Gates or no Gates... For a long time I have tried to convince the flow community to look at raw log side scatter versus the various fluorescence channels, particularly when looking at bacteria or nano and microparticles. Cells with different scatter properties can be an lysis artefacts coincidences or functional cell interactions, blasts.... After all we teach everyone that the cells hug each other for signalling. Therefore it is essential to check the untampered data, ideally looking at log side scatter versus log forward scatter and also to observe the log side scatter versus log fluorescence data. The degree of aggregation can vary depending on the anticoagulant used, sample age, temperature, the lysis method, the number of centrifugation steps... and the patient!!! Certain disorders show very selective cell-cell adhesion. Find enclosed an example were I just pulled up the lin and log scatter pictures (apologies for the orientation to the ones who think the other way round and that the area data are out of scale) and the log side scatter/CD3 picture. Gating on the CD3+ monocytes instantly updates the preview plane from which I then selected the CD4 vs. cd8 picture to show the biased aggregation of cd3+ cells towards CD8+ which shows it neither to be coincidence or random aggregation. The monocyte and lymphocyte-monocyte aggregates can even be separated by area forward scatter (yes it would be nicer if on scale). The power of flow cytometry lies in the multiparametric correlation of data. Whilst the VenturiOne software offers a very fast and convenient way of seeing all the correlations at once there is no excuse for not generating at least the raw side scatter versus fluorescence plots on any conventional software package with either Gates - or Jobs technology. Gerhard Nebe-von-Caron Research Scientist and Biomedical Engineer SPD-Spark Swiss Precision Diagnostics Priory Business Park Bedford, MK44 3UP, UK Tel +44(0)1234-835474 Fax +44(0)1234-835002 mailto:g.nebe-von-caron@spdspark.com -----Original Message----- From: Sylvie Amu [mailto:sylvie.amu@rheuma.gu.se] Sent: 05 February 2008 22:25 To: Cytometry Mailing List Subject: Re:Doublets? Halo We do see the same phenomenon running our samples in CantoII. We work with human blood and are staining for B cells (CD19) and T cells (CD3) and see a population at the same location you are describing being double positive CD19+CD3+, doublets. This population will disappear if you gate on single cells firs ( FSC-A vs FSC-H) and then continue your analyses. This has been our approach. Hope it helps. Regards Sylvie Amu, PhD student Dept of Rheumatology and Inflammation Research Gothenburg University Sweden On Feb 1, 2008, at 7:14 AM, Rudensky, Bernard wrote: Many times we find a second small population on the fsc vs ssc plot which has the ssc properties of lymphocytes but higher fsc values approaching that of monocytes. By fluorescent markers this population appears to be lymphocytes with the same phenotype as the major lymphocyte population. We do not see this with every assay but the phenomenon seems to be experiment dependent as every tube in the same experiment gives the same picture. The Canto technician thinks that the second population represents doublets. Does anyone have experience with this phenomenon? Is there a way to avoid it? Can it affect diagnostic results? Your input would be most appreciated. Professor Bernard Rudensky Clinical Laboratories Shaare Zedek Medical Center Jerusalem, Israel 91031 IMPORTANT: The contents of this email and any attachments are confidential. They are intended for the named recipient(s) only. If you have received this email in error, please notify the system manager or the sender immediately and do not disclose the contents to anyone or make copies thereof. *** eSafe scanned this email for viruses, vandals, and malicious content. *** Shaare Zedek Medical Center http://www.szmc.org.il This attachment - 'doublets.jpg' - 653.79 KBytes - can be viewed at http://www.cyto.purdue.edu/MD-parts/9259562d6cf9f16209bc0499ba3055ac5286bb12.jpgReceived on Tue Feb 12 14:58:00 2008
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