The sucrose hemolysis test is used as a confirmatory test for paroxysmal nocturnal hemoglobinuria (PNH) when the sugar water test is positive. • Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal disorder of haemopoiesis in which the patient's red cells are abnormally sensitive to lysis by normal constituents of plasma.
In its classical form, it is characterized by haemoglobinuria during sleep (nocturnal haemoglobinuria), jaundice, and haemosiderinuria. Not uncommonly, however, PNH presents as an obscure anaemia without obvious evidence of intravascular haemolysis, or it develops in a patient suffering from aplastic anaemia or more rarely from myelofibrosis or chronic myeloid leukaemia.
characteristic feature of cells belonging to the PNH • Acetylcholine esterase, • Neutrophil alkaline phosphatase, • CD55 (decay accelerating factor or DAF), • Homologous restriction factor (HRF), and CD59 (membrane inhibitor of reactive lysis or MIRL), among others.
CD55, CD59, and HRF all have roles in the protection of the cell against complement-mediated attack. CD59 inhibits the formation of the terminal complex of complement • It has been established that the deficiency of CD59 is largely responsible for the complement sensitivity of PNH red cells.
In the sucrose lysis test, a low ionic strength is thought to lead to the binding of IgG molecules nonspecifically to the cell membrane and to the subsequent activation of complement via the classical sequence. • In addition, the alternative pathway appears to be activated. In each test, PNH cells undergo lysis because of their greatly increased sensitivity to lysis by complement.
Specimen Citrated whole blood: 1 part 0.109 M sodium citrate to 9 parts whole blood.
Principle • Washed red blood cells are incubated in an isotonic sucrose solution containing normal ABO compatible serum. At low ionic concentrations, red blood cells absorb complement components from serum. • Because PNH red blood cells are much more sensitive than normal red cells they will hemolyzed under these conditions. • The normal red blood cells will not. At the end of the incubation period the mixture is examined for hemolysis.
Calculate the percent hemolysis • Percent Hemolysis = (O.D. Test\ O.D. Total) x 100
Interpretation of results • Hemolysis 5% or less is considered negative within normal limits. • Hemolysis of 6 to 10% is thought to be borderline. • Positive results will show greater than 10% hemolysis.
Discussion • Increased hemolysis (generally less than 10%) may be found in some patients with leukemia or myelofibrosis; whereas patients with PNH show 10% to 80% hemolysis (will only rarely be as Iow a 5%). • Results of the sucrose hemolysis test should correlate with the acid serum test.