CHAPTER 4 OSMOTIC FRAGILITY TEST Acknowledgements Addisa Ababa

CHAPTER 4 OSMOTIC FRAGILITY TEST

Acknowledgements • • Addisa Ababa University Jimma University Haramaya University Hawassa 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% Na. Cl 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 Spherocytes – 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 Na. Cl solution and resulting Hemolysis is compared to a 100% standard.

Cont. . • Reagents : • Stock buffered Na. Cl solution (10%). -Na. Cl (powder) 180 g -Na 2 HPO 4 27. 31 g -Na. H 2 PO 4. 2 H 2 O 4. 86 g. . . . -Buffering effect -keep Ph 7. 4 • Make up to 2 L= d. H 2 O • From (the stock solution, prepare first a 10 g/L solution by dilution with water. Dilutions equivalent to 9. 0, 7. 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 -20 O 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 m. L 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 1000 g 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 Na. Cl). 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 Na. Cl concentration (Fig. ) Calculation: Calculate % Hemolysis of each tube % Hemolysis = Abs T- Abs B X 100 Abs Std – Abs. B But, Abs B (tabe 12) = 0. 00 % Hemolysis = Abs T X 100 Abs Std

Figure 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 (200 C, PH 7. 4) Tube No Na. Cl% 1. 0. 10 2. 0. 20 3. 0. 30 4. 0. 35 5. 0. 40 6. 0. 45 7. 0. 50 8. 0. 55 9. 0. 60 10. 0. 65 11. 0. 70 12. 0. 80 Hemolysis (%) 100% 99 -100% 90 -99% 50 -98% 6 -49% 0. 5% 0% 0% 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 37 OC for 24 hours and OFT be repeated • even they give normal or slight increase OFT in the unincubated 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 37 oc for 24 hrs. • 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 Na. Cl thus set up additional hypotonic solution of 9 g/L Na. Cl 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 p. H 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 p. H. • 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. 7 th Ed. Mc. Graw. Hill Co. Inc. 2008. • Dacie, John V and Lewis, S. M. Practical Hematology 10 th Edition Churchill-Livingstone 2006. • Wintrobe, Maxwell M. Clinical Hematology 11 th Edition Lea and Febiger, Philadelphia 2003.