Hello All, Could high flow rate of unobserved cells (under FSC threshold), like RBC, affect side scatter measurements, inducing a decrease in signal? I was looking at lymphocytes in a PB sample (lambda-FITC, kappa-PE, CD19-PecP-Cy5.5, CD10-APC) with incomplete RBC lysis on a BD Canto. I recorded 30.000 events with threshold on FSC at low, medium and high flow rate in 109, 9 and 7 seconds respectively which equals 275, 3333, 4286 cells/sec (see the attached picture). Unfortunately I did not estimate the RBC concentration (at visual inspection it was rather high), and so I don't know the total particle concentration. But RBC under and even above threshold should not be a problem: in a previous tread ("going too fast") about increasing sorting rate using a fluorescence threshold, a comment by dr Shapiro reported about analyzing leukocytes in unlysed whole blood at 50.000 cells/second, with scatter measurements for sizing. BD declares 10.000 cells/sec for acquisition rate on Canto, in my experiments I was under this value. When I analyze purified leucocytes I don't see this phenomenon even at greater rates, so I suppose that the problem is high concentration of unobserved RBC, with very high total particle flow rate, but shouldn't the system be blind to what is under trigger? Aside for my 2006' Canto (and me) be unable to do what dr Shapiro did in mid-1970s, what is the explanation: coincidence, electronic? Why is so affected side scatter (the medians of forward scatter and FL are less decreased)? Pietro Bulian NRCLE IRCSS C.R.O. Aviano (PN) Italy This attachment - 'flow-rate.doc' - 54.27 KBytes - can be viewed at http://www.cyto.purdue.edu/MD-parts/1bc010a6a0f843043995e3eb66bd0bec2138f081.docReceived on Wed May 24 11:58:00 2006
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