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From: Howard Shapiro <hms@shapirolab.com>
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Subject: Re: TO for malaria staining in blood samples (UNCLASSIFIED)
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Donna Wilder wrote-

> 
>  Has anyone used the Thiazole Orange dye from BD(Retic-COUNT) for
> malaria staining in blood samples?  I'm looking for suggestions of dyes
> that are fairly simple to work with on a FacScan. Thank you for any
> input.
> 

The reference on thiazole orange and malaria,  which is fairly ancient
history, is:

Makler MT, Lee LG, Recktenwald D: Thiazole orange: a new dye for
Plasmodium species analysis. Cytometry. 1987; 8(6):568-70.

ABSTRACT

A rapid sensitive method for the determination of Plasmodium falciparum
in in vitro culture is presented. The technique employs a fluorescent
flow cytometer equipped with a 15-mwatt argon laser that emits light at
488 nm and a membrane-permeable fluorochrome thiazole orange (TO) that
stains RNA. Parasitized red cells are stained by suspending them in 1 ml
of phosphate-buffered saline (PBS) containing 10(-5) M of TO and
incubating this mixture for 15 min in the dark at room temperature. The
stained cells may be analyzed fresh or after fixation with 1%
paraformaldehyde/PBS or 0.25% glutaraldehyde/PBS. Alternatively
the cells may be fixed first and then stained. There is excellent
correspondence between the number of fluorescent-labeled parasitized red
cells and Giemsa-stained cells.

(The full text of this Cytometry article is available online as noted by
Paul Robinson in a recent posting.)

---------------------------------------------------------------------------

The abstract is correct in stating that thiazole orange (TO) stains RNA;
the dye also stains DNA. At the time Makler et al were writing, the
previous  papers published on flow cytometry of P. falciparum and other
species of Plasmodia had used either Hoechst dyes, which are
DNA-selective and require UV excitation, unavailable on most cytometers,
or acridine orange (AO), which stains DNA and RNA. The advantage of TO
over AO is that fluorescence background is lower, since TO enhances
fluorescence by a factor of over 1,000 on binding to nucleic acid,
whereas AO is slightly quenched on binding.

Interestingly enough, Donald Hare, using AO as a DNA/RNA stain as
developed by Darzynkiewicz et al in the 1970s, showed in 1986 that
nucleic acid content measurements by flow cytometry sufficed to identify
all stages of development of P. falciparum, making it theoretically
possible to automate malaria diagnosis (dispensing with the
morphological information on which visual diagnosis from Giemsa-stained
smears must be based), but impractical due to the high cost and
complexity of flow cytometry equipment. Unfortunately, very few people
working on malaria read (past and present tense) the Journal of
Histochemistry and Cytochemistry. The relevant references are: Hare JD,
Bahler DW: Analysis of Plasmodium falciparum growth in culture using
acridine orange and flow cytometry. J Histochem Cytochem 1986;
34:215-220, and Hare JD: Two-color flow-cytometric analysis of the
growth cycle of Plasmodium falciparum in vitro: identification of cell
cycle compartments. J Histochem Cytochem 1986; 34:1651-1658. The full
articles are available at no cost at www.jhc.org.

The P. falciparum genome was sequenced some years ago; the haploid
genome is about 23 MBp in size, and has the highest percentage of (A+T)
of any known organism (80%). In clinical falciparum malaria, the most
common intraerythrocytic forms are small rings, which are haploid;
haploid crescent gametocytes are also present, while later trophozoites
and schizonts (2c to 16c DNA content) are seldom seen in peripheral
blood. In malaria due to P. vivax, and to the less common P. malariae
and P. ovale, blood contains a much larger proportion of later
trophozoites and schizonts. The sequence of P. vivax should be published
 in late 2007 or 2008; it contains somewhat more DNA than P. falciparum,
probably about 30 MBp.

In my lab, we have looked at a variety of nucleic acid stains from
Molecular Probes/Invitrogen as stains for P. falciparum in culture. SYTO
13, which stains both DNA and RNA, gives relatively good signals. I
suspect, however, that the best dye to use would be one of Molecular
Probes's DNA-selective dyes, either Pico Green or Vybrant DyeCycle
green. The latter will definitely stain intracellular nucleic acid
without the need to fix or permeabilize cells; both dyes are related
chemically to TO, and enhance fluorescence at least 1,000 fold on
binding to DNA. We have not had the opportunity to try either on malaria
cultures, due to a singular lack of enthusiasm on the part of NIH in
funding our work in this area.

What we are actually trying to do is use a very small, simple, rugged,
inexpensive fluorescence imaging system, with a high-intensity LED for
excitation and an inexpensive CMOS digital camera chip for detection,
for malaria diagnosis; this would be an ideal apparatus for use in most
places where malaria is prevalent. We have already shown that the same
instrument can readily detect signals from DNA stains in individual
bacteria, with genome sizes on the order of 1/10 the size of Plasmodium
genomes (the aim there is rapid detection and 24-36 hour drug
susceptibility testing of M. tuberculosis), so we do not anticipate any
significant problems in detecting the malaria parasites. The instrument
works at low magnification, and can collect signals from an area of a
slide as big as 10 x 10 mm (the area of a typical Giemsa smear) without
any need for stage motion or focus control. The published references on
the hardware are: Shapiro HM: "Cellular astronomy" — a foreseeable
future in cytometry. Cytometry 2004; 60A:115-124, and Shapiro HM,
Perlmutter NG: Personal cytometers – slow flow or no flow? Cytometry.
2006; 69A:620-630.

If you and your colleagues at WRAIR have any interest in this, I'd be
happy to talk to you about it, or about cytometry of malaria in general.
You can reach me at 617-965-6044 (home/office) or 617-283-6092
(cellular); the cellular phone will be the best bet for the next week or so.

-Howard