2.0 Hematopoiesis.docx

**Hematopoiesis** **-** process of blood cell production in the blood forming organs. \- encompasses cellular proliferation, differentiation, morphogenesis, functional maturation and death. \- Hematopoiesis is the continuous, regulated process of renewal, proliferation, differentiation, and matur...

**Hematopoiesis** **-** process of blood cell production in the blood forming organs. \- encompasses cellular proliferation, differentiation, morphogenesis, functional maturation and death. \- Hematopoiesis is the continuous, regulated process of renewal, proliferation, differentiation, and maturation of all blood cell lines. HSC - A hematopoietic stem cell (HSC) is capable of self renewal (i.e., replenishment) and directed differentiation into all required cell lineages **Cellular Poliferation** 1. **Mitosis** -- all lineage, division of nucleus 2. **Endomitosis** -- lineage of megakaryocyte (platelet), shattered pieces - No increase in the number of cells, but the cell gets bigger **Morphogenesis** -- change in structure, production of granules **Functional maturation** -- not capable of performing function of normal cell. **Hemoglobinization** -- nucleus is being excluded, normal cells of RBC (120 days) **Complement of spec. Granules** -- mature cells of WBC, protection against pathogens and invaders SPECIFIC TERMS Erythropiesis -- RBC Leukopoiesis -- WBC Granulopoiesis -- Granulocytes Lymphopoiesis -- Lymphocytes Megakaryopoiesis -- platelets Monocytopoiesis -- monocytes A. **PRENATAL HEMATOPOIESIS** 1. **Mesoblastic Stage/Mesenchymal Stage** Start: 14^th^ day to 1^st^ month of gestation (turgeon) 19^th^ day (Rodak's) Peak: 1^st^ -- 3^rd^ month Persist: 3^rd^ month of gestation Site: Mesenchyme/ erythroblastic islands/ blood islands of yolk sacs (outside the embryo) \*Pre-natal blasts are larger than post-natal\* **Primitive** **Erythroblasts** -- formed intravascularly, megaloblastic, retain their nuclei, contain Embryonic Hemoglobin (Hb) **Embryonic Hemoglobin** 1. Hb Gower I - 2 zeta, 2 epsilon 2. Hb Gower II -- 2 alpha, 2 epsilon 3. Hb Portland -- 2 zeta, 2 gamma \*needed for oxygen delivery in developing embryonic tissues **Hemoglobin** 1. Embryonic 2. Fetal (HbF) 3. Adult (HbA) 2. **Hepatic/ Extramedullary Stage** - 3^rd^ month, yolksac discontinue, **FETAL LIVER** becomes active - No longer persist in the adulthood **Main Hematopoietic Organ** 1. Embryonic Fetal Life -- Liver 2. Adult Life -- Bone Marrow **Fetal Hemoglobin** -- (2 alpha, 2 gamma) fetal hemoglobin (Hb F) is the predominant hemoglobin, but detectable levels of adult hemoglobin (Hb A) may be present **Simultaneously occurring in hepatic phase:** The developing spleen, kidney, thymus, and lymph nodes contribute to the hematopoietic process during this phase. a\. The thymus - the first fully developed organ in the fetus, - becomes major site of T cell production b\. kidney and spleen: - produce B cells. c\. megakaryocyte production 3. **Medullary/ Myeloid stage** - **occurs in the medulla or inner part of the bone cavity** - Site: Parenchyme of bone marrow (medullary cavity); consists of venous sinuses lined by endothelial cells and cords of hematopoietic tissue(stem cells, precursor cells, blasts cells) Start: 4^th^ month Site: bone marrow parenchyma lined by endothelial cell Between 5^th^ and 6^th^ month: : BM assumes role as primary site of hematopoiesis Bone marrow: - Consists of hematopoietic cells (erythroid, myeloid, lymphoid and megakaryocyte, adipose, osteoblasts/clasts, stroma - HSC are compartmentalized in the cords 2 COMPONENTS: A. RED MARROW - All red marrow during the first few years of life, progressively becomes confined to the axial skeleton and proximal ends of the long bones in adults - Composed of developing blood cells and progenitors - By age 18, red marrow is found only in the vertebrae, ribs, sternum, skull bones, pelvis, and to some extent the proximal epiphyses of the femur and humerus b\. YELLOW MARROW \- Hematopoeitically inactive [Detectable at this stage:] B. **POST-NATAL HEMATOPOIESIS** 1. **Medullary --** continuation of pre-natal 2. **Extramedullary** - - - - \*Retrogession: The process of replacing the active marrow by adipocytes (yellow marrow) - Yellow marrow is capable of reverting back to active marrow in cases of increased demand on the bone marrow, such as in excessive blood loss or hemolysis 18 yrs old -- diaphysis is completely filled with yellow marrow 1. **Medullary Stage** a. **Red Bone Marrow** -- appendicular and axial skeleton/ flat and irregular bones (sternum, ribs, pelvis, iliac crest of pelvis, skull, vertebrae), developing blood cells, Iliac crest of pelvis -- Puncture site b. **Yellow Marrow --** Filled with fats, fat cell production begins t 4yrs of age and completed at 18yrs of age - **The bone marrow contains developing erythroid, myeloid, megakaryocytic, and lymphoid cells.** 2. **Extramedullary Hematopiesis** - Outside the confines of the bone marrow - In certain abnormal circumstances, the spleen, liver, and lymph nodes revert back to producing immature blood cells a. **Lymphatic System** b. **Spleen** - Maturation of Lymphocytes and monocytes - Reservoir of blood cells which can be released from the pulp(red pulp-RBC) into the circulation on demand. - \*monocytes are not contained in the peripheral blood, it migrates to the tissues where they become macrophage (mononuclear phagocyte system) - \*RTS Reticuloendothelial System member -- functions to destroy cellular components which the body can still use (iron and globin) c. **Liver** - Not really for differentiation and maturation. - Stores intrinsic factor produced by the stomach. d. **Stomach** - Absorption of Vitamin B12. produce intrinsic factor. Without intrinsic factor, Vitamin B12 is destroyed by the HCl of the stomach. - Vit B12 is a normal requirement for the normal erythropoiesis (synchronous maturation, nucleus and cytoplasm, if not there will be a disorder. megaloblastic anemia or pernicious anemia) 1. Malabsorption 2. Intrinsic factor 3. Worms (parasites) C. **THEORIES OF BLOOD CELL PRODUCTION** 1. **Monophyletic/Unitarian** 2. **Polyphyletic/Polytarian** The first recognizable and most primitive blood cells are already committed to develop into a specific cell line. D. **STEM CELLS AND PROGENITORS** **Marrow hematopoiesis is divided into three major compartments or cell types:** 1. **Hematopoietic Stem Cells (HSC)** - CFU-S (Colony forming Unit - Spleen) - Earliest cells/primitive cells - Give rise to multipotent progenitors 1. Differentiate to divide by mitosis to give rise to two multipotential pathways. 2. Pluripotent/multipotent/totipotent- capable to become many cell types. 3. Have the morphology of blasts. 4. Express CD34 but lacks MHC Class II antigen (HLA-DR) 2. **Multipotent Progenitors** - - - - 3. **Lineage** - **morphologically recognizable, lineage-specific precursor cells** - Commited and Restricted precursor - CD34 -- use serum containing CD, recognized due to additional expression of antigens. E. **GENERAL CHARACTERISTICS OF BLASTS** 1. **Size** -- generally large with high nuclei and cytoplasmic ratio 2. **Cytoplasm** -- basophilic (RNA), small in amount, no granules 3. **Nucleus** -- large and round to oval in shape **F. HEMATOPOIESIS** - Pluripotent stem cell is the first in a sequence of steps of hematopoietic cell generation and maturation - **multipotential hematopoietic stem cell:** progenitor of all blood cells - Stem cells have the capacity for self-renewal as well as proliferation and differentiation into progenitor cells - Hematopoietic cells can be divided into three phases according to cell maturity: - Primitive, multipotential cells. The most immature group capable of self-renewal and differentiation into all blood cell lines. - Intermediate cells. This group consists of committed progenitor cells destined to develop into distinct cell lines. - Mature cells. The most developed group with specific functions. - The multipotential stem cell is the progenitor of two major ancestral cell lines: - Lymphocytic - The lymphoid stem cell is the precursor of either mature T cells or B cells/plasma cells - Nonlymphocytic cells. - The nonlymphocytic (myeloid) stem cell progresses to the progenitor colony-forming unit, granulocyte-erythrocyte-monocyte-megakaryocyte (CFUGEMM) - The unit colony of CFU-GEMM leads to the development of distinct subsets of committed progenitor cells. - **CFU-GEMM: expresses CD34 and CD33** - CFU-granulocyte macrophage/monocyte (CFU-GM) - CFU-eosinophil (CFU-Eo) - CFU-basophil (CFU-B) - CFU-megakaryocyte (CFU-MegE) - Burst-forming unit-erythroid (BFU-E) - CFU-E - CFU-Meg - These cells then give rise to more differentiated progenitors, comprising those committed to two lineages that include T cells and natural killer cells (TNKs), granulocytes and macrophages (GMs), and megakaryocytes and erythroid cells (MEPs) - Ultimately, these cells give rise to unilineage committed progenitors for B cells (BCPs), NK cells (NKPs), T cells (TCPs), granulocytes (GPs), monocytes (MPs), erythrocytes (EPs), and megakaryocytes (MkPs) - The formation and development of mature blood cells from the bone marrow multipotential stem cell is controlled by growth factors and inhibitors as well as the microenvironment. Cytokines and growth factors that support the survival, proliferation, or differentiation of each type of cell are shown in *red*  **\ ** **G. Cytokines and Growth Factors** - - - - - - - - \ **THE INDIVIDUAL CELLS OF HEMATOPOIESIS** **A. Erythrocytic Series** - Erythrokinetics -- production, maturation, and death - Erythron -- complete/entire set or population of mature erythrocytes and their precursors in the blood and n=bone marrow. - From BFU-E to erythrocyte 3 phases in RBC/erythrocyte life cycle: - Production -- in bone marrow(adult erythropoiesis) - Release from marrow to circulation - Destruction \*Production, maturation and destruction are related to activities of macrophages, which engulf senescent/old RBCs. 3 phases of erythrocyte life: production, release from marrow to circulation, destruction \*Erythroblastic Islands -- consist of 1 iron rich macrophage serving as the nurse cell that supplies developing normoblast. Maturation occurs for approximately 5 days while destruction takes place after a life span of 120 days/3months.both processes are linked to the activity of macrophages. \*1% of matured erythrocyte -- normal rate of destruction (senescent RBCs) \*10-15% of precursor cells in bone marrow(destruction per day) \*Production Maturation (5days) Release **MATURATION OF ERYTHROCYTES** - **BFU-E** -- youngest lineage committed/restricted \- confined in the bone marrow - **CFU-E** In the presence of EPO (lineage-specific glycoprotein produced in the *renal* *peritubular interstitial cells)* - **Rubriblast/Pronormoblast/Proerythroblast**  **-** Blast of erythrocytic series \- Earliest/youngest morphologically recognizable cell/precursor \- divides to give rise to 2 prorubricyte \- Size: 12-20um \- N/C ratio: 8:1 \- Nucleoli: 1-2 \- Nucleus: 80% of the cell; fine chromatin; 1-2 nucleoli; has largest nucleus \- Cytoplasm: intensely basophilic (concentration of ribosomes and RNA); non-granular; thin rimmed cytoplasm; (The Golgi complex may be visible next to the nucleus as a pale, unstained area) -The pronormoblast begins to accumulate the components necessary for hemoglobin production. - **Prorubricyte/Basophilic normoblast/ Basophilic erythroblast** **-** Cytoplasm is more intensely basophilic than the cytoplasm of pronormoblast (Abundance of RNA) **-** divides to give rise to 4 rubricyte **-** Size: 12-17um \- N/C ratio: 6:1 \- Nucleus: 75% of the cell; chromatin slightly coarse; Nucleoli not seen/not usually visible but present \- Cytoplasm: more deeply basophilic than blast; non-granular \- Detectable hemoglobin synthesis occurs - **Rubricyte/Polychromatophilic normoblast/ Polychromatophilic erythroblast** **-** hemoglobinization of cell starts **-** start of RNA/basophilia decrease (mixture of blue and pink) **-** Last mitotic stage/last stage capable of mitosis **-** Size: 10-15um \- N/C ratio: 4:1 to 1:1 \- Nucleus: round and eccentric; chromatin very coarse and condensed or clumpy \- Cytoplasm: Blue-gray or pinkish-gray; polychromasia is due to blue RNA mixed with red HB. \- Hemoglobin synthesis increases - **Metarubricyte/Orthochromatic normoblast/ Orthochromatic erythroblast\\** **-** last nucleated stage **-** 1^st^ non-mitotic stage (because of the condensation of the chromatin.) **-** don't divide but differentiates/matures into reticulocyte (nucleus is extruded) **-** Size: 7-12um \- N/C ratio: 1:2 -Nucleus: small, pyknotic (eventually extruded) \- Cytoplasm: Pink-orange due (due to nearly complete hemoglobin production)( The residual ribosomes and RNA react with the basic component of the stain and contribute a slightly bluish hue to the cell) - **Reticulocyte/Polychromatophilic erythrocyte** **-** formed in bone marrow and stay as retic (1-2 days in bm) and will be released into the blood and remain as retic for 1 day. After a day, it becomes mature erythrocyte. (2-4 days as a retic) **-** \#of retic corresponds to amount of loss of destruction **-** retic count -- measure of bone marrow production of RBC or measure of effective erythropoiesis **-** Size: 7-10um \- N/C ratio: \-- -Nucleus: anucleated young erythrocyte that has extruded nucleus, but there's still remnants of DNA in the form of masses of ribosomes that appear reticular after supravital stain (blue reticulum) (By the end of the polychromatic erythrocyte stage, *the cell is the* *same color as a mature RBC, salmon pink.* It remains *larger than a mature cell,* however) - During the first several days after exiting the marrow, the polychromatic erythrocyte is retained in the spleen for pitting of inclusions and membrane polishing by splenic macrophages, which results in the biconcave discoid mature RBC - **Mature erythrocyte** **-** Size: 6-8um \- N/C ratio: \-- -Nucleus: no nucleus \- Cytoplasm: salmon pink with central pale area \- biconcave disc \- remain active in circulation for 120 days after which is removed by spleen \- delivers oxygen to tissues, releases it, and returns to the lung to be reoxygenated \- contains hemoglobin, the oxygen carrying component \- The cell's main function of oxygen delivery throughout the body requires a membrane that is flexible and deformable, that is, able to flex but return to its original shape. Deformability is crucial for RBCs to enter and subsequently remain in the circulation. **\ ** **B. Granulocytic Series** **Physiologic compartments of developing leukocytes:** - Proliferating of mitotic pool -- myeloblast, promyelocyte and myelocyte - Maturation and storage pool -- metamyelocyte, bands/segmenters, mature granulocytes - Circulating pool - 50% granulocytes circulating in blood - Marginating pool --50% closely aligned to walls of BV that undergo diapedesis when there is infection - **CFU-GM** - multipotent progenitor - **CFU-G** -- earliest lineage committed cell of granulocytic series - **Myeloblast** **-** Earliest recognizable cell **-** can be mistaken for a large lymphocyte (chromatin pattern and nuclear membrane to differentiate) **-** **Size**: 15-20um \- **N/C ratio:** 4:1 \- **Nucleus:** Large; round, oval or quadrangular; very fine, uniform chromatin pattern; delicate nuclear membrane; 2-5 distinct nucleoli \- **Cytoplasm:** Pale, clear blue, and without visible granules (Type 1 blast have no visible granules, Type II blasts have few primary granules) - **Promyelocyte** \- stage where granules start to appear **-** **Size**: 15-21um \- **N/C ratio:** 3-2:1 \- **Nucleus:** Round or oval and eccentric; Chromatin slightly coarse; 2-3 nucleoli; large golgi complex pushes the nucleus to the periphery \- **Cytoplasm:** Rich in RNA (basophilic); contains primary or non-specific or azurophilic granules (blue to reddish-purple staining property) - **Myelocyte** - Last stage capable of mitosis - Earliest recognizable stage for eosinophils and basophils - Size: 12-18um - N/C ratio: 2-1:1 - Nucleus: Fine, Dispersed chromatin (early); more condensed chromatin pattern (late); nucleoli not usually seen - Cytoplasm: Secondary or specific granules start to appear; few azurophilic granules remain - **Metamyelocyte** - Maturity of cell: nuclear segmentation Size: 12-18um - Size: 10-15um - N/C ratio: 1:1 - Nucleus: Indented or kidney-shaped (indentation is \ - **Band/Stab** - Indentation is \1/2 width of nucleus but no fine thread filaments between the lobe - Size: 9-15um - N/C ratio: 1:1 - Nucleus: Elongated or curved or band-shaped without a thread-like filament; chromatin coarse and clumped - Cytoplasm: Moderate to abundant amount giving a low N/C ratio; full complement of specific granules - **Mature Granulocytes/ Mature Segmenter/ Polymorphonuclear** (the more lobes, the more mature it is) a. **Neutrophils** - Size: 9-15um - N/C ratio: 1:1 - Nucleus: Segmented with 2-5 lobes and a fine thread filament between lobes; chromatin coarse and clumped - Cytoplasm: Contains bud-shaped lilac pink granules (barely visible but give the cytoplasm a pink/violet color) b. **Eosinophils** - Size: 9-15um - N/C ratio: 1:1 - Nucleus: Usually with 2 lobes; chromatin coarse and clumped - Cytoplasm: Pink and contains large corpuscular (or globular), orange-red granules c. **Basophils** - Size: 10-16um - N/C ratio: 1:1 - Nucleus: Generally unsegmented or bilobed; indistinct covered with large granules; condensed but smudged chromatin - Cytoplasm: Colorless to rose or lavender colored and contains corpuscular, dark purple to blue-black granules that are water-soluble **\ ** **C. MONOCYTIC SERIES** - **CFU-GM --** multipotent progenitor - **CFU-M** - **Monoblast** - Not the earliest recognizable cell because it is indistinguishable from myeloblast of granulocytic series. Differentiate by using morphologic criterion; 3 tests: peroxidase, specific esterase, and Sudan: Mono: (-), Myelo (+) - Size: 12-20um - N/C ratio: 4-3:1 - Nucleus: Ovoid or round; light blue-purple in color; fine, lacey chromatin; 1-2 nucleoli - Cytoplasm: Moderately basophilic to blue-gray; non-granular - **Promonocyte** - Earliest morphologically recognizable cell - Size: 12-20um - N/C ratio: 3-2:1 - Nucleus: Oval; may have single fold or fissure; fine chromatin; 1-5 nucleoli - Cytoplasm: Blue-gray; may contain fine, dust-like azurophilic granules (may/may not be seen); vacuoles variable - **Monocyte** - Largest cell in the blood - Have a relatively low count in blood because they migrate in tissues and become macrophage - Size: 12-18um - N/C ratio: 2-1:1 - Nucleus: Round, kidney-shaped or may show slight lobulation or brain-like convolutions; chromatin not clumped; no nucleoli are visible (linear chromatin pattern that does not show clumping) - Cytoplasm: Abundant; blue-gray; contains many azurophilic granules giving a ground glass appearance; pseudopods and vacuoles common **MACROPHAGE** - Either fixed or wandering - Fixed: Kupffer cells (liver), Langerhans cell, microglial, alveolar - Wandering: can go into circulation - Phagocytose organism or senescent - Size: 18-50um - N/C ratio: 1:2-4 - Nucleus: Eccentric; egg yolk shaped; spongy chromatin pattern - Cytoplasm: Contains coarse azure granules; vacuolated and may contain ingested materials; pseudopods also common; royal blue in color **D. LYMPHOCYTIC SERIES** - Primary Lymphoid organs - bone marrow and thymus - responsible for continuous antigen independent lymphopoiesis to replenish what is continually loss - Secondary Lymphoid organs - lymph nodes, spleen, tonsils, Peyer's patches, and various lymphatic aggregates - Antigen-dependent - Only take part in lymphopoiesis if there's invading foreign body - **Lymphoid Progenitor** - **Lymphoblast** - Size: 10-18um - N/C ratio: 4:1 - Nucleus: Round or oval; chromatin pattern coarse; 1-2 nucleoli; no clumping yet - Cytoplasm: Smooth, no granules present; moderate to dark-blue - **Prolymphocyte** - Size: 10-18um - N/C ratio: 4:1 - Nucleus: Round, oval, or slightly indented; chromatin more clumped; may contain 1-2 nucleoli - Cytoplasm: Moderate to dark-blue (intensely basophilic); Usually nongranular but may contain azurophilic granules - **Lymphocyte** - Size: 8-16um - N/C ratio: 4-2:1 - Nucleus: Round or oval; may be slightly indented; chromatin pattern dense and clumped; no nucleoli - Cytoplasm: Pale or moderate to dark-blue; May or may not contain a few azurophilic granules **Antigen-dependent Maturation Series (not seen in bone marrow)** - **Mature B-cell** - **Plasmablast** - Size: 18-25um - N/C ratio: 2:1 - Nucleus: Eccentric; nuclear chromatin is more clumped; multiple nucleoli may or may not vbe visible - Cytoplasm: Abundant; basophilic; nongranular - **Proplasmacyte** - Size: 15-25um - N/C ratio: 1:1 - Nucleus: Eccentric; chromatin is coarser and densely stained - Cytoplasm: More abundant; intensely basophilic; hof or perinuclear are often seen - **Plasma Cell** - Size: 8-20um - N/C ratio: 1:1 - Nucleus: Eccentric; condensed and coarse chromatin; no nucleoli visible - Cytoplasm: Deeply basophilic; large-well-defined hof next to nucleus and well-developed rough ER **E. MEGAKARYOCYTIC SERIES** - **CFU-Meg** - **Megakaryoblast** - Size: 20-50um - N/C ratio: 10:1 - Nucleus: Fine chromatin; multiple nucleoli that generally stain blue - Cytoplasm: Small to moderate amount; blue (darker than myeloblast); nongranular - **Promegakaryocyte** - Size: 20-50um - N/C ratio: 4-7:1 - Nucleus: Irregularly shaped; may show slight lobulation; coarse chromatin; multiple nucleoli visible - Cytoplasm: More abundant but less basophilic; granules begin to from; Demarcation Membrane System (DMS) develops.---gives rise to membrane system/compartments of platelets - **Granular Megakaryocyte** - Size: 30-90um - N/C ratio: 1-2:1 - Nucleus: Multiple nuclei or multilobulated nucleus; coarser chromatin; no nucleoli - Cytoplasm: Abundant; pinkish-blue; diffusely granular and has an irregular peripheral border - **Mature Megakaryocyte/Metamegakaryocyte** - Size: 40-120um - N/C ratio: 1:1 - Nucleus: Multiple nuclei or multilobulated nucleus; no nucleoli are visible - Cytoplasm: Contains coarse clumps of granules aggregating into bundles which bud off from the periphery to become platelets (2000-4000 platelets) - **Megakaryocyte** -- largest cell in the bone marrow, exemption to the rule that when a cell matures it gets smaller. - **Thrombocytes (cytoplasmic fragments)** - Size: 1-4um - N/C ratio: \-- - Nucleus: \-- - Cytoplasm:Light blue to purple; very granular; consists of 2 parts: chromomere and hyalomere \* Outer hyalomere: nongranular pale blue that surrounds the centromere \*Inner chromomere