SUCROSE HEMOLYSIS TEST

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SUCROSE HEMOLYSIS TEST

• Mr. Mohammed A. Jaber

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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.

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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.

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Introduction

• The characteristic feature of cells belonging to
  the PNH clone is that they are deficient in
  several cell-membranebound proteins including
  red cell
• Acetylcholine esterase,
• Neutrophil alkaline phosphatase,
• CD55 (decay accelerating factor or DAF),
• Homologous restriction factor (HRF), and
• CD59 (membrane inhibitor of reactive lysis or
  MIRL).

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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.

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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
• Sugar water test.
• Sucrose lysis test.
• The acidified-serum Ham test.

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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.

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Reagents and Equipment

• Sucrose solution (isotonic).
• Cyanmethemoglobin reagent. ( drabkins reagent)
• Test tubes.
• ABO compatible serum (or serum from type AB
  blood) from a normal donor.
• Sodium chloride, 0.85 w/v.
• Pipets, Spectrophotometer. 540 nm.
• Specimen must be fresh.
• Citrated whole blood 1 part 0.109 M sodium
  citrate to 9 parts whole blood.

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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.

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Procedure

• Prepare washed cell
• 1 mL patient blood .
• Add normal saline ( sodium chloride 0.85)
• Mix , centrifuge at high speed for 5 min.
  carefully remove supernatant.
• Repeat step 2,3.

Washed cell
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Prepare washed cell 50 solution

• Prepare washed cell 50 solution
• Add from W.C tube 3 drop of cells
• 3 drop of N.S, mix.

W.C 50
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Prepare blood-sucrose tube, Blank1 tube
Blank1 blood-sucrose Reagent
1.7 ml 1.7 ml Sucrose solution. 1.
0.1ml 0.1ml ABO Compatible serum. 2.
0.2ml The 50 W.C, Mix by inversion 3.
Incubate at R.T 30 min. 4.
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Prepare of Total ,Test, Blank tube
Test Blank Total Reagent
4750 µl 4750 µl 4750 µl Drabkins 1.
----- ----- 250 µl, mix incubation 10 min. After complete 30 min. incubation, remix blood-serum tube. 2.
----- 250 µl, mix incubation 10 min. ----- After complete 30 min. incubation, remix Blank1 tube. 3.
250 µl, mix incubation 10 min. ----- ----- After complete 5 min. centrifuge of remaining blood-sucrose tube, from the supernatant. 4.
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procedure

• 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.
• Calculate the percent hemolysis for each specimen
  as shown below.

Percent Hemolysis O.D. Test x 100
Percent Hemolysis O.D. Total x 100
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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.

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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.