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Fragility of the Red Cells or Osmotic Fragility 11

الكلية كلية طب الاسنان     القسم  العلوم الاساسية     المرحلة 2
أستاذ المادة وسن نجم عبد السادة الربيعي       22/02/2019 23:35:56

fragility of the red cells or osmotic fragility

osmotic fragility :water moving through a red blood cell membrane in solutions with three different concentrations of solute. the isotonic (normal) solution has the same concentration as the cell, and the water moves into and out of the cell at the same rate. the hypotonic (dilute) solution causes the cell to swell and eventually hemolyze (burst) because of the large amount of water moving into the cell the hemoglobin of the hemolyzed red cells dissolves in the plasma, coloring it red. the hypertonic (concentrated) solution draws water out of the cell, causing it to shrink (fig.1,2).
a 0.9% sodium chloride solution is isotonic with plasma. when osmotic fragility is normal, red cells begin to hemolyze when suspended in 0.48-0.50 saline and hemolysis is completed in 0.35. erythrocytes have a large surface: volume ratio that allows them to deliver oxygen more efficiently, maintain flexibility to travel in small or obstructed areas of the body, and expand without lysis when the osmotic concentrations of the body are not isotonic .
when a red cells membrane surface decreases and its volume remains the same or increases, the cell becomes more turgid and less deformable. spherocytes, which have a decreased surface to volume ratio demonstrate an increase osmotic fragility. this is due to their inability swell in a hypotonic medium before leaking hemoglobin. sickle cells, target cells are relatively resistant to osmotic change and therefore demonstrate a decreased fragility (fig.3) .

the principle


purpose
to measure the ability of the erythrocytes in a specimen to withstand a range of osmotic concentrations without lysing .































figure (1): osmotic fragility of the red blood cell.

apparatus and reagents
1.blood sample .
2.test tubes and rack .
3.distilled water and 1% nacl solution .
4.pipettes 5 ml .
5.volumetric flask 100 ml .


figure (2): erythrocyte membrane shape changes associated with the osmotic environment.

normocyte


crenated cells


sickled cells


target cells


schistocytes


elliptocytes

figure (3) :examples of erythrocyte morphologies


procedure
1.prepare the rack containing twelve (12) clean dry test tubes and label them from 1 to 12 .
2.with 5 ml pipette add 1% nacl solution (place 1 gm of nacl crystals in a 100 ml volumetric flask and fill to the mark with distilled water, stir to completely dissolve nacl), and with the other 5 ml pipette add distilled water into this series of tubes according to the following table :





table (1):
tube 1% nacl (ml) distilled water (ml) final concentration
1 4.25 0.75 0.85
2 3.5 1.5 0.7
3 3.25 1.75 0.65
4 3.0 2.0 0.6
5 2.75 2.25 0.55
6 2.5 2.5 0.5
7 2.25 2.75 0.45
8 2.0 3.0 0.4
9 1.75 3.25 0.35
10 1.5 3.5 0.3
11 1.25 3.75 0.25
12 0.75 4.25 0.15

3.mix the contents of each tube .
4.add one droping of blood (50µl) into each tube and mix gently .
5.let the tubes sit at room temperature for 30 minutes .
6.centerfuge at 2000 rpm for 5 minutes .
7.note which tubes show initial and complete hemolysis. initial hemolysis is recognized by faintly pink supernatant. complete hemolysis is seen as ared supernatant and there will be no red cells visible at the bottom. now examine a droping from each tube under the microscope. determine the concentration of salt solution at which hemolysis has just started, and the concentration at which hemolsis is completed.
8.this test may be quantitated by measuring each tube on the spectrophotometer. if this done, two additional tubes are necessary, the first is a blank containing 50µl of blood to which 5 ml of 0.9% nacl is added, which will result in no hemolysis. the second blank is for complete (100%) hemolysis and is obtained by adding 50µl of blood to 5 ml of distilled water.



9.after centrifugation ,the supernatant of each tube is removed ,and its optical density (o.d.) is read in a spectrophotometer using a 540 nm. the percentage of hemolysis in each tube is calculated using the following equation :

¬¬¬¬¬¬¬¬¬¬¬¬¬ o.d. (unknown) ? o.d.(0.85% tube)
% hemolysis = ———————————————— × 100
o.d. (second blank) ? o.d.(0.85% tube)

an osmotic fragility curve may be drawn by plotting the percent hemolysis in each tube against the corresponding concentration of nacl solution .

note :fresh heparinized blood is recommended edta, oxalate or citrate should not be used because of the additional salts present. this test should be performed immediately because cell shape and osmotic conditions change with time.

table (2):examples of initial and complete hemolysis in various conditions follow :
examples initial hemolysis
(% nacl) complete hemolysis
(% nacl)
normal 0.45 0.35
hereditary spherocytic anemia 0.65 0.45
acquired hemolytic anemia 0.50 0.40
thalassemia 0.35 0.20
sickle cell anemia 0.35 0.20




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