CHAPTER 10 – BLOOD (part 2)

HISTOLOGY
A TEXT AND ATLAS
SEVENTH EDITION, 2016

Michael H. Ross, PhD (deceased)

Wojciech Pawlina, MD
Hemopoiesis
Hematopoiesis
Generation of new blood cells

Includes :
◦ Erythropoiesis – Red blood cells
◦ Leukopoiesis – White blood cells
◦ Thrombopoiesis - Platelets

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Blood cells
◦ have a limited life span
◦ RBC – 120 days
◦ Platelets – 10 days
◦ WBC – variable
◦ Go out of the circulation → tissue (perform function & complete life span)
◦ Continuously produced and destroyed

Ultimate objective of hemopoiesis

◦ Maintain a constant level of different cell types found in the peripheral
blood

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Bone Marrow
Principal site of hemopoiesis in adults

Occupies the
◦ medullary cavities of long bones
◦ Spongiosa of
◦ Vertebrae
◦ Ribs
◦ Sternum

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Phases of Hemopoiesis
Early embryonic life
◦ No marrow cavities
◦ Skeleton is cartilagenous

Mesoblastic / Yolk sac phase

◦ Begins in the 3rd week of gestation
◦ Earliest formation of blood cells
◦ Characterized by formation of “blood islands” in the wall of the
yolk sac of the embryo

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 Hepatic phase
Early in fetal development - 6 weeks of gestation
Hemopoietic centers appear in the liver
Liver – major blood forming organ in the fetus during the 2nd
trimester
Blood cell formation is largely limited to erythroid cells
Some leukopoiesis occurs in the liver

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 Myeloid / Bone Marrow phase
 Begins during the 2nd trimester – clavicle
 Cartilage is replaced by bone
 Blood cell formation in
 liver – decline
 In the medullary cavities of the developing bones
 Continuous throughout post natal life
 After birth → same as in the adult

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The study of bone marrow
1. Bone marrow is usually removed by suction
from the iliac crest.
2. Small fragment of the semi-solid marrow is
placed between two glass slides → pulled across
each other → spreading the cells of the marrow
into a thin film
3. Smear is stained with Wright’s or Giemsa stain
◦ Good for distinguishing the granules, cytoplasm and
nuclei.
◦ Nucleoli - pale

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Bone marrow smear at low magnification
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Monophyletic Theory of Hemopoiesis
Blood cells are derived from a common hemopoietic stem
cell

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Common Hemopoietic Stem Cells (HSC)
AKA : Pleuripotential stem cell (PRSC)
Capable of :
◦ Differentiating into all the blood cell lineage
◦ Self renewal
Have the potential to differentiate into multiple non-blood cell lineage
During embryonic development
◦ Present in circulation → undergo tissue specific differentiation
Isolated from :
◦ Umbilical cord blood
◦ Fetal liver
◦ Fetal and adult bone marrow
Cell surface markers : LIN-, CD34+, CD90+ and CD38-
Identification : Immunocytochemical methods

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 Hemopoietic Stem cells

Give rise to
Multipotential Progenitor cells

Common Myeloid Progenitor Common Lymphoid Progenitor

- CMP - CLP
- Formerly AKA : CFU-GEMM - Formerly AKA : CFU-Lymphoid
Differentiate into Differentiate into

Specific Lineage-Restricted Progenitor T-cells B-cells NK cells

Megakaryocyte/Erythrocyte Granulocyte/Monocyte
Progenitor (MEP) Progenitor (GMP) or CFU-GM

Megakaryocyte-committed Erythrocyte-committed
Progenitor cell (MKP) or CFU-Meg Progenitor cell (ErP) or CFU-E
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 Granulocyte/Monocyte Progenitor (GMP) or CFU-GM

Neutrophil Eosinophil Basophil/Mast cell

Progenitor Progenitor Progenitor (BMCP)
(NoP) or CFU-G (EoP) or CFU-E

Basophil MCP in
Neutrophilic Eosinophilic
Progenitor cells GI mucosa
Lineage Lineage
(BaP) or CFU-Ba

Can give rise to

Dendritic Cells Monocyte Progenitor
(antigen-presenting cells) (MoP) or CFU-M

Monocyte Linage

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Figure 10.16

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•Stem cells and progenitor cells
•Morphologically indistinguishable
from each other
•Small round cells
•Centrally placed nucleus
•Cytoplasm – scanty and basophilic

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Development of Erythrocytes
Erythropoiesis
Under the influence of
◦ Erythropoietin
◦ IL-3
◦ IL-4
Common Myeloid Progenitor (CMP) →
Megakaryocyte/Erythrocyte Progenitor (MEP) → Erythrocyte-
committed progenitor (ErP or CFU-E) → Proerythroblast

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CFU – E →
PROERYTHROBLAST (Rubriblast)
◦ Earliest recognizable stage of erythrocyte lineage
◦ Not easily identified in routine bone marrow smears
◦ Up to 16 um in diameter (12-20 um)
◦ Cytoplasm
◦ Moderate – rim
◦ Basophilic – presence of free ribosomes
◦ Nucleus
◦ Spherical
◦ Large
◦ 1-2 nucleoli

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Mitosis →
BASOPHILIC ERYTHROBLAST (Prorubricyte)
◦ Smaller
◦ Nucleus
◦ Smaller (10-16 um)
◦ More heterochromatic
◦ Condensed
◦ No nucleolus
◦ Cytoplasm
◦ Intensely basophilic – larger free polyribosomes
◦ Synthesize hemoglobin

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POLYCHROMATOPHILIC ERYTHROBLAST
◦ Cytoplasm
◦ Blue-gray to blue-green or lilac (acidophilic and basophilic staining)
◦ Due to
◦ increased hemoglobin particles
◦ Decreased polyribosome
◦ Smaller
◦ Nucleus
◦ Coarse heterochromatin granules
◦ Checkerboard pattern
◦ Lack nucleolus

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ORTHOCHROMATOPHILIC ERYTHROBLAST
(Normoblast)
◦ Slightly larger than mature erythrocyte
◦ No longer capable of division
◦ Cytoplasm
◦ Eosinophilic
◦ Increased hemoglobin
◦ Decreased ribosome
◦ Nucleus
◦ Smaller
◦ Compact
◦ Densely stained
◦ Extruded

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 RETICULOCYTE
 Polychromatophilic Erythrocyte
 Immature erythrocyte
 Enter the lumen of sinusoid → complete
maturation in circulation

 Cytoplasm
 Eosinophilic
 Slight blue tint
 Small number of basophilic ribosome remaining

 Number in PBS :
 Normal : 1-2% of total erythrocyte count
 measure rate of erythropoiesis
 Increased RBC enter blood stream → increased
reticulocyte
 Regulated by erythropoietin

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Mature Erythrocyte
◦ Bi-concave

The cells become progressively smaller

The cytoplasm changes from blue to pink

The nucleus become smaller and more

condensed → ultimately lost

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 BASOPHILIC ERYTHROBLAST

ORTHOCHROMATOPHILIC ERYTHROBLAST

PROERYTHROBLAST

POLYCHROMATOPHILIC ERYTHROBLAST

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 proerythoblast

Basophilic Polychromatophilic Orthochromatophilic

normoblast normoblast normoblast

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Mitoses occur in :
◦ Proerythroblast
◦ Basophilic erythroblast
◦ Polychromatophilic erythroblast

Pro-erythroblast → mature RBC

◦ 7 days

Nearly all erythrocytes are released into the circulation as soon as they are
formed

Bone marrow is not a storage site for erythrocytes

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 Life span – 120 days
Senescent RBC → degraded in macrophage system of the spleen, bone
marrow and liver
RBC

Heme

Release iron globin

Degraded to bilirubin
Hydrolyzed to amino acid
Enter iron-storage Bound to albumin
pool (spleen) – form of Enter metabolic pool
hemosiderin / ferritin
Released to blood stream Re-used
Stored & re-used for
hemoglobin synthesis liver
Excreted as bilirubin glucuronide of bile
conjugated

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Thrombopoiesis
BM of healthy adult produce about 1 x 1011 platelets daily
◦ May increase with demand

Requires the support of

◦ Interleukins
◦ Colony stimulating factors
◦ Hormones

Megakaryocyte/Erythrocyte Progenitor (MEP)

→megakaryocyte-committed progenitor (MKP or CFU-Meg)
→Megakaryoblast

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MEGAKARYOBLAST
◦ 50 um in diameter
◦ Nucleus
◦ Non-lobed
◦ Many nucleoli
◦ Cytoplasm
◦ Basophilic
◦ Devoid of granules
◦ No evidence of platelet formation

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 Successive Endomitosis occur
 DNA undergo multiple replication without
division of cytoplasm
→ Giant polypoid Cell
MEGAKARYOCYTE
* largest cell type of the marrow
* 100 um
* Nucleus – multilobular
* Cytoplasm
- basophilic
- many small azurophilic
granules

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MEGAKARYOCYTE
◦ Platelet producing
◦ 50-70 um
◦ With complex multilobed nucleus
◦ Scattered azurophilic granules

Azurophilic granules
◦ Arranged in clusters
◦ Separated by aisles of granule-free cytoplasm

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Platelet Demarcation Channel
(examined with EM)
◦ The peripheral cytoplasm of the
megakaryocytes appear to be divided into
small compartments by invaginations of
plasma membranes.

◦ outline areas that will form individual

platelets (fragmentation of the cytoplasm)

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1 megakaryocyte – release as many as 8000 platelets
Degenerate → replaced

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Thrombocytopenia
◦ Decreased platelet count
◦ Increases the risk of bleeding
◦ In cancer patients – limits the dose of chemotherapeutic agents

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Granulopoiesis
Development of granulocytes

Originate from the multipotential

common myeloid progenitor (CMP)
stem cell → differentiate into
granulocyte/monocyte progenitor
(GMP)

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 Granulocyte/Monocyte Progenitor (GMP) or CFU-GM
Induced by IL-3
and IL-5 Lack IL-5

Neutrophil Eosinophil Basophil/Mast cell

Progenitor Progenitor Progenitor (BMCP)
(NoP) or CFU-G (EoP) or CFU-E

Basophil MCP in
Neutrophilic Eosinophilic
Progenitor cells GI mucosa
Lineage Lineage
(BaP) or CFU-Ba

Can give rise to

Dendritic Cells Monocyte Progenitor
(antigen-presenting cells) (MoP) or CFU-M

Monocyte Linage

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MYELOBLAST
◦ First recognizable cells that begin the
process of granulopoiesis
◦ Large - 16 um (14-20 um)
◦ Nucleus:
◦ Large
◦ With finely dispersed chromatin
◦ 3 or more nucleoli
◦ Cytoplasm
◦ Basophilic
◦ No specific granules

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PROMYELOCYTE
◦ The only cells that produce
azurophilic granules
◦ Do not exhibit sub-types
◦ 20 um
◦ Nucleus
◦ Large
◦ With finely dispersed chromatin
◦ 3 or more nucleoli
◦ Cytoplasm
◦ More basophilic
◦ With numerous azurophilic granules

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MYELOCYTE
◦ Smaller
◦ Nucleus
◦ Becomes increasingly heterochromatic
◦ Elliptical or indented
◦ Cytoplasm
◦ Less basophilic
◦ Many azurophilic granules
◦ First to exhibit specific granules
◦ 3 kinds
◦ Difficult to distinguish

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METAMYELOCYTE
 Neutrophilic
 Cytoplasm – 2 granules
 Azurophilic – stain deeply
 Specific – stain faintly
 S:A ratio = 2:1
 Nucleus – heterochromatic
 Indentation → kidney bean-shaped
 Eosinophilic
 Nucleus – indented
 Large eosinophilic granules
 Basophilic
 Rare
 Difficult to preserve granules
 Nucleus – deeply indented
 Large basophilic granules

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BAND → Segmented /Mature
◦ Rarely if ever observed in the eosinophil and basophil line

Development time : 18 days

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Granulopoiesis
In the bone marrow – 2 weeks
◦ Mitotic / Proliferative phase – 1 week
◦ Post-mitotic phase – 1 week
Neutrophil → Blood → CT
◦ Random
◦ Circulate for only a few minutes → 16 hours
◦ In the CT
◦ 1-2 days
◦ Destroyed by apoptosis → phagocytosis by macrophages

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Reservoir of Neutrophils
◦ Bone marrow
◦ Maintain a large reserve of fully functional neutrophils
◦ Free circulating Pool
◦ In the vascular compartment
◦ Marginated Pool
◦ In the small blood vessels

Size of reserve – depend on

◦ Rate of granulopoiesis
◦ Life span of neutrophil
◦ Rate of migration to bloodstream and CT

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Development of Monocyte
MONOBLAST
◦ Similar in appearance to myeloblast

PROMONOCYTE
◦ Large 16-18 um
◦ Nucleus
◦ Euchromatic
◦ Cytoplasm
◦ Basophilic
◦ Devoid of granules

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MONOCYTE
◦ Enter the circulation
◦ Stay for no more than 36 hours
◦ Tissues (Macrophages)

Transformation from MoP →

Monocyte
◦ 58 hours

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 CFU - L LYMPHOBLAST mitosis

PROLYMPHOCYTE
•Nuclei
•deeply staining
•Condensed chromatin
•Cytoplasm
•Small amount
•basophilic

Enter Circulation
Remain in BM

During embryonic
& early post-natal life Spleen / LN Divide throughout life

Thymus Complete their Produce lymphocyte

differentiation

Proliferate /
Circulation
Differentiate B-lymphocytes

T-lymphocytes
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Red – eosinophils
Yellow – neutrophilic metamyelocyte
Blue – orthrochomatic normoblast

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Red Bone Marrow
 Lies entirely within the
Spaces of bone
Medullary cavity of young long
bones
Spaces of spongy / cancellous
bones

 One of the body’s largest organ

3.4 to 4.6% of the total body
weight

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Bone Marrow
• consists of
• blood vessels
• Sinusoids
• specialized units of blood vessels
• provide the barrier between the hemopoietic compartment and the peripheral circulation
• closed circulation system; newly formed blood cells must penetrate the endothelium to enter the
circulation.
• interposed between arteries and veins (normally occupied by capillary
• sponge-like network of hemopoietic cells

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Bone Marrow
 2 forms
Based on appearance on gross examination.
Red
 Active / hematogenous
 Presence of blood and blood forming cells
Yellow
 Inactive
 Consist mainly of adipose cells
 Chief form of bone marrow in the medullary cavity of adult bones that are no longer
hemopoietically active
 such as the long bones of the arms, legs, fingers, and toes

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 At birth  By 10 years old
Red marrow found throughout the Red marrow persist only in
skeleton  Vertebrae
 Ribs
 Pelvis
 After 6 years old  Proximal ends of humerus and femur
Gradually replaced by yellow  Shoulder girdle
marrow

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• Even in hemopoietically active bone marrow in adult humans
• the ribs, vertebrae, pelvis, and shoulder girdle
• about half of the bone marrow space is occupied by adipose tissue and half by
hemopoietic tissue

• The yellow bone marrow retains its hemopoietic potential

• when necessary (after severe loss of blood) → can revert to red bone marrow
• by extension of the hemopoietic tissue into the yellow bone marrow and
• by repopulation of the yellow bone marrow by circulating stem cells

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 Examination of Bone Marrow
• essential for diagnosis of bone marrow disorder
• by :
• Bone marrow aspirate
• Bone marrow core needle (trephine) biopsy
•Indications :
• unexplained anemia (low erythrocyte counts)
• Abnormal peripheral blood smear morphology
• diagnosis and staging of hematological malignant disorders (e.g., leukemia)
• Suspected bone marrow metastases

•Final diagnosis – based on combination of :

• Clinical findings
• Several diagnostic procedures (PBS, BMA & trephine & immunophenotyping, molecular and genetic studies)

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 Bone Marrow Aspiration
• Needle inserted through the skin until it penetrates the
bone

• Preferred anatomical sites

• Posterior part of the iliac crest (hip bone)

• apply negative pressure → obtain small amount of bone

marrow → spread as a smear on glass slide → stain →
examine with microscope

•Assess individual cell morphology

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Bone Marrow Core Biopsy
• intact bone marrow is obtained
•Small incision is made in the skin → biopsy
needle to pass into the bone → with rotating
motion → pulled out with small, solid piece of
bone marrow inside → needle is withdrawn →
core sample is removed from the needle →
fixative → biopsy
• provide analysis of bone marrow architecture
•Used to :
• Diagnose and stage different types of cancer
• Monitor the result of chemotherapy

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