It is used in the diagnosis of hereditary spherocytosis and the screening for thalassemia.
Hereditary spherocytosis is a disorder in which red blood cells are defective because of their round, ball-like (spherical) shape. These cells are more fragile than normal.
Principle:
Red cells will be haemolysed when placed in a hypotonic saline solution. Time and extend of complete haemolysis will depend on the red cell membrane resistance and size and shape of the red cells. Spherocytes will lysed quite quickly in slightly hypotonic solution. Hypochromic microcytic cells will be more resistant to hypotonic solution.
Procedure:
1) Deliver 5ml of 0.9, 0.75, 0.70, 0.65, 0.6, 0.55, 0.50, 0.45, 0.4, 0.35, 0.30, 0.25, 0.2, 0.1% of hypotonic saline solutions into test tubes.
2) 50μl of blood to each tube and mix.
3) Stand tubes for 30mins at room temperature and centurifuge.
4) Pipette the supernatants to cuvette and read absorbance at 540nm.
Note: Supernatent from 0.90% NaCl is used as a blank.
Interpretation of results:
- In normal subjects an almost symmetrical sigmoid curve results.
- Subject with decrease resistance to lysis will show fragility curve that shift to the left.
- Subject with increase lysis will show fragility curve shift to right
(taken from http://www.suite101.com/article.cfm/medical_student/40859)
Clinical Significance
The ability of normal red cell to withstand hypotonicity results from its biconcave shape which allows the cell to increase its volume by about 70% before the surface membrane is stretched, and once this limit is reached lysis occurs.
Spherocytes have an increased volume to surface area ratio; their ability to take in water before stretching the surface membrane is thus more limited than normal and therefore is more susceptible to osmotic lysis.
Decreased osmotic fragility indicates the presence of flattened red cells in which the volume to surface area ratio is decreased. Such a change occurs in iron deficiency anemia and thalassemia in which the red cells with low MCH and MCV are resistant to osmotic lysis.
The ability of normal red cell to withstand hypotonicity results from its biconcave shape which allows the cell to increase its volume by about 70% before the surface membrane is stretched, and once this limit is reached lysis occurs.
Spherocytes have an increased volume to surface area ratio; their ability to take in water before stretching the surface membrane is thus more limited than normal and therefore is more susceptible to osmotic lysis.
Decreased osmotic fragility indicates the presence of flattened red cells in which the volume to surface area ratio is decreased. Such a change occurs in iron deficiency anemia and thalassemia in which the red cells with low MCH and MCV are resistant to osmotic lysis.
Eunice
0503245C
13 comments:
heya eunice....guess it's not too late to ask qn....wanna ask u..u mention that time and extend of complete haemolysis will depend on the red cell membrane resistance..what's the cause of the resistance and also hypochromic microcytic cells will be more resistant to hypotonic solution..may i ask y is this so??..apart from that i also wanna ask u what are the actions you guys take if the pellet was accidentally pipetted together with the supernatant?..tt's all...njoy ur last few wk of SIP...:)
nur zahirah tg02
Hey eunice,
Just wanna ask whether there are any other follow up tests after the Osmotic Fragility Test is done? What are the other methods used for the screening of thalassemia?
Thanks,
Azhar
Hi there!
may i know why supernatant from 0.9% NaCl is used as a blank when u read absorbance? Thanks!!
Kangting 0503331A
Hello Eunice!
Why is it so that red cells with low MCH and MCV are resistant to osmotic lysis?
June, TG02
Hey eunice,
How do you convert the absorbance reading into percentage of lysis?
Thanks.
Adrian TG01
HI!
I noticed that there are alot of different concentration of saline used, some with very little differences like 0.75 and 0.70. Why do u need so many different concentrations? And also, the other qns is same like kangting. Why u use supernatant from 0.9% NaCl as a blank?
Thanks
Charmaine
TG01
Hi Eunice
I have a simple question.
While I do acknowledge that hereditary suggests that spherocytosis is passed from one generation to another, my question is:
Is there a specific group (ethnicity, sex, age group) that is more susceptible to hereditary spherocytosis and/or thalassemia?
Desmond Heng
0503179D
TG02
hi eunice,
when doing this test is there any possibility of false positive result? there might be a slight fluatuation to the graph which we may not notice if we didn't see carefully.
Lizzie
Hey eunice,
Just wondering why is it measured at 540nm? any specific reasons for this being a preset (is it?) value?
Also, if there are varying concentrations of NaCl solutions used, why is only the highest (ie 0.9%) taken as blank instead of blanking at every respective NaCl concentration? Won't this lead to any inaccuracy?
Thanks!
- Debta, TG02
Helloo sista,,,
what if the person have anisopoikocytic cells? How to detect hereditary spherocytosis in this case?
Thank u...
Pei Shan
TG02
Hi Eunice,
Hope you don't mind this question poping up too late.In the graph you mention that subjects with decrease resistance to lysis, the curve will shift left but wouldn't it be shifting right, since it is also prone to lyse?
Avery
TG02
Can somemone remind Eunice to reply the questions? If no reply by next week, I will consider her posting as invalid. Ms Chew
Qns: other follow up tests after the Osmotic Fragility Test is done? What are the other methods used for the screening of thalassemia?
Ans: there are no follow up tests fo OF as this is done as requested. screening of thalassaemia is usually done by running FBC to check for low MCV, MCHC, HbH test, and quantitation of HbA2 and F value.
Qns: why supernatant from 0.9% NaCl is used as a blank when u read absorbance
Ans: at 0.9% there will be no lysis as our physiological concentration for NaCl is around 0.85-0.8%.
Qns: Why is it so that red cells with low MCH and MCV are resistant to osmotic lysis?
Ans: red cells that have low MCV and MCH would mean that they are microcytic and hypochromic thus will have a biconcave shape as compared to spherocytes, and so will allow the cell to increase its volume more than spherocytes.
Qns: How do you convert the absorbance reading into percentage of lysis?
Ans: The absorbance reading will be converted into percentage using a program in the lab.
Qns: Why do u need so many different concentrations?
Ans: This is used in order to plot a curve as seen in the diagram.
Qns: when doing this test is there any possibility of false positive result?
Ans: yes. false positive may occur such as due to pipetting errors thus usually a negative and positive control will be done at the same time.
Qns: why is it measured at 540nm?
Ans: the absorbance measured is according to the lab protocol..not sure if its got any specific reason.
Qns: time and extend of complete haemolysis will depend on the red cell membrane resistance..what's the cause of the resistance and also hypochromic microcytic cells will be more resistant to hypotonic solution..may i ask y is this so?
Ans: The cause of resistance is due the the RBC membrane stucture. For a normal red cell, its biconcave shape will allow the cell to increase its volume to app 70% before it lyse whereas spherocytes had limited volume to stretched its size and so will be more susceptible to lysis.
Qns: the actions you guys take if the pellet was accidentally pipetted together with the supernatant?
Ans: When the pellet is pipetted with the supernatent, it will be centrifuged again. however this hardly occurs.
p.s. : very sorry for the late reply..
eunice
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