RE: [Histonet] cytospin preps and plus slides
What is the overall volume of sample that you are actually receiving? If
you are getting at least several mL of sample from your client, you might
want to spin it down in a standard centrifuge prior to running it on the
Cytospin -- this would allow you to concentrate the specimen and possibly
use it in its entirety (i.e. spin it down, discard the supernatant,
resuspend the pellet, and then pipette the sample into the sample chambers
that are to be run on the Cytospin).
Also, what type of sample chambers are you using on your Cytospin? For
example, are you using the single white Cytofunnel, single brown Cytofunnel,
double Cytofunnel, TPX chambers or Megafunnel? Also, what are your sample
volumes that are being pipetted into the sample chambers? Each type of
funnel has optimal volume ranges that should be observed. I would suggest
referring back to the instructions for your particular type of funnel.
You are using a standard cytology protocol (1000 RPM for 5 minutes) that
should be sufficient. I would not recommend increasing the time (you don't
want to "overspin" the cells and produce a drying artifact)...but you DO
have a little room to "tweak" the RPM a bit. I would try increasing it
slightly to 1100 or even 1200 and see what that does for you.
If you feel that actual cell RECOVERY is not so much the issue and that it
might be more of an issue with cellular ADHESION (to the slide during
staining), would it be possible to get your client to use a fixative other
than 10% NBF? When I worked in the lab, we often supplied doctors' offices
with pre-filled collection cups in which they would subsequently send their
samples to us. You would probably find greater success in terms of actual
cellular adhesion if a cytology fixative containing Carbowax was utilized.
This would allow you to actually let the slides air dry prior to staining
(the Carbowax would coat the deposited cells and prevent any air-drying
artifact). The combination of the Carbowax and allowing the slides to dry
prior to running them through a staining protocol should significantly
improve adhesion (as a wet slide will sometimes have some of its cells
washed away during staining).
These are just a few pointers and thoughts to ponder -- I hope that they
Sherri L. Anderson BS, HTL(ASCP)
Thermo Electron Corp.
>From: "Elizabeth Chlipala"
>Subject: [Histonet] cytospin preps and plus slides
>Date: Wed, 3 Aug 2005 12:42:30 -0600
>I'm working on a project for a client who is trying to isolate prostate
>epithelial cells. I'm getting the cells submitted to me fixed in 10%
>NBF in eppendorf tubes. I'm using a cytospin for 5 minutes at 1000 rpm
>and plus slides. The problem is that I'm not getting the correct yield
>of cells. They check the number of cells in the prep prior to me
>receiving them. They are submitting numbers of cells in the thousands,
>but when I prepare the cytospin slides, I'm only getting cells numbering
>in the hundreds and sometimes less. Does anyone have any suggestions?
>Thanks in advance
>Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
>Premier Laboratory, LLC
>P.O. Box 18592
>Boulder, Colorado 80308
>Office: (303) 735-5001
>Fax: (303) 735-3540
>Ship to Address:
>University of Colorado
>MCDB, Room A3B40
>Boulder, Colorado 80309
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