OSMOTIC FRAGILITY TEST AND PROCEDURE

OSMOTIC FRAGILITY TEST AND PROCEDURE

OSMOTIC FRAGILITY TEST

PROCEDURE FOR  OSMOTIC FRAGILITY TEST

OSMOTIC FRAGILITY TEST PROCEDURE

TECHNIQUE FOR OSMOTIC FRAGILITY TEST

OSMOTIC FRAGILITY TEST INTERPRETATION

OSMOTIC FRAGILITY TEST

This test may be required to investigate haemolytic anaemias due to red cell membrane defect, e.g. spherocytosis. Principle The test determines the resistance of red cells to haemolysis in various concentrations of hypotonic saline solutions (Fig. 6.3). The ability of erythrocytes to absorb water without lysis depends on the ratio of volume to surface area of the cells. In the normal red cells, the volume may increase up to 70 % before lysis can occur. Spherocytes have reduced surface to volume ratio and therefore absorb less water.

principal of osmotic fragility test

 

Specimen Serum 

Technique 

(i) Mix one drop of patient's serum with one drop of well mixed latex suspension coated with DNP, on a slide. 

(ii) Rotate for one minute and examine for agglutination. 

(iii) Run a known positive control in the same way along with the test. Result Agglutination appearing within one minute indicates a positive test.

Reagents

1. The stock solution of sodium chloride osmotically equivalent to 10 % is prepared as follows.

Sodium chloride 90gm

Disodium hydrogen phosphate 13.65 g 

Sodium dihydrogen phosphate 2.34 g

Distilled water  1 liter

Specimen 

Heparinised venous blood or defibrinated blood should be used. Oxalated, citrated or EDTA blood should be avoided because additional salts may interfere with the test. The test should be carried out immediately. If delay is expected, the blood should be preserved at 4°C and the test performed within six hours. 

Technique 

 (i) Dilute the stock solution of sodium chloride 1:10 with distilled water to obtain a 1 % solution.

(ii) take 12 tubes and prepare dilutions as follows:

(ii) take 12 tubes and prepare dilutions as follows:
Table no	1% nacl (ML)	Distilled water(ml)	Final concentration
1	4.5	0.5	0.9
2	3.75	1.25	0.75
3	3.75	1.75	0.65
4	3.25	2	0.6
5	2.75	2.25	0.55
6	2.5	2.5	0.5
7	2.25	2.75	0.45
8	2	3	0.4
9	1.75	3.25	0.35
10	1.5	3.5	0.3
11	1	4	0.2
12	0.5	4.5	0.1

(iii) Add 0.05 ml of the blood to each of the tubes and mix immediately by gently inverting several times. 

(iv) A blood sample from a normal individual is run in parallel as a control. 

(v) Stand at room temperature (18-20°C) for 30 minutes. 

(vi) Remix and centrifuge. 

(vii) The amount of lysis in each tube is determined colorimetrically using a green filter, at 540 nm. The first tube in the series serves as a blank (0 % lysis) as it contains 0.9 %. isotonic saline. The 12th tube containing 0.1 % saline serves as 100 % lysis since this gives complete lysis. 

Calculation

Plot a graph of % lysis against concentration of sodium chloride (Fig 6.4) Record the results as

1. Complete lysis or maximum resistance as the highest concentration of saline showing complete lysis.

2. Initial lysis or minimum resistance as highest concentration of saline in which lysis appears to be just detectable.

Normal range of osmotic fragility is indicated as shown below:
Concentration of sodium chloride	% Lysis
0.1	100
0.2	100
0.3	97-100
0.35	90-99
0.45	50-90
0.5	5-45
0.55	0-6
0.6	0
0.65	0
0.7	0
0.75	0
0.8	0
0.85	0

The normal fragility curve should fall in the shaded area shown in the graph (Fig. 6.4).

Alternatively, the osmotic fragility may be expressed as the concentration of NaCl causing 50 % haemolysis, Mean Corpuscular Fragility (MCF), from the graph. The normal range for MCF is 0.4 to 0.45 g/100 ml.

Note The osmotic fragility is increased when the lysis starts at a concentration higher than 0.5 % and is decreased when 100 % lysis starts at a concentration lower than 0.3 %. 

Interpretation Various haematological disorders show characteristic changes in osmotic fragil. ity. Osmotic fragility is increased (i.e. resistance reduced) in hereditary spherocytosis, in ABO haemolytic disease, and haemolytic disease of the new-born. It is reduced (i.e. resistance increased) in thalassaemia, iron deficiency anaemia, liver disease and splenomegaly.