Unit III- Hematopoiesis I

Question 1

Preparation of a blood smear

Answer 1

• place a drop of blood on a microscope slide
• take a second slide and place it in contact with first slide at an angle of 45 degrees
• move second side over the first until it makes contact with the drop of blood
• spread blood by moving oblique slide uniformly along fixed slide
• air dry
• fixed dried smear in methanol
• stain with Romanovsky stain

Question 2

Romanovsky Type Blood Stain

Answer 2

• basic: methylene blue (RNA), stains cytoplasmic with cytoplasmic RNA heavenly blue
• azure B (DNA and GAGs)- stains nuclei purple, stains cytoplasmic granules of basophils and lysosomes crimson

acidic:
eosin- (proteins), strains hemoglobin of rbc red pink

variations: Wright’s, Giemsa, May-Grunwald

Question 3

Regions of typical blood smear

Answer 3

• beginning or head: red blood cells overlap, leukocytes appear to have shrunk
• end or tail-cells will appear over-stretched, distorted or broken
• middle- red blood cells slightly separated, leukocytes will spread but intact, area of choice for study
• observe in oil immersion 100x for detailed study of cell

Question 4

General considerations of blood cells

Answer 4

• life span is relatively short
• they must be continuously produced
• production of blood cells is known as hematopoiesis (granulopoiesis- development of granulocytes, erythropoiesis- development of eythrocytes)
• hematopoietic organs- bone marrow, lymphoid organs, liver is a major hematopoietic organ in the fetus

Question 5

Monophyletic/ Polyphyletic theory

Answer 5

Mono- all blood cells arise from a common pluripotential stem cell- most widely held theory today
-Poly- each type of blood cell arises from its own stem cell- not a widely help theory today

Question 6

blasts

Answer 6

• precursor cells
• each blast gives rise to one type of blood cell (monopotential)
• morphology:
• relatively large cell with a diameter of 10-15 um
• contains a large euchromatic nucleus (several nucleoli may be seen, large nucleo-cytoplasmic ratio)
• numerous ribosomes in the cytoplasm- cytoplasm will appear pale blue in blood smear
• no cytoplasmic granules

Question 7

Neutrophil differentiation

Answer 7

• condensation of nuclear chromatin (disappearance of nucleoli)
• lobulation of nucleus
• appearance of cytoplasmic granules (primary and secondary)
• decrease in cytoplasmic basophilia
• decrease in descriptive stages: blast (myeloblast), neutrophilic promyelocyte, neurophilic myelocyte, neutrophilic metamyelocyte, neutrophilic ban, mature neutrophil
• normally blasts, promyelocytes, myelocytes and metamyelocytes are found only in bone marrow
• bads and adult neutrophils appear in peripheral blood

Question 8

Neutrophilic promyelocyte

Answer 8

• relatively large cell- 10-15 um
• spherical nucleus with slight chromatin condensation
• nuclei are often observed
• cytoplasmic granules- primary (azurophilic) granules, primary lysosomes- contain hydrolytic enzymes (acid phosphatase +), will be numerous at end of promyelocyte stage
• capable of mitosis

Question 9

Neutrophilic myelocyte

Answer 9

• -round or oval nucleus
• more heterochromatic
• no longer makes azurophylic granules
• appearance of specific granules- secondary granules appear and increase in number, contain lysozyme and lactoferrin, cause cytoplasmic color change from heavenly blue to salmon
• capable of mitosis
• ability to replicate DNA and to synthesize RNA gradually decreases- chromatin becomes more condensed, cytoplasmic basophilia decreases

Question 10

Neutrophilic metamyelocyte

Answer 10

-no longer able to synthesize nucleic acids (DNA and RNA)
-no longer able to undergo mitosis
-morphology:
indented nucleus
chromatin is more condensed
no evidence of cytoplasmic basophilia
cytoplasmic granules- numerous small secondary granules, few large primary granules

Question 11

Neutrophilic band

Answer 11

• morphology
• further bending of nucleus (indentation exceeds 1/2 the diameter of round nucleus)
• chromatin is quite condensed
• cytoplasm resembles that of mature neutrophil
• can be found in normal peripheral blood
• 1-5% of total leukocytes
• percentage of bands in peripheral blood is a rough indicator of the rate of neutrophil production- “shift to the left”
• incapable of mitosis

Question 12

Mature neutrophil

Answer 12

-when the segments between lobes have become thin heterochrmoatic filaments, the band has differentiated into a mature neutrophil

Question 13

Kinetics of neutrophil production

Answer 13

• process takes 9-14 days (blast to adult)
• cells spend less than 1 day in circulation- exchange between circulating and marginating pools
• cells leave blood vessels and enter surrounding tissues (diapedesis)- paracellular and transcellular
• live in the surround tissue about 5 days
• total life span - 15-20 days

Question 14

Eosinophil and basophil productuin

Answer 14

• during myelocyte stage specific granules appear in developing eosinophils and basophils
• these granules are larger than specific granules of neutrophils
• eosinophilcs- specific granules are almost black when they first appear, become pink-red as maturation continues
• basophils- specific granules stain purple in smears

Question 15

Red bone marrow

Answer 15

• site of hematopoiesis
• location: flat bones of body- sternum, vertebrae, ribs, clavicles, bones of pelvis, bones of skill
• composition: blood vessels, discontinuous sinusoids, cords of hematopoietic cells- site of blood cell maturation

Question 16

Hematopoietic stem cell niche

Answer 16

• interactive structural unit- localized supporting cells, ECM (fibronectin, laminin, agrin), soluble factors derived from supporting cells
• nurtures stem cells and maintains their properties
• facilitates activity of stem cells
• found in association with spongy bone- osteoclasts create spaces in bone surface, osteoblasts required for stem cell localization, endothelial cells, pericytes, bm macrophages may also be involved
• alterations may lead to myeloproliferative disease- preleukemic condition
• other stem cells

Question 17

Yellow bone marrow

Answer 17

• not active in hematopoiesis
• location: medullary cavities of all other bones in the adult
• consists mostly of adipose cells
• functions: storage of reserve energy, reserve of hematopoietic tissue

Question 18

Erythrocyte blast

Answer 18

• each blast gives rise to one type of blood cell (monopotential)
• morphology:
• relatively large cell with a diameter of 10-15 um
• large euchromatic nucleus- several nucleoli may be seen, large nucleo-cytoplasmic ration
• numerous ribosomes in the cytoplasm- cytoplasm will appear pale blue in blood smear
• no cytoplasmic granules

Question 19

Erythrocyte Differentiation

Answer 19

• decrease in cell volume
• decrease in nuclear diameter
• increase in heterochromatin
• disappearance of nucleoli
• loss of nucleus
• decrease in cytoplasmic basophilia
• increase in cytoplasmic eosinophilia- amount of hemoglobin increases

Question 20

Descriptive stages

Answer 20

1) blast (erythroblast)
2) basophilic erythroblast
3) polychromatophilic erythroblast
4) normoblast
5) reticulocyte
6) orthochromatic erythroblast
7) mature erythrocyte (red blood cell)

Question 21

Basophilic erythroblast

Answer 21

• cell is smaller than a blast
• nucleus- smaller than blast, checkerboard appearance of chromatin, nucleoli will disappar
• cytoplasm- intensely basophilic (navy blue), large increase in free ribosomes, preparing to produce globin portion of hemoglobin
• lasts 1-2 days
• capable of 1-2 mitotic divisions

Question 22

Polychromatophilic erythroblast

Answer 22

• morphology- size of cell continues to decrease
• nucleus- smaller than that of basophilic erythroblast, further condensation of chromatin, nucleolus will be lost
• cytoplasm-gradual shift from intense basophilia to intense acidophilia- due to increase in amount of hemoglobin and decreases in number of polysomes- hemoglobin binds to anionic dye eosin, RNA of ribosomes binds cationic dye methylene blue, cytoplasm has a double staining reaction
• lasts about 3 days
• capable of 3-4 mitotic divisions

Question 23

Normoblast

Answer 23

• no longer capable of mitosis
• morphology:
• smaller than polychromatophilic erythroblast
• nucleus-smaller than that of polychromatophilic erythroblast, totally heterochromatic
• cytoplasm-faintly polychromatophilic, mostly pink with a hint a blue, due to presence of lots of hemoglobin and a few remaining polyribosomes

Question 24

Reticulocytes

Answer 24

• 80% of normoblasts
• extrude nucleus (unequal cytokinesis)
• cytoplasm contains a few polyribosomes- residual ribosomal RNA can be stained with brilliant cresyl blue
• can remain briefly in bone marrow
• may be released into peripheral circulation- after 1 day in circulation residual RNA is lost, cell becomes mature red blood cell
• reticulocytes make up about 1% of total number of red blood cells
• normoblast-reticulocyte period lasts 3 days

Question 25

Orthochromatic erythroblasts

Answer 25

- 20% of normoblasts become this - lose residual RNA before they extrude nuclei - small heterochromatic nucleus - bright eosinophilic cytoplasm - not normally released into peripheral circulation

Question 26

Kinetics of erythrocyte production

Answer 26

- process takes from 8-9 days - most of the time is spent in bone marrow - 1-2 days as basophilic erythroblast - 3 days as polychromatophilic erythroblast - 3 days as normoblast-reticulocyte - 1 day for reticulocyte-erythrocyte transition

Question 27

Distribution of erythrocytes

Answer 27

- number of circulating mature red blood cells is about 20X the number of immature forms of bone marrow - ready reserve: - located in red bone marrow - reticulocytes that slightly outnumber circulating reticulocytes

Question 28

Erythropoietin

Answer 28

- glycoprotein hormone - synthesized in kidney cortex (endothelial cells of peritubular capillary plexus - increases rate of mitosis in developing red blood cells- blasts, basophilic erythroblasts, polychromatophilic erythropoietin - increases RNA synthesis in developing rbcs - hypoxia stimulates the synthesis of erythropoietin (hemorrhage, hemolysis, high altitude, compromise of pulmonary function)

Question 29

Reticular cells (adventitial cells)

Answer 29

- morphology- large cells with many cytoplasmic processes - contain lots of ingested material in their cytoplasm - large pale staining nucleus erythroblastic islands- polychromatophilic erythroblasts clustered around reticular cells - play a trophic role in the maturation of erythrocytes- supply nutrients and growth factors to developing rbcs - phagocytose extruded normoblast nuclei

Question 30

Plasma cells

Answer 30

- can be observed in bone marrow smears - basophilic cytoplasm - negative image of Golgi apparatus - eccentrically placed nucleus - clock face distribution of chromatin

Question 31

Megakaryocyte Differentiation

Answer 31

- cell enlargement - lobulation of nucleus - increase in level of ploidy (32-64 n) - shift in cytoplasmic basophilia to acidophilia - accumulation of azurophilic cytoplasmic granules - formation of platelet demarcation channels

Question 32

Megakaryoblast

Answer 32

- large cell 40-50um in diameter - large oval or spherical nucleus (polyploid) - homogenous basophilic cytoplasm

Question 33

Megakaryocyte

Answer 33

- even larger cell (up to 150um in diameter) - lobulated nucleus - increase in ploidy up to 32-64n (endomitosis) - cytoplasm becomes eosinophilic - azurophilic granules appear in cytoplasm - invaginations of plasma membrane develop throughout cytoplasm- forms demarcation channels, results in partitioning of cytoplasmic fragments to form platelets, platelets enter discontinuous sinusoids in bone marrow - contain a functional repertoire of mRNAs - platelets may be able to produce functional progeny?

Question 34

Model of platelet production

Answer 34

- pseudopod formation - pseudopods elongate forming proplatelets - platelets released from ends of proplatelets - demarcation channels are a membrane reservoir

Question 35

Kinetics of platelet formation

Answer 35

- megakaryocyte maturation takes 4-5 days in humans - platelets then circulate for about 10 days - destroyed in spleen and liver

Question 36

Lymphocyte differentiation

Answer 36

- cell size decrease, chromatin becomes condensed, nucleoli becomes less visible, lymphocyte subsets acquire distinctive cell surface receptors - lymphoblasts reside in bone marrow - some of their progeny migrate to the thymus- will then acquire characteristics of T lymphocytes- after they will populate lymphoid organs- lymph nodes, spleen, tonsils - some progeny remain in bone marrow for a while, they begin to differentiate to B cells, leave bone marrow and migrate to peripheral lymphoid organs where they complete their differentitations- lymph nodes, spleen, tonsils

Question 37

Monocytes

Answer 37

- develop from blasts within red bone marrow - small azurophilic granules appear in the cytoplasm-smaller than primary granules at neutrophilic promyelocyte, they are lysosomes- contain alpha-napthyl acetate esterase - nuclei assume unusal shapes - formation in bone marrow takes 2-3 days - remain in peripheral circulation for 1-2 days - migrate to tissues and become histiocytes (macrophages) - may function as tissue macrophages for 1-3 months