Cytometry for protozoa related diseases.
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ABSTRACT: The laser dyes oxazine 750 (OX750), LD700, and rhodamine 800 (R800) can be used in an instrument employing a low-power helium-neon laser source for flow cytometry of DNA content in ethanol-fixed or detergent-permeabilized cells. Cells in near-isotonic medium are stained with 10-30 microM dye, and fluorescence excited at 633 nm is measured at wavelengths above 665 nm. The dyes do not appear to stain RNA, and the intensity of DNA staining is not changed when 2 microM Hoechst 33342 is added to cells simultaneously with a red-excited dye. The effects on fluorescence of addition of DNA to LD700 or R800 in aqueous solution are strongly influenced by the base composition of the DNA; binding mechanisms remain to be determined.Cytometry 02/1986; 7(1):107-10.
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- Cytometry Part A 06/2010; 77(6):500-1. · 3.71 Impact Factor
Cytometry for Protozoa Related Diseases
Attila T? arnok*
THE great majority of single cell eukaryotic organisms are liv-
ing free where liquid water is present. Particularly in the ocean
they are forming a major part of the phyto- and zooplankton
relying on nutrition by photosynthesis or organic material.
They were initially taxonomically classified as members of the
Kingdom Protista (E. Haeckel, 1866), albeit this classification
has been questioned recently based on phylogenetic and mo-
lecular biology data (1). Presently, single cell organisms are
members of different kingdoms but the term Protista is still
not out of fashion and used as a very descriptive terminology.
Whereas most protozoa are harmless for humans some
cause serious infections, infecting any human tissue and being
the cause of a variety of severe diseases. Clinically relevant pro-
tozoa include intracellular and extracellular parasites invading
and harming the host using a variety of strategies. Some pro-
duce cysts to survive inside or outside the body, others are
spread by insects and proliferate inside infected host cells.
Protozoa are among the major causes for devastating dis-
eases particularly in the resource poor and developing coun-
tries (2). Infections caused by protozoa include, among others,
toxoplasmosis, amoebic meningitis, malaria, trypanosmiasis,
leishmaniasis (Kala-Azar) and amoebic dysentery as well as di-
arrhea caused by Cryptosporidium spp. or Giardia intestinalis
(lamblia). Many of the Protists that are harmful to humans
such as Plasmodium spec. and Toxoplasma spec. are according
to the new taxonomy members of the Chromalveolata
‘‘Kingdom’’ (1). Malaria caused by at least five different Plas-
modium species is, with an estimated number of 300,000 –
500,000 newly infected per year and an annual death toll of
around one million individuals, probably the major enemy.
But how can cytometry support basic research and clinical de-
cision making in protozoan parasite infections? This is
demonstrated in the following four examples.
Plasmodium performs a complex life cycle between differ-
ent hosts and several proliferation cycles within the infected
erythrocytes prior to their release from and destruction of a
red blood cell. Flow cytometry has been proven to be an excel-
lent tool to distinguish between infected and uninfected ery-
throcytes and to determine parasite load. Furthermore, it
allows combined analysis of parasitemia measurement with
functional assays such as oxidative stress (3). In this regard,
red-excitable DNA dyes are ideal for detecting infected cells
due to the availability of cheap instruments equipped with red
lasers and low cellular autofluorescence at red excitation. This
was recently demonstrated by Fu and colleagues (3) for human
and by Jimenez-Diaz and colleagues for murine erythrocytes
using the SYTO 61 DNA dye.
A comparable assay is presented by Gerena and colleagues
from the University of Puerto Rico, San Juan, Puerto Rico
(this issue, page 965). The authors developed a method for
precisely measuring parasite load in erythrocytes of mice
infected with Plasmodium berghei, a parasite infecting rodents,
by a whole blood approach. Their rationale was to have a
mouse test system at hand that can be used for in vivo phar-
macological testing of novel drugs against the parasites. This is
a highly relevant model as Malaria starts to become resistant
to common antimalaria drugs and rapid assays are needed.
The authors tested two red excitable rhodamine DNA dyes
R800 and LD700 that do virtually not stain RNA (4). They
could clearly demonstrate that RNAse treatment is not essen-
tial for unequivocal detection of parasite infected erythrocytes
and that the flow cytometry data highly correlate with manual
microscopic counting and percentage of erythrocytes carrying
GFP labeled parasites. Thereby, the authors provide an impor-
tant easy to use tool for the efficacy testing of novel antima-
Dept. Pediatric Cardiology, Heart Centre, University of Leipzig,
Received 12 September 2011; Accepted 28 September 2011
*Correspondence to: Prof. Attila T? arnok, Dept. Pediatric Cardio-
logy, Heart Centre Leipzig, University of Leipzig, Str€ umpellstr. 39,
04289 Leipzig, Germany.
Published online in Wiley Online Library
© 2011 International Society for Advancement of Cytometry
Cytometry Part A • 79A: 885?886, 2011
Schuck and colleagues from the University of Sao Paulo,
Brazil (this issue, page 959) also focused on Plasmodium,
albeit on Plasmodium falciparum that is infecting humans and
causing the most severe tropical disease, Malaria tropica. It
synchronously replicates in infected red blood cells and the
liver. It induces 48 hour fever intervals when merozoites are
released and is thereby highly lethal. The parasite replication
and release is synchronized by melatonin, a product of
serotonin metabolism, as well as serotonin derivatives and
tryptamine. The authors used an in vitro cocultivation test
system of human erythrocytes and parasites. Parasitemia and
proliferation after treatment with various indolic compounds
were measured by flow cytometry using YOYO-1 as the DNA
dye. They demonstrated clearly that under treatment with all
indolic compounds the number and frequency of multi-
nucleated parasites within infected erythrocytes but did not
substantially affect parasitemia.
Red blood cells are the major targets of Plasmodium spec.
and they interact with the host cell metabolism that can lead
to changes in osmolarity and shape. Fischer and Korzeniewski
from the RWTH University Aachen in Germany (this issue,
page 946) addressed quantitation of shape changes of indivi-
dual red blood cells under shear stress based on microscopic
image analysis. The authors optimized the present image anal-
ysis assays by improving the mathematical approach and using
suspending media with a viscosity closer to that of normal se-
rum. With their improved method they found a wide interin-
dividual distribution of red blood cell parameters within single
blood samples. This method could be of relevance for a label
free analysis of parasitemia in infected persons and may also
be useful in drug discovery.
Another clinically relevant Chromalveolata member spe-
cies is Toxoplasma gondii. Although it is mainly causing Toxo-
plasmosis in cats, humans may be intermediate hosts and can
induce in immune deprived individuals and the unborn,
severe symptoms. Standard diagnostics today are polymerase
chain reaction and tests for T. gondii specific antibodies by
ELISA in body fluids. Some years ago a method for measuring
T. gondii by Laser Scanning Cytometry was presented (6).
Now from the research group of Aldebert and colleagues from
CNRS-US, Grenoble, and the University Hosptal, St. Etienne
in France (this issue, page 952), a flow cytometric and an
image cytometric high-throughput assay was developed to
detect and quantitate cysts of T. gondii in the brain of infected
mice. Cysts from homogenized brains were labeled for micro-
scopic or flow cytomeric analysis by fluorescence-dyed lectin.
Both assays correlated well with manual counting of the cysts,
however, imaging was preferable when cyst counts were low.
The high-throughput microscopy is quantitative, and more
specific and sensitive than flow cytometry. The authors state
that both techniques are preferable to bioassays or PCR when
determination of cyst number is critical. Their assays could be
a new way to analyze Toxoplasma cysts from water or meat.
These highly relevant manuscripts demonstrate that
Cytometry can be of major support in parasite infections by
helping to understanding the biology of hostile protists and by
improving the diagnosis for immediate health care.
2. Shapiro HM, Ulrich H. Cytometry in malaria: from research tool to practical diag-
nostic approach? Cytometry A 2010;77A(6):500–501.
3. Fu Y, Tilley L, Kenny S, Klonis N. Dual labeling with a far red probe permits analysis
of growth and oxidative stress in P. falciparum-infected erythrocytes. Cytometry A
4. Shapiro HM, Stephens S. Flow cytometry of DNA content using oxazine 750 or
related laser dyes with 633 nm excitation. Cytometry. 1986;7(1):107–110.
5. Jim? enez-D? ıaz MB, Mulet T, G? omez V, Viera S, Alvarez A, Garuti H, V? azquez Y,
Fern? andez A, Ib? a~ nez J, Jim? enez M, Gargallo-Viola D, Angulo-Barturen I. Quantitative
measurement of Plasmodium-infected erythrocytes in murine models of malaria by
flow cytometry using bidimensional assessment of SYTO-16 fluorescence. Cytometry
6. Mital J, Schwarz J, Taatjes DJ, Ward GE. Laser scanning cytometer-based assays for
measuring host cell attachment and invasion by the human pathogen Toxoplasma
gondii. Cytometry A 2006;69A(1):13–19.
ofeukaryotes. Curr Biol.
886Cytometry for Protozoa Related Diseases