Principle
This is a screening test for paroxysmal nocturnal hemoglobinuria (PNH) based on the examination of red cell fragility. This test is based on the fact that red cells adsorb complements from serum at low ionic concentrations. Because of their great sensitivity to complement, PNH cells will undergo lysis, but normal red cells do not.
Reagents & Equipment
1) An iso-osmotic solution of sucrose
Sucrose: 92.4 grams
Distilled water: 1000 ml
This solution can be stored at 4°C for 2–3 weeks.
2) Centrifuge
Procedure
1) Place 2 tubes labeled 1 & 2
2) Put 0.85 ml of sucrose solution in each tube.
3) Add 50µl serum of normal to each tube.
4) Add 100 µl of normal cell suspension to tube 1.
5) add 100µl patient cell suspension to tube 2.
6) Incubate at room temperature for ½ hour & then centrifuge.
7) Observe the supernatant for hemolysis.
No lysis in either tube: Negative
Lysis in tube 2- positive for sucrose lysis.
Quantitation
1) Add 5 ml normal saline to each tube.
2) Centrifuge at 3000 rpm for 2 minutes.
3) Read OD of tube 2 against tube 1 as blank (D1).
4) Add 1 drop of triton to tube 2 & mix in vertically 4-5 times to get complete lysis and read OD (D2).
5) Calculate % hemolysis = D1/D2 X 100.
Interpretation
The red cells from some cases of leukaemia or myelosclerosis may undergo a small amount of lysis, almost always < 10%; in such cases, the acidified serum test is usually negative & PNH should not be diagnosed. In PNH lysis varies from 10% -80% but exceptionally as little as 5%. Sucrose lysis and acidified serum lysis of PNH red cells are fairly closely correlated. The sucrose test is typically negative in HEMPAS.