Barren, I appreciate your help. Thank you. I did not run the Giemsa stained RBC's through a flow cytometer. Therefore I don't know the answer. About your other question. Richard Haugland (Molecular Probes) responded that "several of our SYTO dyes have been used this way too but I believe not published. They are excitable at 488 nm." He also mentioned the paper: "Cytometry 1993;14(3):276-80. Flow cytometric screening of blood samples for malaria parasites. Philip H. van Vianen PH, van Engen A, Thaithong S, van der Keur M, Tanke HJ, van der Kaay HJ, Mons B, Janse CJ.Laboratory of Parasitology, Medical Faculty, University of Leiden, The Netherlands." They lysed the erythrocytes and are able to detect parasitemias as low as 0.001%. They use Hoechst 33258, which cannot be detected in a FACScalibur. Samples contain mainly white blood cells and free parasites. When lysing erythrocytes only 5000 events are needed to detect parasitemia levels under 0.01%, and advantage compared to working with whole blood, where ~1000X more events are needed to detect the same level of parasitemia. I would like to know if someone has modified this method and tried it with 488nm excitable dyes? >I may not be of much help., but I am curious if you ever ran the >Giemsa stained RBC's through a flow cytometer. I wonder if the >Giemsa positive staining RBCs ( with parasites) would not show up as >an increase in SSC? > >Another thought, Human RBC's have no nucleus, could you stain with a >DNA dye (one that permeates live cells , Syto dye series from >Molecular Probes?) and then look for RBC's with DNA (i.e. parasite)? > >Pb > >-----Original Message----- >From: Gabriel Alespeiti [mailto:gabriel_alespeiti@nybc.org] >Sent: Thursday, July 25, 2002 3:13 PM >To: Cytometry Mailing List >Subject: Re: anti-malarial antibodies > >Dear colleagues, > >I have read the replies on anti-malarial antibodies and I hope to >get some help from the experts in this field. A researcher in the >Institute is conducting parasitaemia studies of Plasmodium >falciparum infected erythrocytes using Giemsa stained microscopy, a >tedious, time consuming and technically imprecise method. We want to >replace the microscopic counts with a flow cytometry method. >Peripheral blood will be collected in Brazil and will need to be >fixed before shipping. A FACScalibur is available for this work. We >would like to analyze 25 samples a day, the expected parasitemia >level is between 0.05% and 8%. >I tried formaldehyde fixation followed by propidium iodide staining >and even though there is a direct correlation between cytometric and >microscopic determination, the manual counts are significantly >higher than the flow counts. >What I'm looking for is a simple, proven protocol for daily use that >gives directly the % of parasitaemia by flow. In our case, >determination of erythrocyte or parasite antigens; even though >useful, is not necessary. What is important for us is to be able to >detect low infection levels. >Does anybody know, what is the lowest level of infection that can be >detected by flow? >Please post the reply on this site, so other people can benefit from >it. Thank you very much. > > > > > >-- >************************************** >Gabriel E. Alespeiti >Supervisor, Flow Cytometry >Lindsley F. Kimball Research Institute >New York Blood Center >310 E.67th St. Room 2-16E >New York, NY 10021 > >Phone: (212) 570-3346 >Fax: (212) 570-3307 >E-mail:gabriel_alespeiti@nybc.org >**************************************
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