Saturday, 27 October 2012

Osmotic Fragility Test

Photo 1: Osmotic fragility test (Source)
      The osmotic fragility test is a measure of the resistance of erythrocytes to hemolysis by osmotic stress. The test consists of exposing red cells to varying strengths of hypotonic saline solutions and measuring the degree of hemolysis colorimetrically at room temperature. The percentage of hemolysis is plotted on the vertical axis against decreasing saline concentration on the horizontal axis. The test is done to evaluate hemolytic anemia, especially hereditary spherocytosis, evaluate immune hemolytic states.

    The principle of the test is that the normal RBC is able to resist the influx of water to a limited extent. When the cells are placed in a hypotonic solution the RBC absorbs water due to osmotic pressure. The influx of water causes swelling of RBC. A normal RBC is able to swell because of its volume to surface ratio (of 90:136 μm2)

     This stretch-ability is lost in cells such as spherocyte. Once the limit is reached lysis occurs. The increased osmotic fragility of normal cells is also attributed to the Na pump. The lysis is due to an accumulation of sodium which exceeds the loss of potassium. If incubated for long periods the red cell is stressed and pumping mechanism fails, which is due to lack of glucose that supplied energy to pump ultimately causing lysis.

Reagents:

Stock solution consists of buffered sodium chloride,

NaCl- 90g
Na2HPO4- 13.65g
NaH2PO4.2H2O- 2.34g

Procedure:

     Heparinized venous blood is used. The test should be carried out within 2 hours of collection or within 6 hours if kept at 4° C.

1. Deliver 5.0 mL of the saline solutions (grades of saline solution ranging from 9g/L to 1g/L) in test tubes. Add 5.0 mL of water to last tube. Usually 11 tubes are used though more intermediary grades of dilutions may be used.
2. Add to each tube 50μL of well mixed blood and mix immediately by inverting the tubes for several times avoiding foam.
3. Incubate at room temperature for 30 min. The lysis is complete at the end of 30 min at 200 C
4. Remove supernatant and estimate the amount of lysis in a colorimeter at 540 nm. Use as blank the supernatant in tube 1.
5. Assign a value of 100 % lysis to last tube

The % of hemolysis is calculated as


Factors affecting osmotic fragility tests
  1. The relative volumes of blood and saline
  2. The final pH of the blood in saline suspension
  3. The 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. A rise in temperature decreases the fragility, a rise of 5° C being equivalent to an increase in saline concentration of about 0.1 g/L. Osmotic fragility can be described in terms of the saline concentration at which lysis begins (Initial lysis or minimum resistance normally 4.5 - 5.0 g/L) and at which lysis appears to be complete (complete lysis or maximum resistance, normally 3.0 - 3.3 g/L). Median Corpuscular Fragility (MCF) is the concentration of sodium chloride solution causing 50% lysis.

Photo 2: Spherocytes (Source)
     The normal MCF range is 4.0-4.45 g/L. Hereditary spherocytosis may be the result of an autosome dominant transmitted detect in red cell structural proteins. It is associated with a compensated or uncompensated hemolytic state, which is relieved by splenectomy. Most hereditary hemolytic anemias involve mutations of membrane structural proteins, the majority code for abnormal spectrin molecules. Decreased osmotic fragility maybe seen with iron deficiency, other hemoglobinopathy, hemoglobin C disease, likely due to the target cell population, and is characteristic of thalassemia. 
Table 1: Readings commonly seen in various diseases
Fig 1: OFT (Source)
     The osmotic fragility test is most useful in the diagnosis of congenital spherocytosis. In hereditary spherocytosis, there is abnormal morphology due to a lack of spectrin, a key red blood cell cytoskeletal membrane protein. This produces membrane instability that forces the cell to the smallest volume-a sphere. This is shown by increased osmotic fragility, which causes the entire curve to 'shift to the right' or most of it  may be within the normal range, but with a 'tail' of fragile cells. Spherocytes have reduced surface membrane area relative to the RBC volume. Usually the osmotic fragility test results are graphed and reported as shown. Normal osmotic fragility is seen at top right. Abnormal lysis of RBCs in mildly hypotonic solutions is seen at lower right (hereditary spherocytosis).

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