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Practical Hematology Lab - LAB 14 - Sucrose Hemolysis Test Introduction 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. It is characterized by haemoglobinuria during sleep (nocturnal haemoglobinuria), jaundice, and haemosiderinuria.

Introduction PNH is an acquired clonal disorder resulting from a somatic mutation occurring in a haemopoietic stem cell. The characteristic feature of cells belonging to the PNH clone is that they are deficient in several cellmembranebound proteins including red cell: 1. Acetylcholine esterase 2. Neutrophil alkaline phosphatase 3. CD55 (decay accelerating factor or DAF) 4. Homologous restriction factor (HRF) 5. CD59 (membrane inhibitor of reactive lysis or MIRL). Introduction 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, and it has been established that the deficiency of CD59 is largely responsible for the complement sensitivity of PNH red cells. PNH type III red cells have a complete deficiency of CD59, whereas PNH type II red cells have only a partial deficiency, and it is this difference that accounts for their variable sensitivities to complement. Introduction PNH red cells are unusually susceptible to lysis by complement. This can be demonstrated in vitro by a variety of tests e.g: 1.Sugar water test. 2.Sucrose lysis test. 3.The acidified-serum [Ham test].

Introduction A characteristic feature of a positive test for PNH is that not all the patient's cells undergo lysis, even if the conditions of the test are made optimal for lysis. This is because only a proportion of any patient's PNH red cell population is hypersensitive to lysis by complement. This population varies from patient to patient. There is a direct relationship between the proportion of red cells that can be lysed (in any of the diagnostic tests) and the severity of in vivo haemolysis. Reagents and Equipment 1. 2.

3. 4. Sucrose solution (isotonic). Cyanmethemoglobin reagent. ( drabkins reagent) Test tubes. ABO compatible serum (or serum from type AB blood) from a normal donor. 5. Sodium chloride, 0.85% w/v. 6. Pipets, Spectrophotometer. 540 nm. 7. Specimen must be fresh. 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. A.Prepare washed cell 1. 1 mL patient blood . 2. Add normal saline ( sodium chloride 0.85%) 3. Mix , centrifuge at high speed for 5 min. carefully remove supernatant. 4. Repeat step 2,3.

1. Add from W.C tube 3 drop of cells 2. 3 drop of N.S, mix. W.C 50% B.Prepare washed cell 50% solution Washed cell Procedure C. Prepare blood-sucrose tube, Blank1 tube Blank1 bloodsucrose

Reagent 1.7 ml 1.7 ml Sucrose solution. 1. 0.1ml 0.1ml ABO Compatible

serum. The 50% W.C, Mix by inversion 2. 0.2ml Incubate at R.T 30 min. 3. 4. D. Prepare of Total ,Test, Blank tube Test 4750 l

Blank 4750 l Total 4750 l 250 l, mix incubation 10 min. ----- ----- -----

250 l, mix incubation 10 min. ----- 250 l, mix incubation 10 min. ----- ----- Reagent Drabkins After complete 30

min. incubation, & remix blood-serum tube. After complete 30 min. incubation, & remix Blank1 tube. After complete 5 min. centrifuge of remaining bloodsucrose tube, from the supernatant. 1. 2. 3. 4.

procedure 10.Transfer above mixtures to a cuvet and read in a spectrophotometer at a wavelength of 540 nm. Setting the blank al 0.0 optical density. Record the O.D. readings for each sample. 11.Calculate the percent hemolysis for each specimen as shown below. 100 O.D. Test ------------------------ Percent Hemolysis = O.D. Total

Interpretation Of Results 1.Hemolysis 5% or less is considered negative within normal limits. 2.Hemolysis of 6 to 10% is thought to be borderline. 3.Positive results will show greater than 10% hemolysis. Discussion 1.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%). 2.Results of the sucrose hemolysis test should

correlate with the acid serum test.

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