Hemopoiesis

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Haemopoiesis

• • • • Haemopoiesis is the process by which mature blood cells develop from precursor cells. 3 rd week – yolk sac 2 nd month – liver,& spleen 3 rd month – bone marrow.

• In the human adult, haemopoiesis takes place in the bone marrow mainly of the skull, ribs, sternum, vertebral column, pelvis and the proximal ends of the femur& tibia.

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Haemopoiesis

• After birth and on into childhood, erythrocytes, granular leukocytes, monocytes, and platelets are derived from stem cells located in bone marrow. • The origin and maturation of these cells are termed, respectively erythropoiesis, granulopoiesis, monocytopoiesis, and megakaryocytopoiesis. • The bone marrow also produces cells that migrate to the lymphoid organs producing the various types of lymphocytes.

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Bone marrow

• Two types of bone marrow: – Red, cells.

or hematogenous – Yellow bone marrow , due to blood and blood-forming , due to adipose cells. • In newborns , all bone marrow is red and is therefore active in the production of blood cells. • As the child grows, most of the bone marrow changes gradually into the yellow variety. • Under certain conditions, such as severe bleeding or hypoxia, yellow bone marrow converts back into red bone marrow.

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Red bone marrow

• Hemopoietic and consists of: – Stroma: • The stromal cells secrete hemopoeitic growth factors.

• Formed by reticular connective tissue (type III collagen), branching reticulocytes, macrophages, fibroblasts, endothelial cells.

• Also found in the matrix are adhesion molecules like laminin, fibronectin & hemonectin which bind cells to the matrix.

– Sinusoids: • Large thin walled vessels.

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Bone marrow examination

• Components of BM examination: – Assess cellularity – Calculate M/E (myeloid / erythroid) ratio – Evaluation of hematopoiesis – Number of megakaryocytes – Status of iron stores • Two procedures: – Bone marrow aspiration (morphology) – Bone marrow biopsy (architecture and cellularity) 6

BM aspiration needle BM biopsy needle 7

BM aspiration BM biopsy 8

3 types of cells

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Stem cells: – Pluripotent cells , can differentiate into multiple cell, capable of self renewal. 2.

Progenitor cells: – derived from stem cells, reduced potentiality & committed to single cell lineage.

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Precursor cells (blasts):

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initial cells in each lineage, non self renewing & no potential to switch to a different cell type.

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Erythropoiesis

• The process of erythropoiesis is directed towards producing a cell devoid of organelles but packed with haemoglobin. • The first recognizable erythrocyte precursor is known as the proerythroblast , a large cell with numerous cytoplasmic organelles and no haemoglobin. • Further stages of differentiation are characterized by three main features: – decreasing cell size and nuclear extrusion.

– progressive loss of organelles; the presence of numerous ribosomes at early stages accounts for the marked cytoplasmic basophilia (blue staining) this accounts for the which steadily decreases as the number of ribosomes falls. – progressive increase in the cytoplasmic haemoglobin content; increasing eosinophilia (pink staining) of the cytoplasm towards maturity.

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Erythropoiesis

• The differentiation and maturation of erythrocytes involve the formation (in order) of: – Proerythroblasts – Basophilic erythroblasts (early normoblasts) – Polychromatophilic erythroblasts (intermediate normoblast) – Orthochromatophilic erythroblasts (late normoblasts) – Reticulocytes – Erythrocytes 13

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Erythrobalstic island: unit of erythropoiesis

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Proerythroblast (Precursor cell)

• First recognizable cell of erythropoiesis.

• Large cell with pale nucleus with 1-2 nucleoli • Scant basophilic cytoplasm.

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Early Normoblast / Basophilic Erythroblasts

• Strong basophilic cytoplasm due to RNA synthesis.

• Condensed nucleus 18

Intermediate Normoblast / Polychromatic erythroblast

• Hemoglobin starts to form. This gives the cytoplasm an acidophilic (pinkish) color.

• Ribosomes are also present giving appearance of

pink/blue

, polychromatic cytoplasm .

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Orthochromatic or Late Normoblast

• Small cell and cytoplasm uniform pink color with only traces of basophilia.

• At a later stage nucleus is expelled.

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Reticulocytes

• No nucleus, scanty organelles.

• Acidophilic cytoplasm • Occasionally tiny parts of the nucleus persist in the cytoplasm – Howell Jolly bodies.

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Nucleus of the RBC is usually extruded; occasionally it persists with pieces of chromatin in the cytoplasm forming Howell _ Jollie bodies in the cell

Factors affecting erythropoiesis

• Erythropoetin – produced by the kidneys • Vit B12 & Folic acid • Interleukins • The

principal stimulus for increase in RBC production is tissue hypoxia

, which stimulates the kidney to produce and release erythropoietin into the blood. • Erythrocytes develop close to macrophages which transfer ferritin to the developing cells and also destroy the nucleus expelled by the normoblast.

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Erythropoiesis

• There are from three to five intervening cell divisions between the proerythroblast and the mature erythrocyte. • The development of an erythrocyte from the first recognizable cell of the series to the release of reticulocytes into the blood takes approximately 7 days .

• The hormone erythropoietin and substances such as iron, folic acid , and vitamin B12 are essential for the production of erythrocytes. • Erythropoietin is a glycoprotein produced in the kidneys that stimulates the mRNA for globin, the protein component of the hemoglobin molecule.

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MYELOID SERIES

• Myeloblast • Promyelocyte • Myelocyte • Metamyelocyte • Band Cell • Mature PMN 25

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Granulocyte formation

(Granulopoiesis) • The myeloblast is the most immature recognizable cell in the myeloid series. It has a finely dispersed chromatin, and nucleoli can be seen. • In the next stage, the promyelocyte is characterized by its basophilic cytoplasm and azurophilic granules.

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Primary or Azurophilic granules are produced only in promyelocytes.

• The promyelocyte gives rise to the three known granulocytes. 27

Granulocyte formation (granulopoiesis)

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The first sign of differentiation appears in the myelocytes where specific granules

gradually increase in quantity and eventually occupy most of the cytoplasm. • From the myelocyte stage through the metamyelocyte stage to the mature granulocyte forms, the nucleus becomes increasingly segmented. • The immediate precursors of mature granulocytes tend to have an irregular horseshoe or ring-shaped nucleus and are termed stab cells or band forms . 28

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• The appearance of large numbers of immature neutrophils (band cells) in the blood is clinically significant, usually indicating bacterial infection .

• The total time taken for a myeloblast to emerge as a mature neutrophil in the circulation is about 11 days. • Under normal circumstances, five mitotic divisions occur during stages of development.

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Neutrophil granules

Primary (azurophilic) granules 1. Myeloperoxidase 2. Phospholipase A2 3. Lysozyme 4. Acid hydrolases 5. Elastase 6. Defensins 7. Bacterial permeability increasing protein (BPI) • Secondary (specific) granules 1. Phospholipase A2 2. Lysozyme, 3. NADPH oxidase 4. Leukocyte alkaline phosphatase (LAP) 5. Collagenase 6. Lactoferrin • Tertiary granules: – Phosphatases and metalloproteinases (gelatinases & collagenases) 31

Phagocytosis

• Phagocytosis is enhanced if the material to be phagocytosed is coated with certain plasma proteins called “opsonin”, a process called opsonization. • Opsonins are – serum complement (C3b) – immunoglobulin (IgG) • Corresponding receptors on leucocytes (cellular receptors for C3b, Fc portion of IgG) leads to binding. Binding of the opsonized particles triggers engulfment and formation of vacuole which fuses with lysosomal granule membrane (phagolysosome).

• Granules discharge within phagolysosome and extracellularly (degranulation).

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Phagocytosis

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Oxygen dependent killing of bacteria

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Oxygen dependent microbial killing

• Phagocytosis initiates activity of the HMP shunt, causing an oxidative burst and supplying electrons to an NADPH oxidase in the phagosomal membrane.

• Product of the NADPH oxidase reaction is superoxide, which is further converted to hydrogen peroxide by dismutation.

• Hydrogen peroxide may be further converted to the activated hydroxyl radicals. Hydrogen peroxide alone is insufficient.

• MPO (azurophilic granules) converts hydrogen peroxide to HOCl presence of Cl ), an oxidant/antimicrobial agent.

(in 35

Defects in Neutrophils

1. Chronic granulomatous disease of childhood (CGD) 2. Myeloperoxidase deficiency 3. Leukocyte adhesion deficiency (LAD types 1 and 2) 4. Chediak-Higashi syndrome 36

1.Chronic granulomatous disease of childhood (CGD) • Absence of NADPH oxidase activity.

• Marked by phagocytic cells that ingest but do not kill certain microorganisms.

• Catalase positive (Stapylococcus.aureus) organisms: ingested but not killed.

• Enzyme deficient can not produce H2O2.

H2O2 not available as a substrate for MPO.

• MPO-halide system of bacterial killing fails.

• Nitroblue tetrazolium (NBT) dye test  negative • Catalase negative (Streptococci) organisms: ingested and killed.

– Strep produce sufficient H2O2 to permit MPO-halide system to proceed.

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Test for chronic granulomatous disease Normal nitroblue tetrazolium test (NBT) Abnormal nitroblue tetrazolium test (NBT) 38

2. Myeloperoxidase deficiency: – Autosomal recessive – Infections with candida – Usually little clinical consequence.

3. Leukocyte adhesion deficiency: a. LAD type 1: defective synthesis of integrins – Associated with recurrent bacterial infections.

b. LAD type 2: absence of sialyl Lewis X on neutrophils.

– recurrent bacterial infections.

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4. Chediak-Higashi syndrome – Neutropenia, albinism, cranial and peripheral neuropathy and a tendency to develop repeated infections.

– Defects in microtubule polymerization in WBCs • defects in phagocytosis • defects in chemotaxis (impaired motility) • defects in degranulation (fusion of lysosomes with phagosomes prevented) – Morphological: large cytoplasmic granules

(abnormal lysosomes)

in granulocytes and monocytes.

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Maturation of lymphocytes and monocytes • Study of the precursor cells of lymphocytes and monocytes is difficult because these cells do not contain specific cytoplasmic granules or the nuclear lobulation. • Lymphocytes and monocytes are distinguished mainly on the basis of size, chromatin structure, and the presence of nucleoli in smear preparations. 42

Thrombopoiesis

Large cell with a polypoid nucleus. Divide endomitotically (no daughter cells)

Megakaryoblast

Extremely large cells, lobulated nucleus, many mitochondria with golgi bodies.

Megakaryocyte

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Blood platelet structure

• No nucleus • Residues of the endoplasmic reticulum, Golgi apparatus and Mitochondria • Granules with different secretions (clotting factors, ADP, ATP, serotonin, thromboxane, etc.) • Very permeable plasma membrane • Contractile proteins: actin, myosin 44

Thrombopoiesis

• Megakaryocytes lie just outside the sinusoid. • Cytoplasmic extensions penetrate the sinusoid shedding fragments of platelets into the circulation. 45

Clinical anatomy: thrombocytopenic purpura.

In which the no of blood platelets is reduced, the platelets appear to be bound to the cytoplasm of the megakaryocytes, indicating a defect in the liberation mechanism of these corpuscles.

The lifespan of platelets is approximately 10 days.

On giemsa staining,megakaryoblasts show a homogenous basophilic cytoplasm, kidney shaped nucleus with numerouus nucleoli. An important is the formation of “polypoid” nucleus, which contains up to ________ content of normal cell?

(a)5 times the DNA (b)20 times the DNA (c)10 times the DNA (d)30 times the DNA

Which of the following is most frequently noted in a sample of peripheral blood?

(a)Eosinophil (b)Lymphocytes (c)Monocytes (d)Neutrophils (e)platelets

The azurophilic granules of a neutrophil are which of the following cellular elements?

(a)Phagosomes (b)Golgi apparatus (c)Peroxisomes (d)Residual bodies (e)lysosomes

Loss of the nucleus from an erythroblast occurs between which two stages of erythropoiesis?

(a) proerythroblast-basophilic erythroblast (b) Basophilic erythroblast-polychromatophilic erythroblast (c)Polychromatophilic erythroblast-normoblast (d) Normoblast-reticulocyte (e) Reticulocyte-erythrocyte