Name number Study group
Theoretical part
Osmotic fragility test
The cytoplasmic membrane of red blood cells
The red blood cell cytoplasmic membrane not only represents a barrier between the interior of
the erythrocyte and the outer environment, it also plays a crucial role in intercellular
communication and adhesion. Some of its important features are deformability, flexibility as
well as durability which allows cells to move through capillaries (the diameter of which can
be as narrow as half of the diameter of the erythrocyte). Erythrocytes possess all these
features thanks to the cytoskeletal proteins where spectrin plays the central role. Spectrin fibre
is formed by spectrin alpha and beta and further interacts with actin filaments to create an
intracytoplasmic cytoskeleton connected to the inner layer of the cytoplasmic membrane by
protein 4.1 and ankyrin. The older the erythrocyte gets, the less durability, flexibility and
deformability it possesses, and after approximately 120 days it is destroyed in the spleen.
Osmotic resistance
The resistance of erythrocytes to mechanical, chemical and osmotic damage is not identical in
all red blood cells, and therefore some are able to resist higher stress levels than others. This is
dependent on the cell’s age, shape (e.g. spherical), enzyme content (e.g. genetic deficiency of
glucose-6-P dehydrogenase), etc. The osmotic fragility test is a specific method used in the
differential diagnosis of haemolytic anaemias.
Diffusion Movement of particles based on the
concentration gradient
Osmose Movement of solvent molecules through the
semipermeable membrane into a region with
higher solute concentration
Osmotic pressure Pressure required to stop osmosis
Osmolarity Measured solute concentration defined as
the number of osmotic active molecules per
1 litre of its solvent
Osmolality Same as osmolarity but per 1kg; = 2
[Na+
]+[glc]+[urea] = 275-295mmol/kg H2O
Tonicity Represents the effect of osmotic activity in
the relationship with a cell
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Pathology:
 Elevated values of minimal osmotic resistance
 Inherent haemolytic anaemias
 Decreased values of maximal osmotic resistance
 Polycythemia vera
 Thalassemia
 Sickle-cell anaemia
 Fe2+
deficiency
 Status post splenectomy
Isotonic haemolysis
The isotonic haemolysis of red blood cells occurs under two specific conditions:
 Isotonic solution of glucose – glucose is absorbed by cells until the solution becomes
hypotonic and the cells die
 Isotonic solution of urea – urea freely penetrates cells following the concentration
gradient and at the same time the tonicity of the solution decreases (urea does not
respect the semi-permeability of the cytoplasmic membrane)
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Protocol
Osmotic fragility test
Methods
Equipment: Stand with 13 test tubes, solution of 1% NaCl, distilled water, physiological
solution (0.9% NaCl), 2 graduated pipettes (10 ml), dropper, anti-coagulated human blood,
gloves.
Procedure:
1. Twelve test tubes are filled, by using two pipettes, with decreasing amounts of 1%
solution of NaCl and increasing amounts of distilled water according to the table
below. The last test tube is filled with the physiological solution (0.9% NaCl). Always
use a clean pipette for each test tube.
Tube number 1 2 3 4 5 6 7 8 9 10 11 12
NaCl 1%
(ml)
6.3 6.0 5.7 5.4 5.1 4.8 4.5 4.2 3.9 3.6 3.3 3.0
H2O
(ml)
3.7 4.0 4.3 4.6 4.9 5.2 5.5 5.8 6.1 6.4 6.7 7.0
% NaCl 0.63 0.60 0.57 0.54 0.51 0.48 0.45 0.42 0.39 0.36 0.33 0.30
Table for preparation on NaCl solutions of decreasing osmotic pressure
2. Each test tube is carefully mixed.
3. Two drops of blood are placed into each tube and the content is again carefully mixed.
After this, do not move the stand or the test tubes so as not to interrupt the
sedimentation of non-haemolysed red cells.
4. After approx. 2 hours (min. after 30 minutes) of standing at room temperature,
estimate the range of haemolysis by examining the colour of the supernatant solution
above the sediment cells and the intensity of turbidity of the cell suspension. The tube
with the normal physiological solution serves as the control since blood taken some
time ago may already be partially haemolysed.
In the lowest concentrations of NaCl a complete haemolysis occurs, i.e. all red cells
disintegrate. The content of the tube is quite pellucid (a text can read through the tube). At a
certain concentration, however, the haemolysis is not complete, as a portion of the blood cells
(i.e. those with the highest resistance) did not haemolyse, which appears as a slight turbidity
in the lower part of the tube – incomplete haemolysis. The lowest concentration where a small
fraction of cells remained non-haemolysed designates the s.-c. maximal resistance. On the
other end of the concentration scale one finds a tube where the haemolysis is hardly
distinguishable: the solution above the sediment is only slightly coloured by haemoglobin
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released from the least resistant cells. The concentration where osmotic haemolysis just
begins to appear is designated as the minimal resistance. When non-haemolysed erythrocytes
form a non-transparent sediment on the bottom of the test tube with a yellowish fully
transparent NaCl solution above it, such a test is designated as no haemolysis.
The osmotic resistance range is the difference between the maximal and minimal resistance
value.
Results
 Minimal osmotic resistance was observed in ………%NaCl (physiological: 0.4 - 0.44 %)
 Maximal osmotic resistance was observed in………%NaCl (physiological: 0.3 - 0.34 %)
 Mark osmotic resistance range.
Conclusion
Note down all the types of haemolysis known to you (there are 6 types) and explain their
mechanism.
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