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Hi all,
I have a query regarding the preparation of analytical (original) and
duplicate plasma samples,
Is it compulsory to prepare the analytical and duplicate plasma
samples by dividing the same plasma (obtained after centrifugation of
the blood collected at any individual time point) in to two portions?
As per our protocol, we need to collect the 5 ml of blood from the
subject for different time points, but as the 5 ml, vacutainers are
not available,
shall we collect the blood in two different vacutainers(one is 3 ml
and other one is 2 ml) one by one immediately and centrifuge the two
vacutainers, then use the plasma collected from the 3 ml vacutainer
as analytical sample and the plasma collected from 2 ml vacutainer as
the duplicate sample.
Is the above procedure is OK from a regulatory perspective?
Since both the analytical and duplicate samples are not prepared by
dividing the same plasma and there was a difference in the sample
collection time (in seconds only) of both the 3 ml and 2 ml
vacutaines, since we are measuring the circulating blood
concentration, and it will vary from time to time.
Request experts to comment, provide opinion/suggestions/implications
of the above.
Thanks in advance
Navvy.
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The following message was posted to: PharmPK
They are close but not the same sample. Generally, you would collect
in one
tube, spin and split the harvest into two or more aliquots which are
identical and use these as a hedge against freeze thaw, other analytical
issues and to provide identical samples for different analyses (test
article, antibody or other biomarker) Collecting the sample in two
different
tubes is collecting the sample in two different tubes, they are
different in
time- which may become more exaggerated and they are different in
collection
environment. If the object is to keep a reserve sample it might be
best to
harvest then pool the plasma and then aliquot. The good thing is
that it
would remove any differences; the bad thing is that it removes any
differences.
Ed O'Connor, Ph.D.
Laboratory Director
Matrix BioAnalytical Laboratories
25 Science Park at Yale
New Haven, CT 06511
Web: www.matrixbioanalytical.com
Email: eoconnor.at.matrixbioanalytical.com
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Dear Mr. Navvy,
My suggested answers for your questions are as follows:
Is it compulsory to prepare the analytical and duplicate plasma
samples by dividing the same plasma (obtained after centrifugation of
the blood collected at any individual time point) in to two portions?
Yes. Now, regulators are verifying the plasma vials meant for
preservation, ( may be to check the labeling and others etc)
we need to collect the 5 ml of blood from the
subject for different time points, but as the 5 ml, vacutainers are
not available,shall we collect the blood in two different vacutainers
(one is 3 ml
and other one is 2 ml) one by one immediately
You can collect, but of course, document it as protocol deviation
and your Principal Investigator should justify this action will not
have any significant effect on the outcome of the results.
Kindly note that these two time samples for the particular time
point of the all the subjects should be centrifuged simultaneously.
Other members can also comment on this.
T.R.Yegnaraman,
Azidus Laboratories Limited.
Chennai-600 048
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The following message was posted to: PharmPK
Navvy,
Why do you collect 5 ml of blood in first place?
How much plasma volume you use for processing and subsequent analysis?
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Hi,
I have to ask what you will be doing with the 5 mL of blood? It
seems that you will use it to prepare plasma. The 5 mL of blood will
yield 2-2.5 mL of plasma. I am assuming that you will measure the
concentration of drug in the plasma samples. If so, what is the
volume that is used for the assay? If the assay volume is 100 uL,
then one could ask the question of why you need to take 5 mL of
blood. 2 mL may be sufficient.
Regards
Mark
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Dear Navvy,
1. In case the study is a pilot study, you need not have duplicate
samples. You can keep the blood collection/sample = 5 or even 4 ml
2. In case its a pivotal study, you definitely need duplicate samples
and need to preserve one aliquot for verification, should there be a
need to do so. I suggest you try withdrawing 6 ml per sample (the
total blood loss should be preferably be less than 350 ml, if not,
keep this volume less than 450 ml, otherwise you will need to
convince the ethical committee why the blood loss is more or even
need a special permission from the committee. In case the blood loss
is greater than 350 ml, choose volunteers [i presume study is to be
done on Indian volunteers] who have Hb levels 13.5 mg% or more +/-
your standard variation in acceptable ranges, usually 10%)
Let me know if you have any more q's
Dr. Gagandeep Singh
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Regarding the collection of 5 mL of blood in consecutive aliquot
tubes (3 mL and 2 mL) for generation of duplicate plasma samples:
I agree with previous responders that 5 mL of blood, which generates
2-2.5 mL of plasma, seems like a lot for most methods. Perhaps
collection of only 3 mL, to make 1 to 1.5 mL of plasma would suffice,
and the plasma could be split into two aliquots of 0.5 to 0.75 mL
each. Depending on how the protocol is written, this may be OK, or
it may need to be handled by either a protocol amendment or as a
protocol deviation.
If you MUST collect 5 mL, and you have only 3-mL and 2-mL collection
tubes, then this change in the protocol almost certainly needs to be
handled by either a by a protocol amendment (prospective) or as a
protocol deviation (after the fact). It will be important to
document what is done and the justification.
As others have noted, the consecutively collected samples are
actually two different samples. You could write a protocol amendment
(or possibly a deviation) that allows you to collect the plasma from
the 3-mL and 2-mL samples and either combine the plasma and then use
it as needed, or keep the two samples separate and use only one for
analysis, unless the second is required. The first approach (combine
the plasma) is probably better if you either need the full 2-2.5 mL
of plasma, or if there are to be multiple tests from the same sample,
or if you ever need to use the additional volume for reanalysis
purposes. We have used the first approach for the collection of CSF
samples, where it can take several minutes to collect a few mL of
CSF. In either case, it is very important to document and justify
what is decided, make sure the bioanalytical lab has what it needs,
and ensure (with documentation!) that the study manage ment personnel
are aware of the action and consequences.
Best regareds,
Tom
Thomas L. Tarnowski, Ph. D.
Bioanalytical Development
Elan Pharmaceuticals
800 Gateway Blvd.
South San Francisco, CA 94080
thomas.tarnowski.-at-.elan.com
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The following message was posted to: PharmPK
Dear Navvy,
using the 3 ml vacutainer & 2 ml vacutainer will
make it
two different samples and not aliquots . Hence the collected plasma will
not be duplicate in nature. If by chance there is cannula block between
the collections, then what ?
Regards,
Dr Antaryami Maharana
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