Specimen:
Whole blood anticoagulated with EDTA or heparin. Do not use oxalate or citrate anticoagulants. Specimens should be collected and handled aseptically, due to need to incubate sample (see below).
Test Set-Up:
1) Prepare Sodium Chloride solutions of varying concentrations (based on weight per volume), ranging from 0.20% to 0.90%. The pH should be 7.4.
2) Whole blood is added to the reagent tubes and tested under conditions listed as below.
3) After the testing period is complete, the tubes are scanned at 540 nm compared to distilled water, to detect the degree of hemolysis.
Testing is done with the patient sample compared to that of a known normal control:
1) fresh RBCs standing 1 hour at 20° C for 1 hour
2) RBCs incubated at 37° C for 24 hours
Interpretation
Normal osmotic fragility:
1) fresh RBCs start to hemolyze at 0.50% NaCl and is compete at 0.30% NaCl
2) incubated RBCs start to hemolyze at 0.70% and is complete between 0.15-0.40%. This test is more sensitive but less specific.
|
per cent NaCl (wgt/vol)
|
% hemolysis fresh RBCs
|
% hemolysis incubated
|
|
0.20%
|
97-100
|
95-100
|
|
0.30%
|
97-100
|
85-100
|
|
0.35%
|
90-99
|
75-100
|
|
0.40%
|
50-90
|
65-100
|
|
0.45%
|
5-45
|
55-95
|
|
0.50%
|
0-5
|
40-85
|
|
0.55%
|
0
|
15-70
|
|
0.60%
|
0
|
0-40
|
|
0.65%
|
0
|
0-10
|
|
0.70%
|
0
|
0-5
|
|
0.75%
|
0
|
0
|
Increased osmotic fragility (hemolysis occurs at higher salt concentrations):
(1) hereditary spherocytosis (milder forms may behave normally with the fresh sample, but will show increased fragility with the incubated specimen)
(2) spherocytosis due to immune hemolytic anemias
(3) hereditary stomatocytosis
(4) severe pyruvate kinase deficiency
Decreased osmotic fragility (hemolysis occurs at lower salt concentrations):
(1) thalassemia
(2) asplenia
(3) liver disease
(4) reticulocytosis
(5) iron deficiency anemia
Normal osmotic fragility:
(1) secondary hemolytic anemia
(2) paroxysmal nocturnal hemoglobinuria (PNH)
(3) G6PD deficiency
False positives:
(1) infectious diseases such as severe malaria, causing hemolysis independent of salt concentration
(2) bacterial contamination
