SUCROSE HEMOLYSIS SUCROSE HEMOLYSIS TESTTEST

Mr. Mohammed A. Jaber

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.

Introduction Introduction

It is characterized by haemoglobinuria during sleep (nocturnal haemoglobinuria), jaundice, and haemosiderinuria.

PNH is an acquired clonal disorder resulting from a somatic mutation occurring in a haemopoietic stem cell.

Introduction Introduction

The characteristic feature of cells belonging to the PNH clone is that they are deficient in several cell-membrane–bound proteins including red cell:

1.Acetylcholine esterase,

2.Neutrophil alkaline phosphatase,

3.CD55 (decay accelerating factor or DAF),

4.Homologous restriction factor (HRF), and

5.CD59 (membrane inhibitor of reactive lysis or MIRL).

Introduction 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.

Introduction Introduction

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.

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 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.

Introduction Introduction

1. Sucrose solution (isotonic). 2. Cyanmethemoglobin reagent. ( drabkin’s

reagent)3. Test tubes. 4. 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.

Reagents and EquipmentReagents and Equipment

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.

PrinciplePrinciple

A.Prepare washed cell1. 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.

ProcedureProcedure

B.Prepare washed cell 50% solution

1. Add from W.C tube 3 drop of cells

2. 3 drop of N.S, mix.

B. Prepare washed cell 50% solution

W.C W.C 50%50%

ReagentReagent blood-blood-sucrossucrose e

BlanBlank1k1

1. Sucrose solution. 1.7 ml 1.7 ml

2. ABO Compatible serum.

0.1ml 0.1ml

3. The 50% W.C, Mix by inversion

0.2ml

4. Incubate at R.T 30 min.

C. Prepare blood-sucrose tube, Blank1 tube

Reagent Reagent TotalTotal BlankBlank TestTest11.. Drabkin’s 4750 µl 4750 µl 4750 µl

22..

After complete 30 min. incubation, & remix blood-serum tube.

250 µl, mix incubation

10 min.----- -----

33..

After complete 30 min. incubation, & remix Blank1 tube. -----

250 µl, mix incubation

10 min. -----

44..

After complete 5 min. centrifuge of remaining blood-sucrose tube, from the supernatant. ----- -----

250 µl, mix incubation

10 min.

D. Prepare of Total ,Test, Blank tube

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.

procedure

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.

Interpretation of resultsInterpretation of results

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.

Discussion