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HEMATOLOGY 1

HEMATOPOIESIS

Definition: Production, differentiation, maturation, proliferation of blood cells.

Human stem cells: unspecialized cells capable of self-renewal and differentiation

a. Totipotential stem cells

-present in the first few hours after fertilization

-most versatile; can develop into any cell type, can even develop into extra embryonic cells
(e.g. Placental cells)

b. Pluripotential stem cells

-present several days after fertilization

- stem cell that has the potential to differentiate into any of the three germ layers: endoderm,
mesoderm, ectoderm

c. Multipotential stem cells.

-stem cells that have the potential to differentiate into discrete cell types.

- e. g. multipotent blood stem cell (Hematopoietic stem cell): will differentiate into blood cells

PHASES

1. MESOBLASTIC PHASE
Begins: 19th day of embryonic dev’t.

Embryonic development:
Zygote-morula-blastula-gastrula

**Gastrula stage of embryo: with 3 germ layers: Endoderm, Mesoderm, Ectoderm

**Mesoderm: germ layer where PRIMITIVE ERYTHROBLASTS are first produced

a. PRIMITIVE ERYTHROBLASTS: Large, multinucleate and multiucleolated.


-eventually migrate to YOLK SAC of embryo.
-may also surround cavity of yolk sac to form blood vessels (angioblasts)
-important in early hemoglobin synthesis (EMBRYONIC HGB: Gower-1, Gower-2, Portland)
-form YOLK SAC BLOOD ISLANDS

- Mesoblastic phase occurs : intravascularly

b. Yolk sac erythroblasts migrate to aorta-gonad- mesonephros (AGM) region

2. HEPATIC PHASE
Begins: 5 to 7 WOG
characterized by: Definitive Erythropoiesis, Granulopoiesis, monopoiesis, lymphopoiesis ,
megakaryopoiesis

DEFINITIVE ERYTHROBLASTS: give rise to mature erythrocytes


-Hemoglobin F ( major); small amounts of Adult hemoglobin

- Hepatic phase occurs: extravascularly

- liver : major site of hematopoiesis during the second trimester of fetal life

- Hematopoiesis in the aorta-gonad-mesonephros region and the yolk sac disappear during
this stage.

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- Spleen, kidney, thymus, lymph nodes: also perform hematopoiesis along with the liver during
the hepatic stage.

3. MEDULLARY PHASE
Begins: end of 4th MOG / early 5th MOG
Medullary phase occurs in : medulla of bone; bone cavity

Characterized by: Increased Leukopoiesis and production of all adult blood cell types

BONE MARROW

Weight: 1500 g

Location: Medulla of bone; inner cavity of bone (cortical bones)

Forms:
a. Red marrow: Hematopoietically active
b. Yellow marrow: Inactive, composed of adipocytes, Yellow color due to carotene of
adipocytes, also with macrophages and undifferentiated mesenchymal cells

: can still revert back to Red marrow in times of compromise

**Infancy: Red marrow (major); in all bones

** 5-7 yrs: yellow marrow begin to form in long bones (Process is known as
RETROGRESSION)

** 18 yrs: Red marrow restricted to: sternum (MAJOR SOURCE) , vertebrae, scapulae,
pelvis(iliac crest) , ribs, flat bones of skull, and proximal portion of the long bones( femur and
humerus)

OTHER CELLS in BM:


*Stromal cells:
- secrete : semifluid extracellular matrix

*ECF for: anchoring hematopoietic cells in the bone cavity

contents: fibronectin, collagen, laminin, thrombospondin, tenascin, and proteoglycans


(hyauronate, heparan sulfate, chondroitin sulfate, and dermatan)

: Provide support; “nurse cells”

-endothelial cells (BLOOD VESSEL CELLS: broad, flat, single continuous layer along surfaces
of blood vessels; regulate entry and exit of particles to and from BM )

-adipocytes (fat cells: Regulate BM volume; secrete cytokines and Growth factors to stimulate
hematopoiesis )

macrophages and lymphocytes (release cytokines)

osteoblasts (BONE FORMING CELLS)

osteoclasts (BONE RESORBING CELLS)

reticular adventitial cells (fibroblasts; supporting lattice for the developing blood cells.)

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PRINCIPLES OF CELLULAR MATURATION

1. General cell size and nuclear size


-Cells decrease in general size and nuclear size as they mature (except: megakaryocytic)

2. Cytoplasmic characteristics:
a. Amount: Amount of cytoplasm increases upon maturity

b. Color: Cytoplasm loses basophilia ; darker blue to lighter blue, blue-gray, or pink (except:
Plasma cells)

c. Granules: blast cells generally do not contain granules; granules of granulated cells appear
on certain stages and specific granules appear later in the life cycle of maturing cells

d. Vacuolation , secretory products, Pseudopod formation: Appearance of these structures


may indicate maturity

3. Nuclear characteristics
a. Chromatin pattern:

-Immature cells: Fine, loose chromatin, smooth or homogeneous, fine, delicate, lacy or thready

-Mature: Clumped, deeply stained , smudged, or pyknotic (dense or compact) chromatin


pattern

b. Size:

-Immature cells: larger

-Mature: smaller

c. Nucleoli

-Immature cells: visible

-Mature:Invisible

d. Shape:

-Immature cells: round to oval

-Mature: lobulated/ indented/ folded (except: RBC- anucleated )

4. Nuclear: cytoplasmic ratio

-Decreases with maturity

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ERYTHROPOIESIS

*Primitive: occurs in yolk sac

*Definitive: occurs in liver then finally in bone marrow until adulthood

-The process of production and regulation of mature erythrocytes in the peripheral blood.

1. CFU-GEMM will give rise to CFU- MegE

2. CFU MegE will differentiate into BFU-E

3. BFU-E ( grape-like clusters with hemoglobin) : earliest identifiable colony of RBCs

: characterized to undergo apoptosis (“burst” promoting activity) ; G0/ G1 phase of cell cycle-
not actively dividing

4. Erythropoietin (EPO) will promote differentiation of BFU-E to CFU-E (prevent BFU-E


apoptosis) ; CFU-E- S phase of cell cycle- proliferating

*BFU-E to CFU-E= 1 week

5. EPO will promote CFU-E to differentiate into pronormoblasts

*CFU-E to pronormoblast= 1 week

6. Pronormoblast
Aka: Proerythroblast; rubriblast
Size: 12-20 microns

Shape: ovoid

Cytoplasm: Thin rim of dark purplish hue ; scanty; moderate basophilia ; no granules T
Nucleus: fine chromatin pattern ; nucleoli 1-2/ 1-3

N:C ratio: 8:1


*1% of total precursors in BM

*first recognizable stage of erythrocyte maturation sequence

*Pronormoblast-Basophilic normoblast = 24 hours

7.Basophilic Normoblast
Aka: Basophilic erythroblast; Prorubricyte
Size: 10-15 microns

Cytoplasm: Deeper blue; navy blue


Nucleus: Cartwheel pattern: alternating areas of clumped chromatin and parachromatin(clear
spaces around chromatin) ; nucleoli 0-1

N:C ratio: 6:1


*1%-4% of total precursors in BM

*first stage capable of producing hemoglobin

*Basophilic normoblast- polychromatophilic normoblast = 24 hours

8. Polychromatophilic normoblast
Aka: Polychromatic / Polychromic normoblast; polychromatophilic erythroblast; rubricyte
Size: 10-12 microns

Cytoplasm: Murky grayish blue; combination of shades of pink and blue


Nucleus: condensation of chromatin begins to be apparent

N:C ratio: 4:1


*10%-20% of total precursors in BM

*peak stage of hemoglobin production

*polychromatophilic normoblast- orthochromatophilic normoblast = 30 hours

*last stage capable of mitosis

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9. Orthochromatophilic normoblast
Aka: orthochromatic / Orthochromic normoblast; orthochromatophilic erythroblast;
metarubricyte
Size: 8-10 microns

Cytoplasm: Salmon pink with traces of blue shades

Nucleus: structureless, pyknotic

N:C ratio: 1:2


*5%-10% of total precursors in BM

orthochromatophilic normoblast -reticulocyte= 48 hours

* No longer capable of mitosis

* Pyrenocyte: extruded nucleus through pseudopods motion and active contraction

* Loss of vimentin allow movement of nucleus towards periphery of cell until extrusion

10. Reticulocyte

Aka: Polychromatophilic erythrocyte; diffusely basophilic erythrocyte


Size: 8-10 microns

Cytoplasm: Salmon pink with polychromatophilia

Nucleus: none

N:C ratio: n/a


*1% of total precursors in BM

reticulocyte- erythrocyte = 24- 48 hours

* when supra vitally stained : may show precipitated ribosomes/ RNA fragments of precipitated
ribosomes

* Still with organelles (mitochondria, ribosomes, golgi complex)

* Still capable of heme synthesis because of ribosomes

11. Mature erythrocyte

Size: 6-8 microns

Cytoplasm: Salmon pink ; central pallor occupying 1/3 of the cell

Nucleus: none

N:C ratio: n/a


Lifespan: 120 days

LEUKOPOIESIS
-production and regulation of production of granulocytes

3 pools/ clusters of developing neutrophils

1. Stem cell pool- stem cells (pluripotent stem cells)

2. Proliferation pool- myeloid progenitors (CMP/CFU-GEMM, myeloblasts; promyelocytes;


and myelocytes.)

3. Maturation pool- cells ready to be released but still undergoing nuclear maturation
(metamyelocytes, band neutrophils, and segmented neutrophils. )

**Proliferation pool-Maturation pool= 6 days

Maturation pool- release to PB= 4-6 days

HSC, CMP, GMP= cannot be distinguished under light microscope; must use flow cytomery

*Granulopoiesis

1. CFU-GEMM—-> CFU-GM

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2. CFU-GM——-> CFU-G (***G-CSF)

3. CFU-G ——> Myeloblast

4. Myeloblast
*earliets recognizable granulocyte precursor

*may have Auer rods

*0-3% of total precursors in BM


Size: 10-18 microns/ 14-20 microns

a. Type I myeloblast

Cytoplasm: basophilic with no granules

Nucleus: large oval- quadrangular; very fine / reticular nuclear chromatin ; 1-5/ 2-4 nucleoli
N:C ratio: 8:1- 4:1

b. Type II myeloblast:

Cytoplasm: basophilic with dispersed azurophilic granules (<20 )

Nucleus: large oval- round; very fine / reticular nuclear chromatin ; 1-5/ 2-4 nucleoli
N:C ratio: 8:1- 4:1

c. Type III myeloblast:

Cytoplasm: darker staining purplewith >20 azurohilic granules

Nucleus: large oval-round ; darker staining reticular fine nuclear chromatin ; 1-5/ 2-4 nucleoli
N:C ratio: 8:1- 4:1

*** rare in normal BM; common in AML

5. Promyelocyte

Size: 14-20 microns/ 16-25 microns

Cytoplasm: Moderately basophilic

Granules: Heavy Primary azurophilic granules

Nucleus: eccentric, round to oval with a peri nuclear halo/ hof (not in APML); smooth and
starts to clump ; 1-5 nucleoli

N:C ratio: 3:1

5. Myelocyte
* final stage capable of mitosis

* 6-17% of BM

* First stage where distinction between eosinophil , basophil, neutrophil can be seen

Size: 12-18 microns

Cytoplasm: Blue-pink

Granules: specific granules

Nucleus: oval with indentation; slightly clumped chromatin

N:C ratio:: 2:1- 1:1

5. Metamyelocyte
Aka: juvenile cell

* Can no longer divide

* 3-20% of BM

* First stage where distinction between eosinophil , basophil, neutrophil can be seen

Size: 10-18 microns

Cytoplasm: pink

Granules: specific and tertiary granules

Nucleus: indented/ kidney bean ; clumped chromatin

N:C ratio: 1:1

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6. Band
Aka: stab cell

* 9-32% of BM

* First stage where distinction between eosinophil , basophil, neutrophil can be seen

Size: 10-16 microns

Cytoplasm: pink

Granules: specific and tertiary granules ; secretory granules

Nucleus: horseshoe/ sausage ; indentation more than half the diameter of nucleus (same d all
throughout ) ; highly clumped chromatin

N:C ratio: 1:1


* CLSI= included in SEG count

When in doubt= count as SEG

7. Segmented neutrophils

Size: 10-16 microns

Cytoplasm: pink

Granules: specific and tertiary granules ; secretory granules

Nucleus: densely packed chromatin, 2-5 lobes connected by thread-like filaments

N:C ratio: 1:1

Half-life: 7 hours

a. Circulating pool

-major Bfs

b. Marginated pool

-walls of capillaries in tissues

MONOCYTE MATURATION

1. CFU-GEMM—-> CFU-GM

2. CFU-GM——-> CFU-M (*** M-CSF)

3. CFU-M ——> Monoblast

4. Monoblast- normally not present in BM

Size: 12-20 microns

Cytoplasm: deep blue

Granules: none

Nucleus: large round to ovoid ; Naegeli type - chromatin similar to myeloblast ; Schilling type-
reticulum

N:C: 4: 1

4. Promonocyte

Size: 12-18 microns

Cytoplasm: blue-gray

Granules: large and small lilac-azurophilic dust-like granules

Nucleus: elongated/ folded

N:C: 3: 1- 2:1

5. Monocyte
Size: 15-20 microns

Cytoplasm: blue-gray with blebs and vacuoles

Granules: large and small lilac-azurophilic dust-like granules (ground glass)

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Nucleus: folded; horseshoe shaped; lacey chromatin

N:C: 2:1- 1:1

LYMPHOPOIESIS

1. CLP- Precursor B/T cell

2. Lymphoblast
Size:15-20 microns

Cytoplasm: deep blue with azurophilic granules

Nucleus: large oval nucleus with dark purple chromatin aggregates around nuclear periphery ;
1-2 nucleoli

N:C: 4:1

3. Prolymphocyte
Size: 15-18 microns

Cytoplasm: light to dark blue with occasional granules

Nucleus:oval with slight indentation

N:C: 3:1

4. Lymphocyte
Size: 6-10(SMALL) /15-20 (LGL)

Cytoplasm: robin’s egg; sky-blue color

Granules: azurophilic granules / purple granules in LGL

Nucleus: ovoid, eccentric, sometimes indented; dense and clumped

N:C: 2:1- 1:1

MEGAKARYOPOIESIS

1. CFU-GEMM
2. CFU-MegE (***TPO)

3. CFU-Meg / BFU-Meg / LD-CFU

**ENDOMITOSIS- cellular division


without telophase and cytokinesis

** signals: GAT-1 and FOG-1

RUNX1- signals mitosis to


endomitosis —slows Rho-rock
signaling pathway——> suppresses
actin and myosin levels—-> no
cytoplasmic constriction—->
continuous karyokinesis without
cytokinesis

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Hematopoietic growth factors

- soluble/ membrane bound

- Regulators (proliferation and differentiation)

- Action: local or circulates

- Low concentrations

- Synergism; affects >1 lineage

- Prevent apoptosis

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