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Dear All
Why is it strictly necessary to reject hemolyzed samples for bioanalysis?
What are reasons for sample hemolysis?
How to overcome the problem?
--
Regards
Rajendra H. Dhande.
Research Student,
Department of Pharmacology and Toxicology,
Bombay College of Pharmacy, Kalina,
Santacruz (E),
Mumbai-400098
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1) It is not strictly necessary to reject hemolyzed samples unless you are measuring potassium, or some other molecule normally found within red cells. It is necessary to note in a comments area which samples were hemolyzed.
2) Too small of a needle or cannula, too rapid collection or dispensing, too long in storage before harvesting, too little anticoagulant.
3) Care.
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Hi all,
I think heamolysed plasma samples will be a major concern when your drug binds to RBC's. So, it is wise to avoid haemolysed plasma samples in that case. This is from a PK perspective.
Regards
Chakri
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I think haemolysed sample=bad sample is dogmatic and does not follow
common sense. How does one determine partial haemolysis, for example? By
eye? Hold it against the light?
Rather than avoiding haemolysis one could analyse deliberately and fully
haemolysed samples to see whether there is actually something to worry
about in the first place.
Frederik Pruijn
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The following message was posted to: PharmPK
Hi,
If your drug is preferentially partitioning into RBCs, hemolysis can be
cause of concern. This is especially true when only a fraction of your total
samples are hemolyzed. Apart from that, in PK perspective, I do not see any
reason to reject the hemolysed samples.
http://www.bd.com/vacutainer/pdfs/techtalk/TechTalk_Jan2004_VS7167.pdf
is a
good starting point for you to see the causes of hemolysis in analytical
samples.
Sincerely,
Rhishi
Rhishikesh Mandke
Presidential Fellow
Department of Pharmaceutical Sciences
North Dakota State University
Fargo ND 58103 USA
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Dear All,
Samples are critical and expensive, so a mini validation exercise for haemolysed vs normal samples provides you space for go no go decision with the haemolysed sample bioanalysis.
Thanks
Dr Pradeep Sharma
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Dear All,
once the haemolytic effect is proven during method validation, there is no need of rejecting haemolyzed samples for bioanalysis, unless there is no haemolytic effect.
Regards
Shital P.
Bioanalytical Scienetist
Sandoz Pvt Ltd
Mumbai
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Dear Rajendra,
I do not think that you need to discard haemolysed samples during bioanalysis.
What you can do is perform an additional experiment demonstrating haemolysis effect during your method validation. For this you could prepare a few QCs in control haemolysed plasma and your calibrants in normal control
plasma (containing the same anticoagulant ofcourse) and then establish your accuracy and precision for the QC samples. If the QCs are within acceptance criteria you can go ahead with sample analysis using the haemolysed
samples as well.
Warm regards,
Noel
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If you build that into your validation and you show there is no effect
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yes, thats true,
in validation protocol, hemolytic effect test is to be set and has to be proven in validation
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The following message was posted to: PharmPK
Dear Rajendra,
Matrix effects may exist in haemolysed plasma. As suggested by others, you can incorporate testing that in the validation. If this has not been done before and you get haemolysed plasma samples, it is also possible to run additional partial validation.
However, in case of demonstrated matrix effect, I am still wondering about the following:
- is visual inspection sufficient to distinguish haemolysed samples?
- how could we analyse the samples anyway, against appropriate calibration curve?
(As mentioned by someone, samples are highly precious, especially in paediatric studies...).
Best regards
Frederic Massiere
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The following message was posted to: PharmPK
Dear Frederic,
1. You can't distinguish haemolysed samples from normal plasma by visual inspection in some cases.
2. You can run your haemolysed samples against normal plasma calibration curve. At the time of validation you have to running haemolysed quality control samples against normal plasma calibration curve and prove that there will not be any effect if you will get haemolysed plasma samples during actual sample analysis.
If you run those haemolysed samples under a haemolysed calibration curve, concentration will not vary, if you compare those samples with normal plasma CC. Hence analytically there will be no change. Yes, if you talk about Pharmacokinetics point of view, it may vary.
We are doing plasma analysis, assuming that the concentrations of drug are in equilibrium between intra-cellular fluid and in extra-cellular fluid. Now suppose a drug having more binding towards intra-cellular fluid and you are getting haemolysed plasma for that drug, definitely your concentration will vary compare to normal plasma samples. That's why now a day's blood partitioning concepts create major attention.
Regards,
Sudipta Basu
Senior Research Scientist (DMPK)
Sai Advantium Pharma Ltd., Pune, India.
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There is a nice publication out on "Assessing the matrix effects of hemolyzed samples in bioanalysis" by NC Hughes et al, Bioanalysis 2009 1(6) 1057-1066
Useful guidance is given on how to test if hemolysis is a problem for a particular assay and some examples are given on how problems were solved.
Best Regards,
Ronald de Vries
J&J Belgium
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The following message was posted to: PharmPK
The Hughes et all publication was awarded the best paper of 2009 and is
currently available as a free download in this issue of Bioanalysis:
http://www.future-science.com/toc/bio/1/6
["Assessing the matrix effects of hemolyzed samples in bioanalysis" by NC Hughes et al, Bioanalysis 2009 1(6) 1057-1066 - db]
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