Dear Mary-Alice and collegues, I would like to confirm the statement that CD13, CD65 and CD10 are in favor of AML esp. in your clinical context. I want to remind you of my previous statement on this server list concerning the CD65 clones. We have never seen CD65s (VIM2) on ALL. We had seen different patterns before with the CD65 from Immunotech. We have seen several adulthood AML CD10 (J5) positive. Could you tell us the CD65 subgroup of your clone? CD79a has been accused by a german collegue with broad leukemia typing experience to be not totally exclusive for B lineage as it was seen on two AMLs. I do not share this point of view at the moment and still wait for my first CD79a+ AML. I ignore weak reaction of aberrant markers when AML shows unspecific binding via FcR. The unspecific binding may be tricky as not all antibodies even with the same fluorochrome show similar degree of binding. This observation can be made using a broad panel as we do with redundant markers (eg. CD19). I am aware that I give away much of the objectivity of flow cytometry. To add an interesting case from my side: A 54 year old man showed up with acute leukemia de novo (no CML or MDS)on Nov, 17 in a major hospital south of Mannheim. A bone marrow aspirate and a blood film were prepared. Because of CD34-, cy CD22, CD19+, CD10 weak, surface Ig+ from the immunology lab and panoptical staining & light microscopy by a hemopathologist a diagnosis of B-ALL was made. The patient went to a hospital more near by and a second analysis was made. They found CD14, CD19, CD33, CD10, surface Ig+ and TdT positive on november 27 and started treatment for B-ALL. On december 1st they drew a third BM sample and a blood film. The blood film now showed blasts with a very much monocyte like morphology. The reference center at Berlin confirmed B-ALL from the first sample. We got the sample on december 2nd and they asked us to confirm the B-ALL. At this time WBC of the heparinized BM aspirate was about 1000/ul. We tried to prepare a film from that with little success (heparin). We found 25% NRBC, 50% T, 5%, 10% neutrophils and 4% CD14 with low MPox. We used our broad leukemia panel (FITC/PE/PerCP) CD15/14/45, Isotypes, CD10/5/19, CD38/34/45, CD24/20/45, CD4/8/3, DR/CD13/CD16, CD7/33/45, CD1/2/64, CD41/GpA/45, BB4/CD56/45, cyTdT/79a/3, cyIgM/79/45, cyMPox/LF/45. All cells except the monocytes were completely normal in terms of antigen expression density. No blasts with CD45, no TdT. I then asked the collegues to send me their results, BM and blood films. I reviewed the outside FCM printouts (Nov 27 and Dec 1) and got the following result: CD19 weak, CD10 weak, CD33 strong, CD13 weak, DR+, sIg+, CD14+, CD45+ the other markers being normal. Normal T (CD2, CD5, CD7) and B cells (CD19were still present. Already in their first BM the patient had L2 and no L3 morphology, with finely granulated dark blue cytoplasm and granulated chromatin. Some blasts showed a monocytic notch in the nucleus. 5% of the blasts were bigger with monocytic cytoplasm and chromatin densitiy. Already in the blood film some blasts were even more telltale. On Dec 1st the morphology in the blood film was clearly monocytic with little remaining blasts in the BM. The chemotherapy treatment might have induced maturation. Taken together CD45 strong, CD33 strong, CD14 strong, sIgG strong (FcR mediated), CD13 weak, CD19 weak, MPox weak together with morphology are in favor of a monocytic AML (FAB Mx requires cytochemistry). The remainig normal B cells at all times and the missing CD20 and L3 morphology are against B-ALL. Lessons from this? The broad panels help for plausibility checks and the evaluation of all (!!!) markers that are used for whatever reason and (!!!) their intensity and (!!!) the careful review of the blood film together with the bone marrow prep help in nearly all instances. Eg. normal monocytes are CD45+, CD14++, CD15+, CD13+, CD33++, CD2+, CD4(+), CD38+, CD56+, DR+, sIg+, Mpox+ and therefore are stained in most of our tubes. The EGIL group gave 0.5 scoring points for TdT. We have little "wrong stainig " using the Immunotech mixture of TdT-FITC monoclonals and An der Grub lyse (fix & perm). I suggest to express antigen density in relation to normal cells (which are still present in most cases) in all lab reports : ++ = overexpression, + = normal expression, (+)= dim expression +/- expression on more than 50% of the blasts, (+)/- dim partial expression -/+ expression in less than 50% of the blasts, -/(+) dim partial expr. This method is less expensive and more reliable than beads. I further recommend to give the name of the clone and fluorochrome used. I would appreciate your suggestions for a comparison of reactivity of commercial CD clones communicated via this list server. I suggest to start with more critical ones like CD19, CD65, cy CD22, cy IgM, cy Tdt combined with markers where you would expect agreement (like CD4). The clinical benefit would be - the improvement of diagnosis, - of interlaboratory exchange and - the corelation of marker expression to cytogenetic abnormalities which will better define clinical entities in AML compared to the FAB scheme. Thomas Nebe Klinikum Mannheim D-68135 Mannheim, GERMANY Phone: +49 621 383 3485 FAX: +49 621 383 3819
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