MODULE 5 UNIT 2: OSMOTIC FRAGILITY TEST Flashcards
A standard volume of fresh, heparinized blood is mixed with NaCl solutions ranging from [?] (isotonic saline) to 0.0% (distilled water) in 0.05% to 0.1% increments. After a 30-minute incubation at room temperature, the tubes are centrifuged and the absorbance of the supernatant is measured spectrophotometrically at 540 nm.
0.85%
A standard volume of fresh, heparinized blood is mixed with NaCl solutions ranging from 0.85% (isotonic saline) to [?] (distilled water) in 0.05% to 0.1% increments. After a 30-minute incubation at room temperature, the tubes are centrifuged and the absorbance of the supernatant is measured spectrophotometrically at 540 nm.
0.0%
A standard volume of fresh, heparinized blood is mixed with NaCl solutions ranging from 0.85% (isotonic saline) to 0.0% (distilled water) in (?) to 0.1% increments. After a 30-minute incubation at room temperature, the tubes are centrifuged and the absorbance of the supernatant is measured spectrophotometrically at 540 nm.
0.05%
A standard volume of fresh, heparinized blood is mixed with NaCl solutions ranging from 0.85% (isotonic saline) to 0.0% (distilled water) in 0.05% to 0.1% increments. After a (?) at room temperature, the tubes are centrifuged and the absorbance of the supernatant is measured spectrophotometrically at 540 nm.
30-minute incubation
A standard volume of fresh, heparinized blood is mixed with NaCl solutions ranging from 0.85% (isotonic saline) to 0.0% (distilled water) in 0.05% to 0.1% increments. After a 30-minute incubation at room temperature, the tubes are centrifuged and the absorbance of the supernatant is measured spectrophotometrically at (?).
540 nm
(?) is the absorbance in the tube to be measured
Ax%
(?) is the absorbance in the A0.85% NaCl tube (representing 0% hemolysis)
A0.85%
(?) is the absorbance
A0.0%
The % (?) for each % NaCl concentration will be plotted, and an osmotic fragility curve is to be drawn
hemolysis
(?) show initial hemolysis at 0.45% NaCl
Normal biconcave RBCs
100% or complete hemolysis generally occurs between (?) NaCl
0.35% and 0.30%
initial hemolysis begins at an NaCl concentration greater than (?).
0.5%
If the curve is shifted to the (?), the patient’s RBCs have increased osmotic fragility.
left
If the curve is shifted to the (?), the RBCs have decreased osmotic fragility.
right
(?) is found in conditions characterized by numerous target cells, such as thalassemia.
Incubating the blood at (?) before performing the test (called the incubated osmotic fragility test) allows HS cells to become more spherical and is often needed to detect mild cases.
37°C for 24 hours
Patients who have (?) is incubated tend to have mild disease and a low number of spherocytes in the total RBC population.
increased osmotic fragility only when their blood
Griffin and Sanford Method
Counting chamber
Principle
o 12 test tubes are prepared and each tube are numbered from 25 to 14.
o Each tube must contain a specific NaCl concentration (%NaCl= tube number X 0.02) and a drop from an EDTA treated whole blood. o Except tube 25, the amount of distilled water to be added in each tube containing the drop of whole blood and specific concentrations of NaCl are listed in the table.
o This will test the stability of the red blood cells under different concentrations of NaCl.
o As the amount of distilled water in each tube increases, this makes the solution more hypotonic.
Griffin and Sanford Method
: tubes with compact sediments and clear solution
No Hemolysis
: first tube from the left (tube 25) with not so compact sediments and with faint solution
Initial Hemolysis (IH)
: first tube from the left (tube 25) without sediments and with dark red solution
Complete Hemolysis (CH)
Result Interpretation
Normal Values:
o Initial Hemolysis: tube (?)
o Complete Hemolysis: (?)
o Initial Hemolysis: tube 22 (.42 - .44% NaCl)
o Complete Hemolysis: tube 17 (.32 - .34% NaCl)
This test demonstrates the increase fragility of erythrocytes in blood specimens in which the surface area to volume ratios of the erythrocytes has decreased. A series of tubes with increasingly hypotonic sodium chloride (NaCl) solutions will be added with blood.
Griffin and Sanford Method
In each tube, water enters and leaves the (?) until equilibrium is achieved.
RBCs
In (?) NaCl, the amount of water entering the cell is equivalent to the water leaving the cell because the intracellular and extracellular osmolarity is the same.
0.85%
In a (?), more water will enter the cell to dilute the intracellular contents until equilibrium is reached between the cytoplasm and the hypotonic extracellular solution.
hypotonic solution
Griffin and Sanford Method
As this phenomenon occurs, the (?) swell.
cells
As the (?) are subjected to increasingly hypotonic solutions, even more water will enter the (?) until the internal volume is too great and lysis occurs.
RBCs
Because (?) already have a decreased surface area to volume ratio, they lyse in less hypotonic solutions than normalshaped, biconcave RBCs and thus have increased osmotic fragility.
spherocytes
The osmotic fragility test is time-consuming as it requires a blood specimen collected fresh with heparin as its anticoagulant without trauma and NaCl solutions should be made accurately
At room temperature Specimens are stable for 2 hours. However, if refrigerated, specimen is viable for 6 hours
The osmotic fragility test is also non-specific and an increased fragility does not differentiate spherocytosis caused by other conditions, such as burns, immune hemolytic anemias, and other acquired disorders
