2. HEMOLYTIC ANEMIA Is anemia due to shortening of RBCs life span, which is normally around 120 days by a variety of abnormalities. *The bone marrow has the ability to increase the output of RBC to 6-8 folds by: 1. increased RBC production. 2. Expanding the volume of active marrow. 3. Releasing of premature reticulocytes. **If the rate of destruction exceeds the rate of production, Anemia developed.

3. LAB DIAGNOSIS: LAB DIAGNOSIS: 1. Anemia (decreased RBC Count) 1. Anemia (decreased RBC Count) 2. Increased Unconjucated Bilirubin. 2. Increased Unconjucated Bilirubin. 3. Increased Urinary Urobilinogen. 3. Increased Urinary Urobilinogen. 4. Increased Serum LDH. 4. Increased Serum LDH. 5. Reticulocytosis. 5. Reticulocytosis. 6. Neutrophilia & immature Granulocyte lead to Leukoerythroblastic blood film due to expansion of the active marrow. Leukoerythroblastic film contains nucleated RBCs & immature WBCs. 6. Neutrophilia & immature Granulocyte lead to Leukoerythroblastic blood film due to expansion of the active marrow. Leukoerythroblastic film contains nucleated RBCs & immature WBCs. 7. Decreased Serum level of Haptoblobins. 7. Decreased Serum level of Haptoblobins. 8. +ve Urinary Hemosiderine appears 3-4 days after Hemolysis. 8. +ve Urinary Hemosiderine appears 3-4 days after Hemolysis.

4. -Spherocytes are small, dark red cells which suggest autoimmune haemolysis or hereditary spherocytosis. -Sickle cells suggest haemoglobinopathy. -Red cell fragments indicate microangiopathic haemolysis. 9-the blood film :The appearances of the red cells may give an indication of the likely cause of the haemolysis

5. Peripheral smear from a splenectomized patient with recurrent autoimmune hemolytic anemia. The red arrow marks a circulating nucleated red blood cell. The green arrow marks an erythrocyte with coarse basophilic stippling. The blue arrow marks a Howell-Jolly body

10. TYPES OF HEMOLYSIS:

11. I-intravascular haemolysis Less commonly, red cell lysis occurs within the blood stream due to membrane damage by complement (ABO transfusion reactions, paroxysmal nocturnal haemoglobinuria), infections (malaria, Clostridium perfringens), mechanical trauma (heart valves, DIC) or oxidative damage (e.g. drugs such as dapsone )..

12. : When rapid destrction of red blood cells occur,free HB is released into plasma. freeHB is toxic to cells and binding Protein is evolved to minimised this risk.Haptoglobin is an α 2 - globuline produce by liver which bind free HB, resulting in a fall in levels of haptoglobin. Once haptoglobin saturated free HB is oxedised to form metHB Which bind to albumin,in turn forming methaemalbumin Which can be detected spectrophotometically in the Schumm s test.

13. Methaemoglobin is degraded and any free haem is bound to a second binding protein termed haemopexin. If all the protective mechanisms are overloaded, free haemoglobin may appear in the urine. When fulminant, this gives rise to black urine, as in severe falciparum malaria infection.

14. In smaller amounts, renal tubular cells absorb the haemoglobin, degrade it and store the iron as haemosiderin. When the tubular cells are subsequently sloughed into the urine, they give rise to haemosiderinuria, which is always indicative of intravascular haemolysis.

15. II-Extravascular: Physiological red cell destruction occurs in the fixed Reticular- endothelial cells in the liver & spleen, so avoiding free HB in the plasma. in most haemolytic states haemolysis is predominantly extravascular.

16. To confirm the haemolysis, patients’ red cells can be labelled with 51chromium. When re-injected, they can be used to determine red cell survival; when combined with body surface radioactivity counting, this test may indicate whether the liver or the spleen is the main source of red cell destruction. However, it is seldom performed in clinical practice.

18. CLASSIFICATION: 1. Congenital: A. Red cell membrane abnormalities; Hereditary Spherocytosis. Hereditary Elliptocytosis. B. Red cell Enzymopathies: G6PD deficiency. Pyruvate kinase deficiency. C. Hemoglobinpathies: Sickle cell anaemia. Thalassemia.

20. 2. Acquired: A. Immune Hemolytic Anaemia: a. Autoimmune: Warm autoimmune Hemolysis. Cold agglutinin disease. b. Isoimmune c. Alloimmune B. Non-Immune Hemolytic Anaemia: Mechanical trauma, Burns. Infection Chemicals. Drugs.

22. The basic structure is a cytoskeleton 'stapled' on to the lipid bilayer by special protein complexes. This structure ensures great deformability and elasticity; the red cell diameter is 8 μm but the narrowest capillaries in the circulation are in the spleen, measuring just 2 μm in diameter. Red cell membrane defects

24. When the normal red cell structure is disturbed, usually by a quantitative or functional deficiency of one or more proteins in the cytoskeleton, cells lose their elasticity..

25. Each time such cells pass through the spleen, they lose membrane relative to their cell volume. This results in an increase in mean cell haemoglobin concentration (MCHC), abnormal cell shape and reduced red cell survival due to extravascular haemolysis

27. Hereditary Spherocytosis Autosomal dominant disorder. 25% of cases have no Family History presenting new Mutation. The incidence is approximately 1:5000 in developed countries. The pathogenesis varies between families. The Most common abnormality is Defect of Beta Spectrin or ankyrin.

28. Deficiency of Spectrin, which is RBC membrane protein, the RBC loses its biconcave shape & become spherical, more susceptible to osmotic lysis & most of them destroyed by the spleen. The type of hemolysis is extravascular.

29. The severity of spontaneous haemolysis varies. Most cases are associated with an asymptomatic compensated chronic haemolytic state with spherocytes present on the blood film, a reticulocytosis and mild hyperbilirubinaemia. Pigment gallstones are present in up to 50% of patients and may cause symptomatic cholecystitis.

30. CLINICAL FEATURES OF HEMOLYTIC ANEMIA: 1. Anaemia 2. Splenomegally 3. Jaundice 4. Gall stone of pigment type due to increased bile pigment production,present in up to 50% of patients and may cause symptomatic cholecystitis. 5. Chronic leg ulcers

31. The spectrum of clinical severity of HS is broad. Severe cases may present in infancy with severe anemia, whereas mild cases may present in young adults or even later in life. In women, HS is sometimes first diagnosed when anemia is investigated during pregnancy.

32. The anemia is usually normocytic, with the characteristic morphology that gives the disease its name. A characteristic feature is an increase in mean corpuscular hemoglobin concentration (MCHC): this is almost the only condition in which an increased MCHC is seen.

33. 6. Clinical course may be complicated by crisis: Hemolytic Crisis: when severity of Hemolysis increased, rarely seen associated with infection. Megaloblastic Crisis: following the development of Folate deficiency. This may occur in pregnancy. Aplastic Crisis: occurs in association with Erythrovirus infection. This virus directly invades red cell precursor & temporarily switch off red cell production result in sever anemia without retics..

34. INVESTIGATIONS The patient should be screened for features of compensated haemolysis. *Hb level :variable depending on the degree of compensation. *blood film show spherocytes *direct coombs test is negative. *osmotic fragility test may show increased sensitivity to lysis in Hypotonic saline solution. *more specific test flow cytomeetric test detecting of eosin- 5- Maleimide to red cells are now recommended in borderline cases.

35. Spherocytes (small dense cells lacking central pallor) are the morphologic hallmark of hereditary spherocytosis.

36. TREATMENT: 1. Folic acid prophylaxis 5 mg/wk life long (daily orally) 2. splenectomy improve the condition without normalizing red cell. 3. Blood transfusion: crossed matched blood should be given in acute sever crisis carefully and transfused slowly. (Hemolytic transfusion may occur)

37. Indications of splenectomy: Potential indications include moderate to sever haemolysis with complications (anaemia and gall stones), although splenectomy should be delayed until the child is over 6 years of age in view of the risk of sepsis.

40. This enzyme is pivotal in the hexose monophosphate shunt pathway. Deficiencies result in the most common human enzymopathy, affecting 10% of the world's population, with a geographical distribution which parallels the malaria belt because heterozygotes are protected from malarial parasitisation. The enzyme is a heteromeric structure made of catalytic subunits which are encoded by a gene on the X chromosome. Glucose-6-Phosphate dehydrogenase deficiency Red cells Enzymopathies:

41. The deficiency therefore affects males and rare homozygotic females, but it is carried by females. Carrier heterozygous females are usually only affected in the neonatal period or in the presence of extreme lyonisation, producing selective inactivation of one of the X chromosomes

43. CLINICAL FEATURES: 1. Acute drug induced Hemolysis. This occurs with many drugs: Analgesics: aspirin and Phenactin Antimalarial: primaquine, quinine, chloroquin & pyrimethamine Antibiotics: Sulphanamide, Nitrofurantoin, Ciprofloxacin, miscellaneous(Quinidine, Probencid, vitamin K, Dapsone) 2. Chronic compensated Hemolysis 3. Infection or acute illness 4. Neonatal jaundice (feature of B-enzyme) 5. Favism or acute Hemolysis after ingestion of Broad beans.

45. AHA can develop as a result of three types of triggers: (1) fava beans. (2) infections. (3) drugs. Typically, a hemolytic attack starts with malaise, weakness, and abdominal or lumbar pain. After an interval of several hours to 2–3 days, the patient develops jaundice and often dark urine, due to hemoglobinuria.

46. The onset can be extremely abrupt, especially with favism in children. The anemia is from moderate to extremely severe. It is usually normocytic and normochromic.

47. The most serious threat from AHA in adults is the development of acute renal failure (this is exceedingly rare in children). Once the threat of acute anemia is over, and in the absence of comorbidity, full recovery from AHA associated with G6PD deficiency is the rule.

48. LAB DIAGNOSIS: 1. During the attack, there will be evidence of non- Spherocytic intravascular Hemolysis. 2. Blood film: *Bite Cell —» red cell with a bite of membrane missing. *Blister Cell —» red cell with membrane surface blistered. *Irregular small shaped cells. * polychromasia which reflect Reticulocytosis. *Denatured Hb visible as Heinz body within the red cell cytoplasm,if stained with a supravital stain such as methyle violet.

49. 3.* The level of G6PD can be indirectly assessed by screening method, which usually depends on decreased ability to reduce dyes. * Direct assessment is made in those with low screening values. *care must be taken close to an acute haemolytic episode because reticulocytes may have normal enzyme level and give rise to a false Normal result.

50. Acute hemolysis in G6PD deficiency (arrows indicate “blister cells,” and arrowheads irregularly contracted cells)

51. Heinz bodies, bite cells

53. INVESTIGATIONS: Confirmed by estimating level of G6PD activity of red cell but it may be not accurate if there is a considerable Reticulocytosis. There are characteristics of intravascular hemolysis: Hemoglobinuria, hemoglobinemia, ahaptoglobinemia & later hemosedrinuria.

54. TREATMENT: 1. Treat the underlying cause. 2. Stop any precipitating drugs 3. Deal with the underlying infection 4. Acute transfusion may be life saving

55. The acute hemolytic anemia of G6PD deficiency is largely preventable by avoiding exposure to triggering factors of previously screened subjects Favism is entirely preventable in G6PD-deficient subjects by not eating fava beans. Drug-induced hemolysis can be prevented by testing for G6PD deficiency before prescribing; in most cases, one can use alternative drugs.

56. When AHA develops and once its cause is recognized, in most cases no specific treatment is needed. However, if the anemia is severe, it may be a medical emergency, especially in children, requiring immediate action, including blood transfusion If there is acute renal failure, hemodialysis may be necessary, but if there is no previous kidney disease, recovery is the rule

57. ACQUIRED HAEMOLYTIC ANAEMIA: Autoimmune heamolytic anemia: This results from increased red cell destruction due to red cell autoantibodies. The antibodies may be IgG or M, or more rarely IgE or A. If an antibody avidly fixes complement, it will result in intravascular haemolysis, but if complement activation is weak, the haemolysis will be extravascular. Antibody-coated red cells lose membrane to macrophages in the spleen and hence spherocytes are present in the blood

58. The optimum temperature at which the antibody is active (thermal specificity) is used to classify immune haemolysis: 1.Warm antibodies bind best at 37°C and account for 80% of cases. The majority are IgG and usually react against Rhesus antigens. 2.Cold antibodies bind best at 4°C but can bind up to 37°C in some cases. They are usually IgM and bind complement. They account for the other 20% of cases.

59. Warm autoimmune haemolysis The incidence of warm autoimmune haemolysis is approximately 1/100 000 population per annum; it occurs at all ages but is more common in middle age and there is a female excess. No underlying cause is identified in up to 50% of cases.

60. The remainder are secondary to a wide variety of other condition: 1.lymphoid neoplasms: lymphoma, chronic lymphocytic leukaemia, myeloma 2.solid tumours: lung, colon, kidney, ovary, thymoma 3-connective tissue disease: SLE, rheumatoid arthritis 4.drugs: methyldopa, mefenamic acid, penicillin, quinine 5.miscellaneous: ulcerative colitis, HIV

61. Investigations: There is evidence of haemolysis and spherocytes on the blood film. The diagnosis is confirmed by the direct Coombs test. In this, red cells are mixed with Coombs reagent which contains antibodies against human IgG/M/complement. If the red cells have been coated by antibody in vivo, the Coombs reagent will induce their agglutination and this can be detected visually. The standard Coombs reagent will miss IgA or IgE antibodies.

65. Management: -If the haemolysis is secondary to an underlying cause, this must be treated and any offending drugs stopped -It is usual to treat patients initially with prednisolone 1 mg/kg orally. A response is seen in 70-80% of cases but this may take up to 3 weeks; a rise in haemoglobin will be matched by a fall in bilirubin and LDH and Reticulocytes levels. Once the haemoglobin has normalised And the reticujocytosis resolved the corticosteroid dose can be Reduced slowly over about 10 weeks

66. Steroids work by decreasing macrophage destruction of antibody-coated red cells and reducing antibody production -Transfusion support may be required for life-threatening problems. The least incompatible blood should be used but may still give rise to transfusion reactions or the development of further alloantibodies

67. - If the haemolysis fails to respond to corticosteroids or can only be stabilised by large doses, then splenectomy should be considered. This removes a main site of red cell destruction and antibody production with a good response in 50-60% of cases. The operation can be performed laparoscopically with reduced morbidity. -

68. For patients who fail to respond to corticosteroids or for whom splenectomy is not appropriate, alternative immunosuppressive therapy may be considered. This is least suitable for young patients, for whom long-term therapy may carry a risk of secondary neoplasms. The choice of drug is between azathioprine 1-2 mg/kg and cyclophosphamide 2 mg/kg, both orally; induction of a response usually takes 2-3 months.

69. The anti-CD20 (B cell) monoclonal antibody, rituximab, has shown some success in difficult cases.

70. Cold agglutinin disease This is due to antibodies, usually IgM, which bind to the red cells at 4°C and cause them to agglutinate. It may cause intravascular haemolysis if complement fixation occurs. This can be chronic when the antibody is monoclonal, or Acute or transient when the antibody is polyclonal.

71. *cold agglutinins arise 2 clinical settings:- 1-Monoclonal AB as lymphatic neoplasia e.g.: low grade B-cell lymphoma, usually chronic 2-Polyclonal AB in response to infective conditions and usually transient Transient cold agglutinins occurs in infection like Mycoplasma, pneumonia and infectious mononucleosis

72. This affects elderly patients and may be associated with an underlying low-grade B cell lymphoma. It causes a low-grade intravascular haemolysis with cold, painful and often blue fingers, toes, ears or nose (so- called acrocyanosis). The latter is due to red cell agglutination in the small vessels in these colder exposed areas. The blood film shows red cell agglutination and the MCV may be spuriously raised because the automated analysers count aggregates as single cells.. Chronic cold agglutinin disease

73. Treatment -directed at any underlying lymphoma. -if the disease is idiopathic, then patients must keep extremities warm, especially in winter. --Some patients respond to corticosteroid therapy and blood transfusion may be considered, but the cross-match sample must be placed in a transport flask at a temperature of 37 °C and blood administered via a blood-warmer

74. Alloimmune haemolytic anaemia is due to an antibody against non-self red cells. It has two main causes: - an unmatched transfusion of red cells (a haemolytic transfusion reaction) -maternal sensitisation to paternal antigens on fetal cells (haemolytic disease of the newborn) Alloimmune haemolytic anaemia

75. 2-Non-Immune hemolytic anaemia:- A-physical trauma: Physical disruption of RBC may be occurs in number of conditions and it characterized by the presence of RBC fragments on the blood film and marker of intravascular haemolysis these, those conditions.are: 1. Mechanical heart valve:-high flow though incomplete valve or per prosthetic leaks through suture rings holding valve in place result in shear stress damage 2. March Hburia:-vigorous exercise such as prolonged marching or marathon running can cause red cell damage in capillaries in the feet

76. 4-thermal injury: Sever burns cause thermal damage to red cells Characterised by fragmentationand the presence of Microspherocytesin the blood. 3. Microangiopathic hemolytic anaemia Fibrin depositions in the capillaries cause sever red cell disruption. It may occur in wide Varity of conditions like disseminated carcinomatosis, malignant or pregnancy induce Hypertension,hemolytic uraemic syndrome, thrombotic thrombocytopenic purpura.

79. B-infections: * falciprum malaria: may associate with intravascular haemolysis when sever is termed black water fever due to haemoglobinuria *Clostridium perfringens septicemia: in the context of an ascending cholangitis it May cause sever intravascular haemolysis with Spherocytosis due to bacterial productions of lecithinase which destroys the RBC membrane

80. C-Chemical and drugs: These agent lead to haemolysis by oxidant denaturations Of Hb, dapson, sulphsalazine can produce haemolysis. Arsenic gas, copper, chlorates, nitrites, nitrobenzene derivatives may all cause haemolysis

81. Paroxysmal Nocturnal Hemoglobinuria (PNH) PNH is an acquired chronic HA characterized by persistent intravascular hemolysis subject to recurrent exacerbations. In addition to hemolysis, there is often pancytopenia and a distinct tendency to venous thrombosis..

82. This triad makes PNH a truly unique clinical condition. However, when not all of these three features are manifest on presentation, the diagnosis is often delayed, although it can be always made by appropriate laboratory investigations

83. There is no evidence of inherited susceptibility. PNH has never been reported as a congenital disease, but it can present in small children or as late as in the seventies, although most patients are young adult.

84. The natural history of PNH can extend over decades. Without treatment, the median survival is estimated to be about 8–10 years. In the past, the most :common cause of death has been 1- venous thrombosis. 2- followed by infection secondary to sever neutropenia 3- hemorrhage secondary to severe thrombocytopenia..

85. PNH may evolve into aplastic anemia (AA), and PNH may manifest itself in patients who previously had AA. Rarely (estimated 1–2% of all cases), PNH may terminate in acute myeloid leukemia. On the other hand, full spontaneous recovery from PNH has been well documented, albeit rarely

86. Paroxysmal Nocturnal Hemoglobinuria