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2014-03-19 00:09:37
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  1. What is osmotic fragility?  What does it depend on?
    • RBC ability to withstand various salt concentrations
    • dependent on RBC volume, surface area, and membrane function
  2. What is the principle behind the osmotic fragility procedure?
    • A specific amount of blood is introduced into a series of tubes containing different concentrations of buffered salt solutions
    • The ability of the erythrocytes o resist hemolysis is determined spectrophotometrically on the basis of the free hgb present
  3. Describe the osmotic fragility procedure
    • Prepare a working 1%NaCl soln
    • Prepare 2 sets of tubes
    • Label set 1 as patient, and set 2 as control
    • Set up the following dilutions in set 1 of the tubes .85%(1)....15%(12) [total volume 5mL]
    • Transfer 2.5mL of each dilution into Set 2
    • Using heparinized blood add .25mL patient blood to each tube in set 1
    • Using heparinized blood add .25mL control blood to each tube in set 2
    • Mix well and allow tubes to sit at room temp for 30 min
    • Gently mix and centrifuge for 5 minutes at 2000rpm
    • Visually assess where hemolysis begins and at what NaCl concentration is appears complete in both sets of tubes
    • Remove supernatants to cuvettes and read at 540nm
    • Report results in %hemolysis
    • Normal individual has beginning hemolysis at .45% with complete hemolysis at .35%
  4. How are the results for osmotic fragility normally reported?  What are the normal values?
    • %hemolysis
    • Normal individual has beginning hemolysis at .45% with complete hemolysis at .35%
    • Typically reported in graph form in comparison to control results; x-[NaCl], y- %hemolysis
  5. How would you expect a spherocyte to react in an osmotic fragility test?  A drepanocyte?
    • Spherocyte: will begin lysis at much lower concentration
    • already "stretched" and can't take much water
    • Drepanocyte: will begin lysis at much higher concentration
    • has much more room for H2O than normal cell
  6. What are the possible interferences in the osmotic fragility procedure?
    • Wrong anticoagulant (may introduce additional salts)
    • Age of cells (loss of membrane)
    • Inappropriate pH or temperature of soln
    • Jaundice
    • Hemolytic organisms
    • Low RBC or hgb
    • Abnormal RBC morphology

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