Course title: Hematology (1) Course code: MLHE-201 Supervisor: Prof. Dr Magda Sultan

1. Course title: Hematology (1)Course code: MLHE-201 Supervisor: Prof. Dr Magda Sultan Outcome : The student will know : -The types of hemolytic anemias -The diagnosis of hemolytic anemias -The types of hereditary hemolytic anemias. -The diagnosis of hereditary hemolytic anemias -Types of acquired hemolytic anemias -The diagnosis of acquired hemolytic anemias -The laboratory tests needed for diagnosis

2. Hemolytic anemia • Hemolytic anemia= decreased levels of red blood cells (anemia) because of their destruction (hemolysis) • A red blood cell survives 120 days • The spleen is the main organ which removes old RBCs from the blood.

3. Causes of hemolytic anemiascan be either: 1 - hereditary. 2 - acquired.

4. Hereditary Hemolytic anemia • Defects of hemoglobin • Thalassaemia, Sickle cell anemia • Defects of the red cell membrane • Hereditary spherocytosis, Hereditary elliptocytosis • Defective red cell metabolism (enzymes) • G6PD deficiency. P K deficiency

5. Acquired - Immune mediated : Autoimmune, isoimmune, drugs - Microangiopathic: DIC, HUS - Hypersplenism - Miscellaneous: drugs, toxin, infection, burn, chemical

6. Signs of hemolytic anemia: Physical • Symptoms of anemia • Jaundice • Pallor • Splenomegaly / hepatosplenomegaly

7. Laboratory features • Anemia of increased destruction • Normochromic, normocytic anemia • Short RBC survival • Reticulocytes increased • Increased indirect bilirubin • Increased LDH *Peripheral blood smear microscopy: • fragments of the red blood cells and spherocytes • Normoblasts can be present. Bone marrow smear microscopy: Erytrhroid hyperplasia

8. Hereditary Hemolytic anemia • Defects of hemoglobin • Thalassaemia, Sickle cell anemia • Defects of the red cell membrane • Hereditary spherocytosis, Hereditary elliptocytosis • Defective red cell metabolism • G6PD deficiency. P K deficiency

9. Sickle cell anaemia • The abnormalities of the gene may result from substitution of single amino acid (Substitution of glutamic acid by valine ) The Hb is stable when oxygenated state and become unstable and polymerized on deoxygenated state

10. Sickle cell anaemia • Polymerization will lead to precipitation of Hb. The cell become deformed (sickle shape) and very sticky leading to vascular occlusion and small infarction to the affected areas. • Short life span of cells leading to chronic anaemia,

11. Sickle cell anaemia Diagnosis 1-Sickling test 2- Hemoglobin electrophoresis: Increased hemoglobin S (90% Hgb S, 10% Hgb F, small fraction of Hgb A2)

12. HEMOGLOBIN • NORMAL ADULT RBC CONSISTS OF 3 FORMS OF Hb: - HbA - 2 α and 2 β globin chains - HbA2 – 2 α and 2 δ globin chains - HbF - 2 α and 2 γ globin chains

15. Thalassaemia • Means decrease synthesis of one ofthe globin chain which form normal hemoglobin. • (HbA- 2 α and 2 β globin chains • HbA2 – 2 α and 2 δ globin chains • HbF - 2 α and 2 γ globin chains) • . The defect may be in alpha chain ( thalassaemia), • Beta chain ( thalassaemia) or Delta chain ( thalassaemia)

16. Beta Thalassaemia Defective  chain synthesis Excess  chain Precipitation cell membrane damage Circulating Red cell Bone marrow Anaemia Hemolytic Ineffective erythropoiesis Erythropoietin increased blood transfusion  Iron absorption Bone marrow expansion Iron overload skeletal changes & hyper metabolism Complication and death

17. Beta-Thalassemia major laboratory features • Severe anemia • Blood film: microcytic hypochromic , target cells, basophylic stippling, reticulocytes increased and normoblasts . • Marrow: marked erythroid hyperplasia, Shortened red cell survival • Haemoglobin electrophoresis : • Fetal hemoglobin > 90%, HbA absent, HbA2 low/normal/high

18. HEREDITARY SPHEROCYTOSIS • Defective or absent spectrin molecule • Leads to loss of RBC membrane, leading to spherocytosis • Decreased deformability of cell • Increased osmotic fragility • Extravascular hemolysis in spleen

19. Hereditary spherocytosis (HS)Laboratory features- hemolytic anemia- blood smearspherocytes - increasedosmotic fragility time

20. G6PD DEFICIENCYFunction of G6PD

21. Glucose 6-Phosphate DehydrogenaseFunctions • Regenerates NADPH, allowing regeneration of glutathione • Protects against oxidative stress • Lack of G6PD leads to hemolysis during oxidative stress • Infection • Medications • Fava beans • Oxidative stress leads to Heinz body formation,  extravascular hemolysis

22. G6PD DEFICIENCY • DIAGNOSIS: • QUANTITATIVE ASSAY DETECTING LOW ENZYME • TREATMENT – SUPPORTIVE AND PREVENTATIVE

23. Acquired hemolytic anaemia Due to Antibodies directed against RBC membrane = autoimmune hemolytic anemia destruction of RBC in an enlarged spleen

24. Introduction • Increased RBC Destruction – • Short RBC life span <120 days. • Normocytic normochromic, reticulocytosis. • Anemia, Jaundice, marrow hyperplasia • Splenomegaly, increased bilirubin

25. Types of acquired HA • AutoImmune Haemolytic Anemias (+ve Direct CoombꞋs) • Alloimmunehaemolyticanemias • Drug-induced immune haemolytic anemias

26. Assesment of HA Clinicalfeatures:-pallor-jaundice-splenomegaly

27. Laboratory features:1. Laboratory features- normocytic, normochromic anemia - reticulocytosis - antiglobulin Coombs’ test is positive2. Blood smear - anisopoikilocytosis, spherocytes - normoblasts - schistocytes3. Bone marrow smear - erythroid hyperplasia

28. DIRECT ANTIGLOBULIN TEST (DAT)Coomb′s test

29. Procedure of DAT • Take 2-3 drops of blood to be tested in a clean labeled tube. • Wash the red cells 3-4 times in a large volume of saline to remove free globulin molecules. Remove all supernatant after each wash. Completely decant the final supernatant wash. • Add 2 drops of polyspecific AHG serum in 1 drop of sensitized washed red cells or in 1 drop of 3-5 % suspension of sensitized cells immediately. • Mix, Centrifuge at 1000 rpm for 1 minutes immediately. • Gently shake the tube to dislodge the cell button and see for agglutination, use optical aid if needed. Record the result. • Add 1 drop of IgG coated red cells to a negative test. Mix, centrifuge at 1000 rpm for 1 min. Immediately look for agglutination. If a negative result (no agglutination) is obtained the test result is invalid and whole test should be repeated. If agglutination is obtained, the result is valid.

30. Indirect antiglobulin (coomb′s ) test

31. Procedure: • Place 2-3 drops of the test serum in a tube. Serum should be fresh for detecting complement components and complement binding antibodies, otherwise, fresh AB serum should be added to it. • Add 1 drop of 3-5% suspension of washed O Rh (D) positive red cells to the serum in the tube. • Mix and incubate at 37°C for 30-40 minutes. • Centrifuge at 1000 rpm for 1 minutes. • Examine for hemolysis and/or agglutination. Use optical aid if necessary. Agglutination at this stage indicates the presence of saline (complete) antibodies. • If no agglutination is seen, wash cells 3-4 times in large volume of saline. Decant supernatant in each wash as completely as possible.

32. Procedure: • Add 2 drops of AHG serum to the cells. • Mix and centrifuge at 1000 rpm for 1 minutes immediately. • Gently shake the tube to dislodge the button and examine for agglutination, using optical aid. Record the result. • Add 1 drop of IgG coated red cells to any test that is negative. Mix and centrifuge at 1000 rpm for 1 minutes. Look for agglutination. If there is no agglutination, the test result is invalid and the whole test is repeated. If agglutination is obtained the result is valid. • Auto control should be kept with IAT.

35. Training questions :What are the tests of hemolysis ? • How to diagnose Autoimmune hemolytic anaemia ? • Reference book : • Essential Hematology . • Dacie .