CHAPTER 4

OSMOTIC FRAGILITY TEST

 Acknowledgements

• Addisa Ababa University

• Jimma University
• Haramaya University
• Hawassa University
• University of Gondar
• American Society for Clinical Pathology
• Center for Disease Control and Prevention-Ethiopia

2
 Objectives

• Define osmotic fragility test

• Describe the procedure of the osmotic fragility test
• Interpret findings of the osmotic fragility test
• Discuss the purpose of performing an osmotic fragility
test
• Correlate hematologic disorders associated with
decreased and increased osmotic fragility test findings
 Outline

• Osmotic fragility definition

• Osmotic fragility procedure
• Reporting of OFT results
• Interpretation of osmotic fragility test findings
• Sources of error
 Osmotic Fragility Test

 Measures the resistance of RBCs to hemolysis by osmotic stress.(The

osmotic fragility test (OFT) is used to measure erythrocyte
resistance to hemolysis while being exposed to varying levels of
dilution of a saline solution.)

 Determines the resistance of the red cell to hemolyis in varying

concentrations of hypotonic solutions.

 Gives an indication of the surface area: volume ratio of erythrocytes

SA:V ratio

 Results of OFT are increased in blood sample ( erythrocytes) with

decrease SA / V ratio

 Useful in the diagnosis & confirmation of thalassemia & hereditary

spherocytosis.
 Osmotic Fragility Test Cont’d
 To maintain viability, the cells must be suspended in a
solution with an osmotic concentration relatively equal to
their interior osmotic concentration:
ISOTONIC :0.85% NaCl solution.
• Normal healthy erythrocytes can tolerate a broad range
of Hypotonic osmotic concentrations (0.85 to 0.55%)
due to their ability to expand or collapse as the
environment changes.
• This results from RBCs BICONCAVE shape (allows the
cell to increase its volume by about 70%
 Cont..
– Large flat cells such as target cells, have the ability to
take in more water than normal RBCs, resulting in a
decreased osmotic fragility.
– Is a screening test for the diagnosis of anemias

Fig Target cells

 Clinical Significance
1. Increased Surface – To – Volume Ratios
• it is more resistant to hemolysis and has decreased
fragility
• The larger the amount of red cell membrane (surface
area) in relation to the size of the cell, the more fluid
the cell is capable of absorbing before rupturing
 Iron-deficiency anemia
 Thalassemia
 Sickle cell anemia
 Liver disease and any condition associated with the presence
of target cells
 Cont.…
• As the red cell takes in fluid it becomes more
round (spherocytic). It therefore follows that
the spherocyte has the smallest surface area for
its volume, ruptures the most quickly, and has
increased fragility.
 Clinical Significance Cont’d
2. Decreased Surface – To – Volume Ratios
Increased osmotic fragility (decreased
resistance) is found in
 hemolytic anemias
 hereditary spherocytosis
 And whenever spherocytes are found.
 OFT cont..

Spherocytes have:
– a decreased SA / V ratio
– thinner surface membrane
– Lost ability to expand under osmotic pressure

Spherocytes
 Principle
In the osmotic fragility test, whole blood is added to varying
concentrations of buffered sodium chloride solution and allowed to
incubate at room temperature.

The amount of hemolysis in each saline concentration is then

determined by reading the supernatants on a spectrophotometer.
 Cont..
Reagents :-
• Stock buffered NaCl solution (10%).

-NaCl (powder) 180g

-Na2 HPO4 27.31g
-NaH2PO4.2H2O 4.86g .......

-Buffering effect-keep Ph 7.4

•Make up to 2L= d.H2O

•From (the stock solution, prepare first a 10 g/L 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, 1.0 g/L are then prepared.
 OFT cont..
When RBCs are put in hypotonic solution the cells take up
water and swell until an equilibrium is attained and then
the cell ruptures
– Normal hemolysis is proceeded by a phase in which
the red cells assume a spherical shape
– Lysis occurs (Na Cl) < 0.55%
 Osmotic Fragility Test Method
• Heparinized venous blood is used.
• Oxalated or citrated blood is unacceptable due to
additional salts added to it.
• The test should be carried out within 2 hours of collection if
kept at room temperature (15-20O C) or within 6 hours if
kept at 4° C.
 Osmotic Fragility Test Method
Note:
• Procedure requires a minimum of equipment
• The critical point is not that the amount be exactly 50 μl,
but rather that the amount added to each tube must be the
same.
• The sigmoid shape of the normal OF curve indicates that
normal red cells vary in their resistance to hypotonic
solutions.
• Indeed, this resistance varies gradually (osmotically) as a
function status of red cell age, with the youngest cells
being the most resistant and the oldest cells being the
most fragile. The reason for this is that old cells have a
higher sodium content and a decreased capacity to pump
out sodium.
Tube No Ml of 1% Ml of dist. % buffered
NaCl water NaCl

1 0.50 4.50 0.10

2 1.00 4.00 0.20
3 1.50 3.5 0.30
4 1.75 3.25 0.35
5 2.00 3.00 0.40
6 2.25 2.75 0.45
7 2.50 2.50 0.50
8 2.75 2.25 0.55
9 3.00 2.00 0.60
10 3.25 1.75 0.65
11 3.5 1.50 0.70
12 4.00 1.00 0.80
Procedure
1. Prepare dilutions of buffered sodium chloride and place in the appropriately labeled test tube (Table).
 Osmotic Fragility Test Method
1.Deliver 5.0 ml of the 11 saline solutions in test tubes. Add 5.0
mL of water to tube 12. The 12 dilutions are set up in duplicate.
2. Add to each tube 50 µL (microlitres) of well mixed blood and
mix immediately by inverting the tubes for several times
avoiding foam.
3. Incubate at room temperature for 30 min. Mix again and
centrifuge 1000g for 5 min.
4. Remove the supernatants carefully without including cells and
estimate the amount of lysis in a photometer at 540 nm.or
colorimeter provided with a yellow-green filter.
5. Use as blank the supernatant from tube 12 (osmotically
equivalent to 9 g/l NaCl).
6. Assign a value of 100 % lysis to reading with the supernatant of
tube 1 (water).
Note RCF or G = 1.12 x Radius x (rpm/1000)2
 OFT Method Cont’d and Calculation
7. Express the readings from the other tubes as a percentage of the
value of tube 1
8. Plot the results against the NaCl concentration (Fig.)
Calculation: Calculate % Hemolysis of each tube

% Hemolysis = Abs T- Abs B X 100

Abs Std – AbsB

But, Abs B (tube 12) = 0.00

% Hemolysis = Abs T X 100

Abs Std
Figure Osmotic fragility curves. Osmotic fragility curves of patients suffering from the following:

sickle cell anemia, β-thalassaemia major, hereditary spherocytosis, and “idiopathic” warm

autoimmune hemolytic anemia. The normal range is indicated by the unbroken lines (Dacie and

Lewis 2006)
 Example Calculation

Abs. Tube- 1 (100% STD) = 0.40

Abs. Tube. 5 = 0.20

% Hemolysis of tube 5 = 0.20 X 100 = 50%

0.40
 Interpretation of OFT Results

– Saline concentration at which Hemolysis begins

0.45 – 0.50%
– Saline concentration at which 50% RBC lyse( MCF)
0.40 – 0.45%
– Saline concentration at which Hemolysis is complete
0.30 – 0.35%
 Reporting of Results

• Report Red cell fragility test results using a curve on a

linear graph as increased, decreased, or normal

• Inspect the entire fragility curve

-Include the normal control
-Indicate the concentration of Salt in which;
(1) The highest concentration at which Hemolysis begins
(2) The highest concentration Hemolysis is complete
(3) 50% hemolysis occurred
-Median corpuscular fragility [MCF]
 OFT Normal Range
• Normal Range (200C, PH 7.4)
Tube No NaCl% Hemolysis (%)
1. 0.10 100%
2. 0.20 100%
3. 0.30 99-100%
4. 0.35 90-99%
5. 0.40 50-98%
6. 0.45 6-49%
7. 0.50 0.5%
8. 0.55 0%
9. 0.60 0%
10. 0.65 0%
11. 0.70 0%
12. 0.80 0%
 OFT after Incubation
• Normal OFT at room temperature does not rule out
hereditary spherocytosis because patients who are
mildly affected may have fewer than 1 - 2% spherocytes
in the total RBC population.
• Diagnose should not be based on morphologic grounds
• Therefore, patient blood samples should be incubated at
370c for 24 hours and OFT be repeated

• even they give normal or slight increase OFT in the un-

incubated OFT.
 Cont..
• Increasing the difference between a normal and abnormal
result is usually possible by increasing the susceptibility of
red cell to osmotic lysis by prior incubation of the blood
at 37oc for 24hrs.
• During incubation RBCS (HS cells) become metabolically
deprived and tend to lose membrane surface because of
their relative less membrane stability
 Cont..

• Both normal subjects & HS patients will have increased

OF after incubation, but the effect is more marked for

patients with HS.

• In HS lysis may occur in 8-9 g/L NaCl thus set up

additional hypotonic solution of 9 g/L NaCl

 Osmotic Fragility Test
Sources of Errors
1. Relative volumes of blood and saline.
2. Final pH of the blood in saline suspension.
3. Temperature at which the tests are carried out.
•A proportion of 1 volume of blood to 100 volumes of
saline is chosen to render the effect of the plasma on the
final tonicity of the suspension negligible.
•The fragility of the red cells is increased by a fall in pH.
•Increase in temperature decreases the fragility, a rise of
5° C being equivalent to an increase in saline
concentration of about 0.1 g/L
Summary/Review Questions

1. Does a normal OFT rule out hereditary spherocytosis?

2. What is the significance of measuring osmotic fragility?
3. How do you report and interpret the OFT results?
4. What are the sources of error associated with the OFT?
 Bibliography

• MA Lichtman, E Beutler, U Seligsohn, K Kaushansky,

TO Kipps (Editors). William’s Hematology. 7th Ed.
McGraw-Hill Co. Inc. 2008.
• Dacie, John V and Lewis, S.M. Practical Hematology 10th
Edition Churchill-Livingstone 2006.
• Wintrobe, Maxwell M. Clinical Hematology 11th Edition
Lea and Febiger, Philadelphia 2003.