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

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.

Causes of hemolytic anemias can be either:

1 - hereditary.2 - acquired.

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

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

Signs of hemolytic anemia: Physical

• Symptoms of anemia• Jaundice• Pallor• Splenomegaly / hepatosplenomegaly

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

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

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

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,

Sickle cell anaemia

Diagnosis 1-Sickling test2- Hemoglobin

electrophoresis:Increased hemoglobin S(90% Hgb S, 10% Hgb F, small fraction of Hgb

A2)

Sickle cell anaemia

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

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)

Thalassaemia

Beta ThalassaemiaDefective chain synthesis

Excess chain Precipitation

cell membrane damageCirculating Red cell

Anaemia

Bone marrow expansion

skeletal changes & hyper metabolism

Erythropoietin increased

Ineffective erythropoiesis

Bone marrow

blood transfusion

Iron overload

Complication and death

Hemolytic

Iron absorption

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

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

Hereditary spherocytosis (HS) Laboratory features - hemolytic anemia - blood smear spherocytes - increased osmotic fragility time

G6PD DEFICIENCYFunction of G6PD

G6PD

GSSG 2 GSH

NADPH NADP

2 H2O H2O2

6-PG G6P

Hgb

Sulf-Hgb

Heinz bodies

Hemolysis

InfectionsDrugs

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

G6PD DEFICIENCY DIAGNOSIS : QUANTITATIVE ASSAY

DETECTING LOW ENZYME

TREATMENT – SUPPORTIVE AND PREVENTATIVE

Acquired hemolytic anaemia

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

Introduction Increased RBC Destruction – Short RBC life span <120 days. Normocytic normochromic,

reticulocytosis. Anemia, Jaundice, marrow

hyperplasia Splenomegaly, increased bilirubin

Types of acquired HA AutoImmune Haemolytic Anemias(+ve Direct CoombꞋs) Alloimmune haemolytic anemias Drug-induced immune haemolytic

anemias

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

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

DIRECT ANTIGLOBULIN TEST (DAT)Coomb′s test

Procedure of DAT1. Take 2-3 drops of blood to be tested in a clean labeled tube.2. 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.

3. 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.

4. Mix, Centrifuge at 1000 rpm for 1 minutes immediately.5. Gently shake the tube to dislodge the cell button and see

for agglutination, use optical aid if needed. Record the result.

6. 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.

Indirect antiglobulin (coomb′s ) test

Procedure:1. 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.

2. Add 1 drop of 3-5% suspension of washed O Rh (D) positive red cells to the serum in the tube.

3. Mix and incubate at 37°C for 30-40 minutes.4. Centrifuge at 1000 rpm for 1 minutes.5. Examine for hemolysis and/or agglutination. Use

optical aid if necessary. Agglutination at this stage indicates the presence of saline (complete) antibodies.

6. If no agglutination is seen, wash cells 3-4 times in large volume of saline. Decant supernatant in each wash as completely as possible.

Procedure:7. Add 2 drops of AHG serum to the cells.8. Mix and centrifuge at 1000 rpm for 1

minutes immediately.9. Gently shake the tube to dislodge the

button and examine for agglutination, using optical aid. Record the result.

10. 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.

11. Auto control should be kept with IAT.

Training questions :What are the tests of hemolysis ? How to diagnose Autoimmune hemolytic

anaemia ?

Reference book : Essential Hematology . Dacie .