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Definitions

• Osmotic pressure: is the pressure applied by a


solution to prevent the inward flow of water
across a semipermeable membrane.
• A semipermeable membrane:
also termed (a selectively-permeable membrane,
a partially-permeable membrane or a
differentially-permeable membrane), is a
membrane that will allow certain molecules or
ions to pass through it by diffusion and
occasionally specialized "facilitated diffusion"
• Diffusion:
describes the spread of particles through random motion
from regions of higher concentration to regions of
lower concentration
• Active transport
• Cytolysis, or osmotic lysis:
occurs when a cell bursts due to an osmotic imbalance
that has caused excess water to move into the cell. It
occurs in a hypotonic environment, where water
diffuses into the cell and causes its volume to increase.
If the volume of water exceeds the cell membrane's
capacity then the cell will burst.
• osmotic concentration:
concentration of osmotically active particles in
solutions.
• Osmolarity:
is the measure of solute concentration, defined
as the number of osmoles (Osm) of solute per
liter (L) of solution (osmol/L or Osm/L).
• osmotic fragility test:
is a test to measures red blood cell (RBC)
resistance to hemolysis when exposed to a
series of increasingly dilute saline solutions.
• Factors affect the osmotic fragility:
1. Cell membrane permeability.
2. Surface-to – volume ratio.

Why the Test is performed?


• This test is performed to detect thalassemia and
hereditary spherocytosis.
• Hereditary spherocytosis is a common disorder in which red
blood cells are defective because of their round, ball-like
(spherical) shape. These cells are more fragile than normal.
• Cells that are flatter than normal are more likely to expand,
and thus have decreased osmotic fragility.
• Thalassemia is an inherited condition that affects the portion
of blood (hemoglobin) that carries oxygen.
• Some red blood cells in patients with
thalassemia are more fragile than normal, but a
larger number are less fragile than normal.
• When spherocytes (HS) are suspected on the
basis of an elevated mean corpuscular
hemoglobin concentration or on examination of
a peripheral blood smear, the osmotic fragility
test may be used to confirm the presence of
spherocytes.
• The test does not distinguish between
spherocytes in HS and in acquired autoimmune
hemolytic anemia.
• Osmotic fragility decreased in:
Thalassemia.
Iron deficiency anemia.
Sickle cell anaemia.

• Osmotic fragility of red cells increased in:


Hereditary spherocytosis.
Acquired spherocytosis
Isotonicity
• Isotonic solutions contain equal concentrations
of impermeable solutes on either side of the
membrane.
Hypertonicity
• A hypertonic solution is a solution having a
greater solute concentration than the cytosol.
• It contains a greater concentration of
impermeable solutes on the external side of the
membrane.
• When a cell’s cytoplasm is bathed in a
hypertonic solution the water will be drawn into
the solution and out of the cell by osmosis.
• If water molecules continue to diffuse out of the
cell, it will cause the cell to shrink
Hypotonicity
• A hypotonic solution contains a lesser
concentration of impermeable solutes than the
solution on the external side of the membrane.
• When a cell’s cytoplasm is bathed in a
hypotonic solution the water will be drawn out
of the solution and into the cell by osmosis.
• If water molecules continue to diffuse into the
cell, it will cause the cell to swell, up to the
point that lysis )rupture) may occur
Test
• Purpose:
1- To aid diagnosis of hereditary spherocytosis &
Thalassemia.
2- To supplement a stained cell examination to detect
morphologic RBC abnormalities.

• Material:
 Specimen: whole blood
 Collection Medium: Na Heparin tube or Lithium
Heparin tube.
 Minimum: 5 ml whole blood.
 Rejection Criteria: Hemolyzed specimen.
 Methodology: Spectrophotometer.
Procedure
)%( .Final conc D.W. (ml) 1%Nacl(ml) Test tube

1.00 0.0 10.0 1


0.85 1.5 8.5 2
0.75 2.5 7.5 3
0.65 3.5 6.5 4
0.60 4.0 6.0 5
0.55 4.0 5.5 6
0.50 5.0 5.0 7
0.45 5.50 4.5 8
0.40 6.0 4.0 9
0.35 6.0 3.5 10
0.30 7.0 3.0 11
0.20 8.0 2.0 12
0.10 9.0 1.0 13
0.00 10.0 0.0 14
• Then we divide every volume in 2 tubes so now
we get 28 tubes.
• Add 50 micron of whole blood to every tube.
• let the tubes at R.T for 30 min at 2500 rpm.
• Well mixing by using the vortex.
• Centrifuge for 5 minutes at 2500 rpm.
• Now we will measure the absorbance in the tubes
by using spectrophotometer (540 nm).
• calculate the % of hemolysis.
Result
• % of hemolysis =
(Abs of tube / Abs of tube 14) * 100%

• Normal Range:
Hemolysis begins 0.45% and complete 0.35%

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