Here may be some relevant papers:
(Several of our SYTO dyes have been used this way too but I beliefve not
published. They are excitable at 488 nm.)
Cytometry 1993;14(3):276-80
Flow cytometric screening of blood samples for
malaria parasites.
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.
An automated method for the detection and
estimation of malaria parasites in blood samples using flow cytometry is
presented.
In a single-step procedure 50 microliters of
blood sample was collected in 1 ml of lysis solution containing
formaldehyde, causing
red blood cells to lyse while parasites and white
blood cells are preserved. Thus prepared, samples could be transported
and
remained stored in lysis solution until flow
cytometric analysis was performed. The cells were stained for DNA with
the fluorescent
dye Hoechst 33258 and subsequently analyzed by a
FACStar flow cytometer. Parasites and white blood cells were
distinguished
and counted based on blue Hoechst fluorescence
and forward scattering. Since red blood cells were lysed, parasite
numbers
were given related to the number of white blood
cells similar to what is done in microscopic examination of thick blood
smears. In
dilution experiments with animal and human
material, parasite counts by flow cytometry correlated very well with
the theoretically
calculated numbers (regression coefficients of >
0.94). In human material parasitemias of approximately 0.005% were
detected.
In a pilot study, 700 samples were collected in
Thailand and screened by microscopic examination of thick smears and by
flow
cytometry; 29 were found positive by combining
both methods, 2 were missed by flow cytometry, and 20 were missed by
microscopists in the field. After microscopic
reexamination in the central laboratory, 15 of these 20 were found
positive, 5
remained unconfirmed.
Methods Cell Biol 1994;42 Pt B:295-318
Flow cytometry in malaria detection.
Janse CJ, Van Vianen PH.
Laboratory of Parasitology, University of Leiden,
The Netherlands.
Am J Trop Med Hyg 1990 Dec;43(6):602-7
Automated flow cytometric analysis of drug
susceptibility of malaria parasites.
van Vianen PH, Thaithong S, Reinders PP, van
Engen A, van der Keur M, Tanke HJ, van der Kaay HJ, Mons B.
University of Leiden, The Netherlands.
A method is described for the fully automated
reading of Plasmodium falciparum drug susceptibility tests. Cultured
material was
fixed and could be stored for greater than or
equal to 6 months until analysis. The parasites were stained for DNA
with the
fluorescent dye Hoechst 33258 and analyzed by
flow cytometry. The procedure was done in 96-well microtiter plates,
after which
the material was directed through the sensing
region in the flow cytometer. The data resulting from the analysis were
stored by
microcomputer and processed by a program
developed for this purpose. Using this method, a number of different
parameters
describing the growth in culture can be assessed.
Gabriel Alespeiti wrote:
> 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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