AUTOHAEMOLYSIS TEST

AUTOHAEMOLYSIS TEST

If the metabolic activity of the red cells and their cell membrane is defective, it may lead to hemolytic anemia. In some hemolytic anemia’s, the amount of lysis of red cells is reduced by glucose, while in others it is unaffected by glucose. This test is used for the differential diagnosis of hereditary spherocytosis and pyruvate kinase deficiency. When fresh normal blood is incubated for 24 to 48 hours in the presence of sterile glucose, the amount of lysis is markedly reduced.

Specimen Fresh venous blood, either defibrinated or anticoagulated with heparin.

A specimen from a known normal individual should be used as a control.

Reagents 

Sterile Glucose Solution (10 %) Dissolve 10 g of glucose in 100 ml distilled water and sterilise by steaming. 

Aqueous ammonia solution (0.04%) Dilute 0.1 ml (100 ul) of ammonia to 250 ml with distilled water.

Method

1. Keep 1 ml of whole blood at 4°C to be used as a control for 100 % lysis.

2. Centrifuge 1 ml of blood, separate the plasma and store it at 4°C to be used as a blank.

3. Place 1 ml of blood in each of the four sterile tubes (1, 2, 3 and 4).

4. To two tubes (labelled 1 and 2), add 0.1 ml of the glucose solution under aseptic conditions to avoid bacterial contamination.

5. Place all the four tubes at 37°C in an incubator.

6. Gently shake each tube after 24 hours to mix the contents thoroughly.

7. After 48 hours, check macroscopically for bacterial contamination, which may be indicated by turbidity or offensive odour. If not contaminated, pool the contents of two tubes with glucose (1, 2) and the contents of two tubes without glucose (3, 4).

8. Estimate the PCV (haematocrit L/L) of blood in each tube either by Wintrobe's or by microhaematocrit method.

9. Centrifuge the remaining blood in the two tubes to separate the plasma.

10. Dilute the pre-incubation plasma (from step 2) and post-incubation plasma with and without glucose (from step 9) 1:10 with 0.04% ammonia.

11. Dilute the blood sample from step 1) 1:200 with 0.04 % ammonia for 100 % lysis.

12. Read the optical densities of diluted postincubation plasma. Use diluted pre-incubation plasma (from step 10) as a blank.

13. Read the optical density of the 100 % lysis control. 

Calculation To summarise,

1. Tubes 1 and 2 contain plasma after 48 hours incubation with glucose. Dilute 1:10 with 0.04 % ammonia

2. Tubes 3 and 4 contain plasma after 48 hours incubation without glucose. Dilute 1:10 with 0.04 % ammonia.

3. Pre-incubation plasma from step 2 acts as blank. Dilute 1:10 with 0.04% ammonia.

4. Blood at 4°C from step 1 acts as 100 % lysis standard. Dilute 1:200 with 0.04% ammonia.

Dilution of plasma Normal range Lysis after 48 hours at 37°C

without glucose 1.0-2.5 %

with glucose 0.25-0.75 %

 Interpretation The autohaemolysis test is positive when haemolysis without glucose is more than 2.5 %. In hereditary spherocytosis, the autohaemolysis test is positive, and is corrected with glucose, showing results within the normal range. In pyruvate kinase deficiency, the autohaemolysis test is positive, but is not corrected with glucose.