Osmotic Fragility Test and NESTROFT

1. Osmotic
fragility test
Presenters : Dr. Sivaranjini
N
Dr. Krina Patel

2. Reagents
 Prepare a stock solution of buffered sodium
chloride, osmotically equivalent to 100 g/l
 In preparing hypotonic solutions for use, it is
convenient to make first a 10 g/l solution from the
100 g/l NaCl stock solution by dilution with water.
 Dilutions equivalent to 9.0, 7.5, 6.5, 6.0, 5.5, 5.0,
4.0, 3.5, 3.0, 2.0 and 1.0 g/l are then made.

3. Preparation of serial dilutions
 Prepare a stock solution dissolving 10g NaCl in
1litre of distilled water;
i.e. 10g in 1000mL or 10x1000 mg in 1000 mL
 1mL of such a solution will contain 10mg NaCl
 From this stock solution prepare further dilutions.

4. Concentration of
solution
Volume of stock
solution (mL)
Volume of
distilled water
(mL)
9.0 9.0  4.5 1.0  0.5
7.5 7.5 3.75 2.5  1.25
6.5 6.5 3.25 3.5  1.75
6.0 6.0  3.0 4.0  2.0
5.5 5.5  2.75 4.5  2.25
5.0 5.0  2.5 5.0  2.5
4.0 4.0  2.0 6.0  3.0
3.5 3.5  1.75 6.5  3.25
3.0 3.0  1.5 7.0  3.5
2.0 2.0  1.0 3.0  1.5
1.0 1.0  0.5 9.0  4.5

5. Procedure
• Deliver 5.0 ml of each of the 11 saline
solutions into 12 test tubes.
• Add 5.0 ml of water to the 12th tube.
• Add to each tube 50 µl of well-mixed
blood and mix immediately by inverting
the tubes several times, avoiding foam.
• Leave the suspensions for 30 min at room
temperature.
• Mix again and then centrifuge for 5 min at
1200 rpm.

6. The sigmoid shape of the normal osmotic fragility
curve indicates that normal red cells vary in their
resistance to hypotonic solutions.

7. Osmotic Fragility after incubating the
Blood at 370C for 24 Hours
 Defibrinated blood should be used, care being
taken to ensure that sterility is maintained.
 Incubate 1 ml or 2 ml volumes of blood in
sterile 5 ml bottles.
 After 24 h, if no infection is evident, after
thoroughly mixing the sedimented red cells in
the overlying serum, estimate the fragility as
previously described.

8.  Because the fragility may be markedly
increased set up additional hypotonic
solutions containing 7.0 g/l and 8.0 g/l NaCl.
 In addition, use a solution equivalent to 12.0
g/l NaCl because sometimes, as in HS, lysis
may take place in 9.0 g/l NaCl.
 In this case, use the supernatant of the tube
containing 12.0 g/l NaCl as the blank in the
colorimetric estimation.

10. Principle

11.  The osmotic fragility of freshly taken red cells
reflects their ability to take up a certain
amount of water before lysing.
 This is determined by their volume-to-surface
area ratio.
 The ability of the normal red cell to withstand
hypotonicity results from its biconcave shape,
which allows the cell to increase its volume by
about 70% before the surface membrane is
stretched; once this limit is reached lysis
occurs.

12. Interpretation of results

13.  Spherocytes have an increased volume-to-
surface area ratio.
 Their ability to take in water before stretching
the surface membrane is thus more limited
than normal and they are therefore
particularly susceptible to osmotic lysis.
 The increase in osmotic fragility is a property
of the spheroidal shape of the cell and is
independent of the cause of the
spherocytosis.

14.  Decreased osmotic fragility indicates the
presence of unusually flattened red cells
(leptocytes) in which the volume-to-surface
area ratio is decreased.
 Such a change occurs in iron deficiency
anaemia and thalassaemia in which the red
cells with a low mean cell haemoglobin (MCH)
and mean cell volume (MCV) are unusually
resistant to osmotic lysis

15.  Reticulocytes and red cells from patients who
have been splenectomized also tend to have
a greater amount of membrane compared
with normal cells and are osmotically
resistant.
 In liver disease, target cells may be produced
by passive accumulation of lipid and these
cells, too, are resistant to osmotic lysis.

16.  The osmotic fragility of red cells that have an
abnormal membrane or have enzyme defects,
increases abnormally after incubation.
 In thalassaemia major and minor, osmotic
fragility is frequently markedly reduced after
incubation, again owing to a marked loss of
potassium.
 A similar, although usually less marked,
change is seen in iron deficiency anaemia.

17.  The increased osmotic fragility of normal red
cells, which occurs after incubation is mainly
caused by swelling of the cells associated
with an accumulation of sodium that exceeds
loss of potassium.
 During incubation for 24 hr., the metabolism of
the red cell becomes stressed and the
pumping mechanisms tend to fail, one factor
being a relative lack of glucose in the
medium.

18. Increased osmotic fragility/ shift to right
Hereditary spherocytosis Entire curve may be ‘shifted to the right’,
or most of it may be within the normal
range
but with a ‘tail’ of fragile cells. After
incubation for 24 h, abnormalities usually
more marked.
Hereditary elliptocytosis As in HS, but in general changes less
marked.
Hereditary stomatocytosis As in HS with large osmotically fragile
cells with low MCHC
Other inherited membrane
abnormalities
Results variable; with milder disorders
curve more likely to be abnormal after
incubation for 24 h
Autoimmune hemolytic
anaemia
Tail of fragile cells roughly proportional to
number of spherocytes; rest of curve
normal (or even left-shifted on account of
reticulocytosis)

19. Decreased osmotic fragility/ shift to left
Thalassemia Osmotic fragility decreased in all
forms of thalassaemia, usually the
entire curve is left-shifted
Enzyme abnormalities OF usually normal. After incubation
for 24 h, there may be a tail of
fragile cells.
Hereditary xerocytosis Increased resistance to osmotic
lysis and increased MCHC
Iron deficiency Curve shifted to left, wholly or
partly, depending on proportion of
hypochromic red cells

20. Modified Osmotic
Fragility Test/ NESTROFT
 Simple and inexpensive test for screening for
ß thalassaemia trait
 It is useful when quantification of haemoglobin
A2 is not possible and standardized
automated analyzers are not available for
accurate measurement of MCV and MCH.

21. Principle of NESTROFT

22.  Normally, red cells put in saline solution begin
to lyse at a saline concentration of 0.4-0.5%
and lysis is complete at 0.32%.
 However, in beta thalssemia trait, due to
alteration in osmotic resistance of the affected
RBC’s due to volume/surface area ratio
changes, lysis begins at a saline
concentration between 0.4-0.35% and it may
not be completed even at 0.1% solution.

23. Reagents
 NESTROFT is done at a saline concentration
of 0.36%.
 0.36% buffered saline (BS) prepared by
diluting 36ml of 1% buffered saline with 64ml
of distilled water (DW) to make 100ml

24. Procedure
 Two test tubes labelled as BS (2ml) and DW
(2ml) are taken and a drop of blood is added
to each of the tubes, which are then left
undisturbed for half an hour at room
temperature.

25. The line is clearly visible through DW tube,
If it is the same in BS tube; it is considered negative,
otherwise test result is interpreted as positive

26.  The tubes are then left undisturbed for 3
hours.
 At the end of 3 hours, the DW tube is usually
seen to be homogeneously pink with no
sediments.
 In the BS tube the negative test shows similar
findings as DW tube where as in a positive
case, a clear supernatant and a sediment
at bottom is observed.

27.  Because the false-positive rate is around 10%,
confirmation of a positive result requires referral of a
sample to a laboratory able to quantitate haemoglobin
A2.
 The test can also be used to screen for alpha
thalassaemia trait, with positive samples being referred
to a reference centre for DNA analysis.

28.  About 50% of samples containing
haemoglobin E also give a positive result.
 This is an advantage rather than a
disadvantage because detection of
haemoglobin E is important in predicting the
possibility of thalassaemia major or
intermedia in compound heterozygotes with ß
thalassaemia.

29. Summary
 The osmotic fragility test gives an indication of
the surface area/volume ratio of erythrocytes.
 Its greatest usefulness is in the diagnosis of
hereditary spherocytosis.
 The test may also be used in screening for
thalassaemia.

30. References
 Dacie and Lewis practical haematology.
 Piplani M. NESTROFT - A Valuable, Cost
Effective Screening Test for Beta Thalassemia
Trait in North Indian Punjabi Population.
Journal of clinical and diagnostic research.
2013.