Re: intrinsic versus extrinsic -Reply

From: Maryalice Stetler-Stevenson (stetler@box-s.nih.gov)
Date: Mon Jan 26 1998 - 11:55:47 EST


        We do not need to remove cytophillic antibody by acid wash or
incubating the cells. We simply wash three times in PBS. In our reported
study of 366 consecutive specimens, we observed cytophillic antibodies in 9
cases(2.5%). However, we were still able to detect monoclonality due to the
combinations of antibodies used(Cytometry-CCC 26:243-252, 1996).
        Maryalice


>Andrea at Dahl-Chase asked:
>
>Is washing with PBA sufficient to ensure that immunoglobulin staining is
>intrinsic and not extrinsic (cytophilic)?
>------------------------------------------
>
>I do not believe that a single wash with PBA (I assume this is PBS with BSA and
>azide) would be sufficient.  Cytophilic Ig is removed either by an acidic wash
>or by incubating at 37 C for 20-30 min. and then washing with warm media 1-3
>times.  We incubate ficol/hypaque purified mononuclear cells at 37 C in RPMI
>with 10% FCS for 30 min and then centrifuge through a cushion of fetal calf
>serum.  On occassion I have just washed F/H cells or lysed whole blood
>once with
>PBA and there was still cytophilic Ig present.
>
>Tony Bakke, PhD
>Director, Clinical Immunology and Flow Cytometry Lab
>Dept of Pathology
>Oregon Health Sciences University
>Portland, OR
>bakkea@ohsu.edu

Maryalice Stetler-Stevenson
Director Flow Cytometry Unit
Laboratory of Pathology, NCI, NIH



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