Hematopoiesis Lecture Notes PDF

HEMATOPOIESIS The Formation and Development of Blood Cells  Hematopoiesis  A continuous, regulated process of blood cell production that includes cell renewal, proliferation, differentiation, and maturation in the blood-forming organs. Hematopoietic Stem Cells (HSCs) ...

HEMATOPOIESIS The Formation and Development of Blood Cells  Hematopoiesis  A continuous, regulated process of blood cell production that includes cell renewal, proliferation, differentiation, and maturation in the blood-forming organs. Hematopoietic Stem Cells (HSCs)  They are the foundation of the adult hematopoietic system. The embryo produces the first adult repopulating HSCs.  They are also referred to as hemocytoblasts. TYPES OF HUMAN STEM CELLS (SC)  1. Totipotent SC- present the first few hours after fertilization. The most versatile type of SC because can develop into any human cell type, including dev't from embryo into fetus/.  2. Pluripotential SC- present several days after fertilization. Can develop into any cell type, except they cannot develop into fetus.  3.Multipotent SC- derived from pluripotential SC, found in adults,but they are limited to specific types of cells to form tissues. e.g.bone marrow SC  Human SC  can produce all types of blood cells, bone, cartilage, adipose/fat cells so are said to be MULTIPOTENTIAL  can differentiate along any cell line( erythrocytic, granulocytic, monocytic, megakaryocytic, lymphocytic)  the main factor that will influence what they will differentiate into is the NEED OF THE BODY. Early Dev't of Blood Cells  Blood cells originate from the mesenchymal tissue that arises from the embryonic germ layer, the mesoderm. The mesodermally derived STRUCTURES ARE:  1. paraaortic splannchnopleure  2. aorta-gonad-mesonephros region  In here production of multipotent progenitor cells and hematopoietic stem cells (HSCs) happens before their appearance in the yolk sac. 3 DIVISIONS OF HEMATOPOIETIC CELLS IN THE BONE MARROW  1. Primitive multipotential cells- stem cells, the most immature group capable of self-renewal and differentiation into all blood cell lines.  2. Intermediate cell or Progenitor cells- committed cell and are denoted by the prefix CFU /BFU. They are unipotential and are already committed to develop into a specific cell line.  3.Mature cells or Precursor cells- the developed group and are already with specific function/s.  STAGES OF HEMATOPOIESIS  Pre-natal Hematopoiesis  Post-natal Hematopoiesis  PRE-NATAL HEMATOPOIESIS I. MESOBLASTIC PERIOD  Starts on 14th day, peaks on the 1st month and persist up to 3rd month of gestation, start of ERYTHROPOIESIS.  Chief site – blood islands of yolk sac  3rd month fetal life = Primitive Erythroblast  Primitive Erythroblast  Formed intravascularly( or within developing blood cells), megaloblastic( bigger in size) , and retain their nuclei  Contains Embryonic Hemoglobin- Gower1, Gower 2, Portland  PRE-NATAL HEMATOPOIESIS II. HEPATIC PERIOD  Starts on the 5th to 7th week of gestation and persist up to birth( wanes- lowers in production)  Chief site – Liver, as well as spleen and lymph nodes, thymus (lesser extent)  Definitive Erythroblasts Formed extravascularly and extrude their nucleus, CONTAINS HbF Continuation of erythropoiesis, 3rd month- starts granulopoiesis & megakaryopoiesis  4th month- lymphopoiesis, 5th month- monocytopoiesis/ monopoiesis  PRE-NATAL HEMATOPOIESIS III. MEDULLARY or MYELOID PERIOD  Starts on the 5th month of fetal life up to adulthood  Bone Marrow is the chief site of hematopoiesis by the end of 24 weeks( 6 months). Measureble levels of Erythropoietin (EPO), Granulocyte- Colony Stimulating Factor, (G- CSF)Granulocyte-Monocyte/ Macrophage – colony stimulating factor( GM- CSF). HbA and HbF are detected.  Cells at various stages of maturation can be seen in all blood cell lineages/ lines. POST-NATAL HEMATOPOIESIS I. MEDULLARY/ MYELOID Bone Marrow – is the only site of erythropoiesis, myelopoiesis and thrombopoiesis IN POST-NATAL LIFE. A. Red Bone Marrow – Hematopoietically active marrow- PRODUCE BLOOD CELLS B. Yellow Bone Marrow – composed primarily of adipocytes (fats) Bone Marrow at Birth 1.5 % of total body weight Bone Marrow (Adult) 4.5% of total body weight Red Bone Marrow Site of Blood Cell Yellow Bone Marrow Formation Red Bone Marrow POST-NATAL HEMATOPOIESIS II. EXTRAMEDULLARY  Blood cell production in hematopoietic tissue other than bone marrow. This is resorted to when the converted yellow marrow to red marrow is insufficient to meet the body’s demands A. Lymph nodes-T & B cells dendritic cells(DC)( antigen-presenting cells), macrophages, plasma cells B. Spleen –T and B cells, plasma cells, macrophages C. Liver-can be hematopoietic when needed  2 characteristics of stem cells 1. Uncommitted 2. Mutipotential and can differentiate along any cell line( depending on the body’s need) Theories of Blood Cell Production A. Monophyletic All blood cells are derived from uncommitted stem cells which are capable of giving rise to several types of cells- the accepted theory B. Polyphyletic The first recognizable and most primitive blood cells are already committed to develop into a specific cell line- not accepted theory  3 compartments of cells in Bone Marrow( BM) 1. Hematopoietic Stem Cells (HSCs)  most immature, can do self-renewal & differ- entiation into any of the cell lineages  Divide by mitosis to give rise to multipotent progenitors  Have the morphology of medium- sized lymphocytes  CD34+ but lacks MHC Class II antigen  Can give rise to CFU-L and CFU- GEMM(myeloid) 3 compartments of cells in the BM 2. Progenitors  Starts to commit, as they mature, acquire more cluster of differentiation (CD), remain CD34+ for identification. CD34 is marker for immaturity  Cells with prefix- BFU or CFU  They are unipotential- committed to differentiate along a single cell line  Cells in the BM 3. Precursor cells  Recognized due to additional expression of antigens (CD38) CD71+ – erythroid CD10+ – B cells CD33+ – myeloid CD7+ – T cells CD5+ - T cells Cells in the BM  The formation & dev't of mature blood cells from the multipotent Stem Cells is controlled by growth factors & inhibitors, and the microenvironment. The microenvironment or locale influences behavior & controls the proliferation of multipotential stem cells.  The bone provides the microenvironment most appropriate for proliferation & maturation of cells.  The BM is encased by endosteum Progenitor Cells  The hematopoietic progenitor cells (HPCs) can be mobilized from the BM to the blood by a wide variety of stimuli, including the hematopoietic growth factors & chemokines.  Individual hematopoietic cytokines can be lineage- specific or can regulate cells in multiple lineages, and for some cell types, e.g. stem cells, the simultaneous action of multiple cytokines is required for proliferative responses. HSC  The HSCs in the BM exist in a highly organized microenvironment composed of stromal cells & an extracellular matrix rich in fibronectin, collagen, & various proteoglycans.  HSCs can be found in umbilical cord blood (UCB) also. UCB hematopoietic cells is used as therapeutic source of autologous & allogeneic transplants for more than 20 yrs. Cryopreservation prolongs the storage time of UCB.  Aside from UCB, BM and peripheral blood are sources of stem cells for therapeutic purposes. Hematopoietic Processes  1. Erythropoiesis- process of red blood cell (RBC) formation. Occurs in erythroblastic islands, which are specialized niches in which erythroid precursors proliferate, differentiate, & enucleate( extrude their nucleus). Each island consists of a macrophage(mother cell surrounded by a cluster of developing normoblasts. Within erythroid niches, cell-cell & cell-extracellular matrix adhesion, positive & negative regulatory feedback. Erythroid cells account for 5%- 38% of nucleated cells in BM. Maturation of Blood Cells  A. Principles of Synchronistic Maturation- when nucleus & cytoplasm mature at the same time resulting in normal/ typical cells. While the following cytoplasmic occur, the nuclear changes also occur:  1. Cytoplasmic Changes  a. Loss of basophilia- Generally, the younger the cell, the higher is the RNA content of its cytplasm. The RNA is the cytoplasmic component which give it basophilia( love for basic dyes) Synchronistic Maturation  b. Production of cytoplasmic granules- this is a special feature of the granulocytes (neutrophil, eosinophil, basophil). In the promyelocyte, the non-specific/ primary granules develop. In the next stage, the early myelocytic stage, secondary granules appear. On the late myelocytic stage,tertiary granules appear. Both secondary & tertiary granules are specific granules At the myelocytic stage then, the 3 types of granulocytes are distinguishible from each other because of the color of their cytoplasmic granules. Synchronistic Maturation  c. Elaboration of Hemoglobin (Hb)- a special feature of the erythrocytic cells. Hb formation starts in the pronormoblasts(1st stage of devt) and seen first in the polychromatophilic normoblast stage, continues in the orthochromic stage, up until the reticulocytic stage. At the reticulocytic stage, only 35% of Hb are formed and is thne one used by the erythrocyte for its function of oxygen delivery to the tissues for its entire life span in the blood of 120 days.  Synchronistic Maturation 2. Nuclear Changes  a. Lobulation of nucleus- a special feature of the granulocytic cells. The granulocytes, increase in in nuclear lobes on matutaion. At maturation, the neutrophil has 2-5 nuclear lobes with an average of 3. The eosinophil, has 2-3 lobes, and mostly are bi- lobed. The basophil does not increase its lobe on maturation, but retains its stab-shaped nucleus.  Synchronistic Maturation b. Changes in the structure and cytochemistry of chromatin material - At an early stage, the nuclear chromatin are fine and linear because of the DNA’s double-helical structure. This is referred to as euchromatin. In maturing stages, the chromatin becomes coarse & clumped and are called the heterochromatin. In stages where, heterochromatin is observed, there are non-staining areas seen and are called the parachromatin. Synchronistic Maturation  c. Extrusion of nucleus- a special feature of the erythrocytic cells. In immature stages, from pronormoblast- orthochromic normoblast, these cells are nucleated. Enucleation/ Nucleolysis, the process of removal of the nucleus happens just before the orthochromic normoblast becomes a reticulocyte, the 5th stage. After 2-3 days as retics in the BM, the cell migrates to the blood a & remains in the blood for 1 day until it becomes an erythrocyte.  Synchronistic Maturation  3. Reduction in size of cells- As cells mature, they undergo mitosis, and the daughter cells become smaller, except the megakaryocytic cells. The megakaryocytic cells become bigger as they mature. This cell line undergo endomitosis/ endoreduplication where these do not occur: late telophase & cytokinesis, hence, one megakaryoblast matures into ultimately one mature megakaryocyte or metamegakaryocyte which is the mother cell of platelets. This cell line results in polyploid cells.  Asynchronistic Maturation Pathologic hematopoiesis results in abnormal nuclear maturation, abnormal cytoplasmic differentiation & abnormal size. Their development is asynchronistic. 1. Abnormal cytoplasmic differentiation- In RBC, this is characterized by persistent cytoplasmic basophilia & late hemoglobinization. Abnormal cytoplasmic inclusion bodies may be found in the cytoplasm of borh erythrocytes and leukocytes, especially in the granulocyes. Asynchronistic Maturation  2. Abnormal nuclear maturation- in leukemia & other severe disturbances, 2 nuclei may be present; one may be diploid & the other polyploid. The nucleus may be hypersegemnted or hyposegemented. In megaloblastic anemia, the megalocytes' nucleus takes longer to mature than its cytoplasm. This is so because the mitotic & intermitotic phases in megalocytes are prolonged when compared to the normal red cells. Asynchronistic maturation  3. Abnormal size - abnormally large cells are frequently seen in benign or malignant proliferation. In the erythrocytic series, the megaloblasts are larger than normal mature erythrocytes. Likewise, abnormally small cells may be found. Erythropoiesis  Maintenance of the circulating red cell mass within the BM limits seen in health is achieved by a feedback mechanism, which senses body oxygen demands & adjusts the rate of erythropoiesis accordingly. This feedback mechanism is mediated by the glycoprotein hormone erythropoietin (EPO) - 38kD. The Sequence :  1. Decrease in circulating RBC mass leads to decreased delivery of oxygen to the tissues & hypoxia develops. Hypoxia is the stimulus for erythropoiesis. Erythropoiesis  2.Tissue hypoxia is sensed by an enzyme-linked mechanism in the kidney and synthesis of EPO by the peritubular interstitial cells of the kidney is stimulated.  3.EPO binds to specific receptors on CFU-E(higher binding activity), BFU-E(very low level of binding or almost none), in the BM resulting in a shortening of cell-cycle time, an increased rate of maturation & an increased rate of release of red cells from the BM.  The target cells of EPO: CFU-E and pronormoblasts Erythropoiesis  4. The increased RBC count improves oxygen delivery to the tissues, the hypoxia is corrected & EPO synthesis is switched off/ decreased.  Erythropoiesis is influenced ny interleukin 3 which come from T cells.  DAILY: 1 % OF ALL RBC IS DESTROYED, AND REPLACED BY 1% NEW RBCS. THIS IS TO MAINTAIN THE OXYGEN LEVEL NEEDED BY THE BODY Erythropoiesis  Of the 1% destroyed( equivalent to 20 mL of blood,  90 % undergo extravascular lysis- macrophage mediated -senescent rbc are disposed by the splenic macrophages.  10% undergo intravascular lysis- happens in the blood circulation.  Increased intravascular lysis results in hemolytic conditions. Erythrocytic Cell Stages  1. Pronormoblast/ Rubriblast/ Proerythroblast- earliest regognizable stage of the series., 14-20 um indiameter., 1-2 nucleoli,cytoplasm show a lighter - staining area around the nucleus(perinuclear halo), round nucleus, n:C ratio is 8:1. Undergoes 1 mitosis to produce 2 basophilic normoblast.  - 12-17 um,, intensely basophilc(more tham2. Basophilic normoblast/Prorubricyte/ Basophilic erythroblast-n pronormoblast), N:C ratio is 6:1. Nuclear chromatin is slightly coarser. Able to have 2 mitoses giving rise to 8 polychromatophilic normoblast.  3. Polychromatophilic normoblast/ Rubricye/ Polychromatophilic erythroblast- 10-15 um in diameter., blue-gray to pink gray cytoplasm due to the start of Hb production(acidophilic) and the presence of RNA (basophilic) 4:1 is its N:C ratio. Smaller nucleus that that of the previous stage. last mitosis to produce 16 orthochromic normoblast.  4. Orthochromic normoblast/ Metarubricyte? Orthochromic erythroblast- 7-12 um.. pinker than the previous stage, bec of increased Hb product, no more mitosis because nucleus is pyknotic.  5. Reticulocyte- 7-10 um.. Slightly bigger than mature RBC and possess 2 or more reticular dots(RNA) which only stains by supravital stains(staining blood/ cells immediately upon removal from the body. New methylene blue or brilliant cresyl blue) should be used. No more nucleus as it was extruded already before this stage is reached.  6. Erythrocyte/ Mature RBC- 6-8 um in diameter,( round) pink to peach/buff color in a properly Wright- stained smear, anucleated, round with 1/3 central pallor, biconcave on lateral/side view.  Stages in Granulocytic Series:  1. Myeloblast-15-20 um in diameter, the earliest recognizable stage in the lineage. Blue cytoplasm, round or slightly oval nucleus, N:C ratio is 4:1, fine chromatin(euchromatin). Nucleus is red-purple in color and with 2-5 nucleoli. The earliest recognizable cell in the series.  2. Promyelocyte- 15-21 um in diameter. Cytoplasm is pale pink to bluish due to primary granules. Oval or round nucleus. N:C ratio is 3-1 to 2:1. Chromatin is coarser compared to the 1st stage. A little bit bigger than pyeloblast.  3. Myelocyte- 12-18 um in diameter. Cytoplasm is moderate in amount, few specific granules begin to appear (early myelocyte-secondary granules, late myelocyte-tertiary granules. Both are specific granules)At this stage the 3 granulocytes may be differentiated:  a. Neutrophil myelocyte- pinkish specific granules seen near the nucleus.  b. Eosinophil myelocyte- specific granules are red to orange. Granules are bigger than the non-specific & specific granules of neutrophil.  c. Basophil myelocyte- the specific granules are few, large, & stain a dark blue-purple, nucleus is oval/round. Chromatin is coarse/ more condensed, Generally, no ncleoli. Nucleus may be centrally located or may be eccentric. N:C ratio is 1:1.  4. Metamyelocyte- 10-15 um. Nucleus is indented/ kidney-shaped.. Decreased N:C ratio. Full complement of specific granules, with a dew non- specific granules.  a. Neutrophilic metamyelocyte- granules are more pink & more numerous.  b. Eosinophil metamyelocyte- more numerous and brighter orange-red granules  c. Basophil metamyelocye- dark purple to black granules are more numerous.  5. Stab/ Band/ Staff cell- 9-15 um in diameter., Cytoplasm is same as metamyelocyte. Nucleus is elongated/band-shaped, more deeply-indented tham metamyelocyte. Chromatin pattern is more coarse &clumped.  6. Segmented Neutrophil- 9-15 um in diamter. With pink to rose-violet specific granules, with few non-specific granules. Nucleus is not covered by its granules.  Eosinophil- 9-15 um in diameter.  contains the full complemet of large, reddish-orange granules. Nucleus is usually bi-lobed wc is not overlayed by its granules. Nuclear chromatin is coarse and clumped.  Basophil- 10-16 um in diameter. Cytoplasm is slightly pink to colorless. Contains specific dark purple to blue- black granules which are fewer than in eosinophil. Granules are water-soluble.  Nucleus is not coarse as in eosinophil and neutrophil. Unsegmented nucleus or rarely bilobed and is covered by its granules. Granulopoiesis  Granulocytes account for 23%-85% of the nucleated cells in the normal BM. Granulopoiesis can be said as a maturational unit. Early cells are located in the cords & the bone trabeculae. Neutrophils in the BM reside in theproliferating pool & maturation storage pool. Maturing cells spend an average of 3-6 days in the proliferating pool. If needed, cells from the storage pool can exit into the circulation rapidly and will have an average life span of 6-10 hours. Related to granulopoiesis is monocytopoiesis/ monopoiesis. CFU-GM is influenced by GM-CSF & IL3. IL5 influences Granulopoiesis  no more mitosis happen and spends the next 6-14 days in the maturation and storage pools, durin wc it develops into stab cells and segemented neutrophils or PMNs.  The stab/band is the most clinically impt to doctors as an increase (N. V. 2-6%) signifies infection. The increase in band cells is usually acompanied by leukocytosis or an increase WBC count (N.V. = 4500-11000/uL).  Granulopoiesis  Mature neutrophils are divided as: 50% in the circulatory pool & 50 % in the marginal pool. Neutrophils change back and forth the 2 pools in their 10 hours(average) stay in the blood.  A small % of band/ stab cells are continually released to the blood. The average lifspan is 5 days, since they can go out to the tissues (diapedesis) & perform their function in fighting infectious agents, after which they leave via excretions from GIT, GUT,lungs, saliva.  Pus cell- PMN which had been destroyed after its it had phagocytosed infectious organisms.  Abscess- a collection of pus cells.  Pus cells are eventually disposed of by the cells of the RES/ MPS (monocytes and macrophages) Granulopoiesis  INFECTION is the main stimulus for the generation of a larg number of granulocytes. The response to infection entails three mechanisms:  1. Granulocytes ( and monocytes) are recruited into & around the infected tissue by a releazse of chemotactic factors which comes from bacteria, their products, like toxins, or any other infectious organisms. Granulopoiesis  2. Granulocytes (and monocytres ) stored in the BM are mobilized & released into the blood.  3. The response initiates accelerated cellular production to satisfy the increased demand for these cells.  There are 3-5 mitosis that happen in the series over a period of 6-7 days in the BM. The mitosis happens in the myeloblast, promyelocyte and in the myelocyte, wc compose the proliferative compartment. On mits 4th stage, the metamyelocyte, Eosinophils  Matures in the same manner as neutrophil. Its maturation is through the help of Eo-CSF from the T cells. Primarily a tissue cell. For every 1 eos in the blood there are 300-500 in the tissues.Lifespan is several days, much longer than neutrophils.  Diurnal in vriation, since its number in the blood increases during nightime.  Steroids inhibit their release from the BM & enhance their movement to the tissues.  They can move to and from the tissues/ blood.  Metabolically more active than neutrophils.  Secretory products:  1. major basic protein- against helminths  2.histaminase-neutralize effect of histamine, (secretion of basophils, mast cells)so con-  sidered an anti-inflammatory cell.  3.arylsulfatase B  4. phospholipase B  5. Heparin Basophils Manner of maturation is same with neutrophil, eosinophil. Once released from BM to the blood, itremains in circulation same time as neutrophil, before it moves to the tissues where they are long-living.2. Mast cells have roy Mast cell(tissue basophils) and basophils differ in the ff morphological aspects: 1.Mast cells are slightly larger than basophils hils and Mast cells  2. Mast cells have round, oval nucleus while basophils possess stab-shaped nucleus.  3.Mast cells possess granules which are slightly smaller & less soluble than those of basophils.  Common to both basophils & mast cells :  1. secrete histamine, an impt part of inflammation.  2.both have membranes which readily bind IgE, which is followed by degranulation, anaphylaxis. Monocytopoiesis/ Monopoiesis  The monocytes arise from the same bipotential which give rise to the stem cell, the CFU-GM. From this, arise the CFU-M which is the mother cell of all monocytes. The proliferation of monocytes in the BM takes about 55 hours. They mature in BM but leave without no specific order.  The marginal pool of the monocytes in peripheral blood is about 3.5X the size of the circulating pool.  The mature monocyte spends about 12 hours in the peripheral blood before going iout to the tissues. Monocytic Series  1. monoblast- earliest recognizable cell in the series. 12-23 um in diameter. Non-granular cytoplasm which is moderately basophilic to blue-gray, non-granular. Nucleus is ovoid/round wc is light blue purple in color. Chromatin is fine, lacey.Nucleus has 1-2 nucleoli, N:C ratio is 4:1 to 3:1.  2. promonocyte-immature monocyte- 14-18 um in diameter. Cytoplasm is blue-gray and may contain azurophilic granules which is dust-like. Ground glass appearance cytoplasm which is moxderate in amount. Nucleus is oval, may have a single fold or fissure, with 1-5 nucleoli, fine chromatin pattern. N:C ratio is 3:1 to 2:1.  3. Monocyte- a14-20 um in diameter. Cytoplasm is abundant, blue-gray in color. Cytoplasmic outline may be irregular because of the presence of pseudopods.The cytoplasm has many, fine azurophilic granules giving a ground glass appearance. Vacuoles may be present. The nucleus is round/ kidney-shaped or may show slight lobulation. It may be folded over on top of itself, thus showing brainlike convolutions. No nucleoli are visible. Chromatin is fine & lacey(not clumped), arranged in skein-like(loosely coiled & knotted) strands. Macrophages  The macrophages are more active than monocytes and have richer supply of acid hydrolases. They stay for months or longer in the tissues and later die. They do not re-enter the blood altkough an occasional macrophage may enter the blood via the lymphatic system. They are 20-40 um in diameter. Macrophages  Possess eccentric nucleus, egg-shaped, indented or elongated. The chromatin appears spongy & i-2 nucleoli may be seen. Cytoplasm is abundant, sky- blue & has coarse, azurophilic granules. Cytoplasm is usually vacuolated, & may contain phagocytosed materials. The cytoplasmic outline may be irregular because of the presence of pseudopods. Lymphopoiesis  where antigen-independent lymphopoiesis occur. Lymphocytes are developed in these organs and NO ANTIGEN is needed for lympphopoiesis to happen. In the spleen, lymph nodes, MALT( GALT & BALT), antigen -dependent lymphopoiesis happen because ANTIGEN IS REQUIRED for lymphocytes to undergo lymphopoiesis(incrrease in number as in lymphadenitis/lymphadenopathy)  Stages in Lymphocytic Series:  1. Lymphoblast- 10-18 um in diameter, Cytoplasm is agranular, smooth, moderate to dark-blue, may stain deep-blue at the periphery & lighter blue near the nucleus. More abundant cytoplasm than in the myeloblast. Chromatin pattern is somewhat coarse.Nucleus is round or oval in shape, with 1-2 nucleoli. N:C ratio is 4:1.  2. Prolymphocyte- may be same size as lymphoblast/ smaller. Cytoplasm is moderate to dark blue, non- granular, more abundant than the lymphoblast.  Nucleus is round/ oval or slightly indented. Chromatin pattern is more clumped than in the lymphoblast with 1-2 nucleoli.  3. Mature lymphocyte:  a. small lymphocyte-8-10 um, more or less same size as mature RBC. Cytoplasm forms a thin rim around the nucleus moderate to dark-blue color. Nucleus is round/oval & may be slightly indented, with dense & clumped chromatin. No nucleolus.  b. Medkium sized lymphocyte -10-12 um in diameter, Cytoplasm is more abundant than in the small lymphocye, pale to moderately blue. Nucleus is round/oval & may be slightly indented. Chromatin pattern is clumped but not as dense as in the small lymphocyte. No nucleolus visible.  c. Large lymphocyte - 12-16 umin diameter. Cytoplasm is abundant, clear, very pale blue.Nucleus is round/oval and may be slightly indented. Chromatin pattern is cloarse, without nucleolus. Nucleus may be eccentrically located. Morphological and Functional Classes of Lymphocytes  A. Morphologic:  1. Small  2. Medium  3. Large  B. Functional:  1. T cell  2. B cell  3. Null cell/ non-T, non-B/ Large granular lymphocytes (LGL) Morphological Types  1. Small lymphocytes- life span is 4-10 yrs., accounts for about 80% of the total lymphocytes in blood,, size is 1.5x of the size of a normal red blood cell., demostrate the so called “ hand-mirror” apperance during locomotion.  2. Medium sized lymphocytes- accounts for 5-10 % of the total lymphocytes in blood.  3. Large lymphoctes- short-lived, lasts for 3-4 days, accounts for about 15 % of total adult lymphocytes in blood, 2-3X the size of normal RBC.  The major factor that affects the total number of lymphocytes in the body is the AMOUNT OF EXPOSURE TO ANTIGEN( something foreign like bacteria, viruses)  Lymphocytes are capable of blast transformation & mitosis and so can serve as their own stem cell compartment in terms of replacemet when needed.  Morphological transformation of lymphocytes in disease process  Lymphocytes which undergo transformation during a disease process are called the atypical lymphocytes. They are 2-3X bigger than the small lymphocyte in size.  Morphology of atypical or reactive lymphocytes:  cytoplasm usually seem to flow on one side of RBC, or follows the contour of RBC  Changes in atypical lymphocytes after stimulation by antigen  1.increase in size and nucleus becomes less dense & may show one or more nucleoli. Chromatin not clumped compared with normal lymphocyte.  2. cytoplasm becomes basophilic and increase in size as compared to the nucleus.  3.foamy ( holes) or vacuolated or moth-eaten cytoplasm  4.irregularly-shaped nucleus which may be lobulated, clefted and stained reddish to blue purple.  Changes in atypical l. ff antigenic stimulation  5. increased number of azurophilic granules in the cytoplasm. The Downey Classification  1. Downey Type 1- nucleus may be irregularly shaped. Cytoplasm is basophilic & at ytimes foamy.  2. Downey Type II - increased amount of cytoplasm. The peripheral part of the cell becomes more basophilic. Nuclear chromatin becomes more coarse & clumped.  3. Downey Type III- increase in size & shows basophilic cytoplasm. The nucleus has nucleoli.These cells resemble immature lymphocytes & is seen in Burkitt lymphoma patients.S Megakaryopoiesis  Takes place adjacent to the sinus endothelium. Megakaryocytes protrude through the vascular wall as small cytoplasmic processes to deliver platelets into the sinusoidal blood. Megakaryocytes develop into platelets in approximately 5 days.  Megakaryocytes account for about 1% of total nucleated cells in the BM.  Undergo ENDOMITOSIS, no telophase, cytokinesis. Megakaryopoiesis  2 factors which help in their maturation/ development:  1. Megakaryocyte Colony Stimulating Factor (Meg- CSF)- induce the committed cells to proliferate & differentiate into megakaryoblasts.  2. Thrombopoietin (TPO)- induces the differentiation & maturation of megakaryocytes & also influences their size,number of platelets which will be formed from their cytoplasm.  Stages of devt  1.Megakaryoblast- 20-50 um in diameter. Cytoplasm is varying shades of blue, usually darker than the myeloblast, may have small, blunt pseudopods. Cytoplasm is moderate in amount, a narrow band around the nucleus. On maturation, cytoplsm increases, non granular.. Nucleus is round/ oval/ may be kidney-shape with fine chromatin pattern. Nucleus has multiple nucleoli that generally stain blue. N:C ratio is 10:1.  2. Promegakaryocyte- - 20-60 um in diameter. Cytoplasm is more abundant than the previous stage, less basophilic than the blast stage. Granules begin to form in the golgi apparatus region. Nucleus has multiple nucleoli, irregular in shape, may show slight lobulation, chromatin becomes more coarse. N:C ratio is 4:1 to 7:1 depending on the ploidy.  3. Granular megakaryocyte- 30-90 um in diameter. Cytoplasm kis abundant, pinkish-blue in color, very fine and diffusely granular, with irregular peripheral border. The nucleus is small in comparison the the big cell size. Multiple lobulation of nucleus, coarser chomatin than in the previous stage, no nucleolus. N:C ratio is 2:1 to 1:1.  5. Mature megakaryocyte- 40-120 um in diameter. Cytoplas has coarse clumps of granules aggregating into little bundles which bud off from the periphery to become platelets.. Nucleus is multiple or multilobulated. No nucleolus. N:C ratio is 1:1.  5. Platelet- thrombocyte - 2-4 um in diameter, light blue to purple, very granular. The cental part is called the granulomere, and the peripheral part, the hyalomere.  Characteristics of Blasts Cells 1. Size – generally large with high nuclei and cytoplasmic ratio 2. Cytoplasm – basophilic, small in amount, no granules 3. Nucleus – large and round to oval in shape **Chromatin (Euchromatin) – very fine, very smooth, reddish purple, no clumping of chromatin materials **Nucleoli – pale light blue, capable of mitosis  Synchronistic Maturation A. Cytoplasmic changes 1. Loss of Basophilia – decrease in RNA as cell matures 2. Cytoplasmic Granules – special feature of granulocytic series/lines(N,E,B) 3. Elaboration of hemoglobin – special feature of erythrocytic series. seen in polychromatophic pronormoblastic stage,end-retics  Synchronistic Maturation B. Nuclear Changes 1. Structure and Cytochemistry – most reliable criterion 2. Number of Nucleoli – decreases nucleoli and amount of ribosome 3. Shape Granulocytes – Indentation and Segmentation, most number of lobes (mature) Erythrocytes – Pyknotic, and is eventually extruded at orthochromatic stage  Synchronistic Maturation C. Reduction in Cell Size  Special feature of all cell lineage except megakaryocytes General Rule: As the cell matures, the size, basophilia of cytoplasm and number of nucleoli decreases while coarseness of chromatin increases.  Asynchronistic Maturation 1. Abnormal Cytoplasmic Maturation  Persistent cytoplasmic basophilia – should be decrease or lost  Late hemoglobinization – erythrocytic series  Inclusion bodies – (erythrocytes, granulocytes) remnants of cell organelle, microorganism, parasites, iron deposits  Asynchronistic Maturation 2. Abnormal Nuclear Maturation  Polyploidy (megakaryocytic cells) – 2 nuclei in 1 cell, myeloid precursor  Hyposegementation or Hypersegmentation – excess lobes, infectious and toxic substances  Delayed Maturation – megalocyte (blast), smaller, takes longer time to mature than its cytoplasm  Asynchronistic Maturation 3. Abnormal Size  Large atypical lymphocytes  Megalocytes or Macrocytes  Micromegakaryocytes  Regulation of Hematopoiesis 1. Qualitative & Quantitative Cellular Equilibrium Balance between qualitative and quantitative parameters **Qualitative – normal morphology (size and shape) **Quantitative – normal count Regulation of Hematopoiesis  Erythropoietin (EPO) - major regulator of erythropoiesis, stimulates erythroid CFU cells and proerythroblasts/pronormoblasts. encoded on Chromosome 7.HEMA  Thrombopoietin (TPO) - increases platelet production, stimulates megakaryocyte CFU cells  Granulocyte CSF (G-CSF) - increases production of neutrophils, stimulates granulocyte- macrophage CFU cells Regulation of Hematopoiesis  Granulocyte-macrophage CSF (GM-CSF) - increases macrophage production, stimulates granulocyte- macrophage CFU cells  IL3-stimulates hematopoietic SC,promotes growth by early lineages.  The above are the hematopoietic growth factors.  Interleukins (IL) - stimulate B- and T-cell formation, function together with G-CSF and GM-CSF, and other activities. They are the primary messengers & directors of the immune response (IR). As of 2010,there are 35 known ILs. Pls refer to pp. 80-85 0f CLINICAL HEMATOLOGY, Theory & Procedures by Mary Louise Turgeon., 5th edition.  The genes for GM-CSF, M-CSF & IL3 are clustered in the long arm of Chromosome 5. Chromosome 17 is the location of G_-CSF