Erythrocyte Osmotic Fragility Test
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Red blood cells (RBCs)
• Red blood cells or erythrocytes take up oxygen in the lungs and release
it into tissues.
• The erythrocyte is characterized by a biconcave shape giving it an excess of
surface area in relationship to its volume.
• The cytoplasm of erythrocytes is rich in hemoglobin that can bind oxygen
and is responsible for the red color of the cells and the blood.
• Red blood cells are surrounded by a semipermeable
membrane which allows water to pass through
while generally restricting the solutes.
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Hypotonic
watER
hypERtonic
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Tonicity effects on the red blood cell
• 0.9% w/v NaCl solution is said to be isotonic with human blood.
• In a hypeRtonic solution (˃ 0.9% NaCl)
RBCs shRink
• In a hypOtonic solution (˂ 0.9% NaCl)
RBCs swOllen then burst (Hemolysis)
So, in a hypotonic medium, a membrane rupture occurs allowing hemoglobin to exit from the cells.
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Osmotic fragility test principle
RBC
Hypotonic solution (˂ 0.9% NaCl)
Water influx
Swelling/Spheroidal
Critical volume (Normal at 70%)
Membrane leakage
Burst
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Osmotic fragility test procedure
10 mL Hypotonic
NaCl solution
+
0.1 mL blood
NaCl Conc
Hemolysis starts at 0.45% NaCl
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Osmotic fragility test
Definition:
Surface
Volume
• The osmotic fragility test is a measure of the ability of the red cells to take up fluid
without lysing.
• Osmotic fragility is a test to measure RBCs resistance to hemolysis when exposed to a series
of increasingly dilute saline solutions.
• The sooner hemolysis occurs, the greater the osmotic fragility of the cells.
Factors affect the osmotic fragility:
The primary factor affecting the osmotic fragility test is the shape of RBCs, which, in turn, depends
on the:
1. Cell membrane permeability.
2. Surface-to–volume ratio (detect morphologic RBCs abnormalities).
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1. Increased Surface – To – Volume Ratios:
• The larger the amount of red cell membrane (surface area) in relation to the
size of the cell, the more fluid the cell is capable of absorbing before rupturing.
• The RBC is more resistant to hemolysis and has decreased osmotic fragility.
• Examples: Iron-deficiency anemia, Thalassemia, and Sickle cell anemia.
Normal RBCs
Thalassemia
Sickle cell anemia
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Surface
2. Decreased Surface – To – Volume Ratios:
Volume
• The RBC has ball-like (spherical) shape, so there is no space for more fluid to
enter the cell.
• The RBC is less resistant to hemolysis and has increased osmotic fragility.
• Examples: Hereditary spherocytosis and whenever spherocytes are found.
Normal
RBCs
Hereditary
spherocytosis
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Calculation
% Hemolysis
=
OD of sample
--------------------------- x 100
OD of tube #1
Use test tube #1 (distilled water) as a control to represent 100% hemolysis (0% Na Cl).
The results of the test may then be graphed, with the % hemolysis on the Y axis and
NaCl concentration on the X axis.
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[1] represents the
normal range for %
hemolysis.
Normal range:
• Initial hemolysis
Hemolysis begins in 0.45 - 0.5% NaCl solution
• Complete hemolysis
Hemolysis is complete in 0.3 – 0.33% NaCl solution
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Report from graph pattern
1. Normal osmotic fragility: Within shading area
2. Decreased osmotic fragility: Shift of the curve
• to the left if start from 0.0% NaCl
• to the right if start from 0.9% NaCl
3. Increased osmotic fragility: Shift of the curve
• to the right if start from 0.0% NaCl
• to the left if start from 0.9% NaCl
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Normal osmotic fragility curve
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Typical Graphs for RBC Osmotic Fragility
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Given the following OD values and your calculated osmolarity, draw the osmotic fragility curve.
Calculate % hemolysis for test tubes #1 to #6.
Tube
#
NaCl solution
(%)
1
(X axis)
0.9
2
0.6
3
0.5
4
0.4
5
0.3
6
0.0
Optical density
(OD)
%Hemolysis
(Y axis)
Osmolarity
(Osmols/l)
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Any Questions?
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