1. Tabuk University Hematology – 1, MLT 205 Hereditary Spherocytosis
Faculty of Applied Medical Sciences
Department Of Medical Lab. Technology
2nd Year – Level 4 – AY
Hematology – 1, MLT 205
Hereditary Spherocytosis
Mr. Waggas Ela’as, M.Sc, MLT

2. Objectives
To know spherocytes and the difference between them and normal Biconcave red cells.
To review red cell membrane constituents.
To know H.S as one of the Hemolytic anemias.
To know the lab. Diagnosis of H.S and the principle of Osmotic fragility Test.

3. Defect in genes for the skeletal proteins of the red cells membrane
Defect in genes for the skeletal proteins of the red cells membrane . (spectrin, ankyrin and band 3 ).
Parts of the lipid bilayer not supported by skeleton are removed resulting in  surface to volume ratio – causes the cells to become spheroidal.
Less deformable than normal RBCs.
Destruction in micro circulations of the spleen

4. The red cell membrane

5. Normal red blood cells easily change shape to move effectively through the small blood vessels between organs of the body.
In spherocytosis the red blood cells are very round and have difficulty changing this shape. The lack of ability to change shapes makes moving through the small blood vessels difficult. Therefore, the red blood cells stay in the spleen longer than normal.
This lengthy stay in the spleen damages the cell membranes.

6. Then they become spherical
Bone marrow produce normal biconcave discs but they lose membrane in R.E.S = Reticulo Endothelial System (mainly Spleen).
Then they become spherical
Ultimately, Spherocytes are destroyed in the spleen EXTRAVASCULAR HEMOLYSIS
Spectrin deficiency is the most common defect.

7. Spherocyte formation & destruction

9. Clinical Features Fluctuating Jaundice . Anemia
Mild to moderate splenomegaly
Aplastic crises
Gall stones

10. Laboratory Findings : - minimal anemia -reticulocytosis (5 – 20%)
Laboratory Findings : - minimal anemia -reticulocytosis (5 – 20%). -increased mean corpuscular hemoglobin concentration (MCHC), -spherocytes on the peripheral blood smear. -DAT : Direct Antihuman Globulin Test is negative -hyperbilirubinemia -The osmotic fragility test is increased. -Autohaemolysis is increased and is corrected by Glucose.

11. The blood Film Anisocytosis = cells vary in size
Spherocytes.(less in diameter, greater thickness, no central pallor).
Polychromatic cells.

13. Polychromatic red cells are larger than mature erythrocytes and still contain ribosomes and mitochondria, giving the cell a blue tint. When polychromatic RBCs are stained with a supravital dye (new methylene blue or cresyl violet) ribosomes clump to form a blue stained granulars called reticulum. These are called reticulocytes.

14. Supravital stain of blood from a patient with hemolytic anemia
Supravital stain of blood from a patient with hemolytic anemia. The reticulocytes are the cells with the dark blue dots and curved linear structures (reticulum) in the cytoplasm

15. Scanning electron micrograph of a normal biconcave erythrocyte
Scanning electron micrograph of a spherocyte. It is smaller, round, and lacks concavities

16. The osmotic fragility test
Non specific test.
The osmotic fragility test is a measure of the resistance of erythrocytes to hemolysis by osmotic stress. The test consists of exposing red cells to decreasing strengths of hypotonic saline solutions and measuring the degree of hemolysis colorimetrically at room temperature. (15 ° - 20 ° C).

17. Normal results in osmotic fragility test
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 and they are therefore particularly susceptible to osmotic lysis.
Hypotonic solutions Cells will swell
Hpertonic solutions Cells will shrink
Isotonic solutions Cells will remain
Normal results in osmotic fragility test
Fresh blood (g/l NaCl)
Blood incubated 24 hours, 37°C (g/l NaCl)
Initial lysis
5.0
7.0
Complete lysis
3.0
2.0
MCF* (50% lysis)
4.0 – 4.45
4.65 – 5.9
* MCF : Mean Cell Fragility

18. Autohaemolysis Test Is increased and is corrected by Glucose.
when sterile defibrinated blood is incubated at 37°C, normal red blood cells hemolyze slowly; cells with membrane or metabolic defects do so to a greater
extent.
Normal Lysis at 48 hours:
without added glucose, %
with added glucose, 0-0.9%.
In membrane disorders such as HS, the rate of glucose consumption is increased to compensate for an increased cation leak through the membrane

19. DAT : Direct Antihuman Globulin Test
Also called Coomb’s Test.
Red cells coated with antibodies do not agglutinate directly when centrifuged.  These cells are said to be sensitized .
In order for agglutination to occur, AHG is added. (Anti Human Globulin).
This will form a "bridge" between the antibodies coating red cells, causing agglutination.

20. Direct Antihuman Globulin Test

21. Differential diagnosis Other causes of spherocytes in peripheral blood film: 1.Autoimmune hemolytic anaemias. (DAT +ve) 2.Hemolytic transfusion reactions. 3.Thermal injury 4.Clostridial septicaemia, 5.Severe hypophosphataemia 6.ABO incompatibility (neonates)