HEMATOPOIESIS Lecture Notes PDF

HEMATOLOGY 1 PRELIMS LEC 01 - HEMATOPOIESIS INTRODUCTION 2.) HEPATIC PHASE Begins at 5th to 7th gestational weeks haemas...

HEMATOLOGY 1 PRELIMS LEC 01 - HEMATOPOIESIS INTRODUCTION 2.) HEPATIC PHASE Begins at 5th to 7th gestational weeks haemas - blood Before the transition where the yolk sac stops producing Hema 1 - RBCs and WBCs cells, the liver has already started producing cells Hema 2 - platelets and hemostasis Characterized by recognizable clusters of developing Anemia - disease commonly affecting RBCs; leukemia for erythroblasts, granulocytes, and monocytes WBCs Lymphoid cells and megakaryopoiesis Megakaryocytes - where platelets are formed/came from HEMATOPOIESIS The liver can’t do the job alone so other active sites help such as the spleen, lymph nodes, thymus and kidneys Production of cells of the blood - RBCs, WBCs, and Platelets Hemoglobin production, but the 3 embryonic hemoglobins Continuous, regulated, and controlled process of cell are not present anymore as HbF (Hemoglobin F - Fetal production Hemoglobin) becomes dominant in this phase ○ if there is something stopping the production of Globins: 2 alpha and 2 gamma cells, there should be a replacement Detectable labels of HbA (Adult Hemoglobin) ○ the body produces what you need (not too much and not too less), regulated by growth hormones During pregnancy - HbF ○ cell renewal, proliferation, differentiation, when we are born - HbF (Fetal hemoglobin) decrease, HbA maturation (Adult hemoglobin) increase Start immature and mature over time to become functional as soon as we are delivered - HbF is still dominant Main role of mature RBCs - oxygen delivery; immature RBCs 6 months after delivery - increase in HbA1 and HbA2 are not functional ○ HbA1 - 2 Alpha, 2 Beta - 95% hemoglobin as adult ○ if a patient has hypoxia = more RBCs produced ○ HbA2 - 2 Alpha, 2 Delta - 2-3 % ○ leukemia - keeps on producing cells in an ○ HbF - 2 Alpha, 2 Gamma - 1-2% uncontrolled and unregulated process WBC - increased when infection is present in the body 3.) MEDULLARY PHASE ○ if there is no infection already, the production of WBC should be back to normal Occurs in the medulla (inner part) specifically in the bone marrow of newborn 5th month of gestation STAGES OF HEMATOPOIESIS HSCs (hematopoietic stem cells) and mesenchymal cells migrate into the core of the bone that helps synthesize 1.) MESOBLASTIC PHASE mature RBCs Chief site: yolk sac Measurable labels of growth factors that stimulate stem cells Starts as early as the 19th day of gestation to mature ○ mother is not even aware she is pregnant Occurs in most of the bone (flat bones) ○ can last up to 8-12 weeks gestation ○ e.g. sternum, rib cage Embryonic hemoglobins ○ adult - iliac crest, hips ○ RBC are useless if there is no hemoglobin ○ newborn - tibia and femur Mesodermal cells migrate to the yolk sac leading to the development of A. ERYTHROBLAST - immature form of RBC, primary B. ANGIOBLAST - formation of blood vessels * -blast = immature cell * angio- = blood vessels For RBCs to be functional, they always need hemoglobin which will bind and carry oxygen - to differentiate hemoglobin - take note of globin component - pairing: 4 units of heme= 4 chains of globin 3 EMBRYONIC HEMOGLOBINS 1.) Gower 1 - 2 epsilon and 2 zeta chains 2.) Gower 2 - 2 alpha and 2 epsilon chains 3.) Portland - 2 zeta and 2 gamma chains Important feature: cell production occurs intravascularly - surrounded by angioblast; some of the mesoderm cells also go to the AGM (aorta-gonad mesonephros) giving rise to hematopoietic stem cells Garcia | Montano (2023) 1  ADULT HEMATOPOIETIC TISSUES MYELOID TO ERYTHROID RATIO (M.E.) Numerical expression comparing numbers of granulocytes precursors to RBC precursors PRIMARY LYMPHOID ORGANS ○ precursors - immature cells Bone Marrow and Thymus - production of lymphoid cells - lymphocytes - T and B cells NORMAL M:E RATIO ○ 1.5:1 - 3.3:1 SECONDARY LYMPHOID ORGANS ○ 2:1 - 4:1 Lymph nodes, Spleen, and Liver - respond to foreign in BM, there are more WBCs precursors antigens compared to RBCs but in blood, there are more RBCs than WBCs BONE MARROW WBCs only survive for a few hours thus RED - active, capable of producing cells, both have mature it signals the BM to produce more, while and immature forms of blood cells RBC lasts up to 120 days and sends a YELLOW - inactive, incapable of producing cells, contains signal to stop production adipocytes at birth, the bone marrow is very active 90-95%, as ABNORMAL M:E RATIOS they have higher cell counts ○ Infection - 6:1 in adults - equal distribution of red and yellow BM ○ Leukemia - 25: 1 - not only WBCs but all cells transition of red to yellow BM - retrogression ○ Myeloid hyperplasia - 20:1 ○ Is it possible for the transition of yellow ○ Myeloid hypoplasia - 3:20 to red? yes, during blood loss or ○ Erythroid hyperplasia - 1:20 hemolysis ○ Erythroid hypoplasia - 5:1 preferably during BM cell count - 1000 20s-30s - 60% red, 40% yellow Senior years - 60% yellow, 40% red EXTRAMEDULLARY HEMATOPOIESIS Blood cell production in hematopoietic tissue other than the NORMOCELLULAR bone marrow Must have 30-70% hematopoietic cells - combination of Sites: liver, spleen, lymph nodes, thymus mature and immature cells ○ immature cells present in the bone marrow are larger than mature cells - they grow to be smaller STEM CELL THEORY OF HEMATOPOIESIS HYPERCELLULAR - more than 70% All cells are derived from a pool of stem cells that are HYPOCELLULAR - less than 30% self-renewing APLASTIC - aplasia, very few or total absence Pluripotential & multipotential stem cells give rise to committed stem cells for each cell line In cases of Leukemia - hematopoiesis is not controlled HEMATOPOIETIC MICROENVIRONMENT Nurturing and protecting HSCs if one stem cell produces RBC, RBC lang ang Balance in quiescence - self renewal and differentiation of ipro-produce nya HSCs - in the niches Responsible for supplying semifluid matrix (stroma) that serves as an anchor for the developing hematopoietic cells 1.) MONOPHYLETIC THEORY To support HSCs stroma has cells All blood cells derived from a single cell called pluripotent HSCs, can extract more cells coming STROMAL CELLS from this single cell Endothelial cells - regulate flow of particulates and nutrients More accepted theory Adipocytes - fats that secrete steroids - influence cell production of RBCs for bone integrity 2.) POLYPHYLETIC THEORY Macrophages - remove unwanted particulates, cytokines Each blood cell is derived from its own unique Osteoblasts - bone-forming cells, waterbag/ comet stem cell appearance Osteoclasts - bone-destroying cells HEMATOPOIETIC STEM CELLS - committed to only one Reticular cells - supports hematopoietic cells Self-renewing Pluripotent - can derive from one stem cell EXTRACELLULAR MATRIX OF THE MARROW Differentiation Fibronectin Apoptosis Collagen Laminin Thrombospondin Tenascin Proteoglycans Garcia | Montano (2022) 2 CULTURE-DERIVED COLONY-FORMING UNITS (CFUs) LINEAGE SPECIFIC HEMATOPOIESIS ERYTHROPOIESIS STEM CELL CYCLE KINETICS Regulated process for maintaining adequate number of 2.5 billion erythrocytes RBCs in the peripheral blood 2.5 billion platelets A process by which erythroid precursor cells differentiate to 1 billion granulocytes become mature Total = 6 billion cells, per kilo of body weight, DAILY Immature RBCs become mature RBCs 1:1000 nucleated blood cells Main regulator: EPO (Erythropoietin) from the kidneys (primarily) and liver, released during hypoxia REGULATION OF HEMATOPOIESIS ○ binds to the CFU to make sure that stem cells are Erythropoietin (EPO) committed to producing RBCs Thrombopoietin (TPO) Granulocyte CSF (G-CSF) - colony-stimulating factor Granulocyte-macrophage CSF (GM-CSF) Interleukins - signaling mechanism *** monocytes - blood macrophages - tissues CYTOKINES FUNCTIONS Prevent hematopoietic precursor cells from dying Stimulate stem cells to divide, undergo mitosis Regulate cell differentiation CYTOKINES WITH NEGATIVE INFLUENCE Becomes smaller as they mature, starting from a large cell and Inhibits cell production Becomes small primarily because of cell division (mitosis) Transforming growth factor-beta Immature RBCs contain nucleus, mature cells don’t Tumor necrosis factor-alpha Nuclear Cytoplasmic ratio (N:C ratio) decreases Interferons ○ nucleus is extruded Garcia | Montano (2022) 3  Nuclear pattern becomes more coarse and condense, A.) PRONORMOBLAST nucleoli disappears Cytoplasm (basophilic) blue becomes (acidophilic) pink As cells mature, there is a need to synthesize hemoglobin Hemoglobin synthesis happens only to immature RBCs ○ only immature cells contain nucleus ○ mature cells are not capable synthesizing hemoglobin, they not contain nucleus Earliest recognizable stage First morphologically identifiable red cell precursor THREE ERYTHROID PRECURSOR NOMENCLATURE SYSTEM Basophilic (blue) - concentration of ribosomes and RNA Starting to collect substances needed for Hgb synthesis B.) BASOPHILIC NORMOBLAST Detectable Hgb synthesis, already started but invisible Entire cytoplasm is intensely blue C.) POLYCHROMATOPHILIC NORMOBLAST Multiple colors Visible Hgb synthesis - portion of blue and pink area First shows synthesis of Hgb very well Last stage to undergo cell division D.) ORTOCHROMIC NORMOBLAST specific production of RBCs all start from the pluripotential Last stage to contain nucleus hematopoietic stem cells ->(thru stromal cells, cytokine) -> forming Nucleus is in the peripheral area already CFU-GEMM (myeloid progenitor) giving rise to -> BFU-E (first larger Looks like the nucleus is being extruded out colonies to have during erythropoiesis, burst -forming unit erythroid) - Vimentin is lost - responsible for holding the organelles tight larger and contains less receptors from ETO ○ extruded nucleus is called pyrenocyte and will interleukins, kit-ligand - stimulate BFU to mature phagocytize PHSC -> CFU-GEMM -> BFU -> CFU (smaller, more Decreased level of Hgb synthesis receptors for erythropoietin) from BFU to become CFU - takes about 1 week E.) RETICULOCYTE from CFU to become pronormoblast - takes about 1 week from pronormoblast to RBC - takes about 5-7 days from BFU to become RBC - up to 21 or 25? days CFUs all look the same microscopically, always start at the recognizable stage where its characteristics are different from other Last stage that will synthesize hemoglobin cell types Contains remnants of RNA - allows to synthesize normoblast -> reticulocyte = 3 - 5 days hemoglobin ○ reticulocyte stays in the BM for 1 - 2 days Nucleus is totally out ○ after 2 days, reticulocyte goes to the blood Immature form of RBC, but present in the blood ○ matures to become RBC for 1 day Uses supravital stains - to count reticulocyte and see RNA still capable of Hgb synthesis since remnants reticulocyte contains remnants of RNA Counting them is a way to determine bone marrow’s erythropoietic activity *** all immature should be in the bone marrow only, and mature in the blood Garcia | Montano (2022) 4 E.) ERYTHROCYTE stem cell pool - HSC proliferation pool - where they are capable of mitosis, many divisions ○ CMP maturation pool - nucleus develops Mature, functioning RBC A. MYELOBLAST Discocyte, 7-8 micrometers in diameter Has central pallor area - extra surface area Red cells contain hemoglobin NORMOBLASTIC SERIES: SUMMARY OF STAGE MORPHOLOGY Granules are not yet present B. PROMYELOCYTE LEUKOPOIESIS Granules will appear on this stage Start where you see primary granules; a non-specific Division: Myelopoiesis and Lymphopoiesis granules ○ Myelopoiesis - granulocytes: neutrophils, eosinophils, basophils, monocytes C. MYELOCYTE ○ What’s important is the appearance of granules GCS-F growth factors for granulocytes, stages are the same; the difference is in the growth factor eosinophils - interleukin 5 & 3 neutrophils - G CSF ○ Lymphopoiesis - lymphocytes Secondary granules appear MYELOID LINEAGE Last stage to undergo cell division D. METAMYELOCYTE “Juvenile cell” Kidney-shaped cells Synthesis of tertiary granules/gelatinase granules E. BAND CELL NEUTROPHIL DEVELOPMENT Neutrophils share a common progenitor with monocytes Share the same progenitor - granulocyte-monocyte progenitor Immature cells but normally present in the blood Garcia | Montano (2022) 5 F. SEGMENTED NEUTROPHIL EOSINOPHIL DEVELOPMENT Development is similar to that described for neutrophils Appears as reddish to orange granules 3-5 lobes Granules are pink to rose-violet color *** when doubt exists between band cell or segmented neutrophil, ALWAYS count the cell as segmented neutrophil *** neutrophil are the most abundant, and 1st to attack PRIMARY (AZUROPHILIC) GRANULES Formed during the promyelocyte stage Last to be released (exocytosis) Contain: PRIMARY GRANULES ○ Myeloperoxidase Formed during promyelocyte stage ○ Acid Beta-glycerophosphatase Contain: ○ Cathepsins ○ Charcot-Leyden crystal protein ○ Defensins ○ Elastase SECONDARY GRANULES ○ Proteinase-3 Formed throughout remaining maturation Contain: SECONDARY (SPECIFIC) GRANULES ○ Major basic protein (core) - help eosinophil to kill Formed during myelocyte and metamyelocyte stages parasitic organism Third to be released ○ Eosinophil cationic protein (matrix) Contain: ○ Eosinophil-derived neurotoxin (matrix) ○ Beta 2 - Microglobulin ○ Eosinophil peroxidase (matrix) ○ Collagenase ○ Lysozyme (matrix) ○ Gelatinase ○ Catalase (core and matrix) ○ Lactoferrin ○ Beta-Glucuronidase (core and matrix) ○ Neutrophil gelatinase-associated lipocalin ○ Cathepsin D (core and matrix) ○ Transcobalamin I ○ Interleukin-2, -4, and -5 (core) ○ Interleukin-6 (matrix) TERTIARY GRANULES ○ Granulocyte-macrophage colony-stimulating factor Formed during metamyelocyte and band stages (core) Second to be released Contain: SMALL LYSOSOMAL GRANULES ○ Gelatinase Acid phosphatase ○ Collagenase Arylsulfatase B ○ Lysozyme Catalase ○ Acetyltransferase Cytochrome b558 ○ Beta 2 - Microglobulin Elastase Eosinophil cationic protein SECRETORY GRANULES (Secretory Vesicles) Formed during band and segmented neutrophil stages LIPID BODIES First to be released (fuse to plasma membrane) Cyclooxygenase Contain (attached to membrane): 5-Lipoxygenase ○ CD11b/CD18 15-Lipoxygenase ○ Alkaline phosphatase Leukotriene C4 synthase ○ Vesicle-associated membrane-2 Eosinophil peroxidase ○ CD10, CD13, CD14, CD16 Esterase ○ Cytochrome b558 ○ Complement 1q receptor STORAGE VESICLES ○ Complement receptor-1 Carry proteins from secondary granules to be released into the extracellular medium Garcia | Montano (2022) 6 BASOPHIL DEVELOPMENT LYMPHOID LINEAGE Derived from progenitors in the bone marrow and spleen Covered with darkly-stained purplish granules IL-3 (Interleukin-3) - IgE dependent TSLP (Thymic Stromal Lymphopoietin) - non-IgE dependent SECONDARY GRANULES Histamine Platelet-activating factor Leukotriene C4 Interleukin-4 Lymphoid stem cell gives rise to T-lymphocyte and B- Interleukin-13 lymphocyte lineages Vascular endothelial growth factor A T-cell maturation - thymus Vascular endothelial growth factor B B-cell maturation - bone marrow Heparan sulfate Plasma cells - present in marrow, lymphatic tissue, connective tissue MONOCYTE DEVELOPMENT B and T cells are morphologically same, function-wise are different Similar to neutrophil development because both cell types ○ humoral immunity are derived from the GMP To be able to identify the difference between the two, CD Main stimulant: Growth-Stimulating Cytokines Marker testing is performed Monoblast-Promonocyte-Monocyte *** monocytes are the largest, and 2nd to attack LYMPHOCYTE DEVELOPMENT Antigen independent Antigen dependent - there is an antigen-stimulating the production B-cell maturation T-cell maturation 1. B CELLS DIFFERENTIATION INTO MACROPHAGES pro-B, pre-B, and immature B cells In areas of inflammation or infection (inflammatory ○ immature B cells - not yet exposed to antigen macrophages) Antigen “NAIVE” cells As “resident” macrophages in: ○ Liver (Kupffer cells) ○ Lungs (alveolar macrophages) ○ Brain (microglia) ○ Skin (Langerhans cells) ○ Spleen (splenic macrophages) ○ Intestines (intestinal macrophages) ○ Peritoneum (peritoneal macrophages) ○ Bone (osteoclasts) ○ Synovial macrophages (type A cell) ○ Kidneys (renal macrophages) ○ Reproductive organ macrophages 2. T CELLS pro-T, pre-T, and immature T cells Garcia | Montano (2022) 7 3. NK CELLS (Natural Killer) PLATELET SHEDDING CD56+, CD16+, CD3-, CD7+ large granular lymphocytes 4. LYMPHOCYTE SUMMARY MEGAKARYOPOIESIS Maturation series of a hematological cell that is committed for platelet production Production of megakaryocytes Responds to a growth factor called Thrombopoietin and CSF-Meg Megakaryocyte Lineage Progenitors BFU-Meg, capable of mitosis CFU-Meg, capable of mitosis LD-CFU-Meg, not capable of mitosis but will undergo ENDOMITOSIS, where only the nucleus divides but not the cytoplasm As the cell matures; Cell size increases N:C ratio decreases Number of nucleus increases Maturation time: 7 days to release platelets Garcia | Montano (2022) 8