NOT REPORTED AS A SEPARATE
CATEGORY
GRANULOPOIESIS
(MATURE GRANULOCYTES)
• SEGMENTED NEUTROPHIL
• Size: 9-15 m in diameter
• Secretory granules continue to be formed in this stage
• Has 2-5 nuclear lobes connected by a nuclear filament
• Neutrophilia – increased level of neutrophil in the
blood
• Physiologic neutrophilia – a shift of migrated cells to the
circulatory pool usually caused different physiologic
activities and states
• Neutropenia – decreased level of neutrophil in the
blood
• Agranulocytosis or extreme neutropenia is neutrophil
count <0.5x109/L usually caused by amidopyrine and
cephalosporin
GRANULOPOIESIS
(MATURE GRANULOCYTES)

• EOSINOPHIL
• Size: 9-15 m in diameter
• Nucleus: bilobed
• Cytoplasm: contains characteristic refractile,
orange-red secondary granules
• Eosinophilia – increased levels of
eosinophils in the blood
• Trichinosis may possibly produce the
highest eosinophil count
GRANULOPOIESIS
(MATURE GRANULOCYTES)

• BASOPHILS
• Size: 9-15 m in diameter
• Nucleus: lobulated and is often obscured by
its granules; segmented with visible
filaments occur rarely
• Cytoplasm: colorless; contains large
numbers of characteristic large blue-black
granules
• Chromatin pattern: clumped
MONOPOIESIS

• Similar to neutrophil development because both are derived from GMP

• Macrophage colony-stimulating factor (M-CSF) is the major cytokine
responsible for growth and development of monocytes
MONOPOIESIS

• MONOBLAST
• Size: 12-20 m in diameter
• N:C ratio: 4:1 to 3:1
• Cytoplasm: ample variably basophilic
cytoplasm that may be agranular or contain
MPO-negative fine basophilic granules.
• Nucleus: round to oval, central or eccentric,
can show evidence of indentation or folding
• Chromatin pattern: fine
MONOPOIESIS

• PROMONOCYTE
• Size: 12-18 m in diameter
• Cytoplasm: blue, contain scattered azure
granules
• Nucleus: slightly indented or folded
MONOPOIESIS

• MONOCYTE
• Size: 15-20 m in diameter
• Nucleus: round or oval or kidney-shaped,
but more frequently is deeply indented
(horseshoe shaped) or folded on itself
• Chromatin pattern: lacelike or stringy
• Nucleoli: present in roughly half of
circulating monocyte
• Cytoplasm: blue-gray, has azure granules
often referred to as azure dust or a ground-
glass appearance
MONOPOIESIS

• MONOCYTE
• NOTE:
• Small cytoplasmic pseudopods or blebs may
be seen
• Cytoplasmic and nuclear vacuoles may be
present
• Tends to stick to and spread out on glass or
plastic
• Slightly immature whose goal is to enter the
tissue and mature into macrophages,
osteoclast, or dendritic cells
MONOPOIESIS

• MACROPHAGE
• Size: 20-40 m in diameter
• Monocytes found in tissue
• Some functions of macrophage:
• Phagocytosis
• Synthesize nitric oxide
• Release IL-1
• Produce transcobalamin II
LYMPHOPOIESIS

• LYMPHOBLAST
• Similar to other blast cells
• Large, reddish, purple nucleus
• Fine chromatin
• Several nucleoli or 1-2 nucleus
• Cytoplasm is non granular
LYMPHOPOIESIS

• LYMPHOBLAST
• Has 2 categories:
• Small lymphoblast
• 1.0 to 2.5 times the size of a normal
lymphocyte
• Scant blue cytoplasm
• Indistinct nucleoli
• Large lumphoblast
• 2 to 3 times the size of a normal lymphocyte
• Has nucleolar membrane irregularities
• Has prominent nucleoli
LYMPHOPOIESIS

• PROLYMPHOCYTE
• Nucleus is still large with remnants of
nucleoli
• Chromatin is condensed to a mosaic pattern
LYMPHOPOIESIS

• LYMPHOCYTE
• Nucleus: dense, round, oval, or slightly indented
• Small lymphocyte
• 7-10 m in diameter
• Predominant type of lymphocyte in normal adult
blood
• Composed mostly of nucleus with scanty cytoplasm
• Medium/intermediate lymphocyte
• 10-12 m in diameter
• Large lymphocyte
• 11-25 m in diameter
• Has abundant cytoplasm
LYMPHOPOIESIS

• LYMPHOCYTE
• Three physiologic characteristics of
lymphocytes:
• Predestined to migrate
• Heterogeneous group of cells
• Not an obligate end cell
• Obligate end cell – mature cell committed to
perform a function then die
LYMPHOPOIESIS

• LYMPHOCYTE
• Variant lymphocytes
• TYPE I
• Plasmatoid lymphocyte; Turk's irritation cell
• Seen in German measles
• TYPE II
• Infectious mononucleosis cell
• Flared skirt or fried-egg appearance
• See in IM
• Type III
• Finely reticulated chromatin pattern
END