2020+myelopoiesis.pptx

MYELOPOIESIS LEUKOCYTE DEVELOPMENT, KINETICS, AND FUNCTIONS Chap 9 Objectives 1. Describe the pathways and progenitor cells involved in the derivation of leukocytes from the hematopoietic stem cell to mature forms. 2. Name the different stages of neutrophil, eosinop...

MYELOPOIESIS LEUKOCYTE DEVELOPMENT, KINETICS, AND FUNCTIONS Chap 9 Objectives 1. Describe the pathways and progenitor cells involved in the derivation of leukocytes from the hematopoietic stem cell to mature forms. 2. Name the different stages of neutrophil, eosinophil, and basophil development and describe the morphology of each stage. 3. Differentiate the different stages of the series from each other 4. Discuss the important functions of neutrophils, eosinophils, and basophils. 2 4  Leukocytes Leukocyte classifications ◦ Granulocytes - filled with granules with differing staining characteristics and whose nuclei are segmented or lobulated ◦ Eosinophils - filled with granules whose nuclei are segmented or lobulated and stain with the acid stains such as eosins ◦ Basophils - granules that are acidic and stain with basic stains such as methylene blue; 5 Segmented polymorphonuclear neutrophils (PMNs) Segmented polymorphonuclear neutrophils (PMNs) – ◦ granules that react with both acid and basic stains, which gives them a pink to lavender color and nuclear segmentation 6 Mononuclear cells ◦ Mononuclear cells – non segmented nucleus ◦ Lymphocytes ◦ Monocytes 7 FUNCTION Normal range: 4.5 × 109/L to 11.5 × 109/L for adults. Mediate immunity, either innate (nonspecific), as in phagocytosis by neutrophils, or specific (adaptive), as in the production of antibodies by lymphocytes and plasma cells. 8  Hematopoiesis Neutrophils share a common progenitor with monocytes, known as the granulocyte-monocyte progenitor (GMP). 9  Neutrophil Development Cells that are dividing and includes (listed in the order of maturation) Cells with nucleus maturating marrow reserve and are available for release: metamyelocytes, band neutrophils, and segmented neutrophils. Proliferation pool - Common myeloid progenitors (CMPs), also known as colony-forming units–granulocyte, erythrocyte, monocyte, and megakaryocyte (CFU-GEMMs); granulocyte-macrophage progenitors (GMPs);myeloblasts; promyelocytes; and myelocytes. Recognized by surface antigens using flow cytometry. Stem Pool - Hematopoietic stem cells (HSCs) that are capable of self-renewal and differentiation. 10 HSCs, CMPs, and GMPs are not distinguishable with the light microscope and Romanowsky staining major cytokine responsible for the stimulation of neutrophil production is granulocyte colony- stimulating factor, or G- CSF.2, 3 11 Cytokines and Growth Factors  Diverse group of soluble proteins that modulate functional activities of cells.  Some are growth factors and terms are often used interchangeably.  Most are glycoproteins  Responsible for stimulation or inhibition of production, differentiation, or trafficking of mature blood cells or their precursors. 12 Cytokines Examples Interleukins (ILs)  Lymphokines  Monokines  Interferons  Chemokines  Colony-stimulating factors (CSFs) 13 Effect of cytokines on stem cells Some influence stem cells with multilineage potential Some influence differentiation of specific cells types Some work only in combination with other factors Some prevent apoptosis 14 Colony Stimulating Factors Produced by many different cells Have high specificity Active at low concentrations Named for cell lines that respond to their presence GM-csf Meg-csf G-csf, etc 15  Neutrophil Development Cells that are dividing and includes (listed in the order of maturation) Recognized by surface antigens using flow cytometry not with light microscope and Romanowsky stain Hematopoietic stem cells (HSCs) that are capable of self-renewal and differentiation. 18 19  Myeloblast ◦ Size - 14 to 20 μm in diameter N:C ratio – 8:1 to 4:1 Incidence - 0% to 3% of the nucleated cells in the bone marrow ◦ Peripheral Blood – 0% ◦ Nucleus: ◦ round to oval ◦ Nucleoli: 2-5 ◦ Chromatin-fine. ◦ Cytoplasm moderate basophilia, granules – no granules in type I; up to 20 in type II myeloblast Type III myeloblasts have a darker chromatin and a more purple cytoplasm, and they contain more than 20 granules that do not obscure the nucleus. 20 Myeloblast vs Pronormoblast 21 Myeloblast vs Normoblast 22 Myeloblast 23  Promyelocyte ◦ 1% to 5% of the nucleated cells in the bone marrow ◦ Size 16 to 25 μm in diameter ◦ Nuclear features ◦ Shape round to oval - eccentric ◦ paranuclear halo or “hof” (not in the malignant promyelocytes) ◦ nucleus is similar to that described earlier for myeloblasts ◦ chromatin clumping (heterochromatin) may be visible especially around the edges ◦ Nucleoli 1-3 ◦ N:C ratio 3:1 24  Promyelocyte ◦ Larger than the myeloblast cells and measure 16 to 25 μm in diameter ◦ Cytoplasmic features ◦ Granules - evenly basophilic and full of primary (azurophilic) granules (red to purple) ◦ first in a series of granules to be produced during neutrophil maturation ◦ Incidence - 1% to 5% of the nucleated cells in the bone marrow ◦ Peripheral – 0% 25 Promyelocyte 26 Myeloblast vs. Promyelocyte 27  Granules  Primary granules - 1nonspecific, first to appear not unique to any cell type  Formed during the promyelocyte stage  Last to be released (exocytosis)  Contain: Myeloperoxidase Acid β-glycerophosphatase Cathepsins Defensins Elastase Proteinase-3 Others  Secondary - 2- specific – identifies the cell as neutrophilic starts near the golgi  Third to be released  Dawn of neutrophilia  Granules spread later to all the cell  Formed during myelocyte and metamyelocyte stages  β2-Microglobulin Collagenase Gelatinase Lactoferrin Neutrophil gelatinase-associated lipocalin Transcobalamin I Others 28 Granules  Tertiary granules - 3  Formed during metamyelocyte and band stages  Second to be released  Contain: Gelatinase Collagenase Lysozyme Acetyltransferase β2-Microglobulin  Secretory granules (secretory vesicles)  Formed during band and segmented neutrophil stages  First to be released (fuse to plasma membrane)  Contain (attached to membrane): CD11b/CD18 Alkaline phosphatase Vesicle-associated membrane-2 CD10, CD13, CD14, CD16 Cytochrome b558 Complement 1q receptor Complement receptor-1 29  Myelocyte Myelocyte - final stage in which cell division (mitosis) occurs. ◦ Early myelocytes may look very similar to the promyelocytes ◦ except that patches of grainy pale pink cytoplasm representing secondary granules begin to be evident in the area of the Golgi apparatus - dawn of neutrophilia. Dawn of Neutrophilia 30 Myelocyte ◦Cytoplasmic features ◦ Secondary neutrophilic granules slowly spread through the cell until its cytoplasm is more lavender-pink than blue ◦ primary granules decreased, and the cell begins to manufacture secondary (specific) neutrophil granules. ◦ Incidence ◦ 6% to 17% of the nucleated cells in the bone marrow 31 Myelocyte The number of primary granules per cell is decreased, and their membrane chemistry changes so that they are much less visible. Late myelocytes are somewhat smaller than promyelocytes (15 to 18 μm) nucleus has considerably more heterochromatin. Nucleoli are difficult to see by light microscopy 32 Myelocytes 33 Early vs late myelocyte Secondary neutrophilic granules slowly spread through the cell until its cytoplasm is more lavender-pink than blue Late myelocytes are somewhat smaller than promyelocytes (15 to 18 μm), and the nucleus has considerably more heterochromatin. 34 Promyelocyte vs. Myelocyte 35  Metamyelocyte ◦Nuclear features - shape of the nucleus. ◦ The nucleus is indented (kidney bean shaped or peanut shaped), and the chromatin is increasingly clumped. o indentation is less than 50% of the width of the hypothetical round nucleus; ◦ Nucleoli are absent 36  Metamyelocyte ◦Cytoplasmic features o pale blue to pink with few primary granules and many (full complement) secondary granules. ◦ Granules Synthesis of tertiary granules (also known as gelatinase granules) may begin during this stage ◦ N:C ratio 1.5:1 ◦Incidence 3% to 20% of nucleated marrow cells. ◦ Peripheral Blood 0% ◦ cells are no longer capable of division 37 Metamyelocytes 38 Myelocyte vs. Metamyelocyte 39 Neutrophil Development -Band Band ◦ Nuclear features ◦ highly clumped, ◦ nuclear indentation that began in the metamyelocyte stage now exceeds one half the diameter of the nucleus Indentation more than 50% of “round” nucleus 40  Bands ◦Cytoplasmic features – ◦ All evidence of RNA (cytoplasmic basophilia) is absent. ◦ Granules ◦ Tertiary granules continue to be formed during this stage ◦ Secretory granules (also known as secretory vesicles) may begin to be formed during this stage ◦ Incidence ◦ 9% to 32% of nucleated marrow cells ◦ 0% to 5% of the nucleated peripheral blood cells ◦ Reporting ◦ Clinical and Laboratory Standards Institute (CLSI) recommends that bands should be included within the neutrophil counts and not 41 reported as a separate category Metamyelocyte vs. Band 42 Remember Cellular maturation is a continuum ◦ all cells may not demonstrate these characteristic features especially if they are at the point of moving from one stage to another. ◦ example, you may hear someone refer to a cell as being a late promyelocyte or an early myelocyte. ◦ These references suggest that the cell may have some characteristic features of the stage identified but they may also have features of the cell before or after it on the maturational continuum. When in doubt, call it the more mature cell. 43 Identify the Cells 44 Identify the Cells 45 Neutrophil Development (Cont.) Segmented polymorphonuclear What is the N:C ratio? neutrophils (PMN) ◦ Nuclear features - two and five nuclear lobes connected by threadlike filaments Cytoplasmic features -pale blue to pink with rare primary granules and abundant secondary granules ◦ Granules ◦ Incidence 7% to 30% of nucleated cells in the bone marrow ◦ highest numbers in the peripheral blood of adults (50% to 70% of leukocytes in relative numbers and 2.3 to 8.1 × 109/L in absolute terms). ◦ (check inside cover for reference ranges for pediatrics) 46 Band vs Segmented Neutrophil 47  Band vs Seg - Visible chromatin in nucleus - One indented area with no visible chromatin – more of –Indentation less than ½ the width of the nucleus a filament –Indentation more than ½ the width of the nucleus 48 What are these cell? 49 Neutrophil Kinetics Movement of neutrophils and its precursors between the different pools in the bone marrow, the peripheral blood, and tissues. Neutrophil production has been calculated to be on the order of between 0.9 and 1.0 × 109 cells/kg per day. 50 Neutrophil Kinetics (cont.) Proliferative pool in Bone Marrow capable of division ◦ Cell counts ◦ proliferative pool contains approximately 2.1 × 109 cells/kg, 51 Neutrophil Distribution Periphera l Blood Tissu Bone e Marrow CGP Blasts Promye Meta Band Seg MG Myelo Proliferatin P Storage g 52  Maturation pool Maturation pool in the Bone marrow not capable of division ◦ Maturation pool contains roughly 5.6 × 109 cells/kg or a 5- day supply ◦ Myeloblast through myelocyte has been estimated to be roughly 6 days through the maturation pool is approximately ◦ Transit time 4 to 6 days. ◦ Granulocyte release from the bone marrow is stimulated by G-CSF.2, 3 53 Peripheral blood Peripheral blood – Circulating neutrophil pool (CNP) ◦Marginated neutrophil pool (MNP)- localized to the walls of capillaries in tissues such as the liver, spleen, and lung ◦ Ratio of these two pools is roughly equal overall;4 Marginated neutrophils in the capillaries of the lungs make up a considerably larger portion of peripheral neutrophils ◦Half-life of neutrophils in the blood is relatively short at approximately 7 hours. ◦inflammation and infection tend to prolong the neutrophil’s life span through anti-apoptotic signals, Tissue Pool 54 Neutrophil Functions The major function of neutrophils is phagocytosis and destruction of foreign material and microorganisms.  seeking (chemotaxis, motility, and diapedesis) and  destruction (phagocytosis and digestion). Part of the Innate immune system –  Destruction of foreign organisms not antigen specific  No protection against reexposure to the same pathogen Three Major functions 55 Neutrophil Functions Phagocytosis and destruction of foreign material and microorganisms – ◦ Process involves seeking (chemotaxis, motility, and diapedesis) and destruction (phagocytosis and digestion). Seeking action ◦ Chemotaxis - Chemotactic agents - produced by microorganisms, by damaged cells, or by other leukocytes such as lymphocytes or other phagocytes ◦ Motility involves the rolling along the endothelial cells ◦ Diapedesis - transmigration of neutrophils either between or through endothelial Destruction function – ◦ Phagosome phagocytosis ◦ Digestion 56 Margination High shear stress keeps WBCs on the outer edges of the flow of blood through the vessel 57 Chemotaxis and Leukocyte Adhesion Cascade Chemical mediators THEN Capture  microorganisms,  Rolling  damaged cells  Slow Rolling recruit neutrophils to  Firm Adhesion inflammatory site  Transmigration 58 Capture  Chemoattractant activates endothelial cells  Draws leukocytes to a position closer to the endothelium of the blood vessel 59 Slow Rolling  Leukocyte velocity drops dramatically  Requires expression of E-selectin on endothelium and CD18 integrin in lipid bilayer of WBC 60 Firm Adhesion  CD18 (integrin) on the surface of the WBC is required for firm adhesion to the endothelium  E-selectin is the counterpart on the endothelium 61 Transmigration Process where adhered WBC escapes the interior of the blood vessel through the endothelium 62 Neutrophil Functions (Cont.) Neutrophil extracellular traps (NETs) ◦ Structure - extracellular threadlike structures believed to represent chains of nucleosomes from unfolded nuclear chromatin material (DNA). ◦ Function - able to trap and kill gram-positive and gram- negative bacteria as well as fungi. ◦ NETosis - Neutrophils die as a result of antibacterial activity. Secretory ◦ Transcobalamin I or R binding protein -necessary for the proper absorption of vitamin B12. ◦ Source of Cytokines 63