Approach for diagnosis of Hemolytic anemias

Classification of hemolytic anemia

◙-Hemolytic anemia
1-Corpuscular causes
►Congenital
a- Disorders of membrane:
- Spherocytosis
- Ovalocytosis
- Stomatocytosis
b-Enzymopathies:
-G6PD deficiency
-Pyruvate kinase deficiency
c-Hemoglobinopathies:
-Quantitative e.g. Thalassemia
-Qualitative e.g Sickle cell

►Acquired
PNH (Membrane defect)

2-Extracorpuscular causes
►Abnormal plasma constituents
a- Immune hemolytic anemia
-Hemolytic disease of newborn
-Drug immune
-Autoimmune (Warm & Cold)
b-Drugs and toxins
►Abnormal physical environment
a- Blood vessel abnormalities
-Microangiopathic hemolytic anemia
-Mechanical damage by artificial valves
b-Hypersplenism

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Red Cell Membrane
The red blood cell membrane is a phospholipid bilayer composed of proteins
50%, lipids 40% andcarbohydrates 10%.
The membrane skeletonis formed by structural proteins that include:
- α and β spectrin
- ankyrin
- protein 4.1
- actin
Defects in these proteins lead to some abnormalities in the shape of the red cell
membrane i.e.spherocytosis and elliptocytosis.

Hereditary Spherocytosis

Hereditary spherocytosis is caused by the following:

►Ankyrin deficiency or abnormalities
►Spectrin deficiency or abnormalities
►Protein 4.2 abnormalities
These defects lead to loss of the biconcave shape of red blood cells causing
them to become spherical in shape.

Blood Smear
►Micro-spheroycytes
Laboratory findings
►Hb↓, RBC ↓
►Reticulocytes ↑ (due to increased bone marrow activity)
►The classic finding is increased osmotic fragility
►Direct antiglobulin test (Coomb'c test) is –ve

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Hereditary spherocytosis: Red cells are spherical in shape with no central pallor

Hereditary Elliptocytosis
Hereditary elliptocytosis is caused by the following causes:
►α or β spectrin mutants leading to defective spectrin dimer formation
►α or β spectrin mutants leading to defective spectrin-ankyrin associations
►protein 4.1 deficiency or abnormality
►band 3 abnormality
Blood smear
► Elliptocytes
Laboratory findings:
Laboratory findings are similar to those of hereditary spherocytosis.

Hereditary elliptocytosis: The red cells are elliptical in shape.

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INVESTIGATION OF MEMBRANE DEFECTS
■The osmotic fragility test
●gives an indication of the surface area/volume ratio of erythrocytes.
●Itsgreatest usefulness is in the diagnosis of hereditary spherocytosis.
●The test may also be used in screening for thalassemia.
●Red cells that are spherocytic, due to any cause, take up less water in a
hypotonicsolution before rupturing than do normal red cells.

Principle of the test

Small volumes of blood are mixed with a large volumeof buffered saline
solutions of varying concentration. Thefraction of red cells lysed at each saline
concentration is determined colorimetrically. The test is normally carried out at
room temperature (15–25C).

Reagents
● Prepare a stock solution of buffered sodium chloride, osmotically equivalent
to 100 g/l NaCl. This solution can be kept for months at 4C in a well-stoppered
bottle.
●In preparing hypotonic solutions for use, it is convenient to make first a 10 g/l
solution from the 100 g/l Na Cl stock solution by dilution with water.
The following dilutions are prepared: 9.0, 7.5, 6.5, 6.0, 5.5, 5.0, 4.0, 3.5, 3.0,
2.0 and 1.0 g/l .
●It is convenient to make up 50 ml of each dilution.
Method
●Blood sample : Heparinized venous blood .
●The test should be carried out within 2 h of collection with blood stored at
room temperature or within 6 h if the blood has been kept at 4 C.

1. Deliver 5.0 ml of each of the 11 saline solutions into 12 x75 mmtest tubes.
Add 5.0 ml of water to the 12thtube.
2. Add to each tube 50 ul of well-mixed blood and mix immediately by inverting
the tubes several times, avoiding foam.
3. Leave the suspensions for 30 min at room temperature.
Mix again and then centrifuge for 5 min at 1200 g.
4. Remove the supernatants and estimate the amount of lysis in each usinga
spectrophotometer at a wavelength setting of 540 nm.
The supernatant from tube 1 is used as a blank (osmotically equivalent to 9 g/l
NaCl).

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5. Assign a value of 100% lysis to the reading with the supernatant of tube 12
(water) and express the readings from the other tubes as a percentage of the
value of tube
6. Plot the results against the NaCl concentration
Notes
1.The measurement of osmotic fragility is a simple procedure that requires a
minimum of equipment and can give excellent results if carried out carefully.
2.The blood must be delivered into the 12 tubes with greatcare. The critical
point is that the amount added to each tube must be the same.

■The sigmoid shape of the normal osmotic fragility curve indicates that normal
red cells vary in their resistance to hypotonic solutions.
■ Indeed, this resistance varies gradually (osmotically) as a function of red cell
age, with the youngest cells being the most resistant and the oldest cells being
the most fragile.
■The reason for this is that old cells have a higher sodium content and a
decreased capacity to pump out sodium.

Interpretation of Results

▪The osmotic fragility of freshly taken red cells reflects their ability to take up a
certain amount of water before lysing. This is determined by their volume-to-
surface area ratio.

▪The ability of the normal red cell to withstand hypotonicity results from its
biconcave shape, which allows the cell to increase its volume by about 70 %
before the surface membrane is stretched; once this limit is reached lysis occurs.

▪Spherocytes have an increased volume-to-surface area ratio; their ability to

take in water before stretching the surface membrane is thus more limited than
normal→they are more susceptible to osmotic lysis.

▪The increase in osmotic fragility is a property of the spheroidal shape of the

cell and is independent of the cause of the spherocytosis.

▪Decreased osmotic fragility indicates the presence of unusually flattened red

cells in which the volume-to-surface area ratio is decreased. This occurs in iron
deficiency anemia and thalassemia in which red cells with a low mean cell
hemoglobin (MCH) and mean cell volume (MCV) are unusually resistant to
osmotic lysis.

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Sickle Cell Anemia

Sickle cell disease is a hemoglobin disorder in which there is replacement of
glumatic acid by valineat position 6 in the βchain.
Hb S is insoluble and forms crystals when exposed to low oxygen tension causing
the red cellto change into a sickle shape.

Forms of Sickle Cell Disease

Hb SS
■The homozygous state or sickle cell anemia (genotype SS) causes moderate to
severe hemolytic anemia.
■The main clinical disability arises from repeated attacks of vascular occlusion
by sickled RBCs resulting in eventually organ damage.
Note:
Hb SS is composed of two α chains and two βS chains.

Hb AS
The heterozygous state or sickle cell trait (genotype AS) is very common and it
affects millions of people worldwide. It is known as the carrier state.
There are no associated hematological abnormalities. In vivo sickling occurs only
at very high altitudes and low oxygen pressures.
Note:
Hb AS is composed of two α chains, one β chain and one βS chain.
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■ Other Forms of Sickle Cell Disease

■Sickle cell/ Hb C disease

This is a compound heterozygous state for Hbs S and C. It usually results in a
milder formof sickle cell disease.

■Sickle β/thalassemia
This disease arises as a result of inheritance of one Hb S gene and one β
thalassemia gene.

Blood Smear
Sickle cells and target cells

Laboratory Findings
General Tests:
- Hb↓

Screening Tests:
- Solubility test
- Sickling test
Confirmatory Test: Hb electrophoresis

Sickle cell anemia: peripheral blood film showing sickle cells.

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Screening Tests for Sickle Cell Disease
These tests depend on the decreased solubility of Hb S at low oxygen tensions.
All sickle tests, whether positive or negative, must be confirmed by
electrophoresis or HPLC (High performance liquid chromatography)

Sickling Test

Principle
The sickling phenomenon may be demonstrated in a thin wet film of blood
sealed with petroleum jelly/paraffin wax mixture or nail varnish. If Hb S is
present the red cells lose their smooth round shape and become sickled.
Changes should be apparent after 30 -60 min at 37◦ C.
Reagent
The reagent is 2% sodium meta-bisulphite. It should be freshly prepared.
Procedure
1. On a clean slide, add 1 drop of anti-coagulated blood.
2. Add 5 drops of the freshly prepared reagent to the drop of blood.
3. Mix and place a cover slip on top of the slide.
4. Place the slide inside a wet petri-dish and seal with a paraffin wax/ petroleum
jelly mixture or nail varnish.
5. Incubate for 30 min at 37◦ C.
6. Examine under the microscope (x40)
Results
►+ve: A positive result is indicated by the presence of sickled cells.
Hb SS, Hb AS, Hb S/C and Hb S/β thalassemia all give +ve results.

► -ve: A negative result means there are no sickle cells and the cells are round
in shape. Hb AA gives a –ve result.

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Positive sickling test.

Solubility Test
Principle
Sickle cell Hb is insoluble (resist lysis) in the deoxygenated state in a high
molarity phosphate buffer. Thecrystals that form refract light and cause the
solution to be turbid.
Cellscontaining normal Hb, are prone to lysis, and thus the Hb is dissolved giving
a clear solution.
Reagents
Sodium dithionite as a lysing agent.
Procedure
1. Pipette 4 ml of buffer into three solubility test tubes containing sodium
dithionite (the tubesare from the testing kit and they already contain sodium
dithionite).
2. Mix the contents of the tube.
3. Add 50 micro liters of EDTA packed red cells.
4. Mix well and leave to stand for 10-15 minutes.
5. Place the tube on a white rack with narrow black lines and read for turbidity
(see if the linesare clearly visible or not).
6. Record the results.
Results
►A positive result is indicated by a turbid solution through which the black
lines could not beseen.
►A negative result is indicated by a clear solution through which the lines could
be easilyseen.

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Solubility test for sickle cell anemia. The tube on the left shows a –
ve result (clear) whereas the tube on the right represents a
+veresult (turbid).

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