Hemolysis and Fragility Test

Hemolysis and Fragility Test of Red Blood Cells

Hemolysis:

Hemolysis is the destruction of red blood cells.
It is the process, which involves the breakdown of red blood cells (RBCs).
Hemolysis can occur due to different causes and leads to the release of hemoglobin into the bloodstream.
Normal red blood cells (erythrocytes) have a lifespan of about 120 days. After they die they break down and are removed from the circulation by the spleen.

Fragility:

Susceptibility (to be affected) of RBC to hemolysis or tendency to break easily is called fragility (Fragile = easily broken).
Fragility is of two types:
- Osmotic fragility, which occurs due to exposure to hypotonic saline
- Mechanical fragility, which occurs due to mechanical trauma (wound or injury).
Under normal conditions, only old RBCs are destroyed in the reticuloendothelial system.
Abnormal hemolysis is the process by which even younger RBCs are destroyed in large numbers by the presence of hemolytic agents or hemolysins.

Fragility test:

Fragility test is a test that measures the resistance of erythrocytes in hypotonic saline solution.
It is done by using sodium chloride solution at different concentrations from 1.2% to 0.2%.
The solutions at different concentrations are taken in series of Cohn’s tubes.
Then one drop of blood to be tested is added to each tube.
The sodium chloride solution and the blood in each tube are mixed well and left undisturbed for some time.
Results can be analyzed by observing the tubes directly or by centrifuging the tubes after 15 minutes.

Direct observations

Observations after centrifugation

If there is no hemolysis: Cells sediment at the bottom with clear colorless ﬂuid above.
If hemolysis is started: Cell sedimentation is less and the ﬂuid becomes slightly reddish because of the release of small amount of hemoglobin from few hemolyzed RBCs.
If hemolysis is completed: Fluid becomes more reddish without any sedimentation due to release of more amount of hemoglobin from all the hemolyzed cells.

Index for Fragility:

After 20 minutes:

No hemolysis = up to 0.6%
Onset of hemolysis = around 0.45%
Completion of hemolysis = around 0.35% At 0.45%, only the older cells are destroyed because, their membrane is fragile. So, these cells cannot withstand this hypotonicity. But, younger cells are not affected. At 0.35%, even the younger cells are destroyed.

Process of hemolysis:

Normally, plasma and RBCs are in osmotic equilibrium.
When the osmotic equilibrium is disturbed, the cells are affected.
For example, when the RBCs are immersed in hypotonic saline the cells swell and rupture by bursting because of endosmosis.
The hemoglobin is released from the ruptured RBCs.

Conditions when hemolysis occurs:

Hemolytic jaundice
Antigen-antibody reactions
Poisoning by chemicals or toxins
While using artifcial kidney for hemodialysis or heart-lung machine during cardiac surgery (rare occasions).

Hemolysins:

Hemolysins or hemolytic agents are the substances, which cause destruction of RBCs.
The hemolysins are of two types:
- Chemical substances
- Substances of bacterial origin or substances found in body.

Chemical substances:

Alcohol
Benzene
Chloroform
Ether
Acids
Alkalis
Bile salts
Saponin
Chemical poisons like:
- Arsenial preparations
- Carbolic acid
- Nitrobenzene
- Resin.

Substances of bacterial origin or substances found in the body:

Toxic substances or toxins from bacteria:
- Streptococcus
- Staphylococcus
- Tetanus bacillus, etc.
Venom of poisonous snakes like cobra.
Hemolysins from normal tissues.

References:

Online Biochemistry Notes