Recently I read many papers of reputed journals demonstrating the invitro cytotoxcity of synthetic materials, plant extracts, particles by Hemolytic assay, Triton-X100 is generally used as positive control and PBS 7.4 pH as negative control as RBCs suspension is made in that media and used further, I asked many researcher who had published and reported work on that, that how you calculated the value of positive and negative control % hemolysis that you mentioned in your work along with the samples, The answer was that as you well know Triton-X gives 100% hemolysis and PBS gives 0% and some said that visual indication support previous argument too, But I'm curious how to exactly calculate the %hemolysis for control because Normally the method I investigated used the following formula for calculation but I'm not able to calculate the control value,
%hemolysis= (abs. of sample - abs of negative control)/ abs of positive control *100
I believe that the method you described is generally accepted. The purpose is not necessarily to determine the precise amount of hemolysis (which would require measuring the amount of hemolysis in the positive and negative controls if they are not 100 and 0% as expected). Instead, the purpose is to compare the relative hemolytic potencies of compounds.
If you wanted to measure the absolute amount of hemolysis in the negative control, you would measure the concentration of hemoglobin in the plasma after removing all the red blood cells, and compare this to a sample that is known to have zero hemolysis. For the positive control, you would measure the amount of hemoglobin remaining in the red blood cells compared to the amount in whole blood.
I believe that the method you described is generally accepted. The purpose is not necessarily to determine the precise amount of hemolysis (which would require measuring the amount of hemolysis in the positive and negative controls if they are not 100 and 0% as expected). Instead, the purpose is to compare the relative hemolytic potencies of compounds.
If you wanted to measure the absolute amount of hemolysis in the negative control, you would measure the concentration of hemoglobin in the plasma after removing all the red blood cells, and compare this to a sample that is known to have zero hemolysis. For the positive control, you would measure the amount of hemoglobin remaining in the red blood cells compared to the amount in whole blood.
Hi Yasir,
Here's a detailed description on how to set the assay up and how to calculate the results based on your standard and controls.
You will deduct the signal of the blank from both experimental sample and reference standard, and then calculate [test OD / reference OD].
Article Measuring the 50% Haemolytic Complement (CH50) Activity of Serum
Juha Kotimaa Sir thanks for contribution and literature, I have another question that what is the toxicity scale for hemolytic assay, Like up to how much % the hemolysis for plant extracts, drugs and materials is acceptable ?
Adam B Shapiro Sir do you have any reference literature in which the toxicity sacle is mentioned, what is the highest value of % hemolysis that is caused by test sample is acceptable with respect to concentration?
I don't know the answer from references, but my thinking is that no amount of hemolysis is acceptable.
Hi Yasir,
I am not quite sure I follow the purpose of using haemolytic assay to evaluate your compounds or their toxicity.
But I agree with Adam that any haemolysis would be an indication of adverse effect.
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