Osmotic fragility of red blood cells.ppt

hematology

1. OSMOTIC FRAGILITY
OF RED BLOOD CELLS

2. OSMOTIC FRAGILITY TEST
•DEFINITION
•-it is a testthat 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

3. CONT…
•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

4. NORMAL RANGE:
•-hemolysis onset at: 0.45-0.5 % NaCl
•-hemolysis complete at: 0.3-0.33 % NaCl
•FACTORS AFFECTING OSMOTIC FRAGILITY
•-cell membrane permeability
•-surface-to-volume ratio

5. -Hereditary spherocytosis
-Acquired spherocytosis
Hereditary spherocytosisis 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

6. 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

7. •DECREASED OSMOTIC FRAGILITY
•-Thalassemia
•-Sickle cell anemia
•-Iron deficiency anemia

9. PROCEDURE
•We use human erythrocytes that were washed with physiologic
solution and thus do not contain plasma
•Label5eppendorfmicrotubes1–5andpipette1mlofthe
followingsolutionsintothem:
•tube1–physiologicalsolution(non-diluted)
•tube2–physiologicalsolutiondilutedwithwaterintheratio3:1
•tube3–physiologicalsolutiondilutedwithwaterintheratio2:1
•tube4–physiologicalsolutiondilutedwithwaterintheratio1:1
•tube5–physiologicalsolutiondilutedwithwaterintheratio1:5,
containingNH
4
Cl(NH
4
Cldisablesremainingmembranepumps)

10. •Pipette 50μl erythrocyte suspension into each microtube,
gently mix and let stand for 10minutes. Then centrifuge for
3minutes at 3000×gand carefully collect the
supernatants for hemoglobin assay
•Preparefiveglasstesttubes1–5with2mlDrabkin
reagent.Add50μlofeachsupernatantfromtheprevious
stepintothecorrespondingglasstesttubeandmixgently.
•Measuretheabsorbancesofallthesamplesat400nm
againstblankcontainingtheDrabkinreagentonly.

11. •Due to the fact that the blood sample is not fresh, we
observe partial hemolysis even in test tube1. The amount
of hemoglobin determined in this test tube represents the
control value and should be subtracted from the values
obtained for all the tubes. The value of hemoglobin
measured in test tube 5 is the maximum obtainable amount
and we express it as 100% hemolysis. On this basis,
calculate the percentage of hemolysis in test tubes 2–4.
•Usingacalibrationgraph,expressyourresultsasthe
hemoglobinconcentrationinmg/l.