Introduction. Red blood cells are bound by a membrane which allows water to pass through while generally restricting the solutes. This process, called osmosis, causes cells to shrink due to loss of water when placed in hypertonic solutions. On the other hand, red cells absorb water (called endosmosis) when in a hypotonic medium. This results in swelling and ultimately haemolysis as the cell bursts. The osmotic fragility test uses this fact to determine the concentration of solute inside the cell by subjecting it to the haemolytic effects of solutions of different concentrations.
Method. (Please refer to the last article entitled "The Osmotic Fragility Test I")
Results. The experimental results and the calculated percentages are given in Table 1 below.
On plotting the data obtained experimentally, a curve is obtained. This is shown in Figure 1.
Discussion. The shape of the curve is only one aspect of the test. The position of the curve on the axes relative to reference values must also be considered. It is essentially useful to record the concentration of sodium chloride solution causing 50% lysis, i.e. the Median Corpuscular Fragility (MCF). This value is normally 4.0-4.5 g/L. Other useful values include the concentration at which lysis begins (minimum resistance) and that at which lysis appears to be complete (maximum resistance). These are normally 4.5-5.0 g/L and 3.0-3.3 g/L respectively. On adding a sketch of the normal range of relevant values to the same axes, it is observed that the experimental curve lies slightly to the left, outside the normal range. This is depicted in Figure 2. below.
The mean corpuscular fragility (MCF) is obtained as 3.65 g/L of sodium chloride, when the normal range, as indicated earlier, is 4.0-4.45 g/L. The significance of the 'shifting' of the graph to the left may involve an imbalance in intracellular sodium level. The osmotic fragility test is most useful in the diagnosis of congenital spherocytosis (Mukherjee, 1989), but here this is most probably not the case. 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. In these patients, intravascular haemolysis results in haemoglobinaemia, and the circulating haemoglobin is excreted in the urine, imparting it a red colour (Ghai, 1988). Therefore, a urine sample would have been helpful in this case.
