1. Course title: Hematology (1) Course code: MLHE-201 Supervisor: Prof
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 anemias can 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. 2- Hemoglobin electrophoresis: Increased hemoglobin S
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 Anaemia skeletal changes & hyper metabolism
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 smear spherocytes increased osmotic fragility time

20. G6PD DEFICIENCY Function of G6PD

21. Glucose 6-Phosphate Dehydrogenase Functions
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 QUANTITATIVE ASSAY DETECTING LOW ENZYME
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)
Alloimmune haemolytic anemias
Drug-induced immune haemolytic anemias

26. Assesment of HA Clinical features: - pallor - jaundice - splenomegaly

27. Laboratory features: 1. Laboratory features normocytic, normochromic anemia reticulocytosis antiglobulin Coombs’ test is positive 2. Blood smear anisopoikilocytosis, spherocytes normoblasts schistocytes 3. 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 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 .