Dear Andrea,
Some clones are better in a brighter conjugate. CD10 and CD34 are two
of these. We use them in combinations where they are conjugated with PE or
APC. For example CD19 FITC/CD10 PE/CD45 Percp / CD34 APC works well as does
Kappa/Lambda/ CD19 Percp Cy5.5/CD10 APC. Neither is very good in FITC, no
matter the vendor. CD10 in follicular lymphoma may also be dim which we
have seen, so a brighter conjugate also makes sense.
False positive CD34 results are trickier. Most good sales people will
tell you if a particular clone is not good, but only if their customers
ask. You might also consider an extra blocking step with mouse serum to get
rid of FC receptors, use of 7-AAD or PI to detect dead cells which might
non-specifically take up some stains, blocking with unconjugated CD34 to
see if the positive staining disappears(equals real positive) and an IgG
control which I consider vital when not looking as mature lymphocytes, as
non-lymphs have more FC receptors.
Hope this helps,
Paula
>Dear flow group, just a couple of questions for the experts....
>
>CD10: We are currently using Caltag's CD10-FITC (clone 5-1B4) and seem
>to have problem with staining on some of our follicular lymphomas.
>Recently we had a case which was CD10-neg by flow but stained positive
>with Ventanna's IPOX CD10 (clone 56C6). It seems to be Caltag's only
>clone, Coulter has 3 clones (J5 which Coulter recommends, ALB1 and ALB2
>from the Coulter/ Immunotech merger). Since everybody uses a diggerent
>terminology for their clones, can somebody tell me how they compare and
>which clone is the most wiedely used by the flow community?
>
>CD34: We seem to have non-specific staining of CD34-FITC (Caltag's
>clone 581 class III) on monos (we have a gate set around all the
>populations and have noticed this fairly frequently). In the past we
>have ignored this and we able to differentiate this from the real
>specific staining. However, we had a recent case of LPD/B with ?CLL/?HCL
>differentiation, where the monoclonal B-cells showed CD34 positivity
>(TDT-neg, CD20+, CD22+, bright lambda+). Because of the mature phenotype
>I was leaning towards ignoring it, but felt uneasy about totally
>ignoring it. I just happened to have the red CD34 from Coulter (same
>clone 581 class III) and those cells were negative! Has anybody else
>seen this?
>
>Much appreciated!
>Andrea Illingworth
>Dahl-Chase Flow Cytometry
>Bangor, Maine
>207-990-4855
Paula Imus
Flow Cytometry
LP, NCI, NIH
10 Center Drive MSC-1500
Bethesda, Md. 20892-1500
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