Teaching slides for Haematopoiesis & approach to anaemia management

1. Haematopoiesis& Approach to Anaemia Dr (Brig) YD Singh MBBS, MD, FIACM, DIT Professor (Internal Medicine) SKN Medical College & Gen Hospital Pune 411 041

2. Haematopoiesis (1) Process by which formed elements of blood Produced & Regulated through series of steps Pluripotent haematopoietic stem cell Capable of producing red cells, All classes of granulocytes, monocytes, platelets Mechanism to become committed to a given lineage Not fully known 24-Sep-11 Dr (Brig) YD Singh

3. Haematopoiesis (2) Following lineage commitment Haematopoietic progenitor & precursor cells Come under regulatory influence of growth factors and hormones. For red cell production Erythropoietin (EPO) is regulatory hormone EPO is required for Maintenance of Committed Erythroid progenitor cells In absence of EPO hormone Undergo programmed cell death (apoptosis) 24-Sep-11 Dr (Brig) YD Singh

4. Factors Regulating RBCProduction 24-Sep-11 Dr (Brig) YD Singh Regulated process of RBC production is Erythropoiesis

5. RBC Characteristics Mature red cell is 8 micron in diameter Anucleate , Biconcave & discoid in shape Extremely pliable Needs to traverse microcirculation easily Membrane integrity maintained by Intracellular generation of ATP Average RBC lives 100–120 day 1% of all circulating RBC daily replaced Erythron:Organ for red cell production Pool of marrow erythroid precursor cells & large mass of mature circulating RBCs 24-Sep-11 Dr (Brig) YD Singh

6. RBCs : Normal Indices Mean cell volume (MCV) (Haematocrit x10) / (RBC count x 106) =90 ± 8 fL Mean Cell Haemoglobin (MCH) (Hb x 10) / (red cell count x 106) = 30 ± 3 pg Mean cell Hb concentration (MCHC) (Hb x 10) / hematocrit or MCH/MCV = 33 ± 2% 24-Sep-11 Dr (Brig) YD Singh

7. Leukocytes All Leukocytes derived from Common Stem cell in Bone marrow 3/4th of nucleated cells of bone marrow Committed to production of Leukocytes Mediate Inflammatory / Immune Responses Include Neutrophils, T & B Lymphocytes Natural Killer (NK) cells, Monocytes Eosinophils & Basophils These cells have specific functions Antibody production by B Lymphocytes Destruction of bacteria by Neutrophils 24-Sep-11 Dr (Brig) YD Singh

8. Granulocytes Development (1) 24-Sep-11 Dr (Brig) YD Singh

9. Granulocytes Development (2) 24-Sep-11 Dr (Brig) YD Singh

10. Neutrophils 24-Sep-11 Dr (Brig) YD Singh Neutrophil band with Döhle body Normal Neutrophil

11. 24-Sep-11 Dr (Brig) YD Singh Eosinophil Large bright orange granules usually bilobed Nucleus Basophil Large purple-black granules fill the cell & obscure nucleus

12. Normal Bone Marrow 24-Sep-11 Dr (Brig) YD Singh Low Power View

13. Bone marrow 24-Sep-11 Dr (Brig) YD Singh Normoblast with dividing Nucleus Eosinophil Cell Fat Cell Erythrocyte Cell Normoblast Cell Myelocyte dividing Myelocyte Cell Myelocyte Cell Fat Cell Fat Cell

14. Bone Marrow: Erythroid Hyperplasia 24-Sep-11 Dr (Brig) YD Singh

15. Bone Marrow: Myeloid Hyperplasia 24-Sep-11 Dr (Brig) YD Singh

16. Anaemia: Introduction Serum Hblevel or haematocrit < expected value for age & sex WHO criteria Adult men Blood Hbconcentration <13 g/dLor Hematocrit <39% Adult women Blood Hbconcentration <12 g/dL) or Hematocrit <37% 24-Sep-11 Dr (Brig) YD Singh

17. Anaemia: Types Iron-Deficiency Anemia Vitamin B12 Deficiency Anemia Folate-Deficiency Anemia Anemia of Chronic Disease Haemolytic Anaemia AplasticAnemia Myelodysplastic Syndromes 24-Sep-11 Dr (Brig) YD Singh

18. Anaemia: Mechanisms 3 major physiologic mechanisms of anemia Marrow production defects (Hypoproliferation) Reflects absolute or relative marrow failure Erythroid marrow not proliferated appropriately Can result from Marrow damage Iron deficiency Inadequate erythropoietin stimulation 24-Sep-11 Dr (Brig) YD Singh

19. Anaemia: Mechanisms Ineffective erythropoiesis (RBC maturation defects) Nuclear maturation defects associated with macrocytosis & abnormal marrow development Cytoplasmic maturation defects associated with microcytosis and hypochromia, usually from defects in hemoglobin synthesis Decreased erythrocyte survival: blood loss or hemolysis 24-Sep-11 Dr (Brig) YD Singh

20. Anaemia: Symptoms & Signs (1) Often recognized by abnormal results on screening lab tests Signs and symptoms depend on Level of anaemia Time course over which it developed Acute onset Anaemia Chronic Anaemia 24-Sep-11 Dr (Brig) YD Singh

21. Anaemia: Symptoms & Signs (2) Acute anaemia (nearly always due to blood loss or haemoptysis) If Loss of 10–15% of total blood volume Hypotension Decreased organ perfusion Loss of >30% of blood volume Postural hypotension Tachycardia 24-Sep-11 Dr (Brig) YD Singh

22. Anaemia: Symptoms & Signs (3) Loss of >40% of blood volume Hypovolemic shock Confusion Dyspnoea Diaphoresis Hypotension Tachycardia Haemolytic Anaemia Presentation depends on mechanism that leads to RBC destruction 24-Sep-11 Dr (Brig) YD Singh

23. Anaemia: Symptoms & Signs (3) Chronic or progressive anaemia Presentation depends on age of patient Adequacy of blood supply to critical organs Possible Symptom / Sign Fatigue and Loss of stamina Breathlessness (specially on exertion) Pale skin and mucous membranes (Pallor) Palpitation (Tachycardia, after physical exertion) Forceful heartbeat (Heaving Apex beat) High Volume pulse & Systolic flow murmur 24-Sep-11 Dr (Brig) YD Singh

24. Anaemia: Symptoms & Signs (4) In patients with coronary artery disease Anginal episodes may appear or Increase in frequency and severity In patients with carotid artery disease Light-headedness Dizziness may develop 24-Sep-11 Dr (Brig) YD Singh

25. Anaemia: DD (1) Hypoproliferativeanaemias(75% of cases) Marrow damage Infiltration/fibrosis Aplasia Iron deficiency (mild to moderate) Decreased stimulation Inflammation Metabolic defect (Hypothyroidism) Renal disease 24-Sep-11 Dr (Brig) YD Singh

26. Anaemia: DD (2) Maturation disorder Cytoplasmic defects Iron deficiency (severe) Thalassemia Sideroblastic Nuclear defects Folate deficiency , Vitamin B 12 deficiency Drug toxicity Methotrexate & Alkylating agents Alcohol Refractory anemia Myelodysplasia 24-Sep-11 Dr (Brig) YD Singh

27. Anaemia: DD (3) Haemolysis / Haemorrhage Blood loss Intravascular haemolysis Metabolic defect Membrane abnormality Haemoglobinopathy Autoimmune defect Fragmentation haemolysis 24-Sep-11 Dr (Brig) YD Singh

28. Anaemia: Diagnostic Approach (1) 02 questions need to be answered: Type of Anaemia & Cause of Anaemia Careful history Nutritional history Related to diet, drugs or alcohol Family history of anaemia (Genetic) Geographic backgrounds and ethnic origins G 6 PD deficiency Haemoglobinopathies Middle Eastern, Mediterranean, or African origin Exposure to toxic agents or drugs 24-Sep-11 Dr (Brig) YD Singh

29. Anaemia: Diagnostic Approach (2) Physical examination May provide clues to mechanisms / cause of anaemia Infection Blood in the stool Splenomegaly & Lymphadenopathy Petechiae suggest platelet dysfunction. Laboratory assessment Including review of past laboratory measurements to determine time of onset 24-Sep-11 Dr (Brig) YD Singh

30. Anaemia: Diagnostic Approach (3) Physiologic classification / Type of anaemia Reticulocyte index <2.5 & Normocytic, Normochromic anaemia Hypoproliferative Marrow damage: Infiltration / fibrosis Aplasia Decreased stimulation: Inflammation Metabolic defect Renal disease 24-Sep-11 Dr (Brig) YD Singh

31. Anaemia: Diagnostic Approach (4) Reticulocyte index <2.5 & microcytic or macrocyticanemia Maturation disorder Cytoplasmic defects: Iron deficiency, Thalassemia, Sideroblastic Nuclear defects: Folate deficiency Vitamin B deficiency Drug toxicity 24-Sep-11 Dr (Brig) YD Singh

32. Anaemia: Diagnostic Approach (5) Reticulocyte index ≥2.5 Haemolysis / Haemorrhage Blood loss Intravascular haemolysis Metabolic defect Membrane abnormality Haemoglobinopathy Autoimmune defect 24-Sep-11 Dr (Brig) YD Singh

33. AnaemiaAlgorithm 24-Sep-11 Dr (Brig) YD Singh

34. Normal Blood Smear (Wright’s stain) 24-Sep-11 Dr (Brig) YD Singh Normal RBCs, single Neutrophil & few platelets seen

35. Reticulocytes (Supravital Stain) 24-Sep-11 Dr (Brig) YD Singh Reticulocyte count is key to initial classification of anemia Reticulocytes are RBCs recently released from marrow

36. Severe Iron Def Anaemia(Wright’s stain) 24-Sep-11 Dr (Brig) YD Singh Microcytic & Hypochromic RBCs smaller than nucleus of a Lymphocyte + marked variation in size (Anisocytosis) & shape (Poikilocytosis)

37. Macrocytosis 24-Sep-11 Dr (Brig) YD Singh RBCs larger than small Lymphocyte & well haemoglobinized. Macrocytes are oval-shaped (Macroovalocytes)

38. Howell-Jolly bodies 24-Sep-11 Dr (Brig) YD Singh In absence of functional spleen, nuclear remnants are not expelled from RBCs & remain as small homogeneously staining blue inclusions on Wright stain

39. Red cell changes in myelofibrosis 24-Sep-11 Dr (Brig) YD Singh A Teardrop-shaped RBC & a Nucleated RBC is seen. These forms are seen in Myelofibrosis with Extramedullary Haematopoiesis

40. Red cell changes in Thalassemia & Liver disease 24-Sep-11 Dr (Brig) YD Singh Target cells have a bull’s-eye appearance & are seen in Thalassemia & Liver disease

41. Red cell changes in Sickle Cell Disease 24-Sep-11 Dr (Brig) YD Singh Sickle shaped cells are seen in Sickle Cell disease

42. Anaemia: Lab Tests CBC Erythrocyte count Haemoglobin & Haematocrit Reticulocyte count Erythrocyte indices Mean cell volume (MCV) Mean cell haemoglobin Mean cell haemoglobin concentration Leukocyte count Cell differential Nuclear segmentation of Neutrophils 24-Sep-11 Dr (Brig) YD Singh

43. Anaemia: Lab Tests Platelet count Cell morphology Cell size Anisocytosis (variations in cell size) Poikilocytosis (variations in cell shape) Polychromasia Iron supply studies Serum iron Total iron-binding capacity (TIBC) Serum ferritin, marrow iron stain 24-Sep-11 Dr (Brig) YD Singh

44. Hypoproliferative Anaemia: Key Tests Serum iron & iron-binding capacity Serum ferritin, to assess iron stores Evaluation of renal & thyroid function Marrow biopsy or aspirate Detect marrow damage or infiltrative disease Anemia of chronic inflammation shows Low serum iron & Normal or low TIBC Low percent transferrin saturation Normal or high serum ferritin 24-Sep-11 Dr (Brig) YD Singh

45. Hypoproliferative Anaemia: Key Tests Mild to moderate iron deficiency anaemia: Low serum iron level & High TIBC Low percent transferrin saturation Low serum ferritin level Marrow damage by drug, infiltrative disease (Leukaemia / Lymphoma / Aplasia) Peripheral blood and Bone marrow morphology Infiltrative disease or fibrosis Marrow biopsy will likely be required 24-Sep-11 Dr (Brig) YD Singh

46. Maturation disorders Anemia: Tests Vitamin B12 Folate Serum iron and iron-binding capacity Serum ferritin to assess iron stores Haemoglobin electrophoresis 24-Sep-11 Dr (Brig) YD Singh

47. Haemolytic Anemia: Tests Haemoglobin electrophoresis Screen for red cell enzymes Direct or indirect anti-globulin test Cold agglutinin titre 24-Sep-11 Dr (Brig) YD Singh

48. Anaemia Classification Based on defect in RBC production Marrow production defects: Hypo-proliferation Maturation defects: Ineffective Erythropoiesis Decreased survival: Blood Loss / Haemolysis Classification by MCV Microcytic: MCV <80 fL Normocytic: MCV 80–100 fL Macrocytic: MCV >100 fL 24-Sep-11 Dr (Brig) YD Singh

49. Complications: Anaemia High-output Cardiac Failure End-organ ischemia or infarct Myocardial infarction Stroke Hypovolumic shock Death 24-Sep-11 Dr (Brig) YD Singh

50. Management Pearls: Anaemia Anaemia may be Multi-factorial Finding one cause does not mean that no other forms of anaemia are present Iron deficiency may occur with folate / B12 def Producing Dimorphic anaemia Iron deficiency often means Occult blood loss Worms infestation Nutritional 24-Sep-11 Dr (Brig) YD Singh

51. Treatment Approach: Anaemia Mild to Mod Anaemia Initiate treatment when sp diagnosis is made Selection of treatment Determined by cause of anaemia Cause may be multi-factorial Evaluate iron status before starting treatment Rarely anaemia may be so severe RBC transfusions required before specific diagnosis is made 24-Sep-11 Dr (Brig) YD Singh