I CHAPTER 1

ERYTHROCYTE FRAGILITY
TEST

Aim
The aim of this chapter is to study the effect of solutions with
different osmolarities on the integrity of erythrocytes. This is
one of the clinical tests used to examine the fragility of
erythrocytes.

Introduction
When an erythrocyte is placed in a hypotonic sodium chloride
-(NaCl) solution, a net influx of solvent (water) into the cell
-will occur and the cell will swell. If the cell size reaches a
certain point, the cell membrane will become leaky and
haemoglobin will diffuse out (haemolysis). If the NaCl solution
is hypotonic enough, the cell will rupture. The degree of
haemolysis can be measured by determining the absorbance
of the supernatant using a spectrophotometer. The degree of
haemolysis in distilled water is considered maximum (100%),
and the absorbance of this supernatant is taken as "100%
haemolysis". In the erythrocyte fragility test, the erythrocyte
is placed in NaCl solutions of decreasing osmolarity (i.e.
0.85%, 0.75% ). The amount of haemolysis that occurs in
each solution can be determined, and a graph of " % haemolysis
vs NaCl concentration" can be plotted. In an abnormal
erythrocyte that is more fragile than normal, haemolysis will
occur at a higher NaCl concentration (i.e. 'earlier') than normal 11. Repeat the above procedures for each of the following
and the graph will shift to the right. solutions:
In this experiment, we will also examine the fragility of a. 150 mmol/L urea
erythrocytes (haemolysis) in several other solutions to b. 300 mmol/ L urea
understand the principles of diffusion of particles across the c. 300 mmol/L urea +150 mmol/L NaCl
cell membrane. d. 300 mmol/ L glucose

Procedure
1. This experiment can be conducted in groups of 4 students.
2. Label the centrifuge tubes as follows: 0.85%, 0.75%, 0.65%,
0.55%, 0.45%, 0.40%, 0.35%, 0.20%, 0.10% and 0%.
3. Pipette 5 ml of each of the NaCl solution provided into
the appropriately labelled tubes. In the tube labelled 0%,
pipette in 5 ml of distilled water.
4. Using a micropipette, add 0.02 ml (20ul) of blood
(containing an anticoagulant agent) into each tube.
5. Cover the tube with parafilm, mix the contents of each
tube gently and then allow the solution to settle for about
20 minutes at room temperature.
6. Mix the contents in each tube again. Then, centrifuge the
tubes at a speed of 2000 rotations per minute for 5 minutes.
7. Carefully transfer the supernatants into clean and
appropriately labelled cuvettes.
8. Using a spectrophotometer, measure the absorbance of
each supernatant, at a wavelength of 540 nm. For this
procedure, use distilled water as a 'blank' (zero reading
in the meter). Then obtain the absorbance reading of each
supernatant. Compare each of these readings with that
obtained from the 0% NaCl tube (distilled water = 100%
haemolysis).
9. Calculate the % haemolysis for each supernatant as
follows:

10. Plot a graph of % haemolysis vs NaCl concentration. This

represents the osmotic fragility curve for the blood
sample.