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Osmotic fragility
Definition:
Osmotic fragility is a test to measures red blood cell (RBC) resistance to hemolysis when exposed to a series of increasingly dilute saline solutions. The sooner hemolysis occurs, the greater the osmotic fragility of the cells. Factors affect the osmotic fragility:
1. 2. Cell membrane permeability. Surface-to volume ratio.
Material:
Specimen: whole blood Collection Medium: Na Heparin tube or Lithium Heparin tube. Minimum: 5 ml whole blood. Rejection Criteria: Hemolyzed specimen. Methodology: Spectrophotometer.
Procedure
1- We will do this dilution:
Test tube 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1%Nacl(ml) 10.0 8.5 7.5 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.0 1.0 0.0 D.W. (ml) 0.0 1.5 2.5 3.5 4.0 4.0 5.0 5.50 6.0 6.0 7.0 8.0 9.0 10.0 Final conc. (%) 1.00 0.85 0.75 0.65 0.60 0.55 0.50 0.45 0.40 0.35 0.30 0.20 0.10 0.00
Procedure
2- Then we divide every volume in 2 tubes so now we get 28 tubes. 3- Add 50 micron of whole blood to every tube. 4- let the tubes at R.T for - 30 min at 2500 rpm. 5- Well mixing by using the vortex. 6- Centrifuge for 5 minutes at 2500 rpm. 7- Now we will measure the absorbance in the tubes by using spectrophotometer (540 nm). 8- calculate the % of hemolysis.
Result:
% of hemolysis =
(Abs of tube / Abs of tube 14) * 100%
Normal Range:
Hemolysis begins 0.45% and complete 0.35%
Discussion
Interfering factors :
1- Completely fill the collection tube and invert it gently several times to mix the sample and anticoagulant thoroughly. 2- Handle the sample gently to prevent accidental hemolysis. 3- In some cases, RBCs don't hemolysis immediately, incubation in solution for 141 hours improves test sensitivity. 4- Presence of hemolytic organisms in the sample. 5- Severe anemia or other conditions with fewer RBCs available for testing . 6- Recent blood transfusion. 7- Old sample.