Osmotic Fragility of Red Blood Cells
DEFINITION
-it is a test that measures the resistance to hemolysis of
red blood cells (RBC) exposed to hypotonic solutions
RBC are exposed to a series of saline (NaCl) solutions with
increasing dilution
The sooner hemolysis occurs, the greater is osmotic
fragility of RBC
The osmotic fragility : is the resistance to hemolysis of red blood
cells when exposed to hypotonic saline. It is employed as a
screening test for hemolytic anemia.
Isotonic (physiological) solution – 0.9 %
NaCl
RBC burst in hypotonic (< 0.9 % NaCl),
and shrink (crenate) in hypertonic
solutions (> 0.9 % NaCl)
In hypotonic medium a membrane rupture
occurs, allowing hemoglobin (Hb) to exit
from the cells
By measuring Hb concentration, the % of
hemolysis at different NaCl concentrations
can be calculated
INCREASED OSMOTIC FRAGILITY
- Hereditary spherocytosis
- Acquired spherocytosis
Hereditary spherocytosis is a disorder characterized
by a defective RBC membrane and decreased
surface-to-volume ratio
Characteristic round cells (spherocytes) are seen in
blood smear and they are more fragile and break
open in less hypotonic solutions than normal red
blood cells
In hypotonic solutions water enters red blood
cells
Therefore, normal RBC with a biconcave
shape swell and expand their volume
On the other hand, spherocytes cannot
absorb much extracellular liquid and break
very easily
DECREASED OSMOTIC FRAGILITY
- Thalassemia
- Sickle cell anemia
- Iron deficiency anemia
Normal range of fragility
Normally, hemolysis begins in about 0.48% saline. No
cells hemolyze in solutions of 0.5% saline and above.
Hemolysis is complete at about 0.36% saline
Note
When red cells become more fragile, hemolysis may begin at about
0.64% saline and be complete at about 0.44% saline
Apparatus and materials
➢ Test tube rack with 7 clean dry glass test
tubes, marking pencil and dropper
➢ 3ml syringe
➢ 0.9% sodium chloride solution and distilled
water
Procedure
1. Number the test tubes from 1 to 7 with the marking
pencil and put them in the rack.
2. Using the dropper, place the varying number of
drops of normal saline and distilled water in each of
the 7 test tubes as shown in the table below and mix
3. Draw 2 ml of blood from a suitable vein and
gently eject one drop of blood into each of the 7
tubes and mix*
4.Leave the test tubes undisturbed for one hour
then observe the extent of hemolysis in each
tube
No. of test tube 1 2 3 4 5 6 7
No. of drops of 25 16 12 10 9 7 0
NaCl
No. of drops of 0 9 13 15 16 18 25
D.W
Tonicity of NaCl 0.9 0.64 0.48 0.4 0.36 0.28 0
(in%)
While judging the extent of hemolysis from the
depth of red color of supernatant saline, tube#1
(normal saline) and tube#7 (distilled water) will
act as controls, i.e., no hemolysis in normal saline
and complete hemolysis in distilled water
a. The test tubes in which no hemolysis occur, the
RBCs will settle down and form a red dot at the
bottom of the tube, leaving the saline above clear
b. If there is some hemolyis, the saline will be tinged red with
Hb with unruptured cells forming a red dot at the bottom.
The color of the saline will be seen to be increasingly deeper
with decreasing tonicity of saline
c. The test tubes in which there is complete
hemolysis, the saline will be equally deep red
with no red cells at the bottom of these tubes.
Results
▪ Note the start of hemolysis (the onset of fragility)
and record the test tube number. Express your
results in % saline
▪ Note the start of complete hemolysis
