OSMOTIC FRAGILITY
TEST
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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
Osmotic fragility of Erythrocyte
Erythrocyte fragility: refers to the tendency of erythrocytes to
hemolyse (rupture) under stress (commonly osmosis).
Osmotic fragility:- is a test to measure red blood cell (RBC)
resistance to hemolysis when exposed to a series of increasingly
diluted saline solutions.
Hemolysis:- This term refers to the breaking down (bursting) of red
cells resulting in release of Hgb into the surrounding fluid.
The sooner hemolysis occurs, the greater the osmotic fragility of the
cells.
Osmotic fragility of Erythrocyte
Isotonic solution: A solution that has the same
salt concentration as cells and blood.
Movement of water out of the cell is exactly
balanced by movement of water into the cell.
E.g. 0.9% of NaCl (saline)
hypertonic solution (> 0.9 % NaCl): total molar
concentration of all dissolved solute particles is
greater than the concentration in a cell.
Osmotic fragility of Erythrocyte
Hypotonic solution: the total molar concentration of all dissolved
solute particles is less than that of a cell.
If concentrations of dissolved solutes are less outside the cell than
inside, the concentration of water is greater in the outside .
When a cell is exposed to such hypotonic conditions (< 0.9 %
NaCl), there is net water movement into the cell, causing the cell
to burst
Osmotic Fragility Test
It is a test that measures the resistance to hemolysis of red
blood cells (RBC) exposed to hypotonic solutions
RBC are exposed to a series of saline (NaCl) solutions with
increasing dilution.
The sooner hemolysis occurs, the greater is osmotic fragility of
RBC
In hypotonic medium a membrane rupture occurs, allowing
hemoglobin (Hgb) to exit from the cells
Osmotic Fragility Test
By measuring Hgb concentration, the % of hemolysis at
different NaCl concentrations can be calculated
The test consists of exposing red cells to decreasing strengths
of hypotonic saline solutions and measuring the degree of
hemolysis colorimetrically at room temperature (15 ° - 20 ° C).
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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%.
Osmotic Fragility Test
Determines the resistance of the red cell to hemolysis 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.
Why the Test is performed?
This test is performed to detect thalassemia and hereditary
spherocytosis.
Hereditary spherocytosis is a common disorder in which red
blood cells are defective because of their round, ball-like
(spherical) shape. These cells are more fragile than normal
because they are less likely to expand.
Why the Test is performed?
Cells that are flatter than normal are more likely to expand, and
thus have decreased osmotic fragility .
Thalassemia is an inherited condition that affects the portion of
blood (hemoglobin) that carries oxygen.
Why the Test is performed?
The test only indicates that a proportion of the red cells have
decreased surface-to-volume ratios and are more susceptible to
lysis in hypo- osmotic solutions.
Cells with increased surface-to-volume ratios, such as occur in
thalassemia and iron deficiency, may show decreased osmotic
fragility.
For patients with acute hemolysis, a normal red cell osmotic
fragility test result cannot exclude an osmotic fragility
abnormality since the osmotically labile cells may be hemolyzed
and not present.
Why the Test is performed?
Recommend testing during a state of prolonged homeostasis
with stable hematocrit.
To supplement a stained cell examination to detect
morphologic RBC abnormalities.
OFT is not used as a screening test for the diagnosis of anemia
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Osmotic Fragility Test Cont’d
Test Principle
Patient sample and normal red cells are placed in a series of graded
strength of NaCl solution and resulting hemolysis is compared to a
100% standard.
The normal red cells can remain suspended in normal saline (0.9%
NaCl solution) for hours without rupturing or any change in their
size or shape.
But when they are placed in decreasing strengths of hypotonic
saline, they absorb water (due to osmosis) and finally burst
Osmotic Fragility Test Cont’d
The ability of RBCs to resist this type of hemolysis can be determined
quantitatively.
Notes
When RBC's are placed in distilled water they will swell, burst
& the hemoglobin will be released and the hemoglobin will
color the plasma {hemolysis}
If the RBC's are placed in 0.9%NaCl nothing happens to them
because it is isotonic with the cells
If we put the RBC's in a hypertonic solution they will shrink
Factors affecting the osmotic fragility test
The primary factor affecting the osmotic fragility test is the
shape of the red cell, which, in turn, depends on the
1. Volume
2. Surface area
3. Functional state of the red blood cell membrane.
Osmotic Fragility Test Method
Method
Manual osmotic fragility test
Material & instruments
1. Test tubes
2. NaCl with different concentrations
3. Heparinized venous blood
4. Distilled water
Osmotic Fragility Test Method
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.
Procedure
We use human erythrocytes that were washed with physiologic
solution and thus do not contain plasma
1. Label 14 tubes 1–14 and do this dilution:
Test tube 1%Nacl(ml) D.W. (ml) Final conc. (%)
1 10.0 0.0 1.00
2 8.5 1.5 0.85
3 7.5 2.5 0.75
4 6.5 3.5 0.65
5 6.0 4.0 0.60
6 5.5 4.0 0.55
7 5.0 5.0 0.50
8 4.5 5.50 0.45
9 4.0 6.0 0.40
10 3.5 6.0 0.35
11 3.0 7.0 0.30
12 2.0 8.0 0.20
13 1.0 9.0 0.10
14 0.0 10.0 0.00
Osmotic Fragility Test Method
2.Deliver 5.0 ml of the 11 saline solutions in test tubes. Add 5.0 mL
of water to tube 12.
3. Add to each tube 50 µL (microlitres) of well mixed blood and
mix immediately by inverting the tubes for several times
avoiding foam.
4. Incubate at room temperature for 30 min. Mix again and
centrifuge 1000g for 5 min.
5. 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.
6. Use as blank the supernatant from tube 1 (osmotically equivalent
to 9 g/l NaCl).
OFT Method and Calculation
7. Assign a value of 100 % lysis to reading with the supernatant of
tube 12 (water).
8. Express the readings from the other tubes as a percentage of the
value of tube 12
9. Plot the results against the NaCl concentration
Calculation: Calculate % Hemolysis of each tube
% Hemolysis = Abs T- Abs B X 100
Abs Std – AbsB
But, Abs B = 0.00
% Hemolysis = Abs T X 100
Abs Std
Interpretation of OFT Results
% of hemolysis =
(Abs of tube / Abs of tube 14) * 100%
Normal Range:
Saline concentration at which Hemolysis begins
0.45 – 0.50%
Saline concentration at which 50% RBC lyse (Median
corpuscular fragility (MCF))
0.40 – 0.45%
Saline concentration at which hemolysis is complete
0.30 – 0.35%
Discussion
Observation and Results :
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.
Diagnosis 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.
OFT after Incubation
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
OFT after Incubation
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
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Clinical Significance/ Medical applications
A. Increased red cell fragility (increased tendency to hemolysis)
It is seen in the following conditions:
1. Hereditary spherocytosis.
2. Autoimmune hemolytic anemia.
3. Toxic chemicals, poisons, infections, and some drugs (aspirin).
4. Deficiency of glucose 6-phosphate dehydrogenase (G6D).
Clinical Significance/ Medical applications
B. Decreased red cell fragility (increased resistance to hemolysis):
Osmotic fragility decreased in:
Sickle cells (seen in Sickle cell anemia)
Hypochromic microcytic cells(seen in IDA and Thalassemia)
Leptocytosis (target cells)
Clinical Significance/ Medical applications
Interfering factors
Relative volumes of blood and saline.
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.
Final pH of the blood in saline suspension.
The fragility of the red cells is increased by a fall in pH.
Temperature at which the tests are carried out.
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
Interfering factors
Handle the sample gently to prevent accidental hemolysis.
In some cases, RBCs don't hemolysis immediately, incubation in
solution for 141 hours improves test sensitivity.
Presence of hemolytic organisms in the sample.
Severe anemia or other conditions with fewer RBCs available for
testing .
Recent blood transfusion.
Old sample
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?
Thank you
