[Cytometry] Pleural effusion sample prep for sorting

Sian Rizzo RizzoS at cardiff.ac.uk
Fri May 6 05:17:34 EDT 2011


Hi Kathy, we did have a couple of pleural effusions (sorry Ian....) but treated them the same as the ascites. All samples were collected into heparin. This was essential.

We found that storage at room temperature was better than refrigerated if it was necessary to keep overnight. Those that were chilled had lower recovery of cells, with higher cell death than those left on the bench.

Some of the ovarian samples some were heavily contaminated with blood, others with exceptionally high platelets and others were just packed with clusters of tumour cells, while we also had samples where recovery of tumour cells was minimal (e.g. <100,000 from 2L of fluid). There was variable haemolysis, with samples appearing straw coloured through bright red to dark brown. 

We found an initial spin to concentrate the cells followed by ficoll or lymphoprep enriched for tumour cells at the interface. The clinical SOP (for the parallel samples we didn't use) was concentration followed by dynal bead HEA selection, or you could use CD45 depletion.

They are fairly delicate after all the processing so it was essential to use live cell selection when we wanted to perform functional assays with the sorted cells. However, those that made it through the process were able to grow in vitro.

hope that helps, Sian Rizzo



-----cytometry-bounces at lists.purdue.edu wrote: ----- 
To: "Purdue Flow MailList (cytometry at lists.purdue.edu)" <cytometry at lists.purdue.edu>, "Kathy Heel" <kathy.heel at uwa.edu.au>
From: "Ian Titley" 
Sent by: cytometry-bounces at lists.purdue.edu
Date: 06/05/2011 09:55
Subject: Re: [Cytometry] Pleural effusion sample prep for sorting

Hi Kathy

We have looked at cells from ascites http://www.ncbi.nlm.nih.gov/pubmed/21216927 (Rizzo et al Molecular Cancer Therapeutics 10(2) 2011). I was not involved with preparation, I think they were spun and washed and then spun on a ficoll type gradient, washed again, it's all in the publication. From the sorting point of view there were no problems with blockages  - we used a 100um nozzle on a BD FACSVantage. I would have thought the pleural effusions similar but never tried them and I'm sure there is much variability between samples, maybe we got lucky.

>From a biosafety point of view I would have some concern over TB if the samples are fresh, but not sure if TB gets into pleural effusion?

Best wishes

Ian





Ian Titley PhD
Division of Molecular Pathology
Institute of Cancer Research
15 Cotswold Road
Belmont
Sutton
Surrey  SM2 5NG
UK

Tel +44 (0)20 8722 4255



>>> Kathy Heel <kathy.heel at uwa.edu.au> 05/05/2011 04:06 >>>
Hello All,

We've had a request to sort pleural effusions from mesothelioma patients and was wondering if anyone has any experience with these samples and suggestions for sample prep? I've been told the effusions can be very rich in protein and coagulating agents and can be very sticky.

Many thanks
Kathy

Kathy Heel PhD
Assistant Professor
Centre for Microscopy, Characterisation and Analysis (M510)
The University of Western Australia
35 Stirling Highway
Crawley WA 6009
Australia

Ph 61 8 9346 4525
Fax 61 8 9346 3469
Mob 0416 085 960
www.cmca.uwa.edu.au<http://www.cmca.uwa.edu.au>

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