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

 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 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
 CLINICAL SIGNIFICANCE

• Elevated values high OFT= increased V / SA

associated with:
-Hereditary spherocytosis -
-Acquired immune Hemolytic -
Anemia with Spherocytesis
-Hereditary stomatocytrsis
 CLINICAL SIGNIFICANCE
CONT’D
Decreased Values low OFT= decreased V / SA
Associated with abnormal RBC morphology:
Sickle cells (seen in Sickle cell anemia)
Hypochromic microcytic cells(seen in IDA and Thalassemia)
Leptocytosis (target cells)
 OFT CONT..
Spherocytes have:
Spherocytes

• a decreased SA / V ratio
• thinner surface membrane

• higher volume (already spherical)

• Lost ability to expand under osmotic pressure

 TEST PRINCIPLE

• Patient sample and normal red cells are placed in a series of

graded strength NaCl solution and resulting
Hemolysis is compared to a 100% standard.
 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
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.
 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.
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 1 (osmotically equivalent
to 9 g/l NaCl).
6. Assign a value of 100 % lysis to reading with the supernatant of
tube 12 (water).
 OFT METHOD CONT’DAND
CALCULATION
7. Express the readings from the other tubes as a percentageof the value
of tube 12
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 (tabe 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 37OC 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

• Also prepare 12 g / L Na Cl solution to use supernatant as a BLANK

 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.