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The following message was posted to: PharmPK
Dear list,
I would like to measure a drug in lymphocytes. Can anyone point me in
the
direction of a relatively quick and easy method for getting a good
preparation of lymphocytes from whole blood?
On a related topic, can anyone comment on the length of time blood
samples
can be stored and still produce viable PBMCs for drug analysis?
Regards,
Derrick Parsons
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The following message was posted to: PharmPK
Hi, The fastest and easiest way to purify PBMCs from whole blood is to
use
Becton Dickinson Vacutainer CPT tubes (CPT= Cell Preparation Tube part
#362753). These tubes contain the anticoagulant, the density separation
media and are compatible with the standard vacutainer blood collection
equipment. Just collect your whole blood directly into the CPT tube,
spin
it according to the manufacturers directions and the peripheral blood
mononuclear will collect as an identifiable band. We typically get
approx.
one million PBMCs per milliliter of whole blood.
Have a good day,
Michael Branks
Research Scientist
La Jolla Parmaceutical Company
6455 Nancy Ridge Drive
San Diego, Calif. 92121-2249
ofc. (858) 646-6678
fax. (858) 626-2845
mjbranks.aaa.ljpc.com
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The following message was posted to: PharmPK
Dear Derrick,
We have been using BD Vacutainer(BD Vacutainer system,
NJ). You withdraw blood directly in vacutainer tubes
and centrifuge for 15 min to get a layer of PBMCs
(+platelets).
Though we have never stored blood in tubes but
probably you can store blood in tubes for up to 24 h
after collection, according to manufacturer.
website: www.vmdp.com.
hope this helps.
Dr. Sunil bajad (Patil)
Research Associate
Department of Medical Sciences
School of Vet. Medicine
University of Wisconsin-Madison,
2015 Linden Drive west,
Madison 53706, WI,USA
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The following message was posted to: PharmPK
Hi Michael,
Using CPT tubes, though we get PBMCs but I am
concerned about the Platelet contamination. If you
count the cell number by microscope you won't probably
count platelets. We have used cell counter and were
surprised to find that there are approx 100 platelets
for 1 PBMCs.
do you have any experience separating them!
regards,
Dr. Sunil bajad (Patil)
Research Associate
Department of Medical Sciences
School of Vet. Medicine
University of Wisconsin-Madison,
2015 Linden Drive west,
Madison 53706, WI,USA
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The following message was posted to: PharmPK
Dear Derrick,
We use also Vacutainer tubes from Becton. You will have a separation of
Peripheral Blood Mononuclear Cells (PBMCs): mainly lymphocytes (with
also
monocytes).
Regarding the lenght of the time between blood sampling and tube
centrifugation, the most rapid would be the best (within two hours, for
best
recovery). Moreover, you should check the stability of your analyte(s)
in
blood during this period, since degradation may occurs between two
lenghts
of time. You will need to wash (preferably at 4°C) then lyse the cells
(methanol/buffer) before injection of the supernatant in your analytical
system. Check also for analyte stability. Perhaps you will need to
carry out
an extraction step, but intracellular differs largely from plasma
medium.
If you have some trouble in PBMCs counting you can contact me
(henri.benech.-at-.cea.fr). We have developped and validated as a kit a
biochemical method for PBMCs counting in clinical samples applied to
intracellular drug assay.
You can see also some publications:
Development of a direct assay for measuring intracellular AZT
triphosphate
in human PBMCs.
F. BECHER, D. SCHLEMMER, A. PRUVOST, M.-C. NEVERS, C. GOUJARD, C.
GUERRERO,
T. BROSETTE, L. LEBEAU, C. CREMINON, J. GRASSI AND H. BENECH
Anal. Chem.74(16) : 4220-4227 (2002).
Improved method for the simultaneous determination of d4T, 3TC and ddI
intracellular phosphorylated anabolites in human peripheral blood
mononuclear cells using high performance liquid chromatography/tandem
mass
spectrometry.
F. BECHER, A. PRUVOST, C. GOUJARD, C. GUERRERO, J.F. DELFRAISSY, J.
GRASSI
AND H. BENECH
Rapid Commun. Mass Spectrom. 16: 555-565 (2002).
Hope this help
Henri BENECH
CEA
Service de Pharmacologie et d'Immunologie
DSV/DRM/SPI/LEMM
F-91191 Gif-Sur-Yvette Cedex, FRANCE
email: henri.benech.at.cea.fr
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