To determine the osmotic fragility of blood.
PRINCIPLE: Osmotic fragility of red blood cells is assessed by adding blood to hypotonic saline solutions of decreasing concentrations. The resistance which erythrocytes offer to the haemolytic actions of hypotonic saline is used as an index of the osmotic fragility of RBC's.
A series of 10 small test tubes (Khan tubes), sodium chloride solutions of varying strength from 0.25%- 0.7% at a difference of 0.05% sterile lancet, wooden rack, cotton and spirit.
Place the 10 test tubes on the wooden rack labelled serially from 0.7% to 0.25%. Fill the tubes upto three fourth with sodium chloride solutions of corresponding serial strengths. Under sterile precautions, prick the finger tip and add one drop of blood to each test tube. Close the mouth of the test tube with the index finger and mix the contents gently by inverting the tube twice or thrice. Allow to stand for one hour. Examine the tubes from higher to lower concentrations. There should not be any admixture of fluids in different tubes. The finger tip should be wiped in between addition of the blood to the different tubes.
OBSERVATION: In tubes with solutions of higher concentration, blood settles down with a clear, colourless supernatant. As the concentration decreases gradually, the supernatant starts getting a reddish tinge. The strength of the sodium chloride solution in which the reddish tinge is first seen indicates the begginning of haemolysis (most fragile RBC5 are lysed). The solution in which haemolysis is complete (all RBC5 are lysed) is uniformly red, clear and transparent without any sediment. In all the solutions of lower concentration the same picture is seen. Osmotic fragility is expressed as the range of saline concentrations in which, beginning and completion of haemolysis occurs.
DISCUSSION: Osmotic fragility is the ease with which RBC5 undergo lysis, when kept in hypotonic solution. When the red cell is placed in hypotonic saline, water is drawn in by the higher intracellular osmotic pressure. Consequently the cells become spherical and ultimately the red cell membrane gives way with liberation of haemoglobin into the surrounding fluid. The process of liberation of haemoglobin from the red cells is known as haemolysisorlaking of blood.
Factors increasing the osmotic fragility of red cells
Increase in term perature
1. Hereditary spherocytosis
2. Acquired spherocytosis
3. Snake poisoning eg:- Viper, Krait
Conditions where osmotic fragility is decreased:
1. Sickle cell anaemia
2. Iron deficiency anaemia
1. What are the causes of haemolysis in this experiment?
2. What is the role of osmotic fragility test in clinical medicine?
3. Name a few hemolytic agents.
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