Gabriel Alespeiti wrote- >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. I am not sure that anybody is doing routine detection of malaria parasitemia by flow. Propidium iodide would not be the stain of choice to detect parasite nucleic acid, because its fluorescence enhancement on binding is only about 50x. Thiazole orange would be a much better choice; it enhances fluorescence over 1,000x on binding to nucleic acid. The low end of your desired range, 0.05%, is 5 parasitized cells per 10,000 cells, and you ought to be able to count close to 200,000 RBCs in about a minute on a FACSCalibur, so you shouldn't have too much of a problem detecting parasitemia even at that low level. I hope the people who follow this list and work regularly with malaria will chime in. -Howard
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