Osmotic fragility of red blood cells by Pandian M

introduction
Apparatus
Procedure
Precautions
Results

1. Osmotic fragility of red blood cells Pandian. M Dept. of Physiology D. Y. Patil Medical college, KOP.

2. Aim To measure the resistance of red cells to hemolysis in the hypotonic solution

3. Principle Normal red blood cells remain suspended in isotonic solution (0.9% saline ) for hours without any change in their size i.e. without rupturing. But when they suspended in hypotonic solution, they absorb the fluid, swell and ultimately get haemolysed . Their behavior in different hypotonic strengths is observed.

4. Terms fragility and hemolysis Fragility – this term refer to the susceptibility of red cells to being broken down by osmotic or mechanical stresses. Hemolysis – this term refers to the breaking down (bursting) of red cells resulting in release of Hb into the surrounding fluid.

5. NORMAL RANGE Normally haemolysis begins in 0.48% saline. No cells haemolyse in solution of 0.5% saline and above. Haemolysis complete at 0.36% saline. It is also complete in tube 10, 11, & 12. NOTE – when red cells become more fragile, hemolysis may begin at 0.64% saline & be complete at 0.44% When red cells are less fragile, hemolysis starts & complete at lower strengths of saline.

6. Abnormal osmotic fragility Increase in osmotic fragility index occurs in following conditions: Congential spherocytosis , i.e. when the RBCs are spherical. In this condition onset of haemolysis occurs at 0.7% NaCl and it is completed at 0.45% NaCl solution. Autoimmune haemolytic anaemia in which the autoantibodies damage the structure proteins and render the red cells more fragile.

7. Deficiency of glucose 6-phosphate dehydrogenase (G6PD) increases the tendency of red cells to get haemolysed by antimalarial drugs and other agents. Venom of cobra and other insects contains lecithinase which dissolves lecithin from the red cell membranes making them more fragile.

8. Decrease in osmotic fragility index occurs when the RBCs become slender, e.g. in iron deficiency anaemia . In this condition the onset of haemolysis occurs at 0.36% NaCl and is completed at 0.24% NaCl solution.

9. Apparatus Clean Kahn’s tube. Test tube rack. Distilled H 2 O. 0.9% NaCl solution, 0.5% NaCl solution Dropper.

10. Procedure

11. Protocol:- Number of tubes Isotonic saline 1 2 3 4 5 6 7 blank Dis.H 2 O Drops --- 2 4 6 8 10 12 25 0.5% NaCl drops 25 (0.9%) 25 (0.5%) 23 21 19 17 15 13 ----- % is obtained 0.9% 0.5% 0.46% 0.42% 0.38% 0.34% 0.30% 0.26% ----- NOTE :- Use same dropper, after rinsing each time, or use same size dropper which do not change the volume or measuring saline and distilled water

12. Observation Supernatant fluid is straw colored- No hemolysis.

13. Increased surface to volume ratio It is more resistant to haemolysis and has ↓ sed fragility The larger the amt of red cell memb ( surface area ) in relation to the size of the cell, the more fluid the cell is capable of absorbing rupturing – As ?????? Reticulocytes Iron deficiency anaemia Thalassemia Sickel cell anaemia Occurs following splenectomy , in liver disease, polycythemia vera , and conditions in which target cells are present.

14. Factors affecting on the Osmotic fragility The primary factor affecting the osmotic fragility test is the shape of the red cell, which in turn depends on the Volume Surface area Functional state of the red blood cell membrane

15. CONDITION WHICH ALTER THE FRAGILITY FRAGILITY MORE IN Venous blood Stored blood Spherocytosis Sickle cell anaemia

16. FRAGILITY LESS IN Iron deficiency anemia Thalassemia

17. Name of hemolytic agents:- Hypotonic saline Incompatible blood transfusion Snake venom Severe infection Rxn to certain drugs. Aspirin is a common drug that may cause hemolysis at any time.

18. THANK YOU . . .

19. SEDIMENTATION OF RED CELLS 3 STAGES: FIRST STAGE - RBC’S Pile UP ( LIKE STACK OF COINS AND FORM ROULEAUX IN 1 ST 10-15 mins SECOND STAGE - The Rouleaux is heavier start sink in to bottom takes 40-45 mins THIRD STAGE - Packing of massed bunches to bottom this stage last about 10-12 MINS

20. WINTROBES METHOD NORMAL VALUES: MALE : 3-5 mm after one hour FEMALE : 5 – 7 mm after one hour WESTERGREN METHOD: NORMAL VALUES: MALE : 0-9 mm after 1 hour FEMALE :0-20 mm after 1 hour

21. WESTERGREN’S METHOD REQUIREMENTS WESTERGREN TUBE (300mm long/2.5mm bore diameter) CITRATE BULB (3.8 % SODIUM CITRATE) WESTERGREN STAND SYRINGE FOR BLOOD COLLECTION

22. PROCEDURE Blood collected in citrate bulb Draw the blood up to mark zero fix the pipette in vertical position in the rack Read the mm of clear plasma on the top of the blood column at he end of 1 hour.

23. Wintrobe’s method WINTOBE’S HEMATOCRIT TUBE STAND FOR WINTROBE TUBE LONG NOZZLE DOPPER WINTOBE’S BULB ( AMMONIUM AND POTASSIUM OXALATE MIXTURE)

24. PROCEDURE Blood collected in wintrobe’s bulb after venous puncture. Help of long nozzle dropper reach the bottom of the tube fill the blood up to zero mark. Avoid air bubbles and frothing while filling. Fix vertically in the stand Read the height of plasma on the top of the blood column after 1 hour.

25. SIGNIFICANCE OF ESR ↑ in inflammatory conditions used as a prognostic tool ( degree of activity of disease and effectiveness of the treatment) assess progress of disease & the response of the disease to treatment intensity of pyogenic infection

26. VARIATION IN ESR PHYSIOLGICAL ↑ New Born, Female, Pregnancy, Old Age PHYSIOLOGICAL ↓ - MALE PATHOLOGICAL ↑ ACUTE Infections, CHRONIC Infections (E.G. Tb, Rheumatic Fever, Rheumatic Heart Disease Pyogenic Infections, Anaemia , Malignant Tumor) PATHOLOGICAL ↓ Sickle Cell Anaemia Spherocytosis , Polycythemia

27. References MUHS Journal Text book of Practical Physiology G.K.Pal C.L.Ghai A.K.Jain

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