Blood & Hematopoiesis by dr. EHAB MOSAAD

1. Blood & Hematopoiesisbydr. EHAB MOSAAD

2. Blood Specialized connective tissue Composition 1. Plasma 2. Cells A. Erythrocytes B. Leukocytes i. Agranulocytes ii. Granulocytes 3. Platelets

3. Blood Functions 1. Transport A. Gases (e.g., O2, CO2) B. Nutrients and waste products C. Regulatory molecules (e.g., hormones) 2. Homeostasis A. Thermoregulation 3. Protection A. Vascular integrity (e.g., coagulation) B. Immune cells and molecules (e.g., IgGs)

4. Plasma

5. Blood Cells and Platelets RBCs - constitute >99% of blood cells by number Hematocrit - packed RBC volume (% of blood volume) Lifespan - Neutrophils, only 12-14 hours Platelets, ~10 days RBCs, ~4 months (~120 days) Lymphocytes, many years

6. Erythrocytes • Transports O2 (bound to hemoglobin) • Converts CO2 into HCO3- (transported in plasma) • Biconcave disk-shape, deformable, resilient, no organelles • Plasma membrane and spectrin cytoskeleton present • Proteins - mainly hemoglobin (a2b2) and glycolytic enzymes • Quality control - spleen removes old or damaged RBCs

7. Leukocytes Granulocytes Neutrophil (E) Band Neutro. (I) Eosinophil (D) Basophil (J) Agranulocytes Monocyte (F) Lymphocyte (H) Platelets (G)

8. Leukocytes Size - larger than erythrocytes (10 - 18 µm diameter) All - contain non-specific azurophilic (primary) granules that are equivalent to lysosomes Granulocytes - contain specific (secondary) granules that contain specific enzymes for each cell type Agranulocytes - no specific granules present

9. Neutrophil • Most common leukocyte - 49-67% of leukocytes • Azurophilic granules - NADPH oxidase, superoxide dismutase, myeloperoxidase (form bactericidal ions O2-, H2O2, HOCl) • Specific granules (neutral staining) - alkaline phosphatase, lysozyme, phospholipase, and other enzymes • Functions - migration to sites of inflammation, phagocytosis of bacteria, exocytosis of enzymes that attack microorganisms, results in formation of pus (dead neutrophils and cell debris)

10. Eosinophil • Intermediate abundance - 2-5% of leukocytes • Azurophilic granules - lysosomal acid hydrolases • Specific granules (eosinophilic) - eosinophilic cationic protein, major basic protein, eosinophilic peroxidase, histaminase • Functions - migration to sites of infection and release of cytotoxins that combat parasites (protozoa, helminths), migration to sites of allergic inflammation, phagocytosis and endocytosis of antigen-antibody complexes

11. Basophil • Least common leukocyte - <1% of leukocytes • Azurophilic granules - lysosomal acid hydrolases • Specific granules (basophilic) - histamine, heparin, heparan sulfate, chemotactic factors for other granulocytes • Functions - similar to mast cells, upon stimulation it releases preformed and newly synthesized secretory products that initiate, maintain, and control inflammation

12. Monocyte • Largest cell in peripheral blood (18 µm diameter) • Agranulocyte - lysosomal acid hydrolases, myeloperoxidase • Circulates 12 to 96 hours, then enters CT and differentiates • Derivatives - macrophage (Kupffer cell in liver, alveolar macrophage in lung), osteoclast in bone marrow, Langerhans cell in skin (dendritic cell)

13. Lymphocyte • B lymphocyte - humoral immunity (~20-30%) • T lymphocyte - cell-mediated immunity (~60-80%) • Natural killer (NK) cell - cell-mediated immunity (~5-10%) • Agranulocyte - lysosomal acid hydrolases

14. Platelets • Cell fragment - small (~2 µm), irregular disk shape, no nucleus • Structure - granules, mitochondria, glycogen, cytoskeleton • Granules - alpha (clotting factors), dense core (histamine) • Megakaryocyte - produces platelets in bone marrow sinus • Function - clot formation, bind to injured blood vessel wall, aggregate with RBCs forms hemostatic plug (stops bleeding)

15. Hematopoiesis • Yolk sac phase - blood in 'islands' in yolk sac, no leukocytes • Hepatic phase - primitive nucleated RBCs, fetal hemoglobin • Bone marrow phase - mature RBCs, leukocytes, ~12 weeks • Adult phase - pelvis, vertebrae, skull, ribs, ends of long bones

16. Hematopoiesis PPSC

17. Stem Cell Basics • Stem cell - asymmetric cell division yields cells with different fates (one is stem cell; one is transient amplifying cell) • Self-renewal - capacity of stem cell to regenerate itself • Transient amplifying cells - symmetric cell division yields daughter cells with same fate (transient amplifying cells) • Differentiated cells - cells exit cell cycle and differentiate

18. Hematopoietic Stem Cells Pluripotent hematopoietic stem cell (HSC)Undifferentiated cell producing blood cells of all lineages, capable of self-renewal Multipotent HSCUndifferentiated cell producing cells of multiple lineages, limited self-renewal (e.g., myeloid SC, lymphoid SC) Committed progenitor - undifferentiated cell capable of producing cells of one lineage, colony forming units (CFUs)(e.g., erythroid CFU, granulocyte-macrophage CFU)

19. Stages in Erythropoiesis Early Intermediate Late Proerythroblast Polychromatophilic erythroblast Reticulocyte Basophilic erythroblast Orthochromatic erythroblast (normoblast) Erythrocyte

20. Erythropoiesis

21. Leukopoiesis - Myeloid Lineage Band Stage

22. 1. Promyelocyte 3. Metamyelocyte 2. Myelocyte 4. Band Form Myeloid Lineage (Neutrophilic)

23. Myeloid Lineage

24. Pluripotent stem cell Leukopoiesis - Lymphoid Lineage • Lymphoid stem cell gives rise to T-lymphocyte and B-lymphocyte lineages • T-cell maturation - thymus • B-cell maturation - bone marrow • Plasma cells - present in marrow, lymphatic tissue, connective tissue

25. Hematopoietic Growth Factors Erythropoietin (EPO) - major regulator of erythropoiesis, stimulates erythroid CFU cells and proerythroblasts Thrombopoietin (TPO) - increases platelet production, stimulates megakaryocyte CFU cells Granulocyte CSF (G-CSF) - increases production of neutrophils, stimulates granulocyte-macrophage CFU cells Granulocyte-macrophage CSF (GM-CSF) - increases macrophage production, stimulates granulocyte-macrophage CFU cells Interleukins - stimulate B- and T-cell formation, function together with G-CSF and GM-CSF

26. Bone Marrow • Bone marrow cavity - marrow proper and venous sinuses • Bone marrow cells - stromal cells, adipocytes, endothelial cells, macrophages, hematopoietic cells • Blood vessels - nutrient arteries supply marrow cavity • Stem cells and early precursor cells do not leave marrow

27. Bone Marrow

28. Anemia Reduced RBC ProductionFactor Deficiency Iron - required for heme synthesis Vitamin B12 - required for DNA synthesis Folate - required for DNA synthesis Dietary limitations - most common cause Hematopoietic Cell Damage Drugs Radiation Infections Toxins Increased RBC Destruction HereditaryHemolytic Anemias Blood Loss

29. Megaloblastic Anemia Defect in hematopoiesis Peripheral blood - macroovalocytes (large oval RBCs) Bone marrow - megaloblasts Anemia - autohemolysis of megaloblasts, short life span of macroovalocytes Causes - usually deficiency of folic acid or vitamin B-12, which are require for DNA synthesis during cell division Other causes - leukemia, myelofibrosis, multiple myeloma, hereditary disorders, chemotherapy drugs (that affect DNA synthesis, e.g., methotrexate), alcohol

30. Megaloblastic Anemia Peripheral blood - macroovalocytes and hypersegmented neutrophil

31. Megaloblastic Anemia Normal erythroids in bone marrow Megaloblasts in bone marrow

32. Leukemia • Proliferation of leukocytes from hemopoietic tissue • Four major forms Acute Myelogenous Leukemia (AML) Acute Lymphocytic Leukemia (ALL) Chronic Myelogenous Leukemia (CML) Chronic Lymphocytic Leukemia (CLL) • Myelogenous - proliferation of cells of myeloid lineage • Lymphocytic - proliferation of cells of lymphoid lineage • Acute - rapidly progressing, cells are immature and do not carry out their normal functions • Chronic - progresses slowly, more developed cells that carry out normal functions • Blood test - measure leukocyte alkaline phosphatase (LAP)

33. White blood cell growth factors(G/GM-CSF)A practice guideline

34. Precautions of CSF • Adult respiratory distress syndrome, specifically in neutropenic patients with sepsis • Allergic-type reactions • Avoid the simultaneous use of chemotherapy and radiation therapy with filgrastim; DO NOT administer within 24 hours before or after cytotoxic chemotherapy • Possible growth stimulation of tumors • Leukocytosis (monitoring twice weekly recommended) Micromedex

35. Precautions of CSF • Myelodysplastic syndrome or acute myeloid leukemia development when used in patients with congenital neutropenia • PBPC mobilization (data inconclusive) • Risk of development of myelodysplastic syndrome or acute myeloid leukemia when used in patients with congenital neutropenia • Sickle cell disease, severe sickle cell crisis (some resulting in death) • Splenic rupture, some fatal Micromedex

36. Adverse effect of CSF • COMMON • Gastrointestinal: Nausea and vomiting • Musculoskeletal: Bone pain • Other: Influenza-like illness • SERIOUS • Dermatologic: Vasculitis of the skin (rare) • Hematologic: Hemoglobin S disease with crisis • Respiratory: Acute respiratory distress syndrome • Other: Rupture of spleen (rare) Micromedex

37. How to Practice?

38. Goal of G-CSF • Overall survival • Response rate • Treatment related mortality • Antibiotics course and hospital days • WBC count • Placebo for the patient, family, and doctors

39. Clinical Situations • Primary prophylaxis • Secondary prophylaxis • Therapy of patient with neutropenia • CSF to increase chemotherapy dose density and intensity • In older patients • Other situations

40. Primary prophylaxis • Primary prophylaxis is recommended for the prevention of febrile neutropenia(FN) in patients who have a high riskof FN based on age, medical history, disease characteristics and myelotoxicity of chemotherapy regimen. • Special circumstances: relative nonmylelo-suppressive chemotherapy but who have potential risk factors for febrile neutropenia or infection because of bone marrow compromise or comorbidity. JCO 2006;24:3187-3205

41. In 2000 guideline, CSF is recommended in FN risk > 40% as primary prophylaxis. • Adjust the risk to >20% in 2005 update guideline • CSF reduced incidence of FN(1% vs 17%) and hospitalization for FN(1%vs 14%) for breast cancer patient receiving Docetaxel 100mg/m2 q3wk • Meta-analysis in lymphoma showed CSF significantly reduced the relative risk of severe neutropenia (RR 0.67), febrile neutropenia (RR 0.74), and infection (RR 0.74). No benefit on the patients requiring intravenous antibiotics, infection-related mortality, complete tumor response, freedom from treatment failure, or overall survival JCO 2006;24:3187-3205

43. JCO 2006;24:3187-3205

45. CSF reduce the incidence of FN when the rate is approximately 20%. • When available, alternative regimens offering equivalent efficacy, but not requiring CSF support, should be utilized. • Adjustment according to patient’s co-morbidity and general conditions, which make the patient unlikely to be enrolled in clinical trials. JCO 2006;24:3187-3205

46. Secondary prophylaxis • Recommended for patients who experienced a neutropenic complication from a prior cycle of chemotherapy, IN which a reduced dose may compromise disease-free or overall survival or treatment outcome. JCO 2006;24:3187-3205

47. Re-evaluate the goal of chemotherapy. Dose reduction or delay remains an appropriate strategy for the palliative treatment of cancer. • No definite conclusion can be drawn regarding the benefits of secondary prophylaxis on survival, quality of life, or cost. • In breast cancer adjuvant therapy, G-CSF recipients experienced fewer episodes of hospitalization for FN and greater dose-intensity compared to historical controls. JCO 2006;24:3187-3205

48. Patient with neutropenia • Afebrile neutropenia Should NOT be routinely used for patients with neutropenia who are afebrile. • Febrile neutropenia Should NOT be routinely use as adjunctive treatment with antibiotic therapy for patients with fever and neutropenia. JCO 2006;24:3187-3205

49. Febrile neutropenia • Randomized control trial: G-CSF recipients had a shorter period of grade 4 neutropenia (2 vs 3 days), antibiotic therapy (5 vs 6 days), and hospital stay (5 vs 7 days). No survival benefit. • Meta-analysis: CSF recipients had less prolonged neutropenia, less prolonged hospitalization, marginally less infection-related mortality, no significant difference in overall mortality.

50. Febrile neutropenia • CSF is recommended in patient with high-risk feature. • Expected prolonged (10 days) and profound (100/ml) neutropenia, age greater than 65 years, uncontrolled primary disease, pneumonia, hypotension and multi-organ dysfunction (sepsis syndrome), invasive fungal infection, or being hospitalized at the time of the development of fever.