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CHAPTER 4
OSMOTIC FRAGILITY TEST
By: JYOTI BALMIKI
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:
– a decreased SA / V ratio
– thinner surface membrane
– higher volume (already spherical)
– Lost ability to expand under osmotic pressure
Spherocytes
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.

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Hema II Chapter 4_OFT.ppt

  • 1. CHAPTER 4 OSMOTIC FRAGILITY TEST By: JYOTI BALMIKI
  • 2. 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
  • 3. 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
  • 4. Outline • Osmotic fragility definition • Osmotic fragility procedure • Reporting of OFT results • Interpretation of osmotic fragility test findings • Sources of error
  • 5. 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.
  • 6. 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%
  • 7. 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
  • 8. Clinical Significance • Elevated values high OFT= increased V / SA associated with: -Hereditary spherocytosis - -Acquired immune Hemolytic - Anemia with Spherocytesis -Hereditary stomatocytrsis
  • 9. 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)
  • 10. OFT cont.. Spherocytes have: – a decreased SA / V ratio – thinner surface membrane – higher volume (already spherical) – Lost ability to expand under osmotic pressure Spherocytes
  • 11. 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.
  • 12. 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.
  • 13. 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%
  • 14. 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.
  • 15. 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.
  • 16. 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).
  • 17. 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
  • 18. 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)
  • 19. 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
  • 20. 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%
  • 21. 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]
  • 22. 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%
  • 23. 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.
  • 24. 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
  • 25. 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
  • 26. 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
  • 27. 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?
  • 28. 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.