Re: [Histonet] Buffy coat preparation for immuno

From:John Kiernan

Why go to all the trouble of embedding and sectioning a buffy coat specimen? What's wrong with diluting it in saline and then making smears or (better, if you have the equipment) cyto-centrifuge preparations? You'll get lots of slides. They can be fixed in methanol and the WBC stained with any conventional Romanowsky-Giemsa method, or immunohistochemically. You could even fix in formalin, if there's some special reason to do so. (You have to lower the pH of an ordinary blood stain if the fixative was formaldehyde.) 

I hope this message is readable; it is sent as plain text.

John Kiernan
Anatomy, UWO
London, Canada
= = =
----- Original Message -----
From: Stephen KumJew 
Date: Sunday, November 30, 2008 23:30
Subject: [Histonet] Buffy coat preparation for immuno

> Hi from Down Under
>     I was wondering if anyone had a method for 
> preparing buffy coats for
> immuno.    
> What we have done so far
> 1) is spun down whole blood, extracted the buffy coat and made a
> thrombin/plasma clot of the extracted buffy coat (minus RBCs). 
> The clot was
> post fixed overnight in 10% formalin and subsequently paraffin 
> processed.5um Sections were cut and immuno done. The clumping 
> cells did not stain, but
> the single ones on their own did.
> 2) To improve on fixation (assuming this was the problem with 
> the unstained
> clumping cells), the buffy coat was collected and fixed in 10% 
> formalin. The
> fixed buffy coat was washed in 0.9% NaCl twice, and then 
> embedded in both  a
> thrombin/plasma clot (unsuccessful), and  an agar block. 
> The agar block was
> paraffin processed. The subsequent immuno showed good staining 
> in single
> WBC, and in one clump of cells. The other clump showed no 
> staining and had a
> bit of cell damage.
>     Thanks
>     Stephen
>     Pathology
>     Sydney University
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