[Histonet] Cell block preparations

[Tony Reilly]

Hi Charles

A method my EM scientist used many years ago was quite simplistic and he thought it worked well.
You simply centrifuge the specimen,  add a set volume of deionised water, mix to lose the red cells, then add an equal volume of double strength normal saline to create a suspension in normal saline.  
The centrifuge where the heavier uncleared cells go to the bottom of the tube.  I have to say the I have never tried this personally but if the morphology was retained for EM it must be fine for LM.
Having said that I have great respect for Tony Henwood and his method looks much more scientific.

Regards
Tony

Sent from my iPhone

> On 17 Jan 2020, at 8:24 am, Tony Henwood (SCHN) via Histonet <histonet at lists.utsouthwestern.edu> wrote:
> 
> Hi Jennifer,
> 
> I have had excellent success with lysing the red blood cells (using  Isotonic Ammonium Chloride) prior to cell block preparation with thromboplastin-plasma. 
> The lysing solution contains EDTA so you will need to add a few drops of 1% calcium chloride. Method as follows:
> 
> Lysis solution
> Ammonium Chloride        4.5g
> Potassium carbonate      0.5g
> EDTA                     0.0186g
> Distilled water          500mls
> 
> Method:
> 1.    Centrifuge bloody fluid.
> 2.    Remove supernatant and add equal volume of lysis solution.
> 3.    Resuspend and incubate for 5 minutes at 4oC.
> 4.    Centrifuge, if blood still remains, then repeat from step 2.
> 5.     Rinse in Hanks or RPMI, centrifuge.    
> 6.    Mix pellet in a few drops of plasma.
> 7.     Add thromboplastin and a few drops  of 1% Calcium Chloride, mix gently and allow clot to form.
> 8.     Add 10% buffered formalin and fix and process as usual.
> 
> Reference:  
> Kuenen-Boumiester etal (1996) Acta Cytolog 40:475-479
> 
> If you donot use the plasma clot method for cell block preparation, then use your preferred method after step 5.
> The lysis solution can also be purchased commercially from several companies (eg Biolegend). It is commonly used for sample preparation for flow cytometry. Check the SDS to make sure it does not contain formaldehyde.
> 
> 
> -----Original Message-----
> From: Mac Donald, Jennifer via Histonet [mailto:histonet at lists.utsouthwestern.edu] 
> Sent: Friday, 17 January 2020 4:53 AM
> To: Charles Riley <criley at dpspa.com>; Histo List <histonet at lists.utsouthwestern.edu>
> Subject: Re: [Histonet] Cell block preparations
> 
> Acetic acid would work.
> 
> Get Outlook for iOS<https://aka.ms/o0ukef> ________________________________
> From: Charles Riley via Histonet <histonet at lists.utsouthwestern.edu>
> Sent: Thursday, January 16, 2020 8:55:21 AM
> To: Histo List <histonet at lists.utsouthwestern.edu>
> Subject: [Histonet] Cell block preparations
> 
>  EXTERNAL SENDER- Exercise caution with requests, links, and attachments.
> 
> What is the best way to remove excess blood from FNA sample collections before spinning them down into cell blocks?
> 
> --
> 
> Charles Riley BS  HT, HTL(ASCP)CM
> 
> Histopathology Coordinator/ Mohs
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