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.
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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


Defects of the red cell membrane


Thalassaemia, Sickle cell anemia
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
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,
Sickle cell anaemia
Diagnosis
1-Sickling test
2- Hemoglobin electrophoresis:
Increased hemoglobin S
(90% Hgb S, 10% Hgb F, small fraction of Hgb A2)
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
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)
Beta Thalassaemia
Defective  chain synthesis
Excess  chain Precipitation
cell membrane damage
Circulating Red cell
Bone marrow
Anaemia
Hemolytic
Erythropoietin increased
Ineffective erythropoiesis
blood transfusion
 Iron absorption
Bone marrow expansion
skeletal changes & hyper metabolism
Iron overload
Complication and death
Beta-Thalassemia major
laboratory features
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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
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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 DEFICIENCY
Function of G6PD
Infections
Drugs
2 H2O
GSSG
H2O2
2 GSH
NADPH NADP
6-PG
G6P
G6PD
Hgb
Sulf-Hgb
Heinz bodies
Hemolysis
Glucose 6-Phosphate Dehydrogenase
Functions

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Regenerates NADPH, allowing regeneration of
glutathione
Protects against oxidative stress
Lack of G6PD leads to hemolysis during
oxidative stress
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Infection
Medications
Fava beans
Oxidative stress leads to Heinz body
formation,  extravascular hemolysis
G6PD DEFICIENCY


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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
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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 DAT
1.
2.
3.
4.
5.
6.
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.
Indirect antiglobulin (coomb′s ) test
Procedure:
1.
2.
3.
4.
5.
6.
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.
Procedure:
7.
8.
9.
10.
11.
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.
Training questions :
What are the tests of hemolysis ?
 How to diagnose Autoimmune hemolytic anaemia ?
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Reference book :
Essential Hematology .
Dacie .