[Cytometry] Distinguishing Surface vs. Cytoplasmic staining

serhan alkan alkanserhan at yahoo.com
Thu Jul 16 21:47:14 EDT 2009


Hi Prashant,
It is difficult to predict which surface antigen still have similar affinity and binding after fix and perm fixation. It is possible that fix and perm will not change epitope of interest to antibody binding. 
Regarding your specific question about M7 and CD41/61 staining, what most people do is that you could take a very small left-over sample from the CD41/61 tube, put a coverslip and look at under fluorescent microscope. You should be able to notice platelet satellitism which is quite different than nice membraneous staining. 
The staining pattern of CD41/61 due to platelet satelletism gives sharper peak while M7 blasts have a broader expression. More specific and detailed information could be found in a paper published in Blood in 1992 (free to public since early edition).
"False-positive flow cytometric platelet glycoprotein IIb/IIIa expression in myeloid leukemias secondary to platelet adherence to blasts.Betz SA,  et al."
I don't do the cytospin since the cells loose their oval round structure but you could try either method and decide what to do with your cases.

Serhan Alkan, MD
Director of Hematopathology and Molecular Diagnostics
Vitro Molecular Laboratories
Miami



----- Original Message ----
From: Robin Barclay <robin.barclay at ed.ac.uk>
To: Prashant Sharma <prashant.sh at gmail.com>; cytometry at lists.purdue.edu
Sent: Thursday, July 16, 2009 4:40:13 AM
Subject: Re: [Cytometry] Distinguishing Surface vs. Cytoplasmic staining

I would expect a cell surface antigen to remain present and accessible after 
fix/perm (unless that process changes it).  It would be of interest to stain 
before fix/perm with with one fluorochrome conjugate then following fix/perm 
with another fluorochrome conjugate of the same antibody, which might 
discriminate surface and cytoplasmic expression of the same antigen??
Robin Barclay

Dr George Robin Barclay PhD
Consultant Clinical Scientist (Lead Scientist, SNBTS Group)
  & Honorary Senior Lecturer
SNBTS Cell Therapy Group
MRC Centre for Regenerative Medicine
The Chancellor's Building
University of Edinburgh


-- 
The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.


----- Original Message ----- 
From: "Prashant Sharma" <prashant.sh at gmail.com>
To: <cytometry at lists.purdue.edu>
Sent: Tuesday, July 14, 2009 6:06 PM
Subject: [Cytometry] Distinguishing Surface vs. Cytoplasmic staining


> Dear List members,
>
> I had a rather basic question on flow and would be glad to have help: Will 
> a
> cell positive for a certain antigen on surface staining always show that
> antigen as positive after fix-and-perm cytoplasmic staining? Even if the
> antigen is not present in the cytoplasm?
>
> The reason I ask (this biologically unlikely question) is because it was
> suggested somewhere that one way to exclude a false positive CD41 / CD61 
> on
> blasts due to platelet adhesion would be to simply perform cytoplasmic
> staining instead of surface.
>
> My take on this is that since the platelets or their membranes are 
> adherent
> to the cell surface, they would (most likely) still be there after the fix 
> /
> perm steps and would therefore still give a positive signal.
>
> Would you say this last assumption is correct? Or to extrapolate the
> question, if we are doing cytoplasmic staining for an antigen, are we, in
> effect, doing a "cytoplasmic or surface or both" stain?
>
> Thanks!
>
> Prashant
> ____________
> Prashant Sharma, MBBS, MD, DNB
> Senior Resident, DM student
> Hematology Department
> All India Institute of Medical Sciences
> Ansari Nagar, New Delhi, India.
> _______________________________________________
> Cytometry mailing list
> Cytometry at lists.purdue.edu
> https://lists.purdue.edu/mailman/listinfo/cytometry
>
> 

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