RE: buffy coat preparation

Hello Doris

We are am collecting blood into a syringe or transferring blood to
syringe if is collected otherwise. Depending on blood volume it may be 1
to 50 ml volume syringe. We are then leaving the syringe to stand up
(e.g. supported, within a narrow container such as Coplin jar or
beaker).  After 30 min - 2 h (depending on rbc sedimentation rate) we
are replacing the needle for a new one, and bending the needle to U
shape, to have the needle exit downwards. Then very slowly and gently
pushing the piston (in fact having the piston positioned tight on the
bench and in contact with the bench, but moving the syringe itself
slowly down) we are rejecting the initial 2-4 drops of plasma (they may
contain some erythrocytes) and then we are collecting plasma with wbc
into a tube placed underneath the bent needle. This way we are
transferring plasma into the tube, at the end drop by drop, until the
very first drop hanging at the needle's orifice becomes red. Most wbc
are within the last few plasma drops before erythrocytes start to appear
so it is important to collect the final drops and yet not to contaminate
with rbc with the first drop that becomes visibly red.	    

This is an old procedure that we developed many decades ago. Perhaps now
there is something simpler or more rapid. But we still use it since it
provides decent yield and purity of wbc.  

 Zbigniew 

Zbigniew Darzynkiewicz, M.D., Ph.D. 

Professor of Pathology and Medicine 
Director, Brander Cancer Research Institute 
New York Medical College 
BSB, Room 438 
Valhalla, N.Y. 10595 

www.darzynkiewicz.com/zbigniew/ 

-----Original Message-----
From: Wiener, Doris [mailto:Doris.Wiener@moffitt.org] 
Sent: Wednesday, February 20, 2008 12:15 PM
To: cyto-inbox
Subject: buffy coat preparation

 

Can someone send me a protocol for preparing buffy coats from peripheral
blood. We will be collecting large volumes of blood from lymphopenic
patients and want to reduce the number of ficoll gradients we need to
make by concentrating the WBC.

 

Thanks

Doris Wiener


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