From: Frederic Preffer (preffer@helix.mgh.harvard.edu)
Date: Mon Jun 01 1998 - 14:49:03 EST
i agree that maryalice and andreas' concerns are quite valid. Due to the nature of this type of staining, we also tend towards a conservative appproach when making a diagnosis utilizing cCD3 staining [and/or cytoplasmic light chain monoclonality as well]. I am most comfortable when internal patient control cells are available. For example, in assessing cCD3, residual B cells (surface CD19+) need to be negative, relative to the cCD3 expression. We also usually run cCD3 against a 'contrasting colored' surface CD3. Running these on control cells simultaneously, and seeing the cCD3 coexpressed on the sCD3 and not on the CD19+ cells or other mononuclear cells, is reassuring. Luckily, we have not seen too much in the way of neutrophil expression of cCD3, but do see Tdt expression in those cells (described in 1990 by Civin et al J Immunol Meth) However, let me also point out that many of the clonal populations we have detected by aberrant CD3 expression were initially surface CD3 'dim'+ and dim cCD3 as well. When we see this, my first reaction is to request molecular diagnostic work to r/o monoclonality. f preffer At 08:45 AM 6/1/98 -0400, Maryalice Stetler-Stevenson wrote: > > >Andrea, > I am glad you have raised this issue. I too have found that there >are problems with current methods for intracytoplasmic staining. Although >the methods we use are good for most specimens,in some cases there can be >"dim" nonspecific staining that varies from tumor to tumor (i.e. one >patient may have greater non-specific staining than another, I believe >related to the tumor type). I don't interpret something as positive >intracellular unless it is relatively bright compared to other cells in the >same specimen. What I find strange is that many people claim that they have >no problems with, for example cytoplasmic CD3 or TDT. Either they have a >much better technique than the majority or they only call bright positive >and don't worry about missing dim positives on those few cases when it is a >problem. Maybe I am too compulsive, but I won't feel comfortable with >intracytoplasmic untill it is as reliable as surface staining. I would be >interested in hearing any feedback you get directly. How many people are >willing to admit they have had similar cases? Does it bother anyone else? > > > Maryalice > >> >>>To all the clinical "Flow people" out there >>> >>>We have been using Caltag's fix & Perm kit for our cytoplasmic staining. So >>>far we have had good results using TdT, MPO, cCD22, and cCD30. However, >>>recently we had a challenging acute leukemia case with the following >>>phenotype: CD45 mod+, CD7 partially+,CD11b (?)weak+, CD13 (?)weak+, HLA-DR+, >>>CD34+, TdT+, CD38 bright+. Pertinent negatives included MPO, CD33, CD11c, >>>CD14, CD15, CD10, CD19, cCD22, CD2, sCD3, CD4, CD8, CD5, CD56. We signed it >>>out as T-cell acute lymphoblastic leukemia with aberrant myeloid expression >>>mainly because of the TdT/cCD3 positivity combined with the MPO negativity. >>>However, since we had just started using the cCD3, we did some more QC on >>>normal lysed peripheral blood and found striking cCD3 positivity on our >>>monocytes and granulocytes! I am really concerned about this finding and >>>would like to find out from the experts if they have seen this in their >>>labs. I also just received the CAP survey and noted that 25.7% of the labs >>>reported cCD3 positivity on a otherwise pretty straight forward AML. Some >>>of the labs (4.4%) even reported this out as ALL/T-cell type. >>> >>>Does this mean that 25% of the flow labs (including myself) have a problem >>>with cCD3 staining? This would be kind of scary since it means (by trusting >>>the cCD3 expression) that there is a chance of misinterpreting some cases as >>>a T-ALL (with some myeloid expression) instead of an AML (with some CD2 or >>>CD7 expression which is very common in AML's). >>> >>>I also should add that the patient was initially diagnosed at another >>>reference lab with biphenotypic leukemia (they found essentially the same >>>phenotype but also CD19, CD22, CD13 positivity). It sounds very unusual >>>since I have never seen a case with myeloid, T and B markers. We are not >>>sure if some non-specific positivity was overinterpreted or if one can see a >>>very undifferentiated leukemia expressing all three lineages? >>> >>>Your feedback is much appreciated >>>Andrea >>>Dahl-Chase Diagnostic Services/Flow Cytometry >>>333 State Street >>>Bangor, Maine 04401 >>>(207)990-4855 >> > >Maryalice Stetler-Stevenson >Director Flow Cytometry Unit >Laboratory of Pathology, NCI, NIH > > > > ```````````````````````````````````````````` Dr. Frederic I. Preffer preffer@helix.mgh.harvard.edu Department of Pathology- Warren 525A 100 Blossom St Massachusetts General Hospital Boston MA 02114 v(617) 726-7481 fax(617) 724-3164
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