Meet the Red Cell

1. Meet the Red Cell • K. Krishnan MD. FRCP, FACP

2. The red cell • Durability of red cell is remarkable • No nucleus to direct regenerative processes • No mitochondria available for efficient oxidative metabolism • No ribosomes for regeneration of lost or damaged protein • No de novo synthesis of lipids

3. RED CELL SURVIVAL • Survives constant mechanical stress like hydrostatic pressure and turbulence and shear stress • Survives biochemical stress of osmotic and redox fluxes as it travels through the renal collecting system, sluggish vascular system of the spleen, muscle and bone • Survives the ambient oxygen pressures occurring in the lungs • ALL CONSPIRE TO DAMAGE RED CELLS BUT IT SURVIVES FOR 120 DAYS!!!

4. RED CELL SURVIVAL TOOLS • SIMPLE but EXQUISITE • Adaptive membrane structures • Pathways of intermediary energy metabolism and redox regulation and • Ability to maintain Hb in the soluble and nonoxidized form • The membrane and enzymes of the red cell are crafted to protect the cell from external ravages of the circulation and the internal assaults of the massive amount of iron rich and oxidizing protein represented by the hemoglobin molecules

5. Basics of Erythropoiesis • Erythropoiesis is the process of producing red cells. • Regulated by a series of steps beginning with the pluripotent hematopoeitic stem cell • Erythroid cells come from a common erythroid/megakaryocyte progenitor • Needs transcription factors, GATA-1 and FOG-1 (friend of GATA-1)

6. Basics of Erythropoiesis • After lineage commitment is achieved, growth factors and hormones regulate development • Erythropoeitin induces the committed progenitor to expand in number. • Epo is regulated by Oxygen availability

7. Physiological regulation of RBC production by tissue oxygen tension

8. Erythropoeitin • Glycoprotein • Released by specialised cells- the peritubular capillary lining cells in the kidney • Small amount of Epo from hepatocytes • Oxygen tension in the kidney is the stimulus for Epo production • Epo binds to specific receptors on the surface of marrow erythroid precursors

9. Concept of the Erythron • Dynamic organ made up a pool of rapidly proliferating marrow erythroid precursors and a large mass of circulating red blood cells • The size of the red cell mass reflects a balance between production and destruction

10. Elements of Erythropoiesis • Eythropoeitin production • Iron availability • Proliferative capacity of the bone marrow • Effective maturation of the red cell precursors

11. What are these? What stains were used?

12. Reticulocyte Count • An accurate reticulocyte count is key to the initial classification of anemia • Represent new, young, just released red cells • Signature- supravital dye that identifies the ribosomal RNA • Blue or black punctate spots • The residual RNA is metabolised over time • Measure of red cell production

13. Reticulocytes • Reticulocytosis • Acute blood loss • Splenic sequestration • Hemolysis • Immune • Non-immune • Infection • Membrane • Reticulocytopenia • Early iron deficiency • Primary bone marrow failure • Secondary bone marrow failure

14. Use of reticulocyte count • Two corrections necessary • Adjusts reticulocyte count based on the reduced number of circulating red cells (with anemia the reticulocyte percentage is increased but not the absolute number). The reticulocyte percentage is multiplied by the ratio of the patient’s hemoglobin/hematocrit for the age and gender. This provides a reticulocyte count corrected for the anemia • For example, if the reticulocyte count is 8 and hemoglobin is 8, then the corrected reticulocyte count is 8/16 x 8=4 • A further correction of the corrected reticulocyte count (reticulocyte production index) is necessary for an index of marrow production to account for the premature release of reticulocytes from the marrow • Examine smear and see if there are polychromatophilic, macrocytes-these are prematurely released reticulocytes called SHIFT RETICULOCYTES. If no polychromatic red cells are seen second correction is not required • These reticulocytes live in the peripheral blood for a longer time than normal reticulocytes and hence provide a falsely high estimate of daily red cell production • If polychromasia is present, the reticulocyte count corrected for anemia should be further divided by a factor of 2.

15. Functional classification of anemias • Marrow production defect • Hypoproliferative • Red cell maturation defect • Ineffective erythropoeisis • Decreased red cell survival • Blood loss/hemolysis

16. Physiological classification of anemia

17. Hypoproliferative anemias • Serum iron, TIBC, renal and thyroid function, bone marrow biopsy, serum ferritin

18. Microcytic, hypochromic anemia

19. Iron deficiency anemia

20. Microcytic hypochromic red cells • Iron deficiency • Thalassemias • Lead poisoning • Sideroblastic anemias • Anemia of chronic diseases

21. Thalassemic syndromes • Hypochromic microcytic red cells • “Chip munk” facies • Hemolytic anemia • Hepatosplenomegaly • Leg ulcers • Gallstones • High output heart failure • Endocrine dysfunction • Infections

22. Beta-thalassemic syndromes • Microcytes • Bizarre poikilocytes • Tear drop cells • Target cells, nucleated red cells • Extraordinarily folded red cells called LEPTOCYTES containing alpha-globin inclusion bodies

23. What is the hematological defect? • Failure of synthesis of the globin chains either alpha or beta chains • Low supply of globin chains and not enough to form hemoglobin tetramers • Leads to microcytosis and hypochromia • Unbalanced accumulation of the normal chain • Toxic inclusions and intramedullary hemolysis • Eythropoeitin surge but ineffective hematopoeisis • Builds up erythroid masses that does not produce hemoglobin

24. Alpha thalassemia syndromes

25. Alpha-thalassemia syndromes • Hemoglobin H disease

26. Hemoglobin H inclusions • Alpha-thalassemia intermedia • Hemolysis and splenomegaly • Supravital staining • Multiple small inclusions due to excess beta-globin

27. Sickle-beta thalassemia

28. Hemoglobin C disease

29. Blood smear in a 43 year old man with history of a motor vehicle accident 12 years ago. What is this? What may have happened?

30. Stomatocytosis • Slit-shaped central pallor • Usually alcoholic liver disease and other liver diseases • No hemolysis • Hereditary forms due to red cell overhydration • Na and water gain • Hemolysis +

31. Target red cells • Increased membrane surface • obstructive liver disease due to excess lipoprotein, cholesterol and post-splenectomy states • no hemolysis, cells are flexible • Volume loss • Decreased Hb: iron deficiency, thalassemia • Poorly soluble hemoglobin: Hb S, Hb C; interact with membrane and cause water loss

32. Acanthocytosis • Irregularly spiculated red cells with net gain in lipids and an asymmetry between the 2 lipid layers • Causes: • Severe liver disease • Hemolytic and non-hemolytic • Abetalipoproteinemia • Mcleod’s syndrome • Cholesterol loading causes spur cell anemia and severe hemolysis; no hemolysis if not cholesterol loaded

33. What is the abnormality? What is the mechanism?

34. Spherocytosis • Microspherocytosis: deficiency of red cell surface • Immune hemolytic anemias • Hereditary spherocytosis • Heinz-body hemolytic anemia • Clostridial sepsis, Severe burns • Hypophosphatemia

35. Spherocytosis

36. Mechanisms of Spherocytosis • Loss of membrane lipids leading to a reduction in surface area due to deficiencies of red cell-hereditary spherocytosis • Removal of membrane material form antibody coated red cells by macrophages- Immune hemolytic anemia • Removal of membrane associated Heinz bodies with the adjacent membrane lipids by the spleen- Heinz body hemolytic anemia

37. Hereditary spherocytosis Rabbit spleen showing how RBC need to elliptically deform in order to pass through the very narrow slits in the wall of the splenic cords of Billroth and enter the sinusoids from which they can return to the circulation. A microspherocyte, deprived of its excess surface area, cannot ellipitically deform and is thus trapped in the cords.

38. Osmotic fragility test Lower panel: Hereditary spherocytosis-lysis occurs in mildy hypotonic solutions

39. Red cell membrane proteins A model depicting the major proteins of the erythrocyte membrane is shown: α and β spectrin, ankyrin, band 3, 4.1 (protein 4.1), 4.2 (protein 4.2), actin, and GP (glycophorin).

40. Elliptocytosis • Hereditary elliptocytosis • Acquired elliptocytosis • Myelofibrosis • Thalassemic syndromes • Iron deficiency

41. What is this? • Causes?

42. Cold agglutinin diseases • Red cell autoantibodies not cryoglobulins • Causes • Monoclonal • Idiopathic/chronic • B cell disorders • Polyclonal • Benign • Postinfectious-mycoplasma, EBV, HIV

43. Rouleaux • Paraproteinemias

44. Rouleaux and Agglutination

47. Tear drop red cells • Bone marrow infiltration • Fibrosis • Tumors • Granulomas

48. What are these? What stains were used?

49. Basophilic stippling • Hemolytic anemias • Pyrimidine-5’nucleotidase deficiency • Iron deficiency • Thalassemias • Lead poisoning • Diffuse fine or coarse blue dots in the red cell representing usually RNA residue

50. Mechanisms of basophilic stippling • Many small bluish dots in portion of erythrocytes; from staining of clustered polyribosomes in young circulating red cells • Failure to digest/clear residual RNA due to • Acquired and congenital hemolytic anemias • Lead poisoning (lead inhibits pyrimidine 5’ nucleotidase which normally digests residual RNA)