Quantitative measurement of Plasmodium -infected erythrocytes in murine models of malaria by flow...

Quantitative Measurement of Plasmodium-InfectedErythrocytes in Murine Models of Malaria by Flow

Cytometry Using Bidimensional Assessment of

SYTO-16 Fluorescence

Marıa Belen Jimenez-Dıaz, Teresa Mulet, Vanesa Gomez, Sara Viera, Angela Alvarez,Helena Garuti, Yolanda Vazquez, Alejandra Fernandez, Javier Ibanez, Magdalena Jimenez,Domingo Gargallo-Viola, Inigo Angulo-Barturen*

� AbstractFlow cytometry is a powerful tool for measuring parasitemias in murine malaria mod-els used to test new antimalarials. Measurement of the emission of the nonpermeablenucleic acid dye YOYO-1 (at 530 and 585 nm after excitation at 488 nm) allowed theunambiguous detection of low parasitemias (�0.01%) but required prolonged fixationand permeabilization of the sample. Thus, we tested whether this issue could be over-come by use of the cell-permeant dye SYTO-16 with this same bidimensional method.Blood samples from CD1 mice infected with Plasmodium yoelii, Plasmodium vinckei, orPlasmodium chabaudi or from NODscidb2m-/- engrafted with human erythrocytes andinfected with P. falciparum were stained with SYTO-16 in the presence or absence ofTER-119 mAb (for engrafted mice) in 96-well plate format and acquired in TrucountTM

tubes. Bidimensional analysis with SYTO-16 was quantitatively equivalent to YOYO-1.Moreover, by combining SYTO-16 with the use of TER-119-PE antimouse erythrocytemAb and Trucount tubes, the measurement of the concentration of P. falciparum-infected erythrocytes over a range of five orders of magnitude was achieved. Bidimen-sional analysis using SYTO-16 can be used to accurately measure the concentration ofPlasmodium spp.-infected erythrocytes in mice without complex sample preparation.' 2008 International Society for Advancement of Cytometry

� Key termsmalaria; flow cytometry; SYTO-16; bivariate analysis; TrucountTM; P. falciparum; mice

MALARIA is caused by the erythrocytic stages of protozoa of the genus Plasmodium,

which colonize and destroy host’s erythrocytes (1). To counter this disease, murine

models of malaria are essential tools for research (2), particularly for drug discovery

(3). In addition to the standard rodent experimental systems, different murine mod-

els of P. falciparum malaria are currently available (4–6). These are of special interest

for drug discovery because, with the exception of human subjects, these are the only

experimental systems available that allow the evaluation in vivo of the real human

pathogen growing inside human erythrocytes (hE) previously engrafted into immu-

nodeficient mice. Not surprisingly, the peripheral blood of these chimeric mice

[humanized mice (HM)] is a complex mixture of murine erythrocytes (mE) and hE,

in which the hematological effects of massive transfusions of hE and their elimination

from peripheral blood may have important effects. Hence, the specific and quantita-

tive measurement of different erythrocytic subpopulations is crucial in HM models,

particularly when these models are used to establish the relationship between the

amount of an antimalarial drug in blood and the effect on parasitemia through ex-

perimental pharmacokinetic and pharmacodynamic studies (PK/PD). In this kind of

GlaxoSmithKline R & D, ID CEDDDiseases of the Developing World,Tres Cantos, Spain

Received 12 March 2008; RevisionReceived 6 July 2008; Accepted 13August 2008

Grant sponsor: Medicines for MalariaVenture (Geneva, Switzerland); Grantsponsor: Fundaci�on Universidad-Empresa, CITIUS Program (Madrid,Spain).

*Correspondence to: I~nigo AnguloBarturen, GlaxoSmithKline Investigaci�ony Desarrollo, SL, Diseases of theDeveloping World, Drug DiscoveryBiology, Therapeutic Efficacy, c/ SeveroOchoa, 2 Tres Cantos, 28760 Madrid,Spain

Email: [email protected]

Published online 10 September 2008 inWiley InterScience (www.interscience.wiley.com)

DOI: 10.1002/cyto.a.20647

© 2008 International Society forAdvancement of Cytometry

Original Article

Cytometry Part A � 75A: 225�235, 2009

studies, an accurate and precise measurement of the concen-

tration of parasites in blood is essential (7,8).

Flow cytometry provides higher throughput, statistical

power, and precision than microscopic assessment of blood

smears from Plasmodium spp.-infected mice. In spite of this,

microscopy has remained as the gold standard to measure par-

asitemia in murine malarias because of the relatively low spec-

ificity of detection of flow cytometry in these models. To date,

most cytometric techniques available employ nucleic acid dyes

to detect parasite’s DNA/RNA in erythrocytes otherwise lack-

ing nuclei (9–14). However, the content of nucleic acids (re-

sidual RNA or DNA) in noninfected mE is highly variable and

may be similar to the amount of nucleic acids present in

infected erythrocytes. For example, since micronuclei-contain-

ing mE have quantities of DNA comparable to uninucleated

parasites, P. berghei has been used as a standard to define the

region of murine micronuclei in studies of genotoxicity in

mice assessed by flow cytometry (15). This problem of speci-

ficity is particularly marked in the presence of reticulocytosis

during recovery from anemia after treatment of an experimen-

tal infection (16) or in infections by P. vinckei or P. chabaudi,

which do not have preference for reticulocytes (2). In order to

overcome this hurdle, we developed a bidimensional method

of measuring the appropriately compensated emission of the

nucleic acid dye YOYO-1 at 530 � 30 (FL1530) and 585 � 42

(FL2585) nm after excitation at 488 nm (YOYO-1530/YOYO-

1585) (16). The YOYO-1530/YOYO-1585 bidimensional patterns

of staining allowed the distinction between infected and unin-

fected reticulocytes or normocytes and we could unequivocally

determine whether a mouse was infected or not at parasite-

mias as low as 0.01%. Unfortunately, biological membranes

are not permeable to YOYO-1, and a relatively complex sample

preparation is required, which makes this dye unsuitable for

quick and quantitative measurement of the concentration of

infected erythrocytes in blood.

A number of cell-permeant nucleic acid dyes that have

been used to detect Plasmodium spp. in mice are currently

available. These include DNA-selective dyes such as Hoechst

33258 and Hoechst 33342 (9,17–19). However, excitation of

these dyes is achieved through the use of ultraviolet lasers

which are not widely available. Among the non-DNA selective

dyes, hydroethidine (13,20) requires prior oxidation to ethi-

dium to render it capable of binding to nucleic acids (21). Ac-

ridine orange, which is another non-DNA selective dye (22),

has recently been described as a permeable dye for staining

nucleic acids that does not require complex sample processing

(23) despite having long been considered a nonpermeable dye

that is highly susceptible to staining conditions (21). In any

case, the emission spectra of Hoechst, hydroethidine or acri-

dine orange, are different from that of YOYO-1, making them

unsuitable for use in a bidimensional FL1530/FL2585 method.

In contrast, SYTO-16 (i) is a cell-permeant non-DNA selective

cyanine dimer, (ii) has excitation and emission spectra similar

to those of YOYO-1, (iii) is essentially nonfluorescent in the

absence of nucleic acids, and (iv) shows a quantum yield

higher than YOYO-1 (0.65 vs. 0.52 relative to fluorescein,

respectively) (24). Interestingly, SYTO-16 has already been

used to detect erythrocytes parasitized with P. falciparum

in vitro (25,26) and P. berghei in vivo (26,27), although, to the

best of our knowledge, it has only been formally validated to

measure parasitemias in murine models of the hemoparasite

Theileria (28). Moreover, none of the authors cited used a

bidimensional FL1530/FL2585 method to detect parasitized ery-

throcytes in mice. In consequence, we tested whether SYTO-

16 could be used in murine models of malaria in a fluores-

cence bidimensional FL1530/FL2585 method (SYTO-16530/

SYTO-16585) aimed specifically at providing a fast, specific,

and quantitative measurement of the concentration of infected

erythrocytes in HM.

In this article, we describe a flow cytometry dual parame-

ter procedure using SYTO-16530/SYTO-16585 to measure Plas-

modium spp. parasitemias in murine models of malaria that

does not require sample processing and is as sensitive and

accurate as YOYO-1530/YOYO-1585. This method allows the si-

multaneous staining of samples with SYTO-16 and mAbs to

identify specific erythrocytic subpopulations and/or avoid

interfering background signals. Moreover, the method is com-

patible with the use of acquisition tubes containing known

numbers of calibrated beads (TrucountTM), which allow the

measurement of the actual concentrations of infected erythro-

cytes in blood.

MATERIALS AND METHODS

Mice

Pathogen-free 8- to 12-week-old female CD1 (Hsd:ICR)

mice were obtained from Harlan (Gannat, France). A colony

of NOD.Cg-Prkdcscid B2mtm1Unc/J (NODscid/b2m-/-) immunode-

ficient mice was raised and maintained by Charles River Labo-

ratories (L’Arbresle, France). Female mice (8-week old) were

used. The mice were provided with autoclaved tap water and

c-irradiated pelleted diet ad libitum and maintained in the

GlaxoSmithKline Laboratory Animal Science animal facilities

at Tres Cantos (Spain). The animal research complied with

national and European Union legislation and GlaxoSmith-

Kline policy on the Care and Use of Animals and related codes

of practice.

Reagents and Antibodies

Saline solution and Dulbecco’s PBS from Gibco (Paisley,

UK); Giemsa solution from Merck & Co. (Whitehouse Sta-

tion, NJ); SYTO-16 and YOYO-1 from Molecular Probes

(Leiden, The Netherlands); biotinylated rat IgG1 isotype con-

trol, CD71 (biotinylated rat IgG1, clone C2), PE-streptavidin,

PE-conjugated rat IgG2b isotype control, and TER-119 (PE-

conjugated rat IgG2b antimouse erythrocyte) from Pharmin-

gen (San Diego, CA).

Parasites and Infection of Mice

P. yoelii 17X, P. berghei ANKA (both kindly donated by

Dr. E. Dei-Cas and Dr. L. Delhaes from Institut Pasteur, Lille,

France), P. vinckei and P. chabaudi (Malaria Research and Ref-

erence Reagent Resource Center, ATCC, Manassas, VA) were

ORIGINAL ARTICLE

226 Bidimensional SYTO-16 in Murine Malaria

used. P. falciparum Pf3D70087/N9 was generated from P. falcipa-

rum 3D7 as a standardized isolate capable of growing reprodu-

cibly in mice engrafted with hE in GlaxoSmithKline-Diseases

of the Developing World (Tres Cantos, Spain) (6). Experi-

ments in standard murine models (P. yoelii, P. chabaudi, or P.

vinckei) were performed as described previously (16). Experi-

ments with NODscid/b2m-/- mice engrafted with hE and infected

with P. falciparum Pf3D70087/N9 were conducted as described

(6). Briefly, in order to assay P. falciparum in mice, cohorts of

age-matched female NODscid/b2m-/- were injected i.p. daily

with hE throughout the experiment. When �40% chimerism

in peripheral blood was reached (7–10 days after initiation of

injections), the mice were infected with 20 3 106 parasites

obtained from infected donors (i.v. in 0.2 ml of saline or hE

suspension). In this animal model, P. falciparum Pf3D70087/N9

is not cleared from peripheral blood of mice.

Human Blood

Incomplete donations or erythrocyte concentrates of

malaria-negative donors were used, generously provided by

the Spanish Red Cross blood bank in Madrid, Spain. Prior to

injection, blood stored at 48C was washed twice with RPMI

1640, 25 mM HEPES (Sigma) containing 7 3 1023 mM hypo-

xanthine (Sigma) at room temperature. The buffy coat was

removed by aspiration (if required), and erythrocytes were

resuspended at 50% hematocrit in RPMI 1640, 25% decom-

plemented human serum (Sigma) 3.1 mM hypoxanthine.

Finally, the blood suspension was warmed at 378C for 20 min

prior to intraperitoneal injection with 1 ml of hE suspension

(administered daily throughout the experiments).

Measurement of Parasitemia

Flow cytometry. In brief, 2 ll of blood from the lateral tail

vein of mice were rapidly transferred into 0.1 ml/well of saline

containing 2.5 or 5 lM SYTO-16 (for rodent plasmodia and P.

falciparum in HM, respectively, since HM may have a signifi-

cantly higher erythrocyte concentration in blood) and, if

required, 10 lg/ml TER119-PE, in clean V-bottomed 96-well

plates. The samples were incubated for 20 min at room tem-

perature in the dark, after which 10 ll of saline 0.25% glutar-

aldehyde was added to each well. After 5 min of incubation to

allow complete inactivation of plasmodia, 30 ll was trans-

ferred from each well to either normal tubes or TrucountTM

tubes (Becton Dickinson, San Jose, CA) containing 270 ll ofsaline and acquired. Each Trucount tube contains a defined

number of fluorescent beads. The samples were acquired in

FACScalibur or LSRII flow cytometers (Becton Dickinson)

equipped with at least a 15 mW 488 nm air-cooled Argon-ion

laser, exactly as described for YOYO-1 (16). All the experi-

ments described in this article were performed using a FACS-

calibur flow cytometer unless otherwise stated. Erythrocytes

and leukocytes were gated in logarithmic forward/side scatter

dot plots, and green and red fluorescence were collected in

photomultipliers through 530 � 30 (FL1530) or 585 � 42

(FL2585) band-pass filters, respectively. The mean fluorescence

channels in FL1530 and FL2585 were adjusted to equal values in

the first decade of intensity in bivariate logarithmic scale dot-

plots with noninfected control samples. Leukocytes were

excluded during analysis in SSC/FL1530 dot plots because they

show much higher amounts of nucleic acids than parasites.

Compensation of SYTO-16 emission was performed exactly as

described for YOYO-1 (16). This was a critical step to accu-

rately define the region of infected events and must be deter-

mined empirically by comparison of the effect of increasing

compensation of the emission of SYTO-16 in FL2585 in sam-

ples from uninfected and Plasmodium-infected mice. Blood

samples taken 48 h after infection with P. yoelii or P. berghei

were optimal for compensation, since they show normocytes

containing rings and trophozoites, and reticulocytes harboring

all stages of the parasite. General procedures and target pat-

terns of staining are detailed in the Results section referring to

Figures 2A and 2B. The samples were analyzed using either

CellQuest-Pro or BD FACSDiva 5.0 (Becton Dickinson) soft-

ware. In samples with parasitemias[0.1%, the number of ery-

throcytes acquired was that which allowed the gathering of at

least 1,000 events (typically 2,000–5,000, depending on para-

sitemia) in the region established for infected erythrocytes.

For parasitemias below 0.1%, 1.5 3 106 total erythrocytes

were acquired. In order to determine the specifications of the

technique, the minimum number of events in the region of

infected events considered statistically significant was 100,

because it was the minimum number of events which could

provide reliably a recognizable bidimensional pattern of

infected events when compared with uninfected controls.

According to this criterion, the maximum sensitivity achieva-

ble by acquiring 1.5 3 106 total erythrocytes was 0.007%.

The concentration of cells in the acquisition tube was cal-

culated according to the following formula: Erythrocytes/

mlacquisition tube 5 (NE/NB) 3 (Beads/mlacquisition tube); where

NE is the number of erythrocytes acquired and NB is the

number of beads acquired. The actual concentration of ery-

throcytes in blood was calculated by multiplying the concen-

tration of cells in the acquisition tube by the dilution factors

employed.

Microscopy. Microscopic analysis of samples of peripheral

blood from infected mice were performed in blood smears

stained with 10% (vol/vol) Giemsa in saline buffer (0.015 M

NaCl, 0.001 M phosphate buffer, pH 7.0) as described in

Ref. 29.

Statistical Analysis

Experimental differences between the means or medians

of different groups were analyzed by the Student’s t test, Wil-

coxon-signed rank test or one-factor ANOVA, followed by

Dunnett’s posttest. Homogeneity of variances was assessed

using the Levene’s test. Normality of variables was assessed by

Kolmogorov–Smirnov test. All statistical analyses were per-

formed using GraphPad Prism 4.0 (GraphPad Software, San

Diego, CA) or SPSS 13.0 for Windows (SPSS, Chicago, IL).

Probability values P [ 0.05 were considered nonsignificant.

All the experiments described were performed at least twice in

order to ensure the reproducibility of results.

ORIGINAL ARTICLE

Cytometry Part A � 75A: 225�235, 2009 227

RESULTS

Optimization of Staining with SYTO-16

To assess the value of SYTO-16 in murine models of

malaria, the concentration of SYTO-16 that lacked hemolytic

activity was first determined, and the resolution in FL1530between noninfected and infected erythrocytes in our assay

conditions was maximized. These conditions were standar-

dized in order to avoid sample processing before acquisition.

The concentration of SYTO-16 was optimized using P. yoelii

since it has a marked preference to infect reticulocytes (2), and

the background due to noninfected reticulocytes is negligible

48–72 h after infection in the conditions used (16,23).Thus,

2 ll of whole blood obtained from CD1 mice infected with

P. yoelii were added to 100 ll saline (�1.63 106 erythrocytes/ll,23.7% of parasitemia at 72 h after i.v. infection with 6.4 3 106

parasitized erythrocytes) containing increasing concentrations

of SYTO-16 in a 96-well plate format. After 20 min of incuba-

tion at room temperature in the dark, the samples were neu-

tralized with glutaraldehyde for 5 min and acquired 30 ll ofeach sample in Trucount tubes containing a defined number

of fluorescent beads. Although not necessary for the flow cyto-

metry technique, neutralization with glutaraldehyde was

included to guarantee safe manipulation of samples poten-

tially infectious to humans. The fluorescent beads in Trucount

tubes acted as internal controls to test whether the addition of

SYTO-16 significantly reduced the number of erythrocytes per

well in comparison to control wells without SYTO-16, as a

means of assessing the potential hemolytic effect of SYTO-16

on fresh erythrocytes. The highest mean of fluorescence inten-

sity (MFI) ratio between infected and noninfected erythro-

cytes was achieved at concentrations between 2.5 and 5 lM(Figs. 1A–1C). At these concentrations, neither significant

reduction in the number of erythrocytes acquired (confirmed

by microscopy in Neubauer hemocytometer) nor significant

changes in FSC/SSC pattern were observed, compared with

wells that were not exposed to SYTO-16. However, these nega-

tive changes appeared in some samples at 25 lM or higher.

Implementation of the

SYTO-16530/SYTO-16585 Method

As is true for all of the nucleic acid dyes tested in murine

models so far, the one-dimensional assessment of emission of

SYTO-16 in FL1530 may have serious problems of specificity at

relatively low but significant parasitemias. A common finding

in infected mice is exemplified in Figure 2A (upper panels).

This figure shows representative histograms of blood samples

from uninfected control CD1 and P. yoelii-infected mice 24 h

after infection with 107 parasites. The estimated parasitemia,

adjusting the region of infected events with samples of infected

mice with higher parasitemias as shown in Figure 1B, matched

the parasitemia measured by microscopy (1%). However, the

background signal in a sample taken from an uninfected CD1

mouse processed in the same conditions was 0.8%. Previously

we showed that the staining differences between complex cells

invading erythrocytes and residual macromolecular structures

in uninfected erythrocytes were unveiled using YOYO-1530/

YOYO-1585 by addressing the effect of compensation on the

light measured by the FL2585 detector, which was found to be

different for infected and uninfected erythrocytes (16). In the

present study, tests were performed to ascertain whether or

not a similar approach could be employed with SYTO-16. As

shown in Figure 2A (lower panels), in the absence of compen-

sation, SYTO-16530/SYTO-16585 did not allow the discrimina-

tion between uninfected and infected erythrocytes. Conversely,

compensation of emission SYTO-16585 (�48% in our experi-

mental set-up) revealed a clearly identifiable population of

infected erythrocytes. The gates of infected events for SYTO-

16530/SYTO-16585 were established by comparing samples

from uninfected and infected mice when the compensation of

the emission of SYTO-16 in the FL2585 detector was increased

from 0% (Fig. 2A, lower panels) to 48% (Fig. 2B). CD1 mice

were infected with the rodent parasites P. yoelii, P. chabaudi,

and P. vinckei, which exhibit high, indifferent and low prefer-

ence for infecting murine reticulocytes, (2), respectively.

Humanized NODscid/b2m-/- mice with �50% of hE in periph-

eral blood were infected i.v. with P. falciparum. All plasmodial

Figure 1. Establishment of the optimum concentration of SYTO-

16. (A) Gating of mE in logarithmic FSC/SSC contour plots. (B)

Regions used in one-dimension FL1530 to estimate the optimum

concentration of SYTO-16 required maximizing the resolution

between infected and uninfected erythrocytes. The MFI of infected

erythrocytes was measured (Region R2) and divided by the MFI

from the negative population (Region R1). The sample shown

was from a CD1 mouse infected with P. yoelii and stained withSYTO-16 at 2.5 lM. (C) Resolution of infected versus uninfectederythrocytes and measurement of hemolysis. Blood from a P.yoelii-infected CD1 mouse (�106 erythrocytes/sample, 23% para-

sitemia) was stained with increasing concentrations of SYTO-16

in a 96-well plate format. The figure shows the ratio of MFI from

the P. yoelii-infected SYTO-165301 population (MFIinfected) with

respect to the uninfected SYTO-16530 population (MFIuninfected)

(left Y-axis) and the percentage of erythrocytes recovered (relativeconcentration of erythrocytes in each sample with respect to con-

trol without SYTO-16, right Y-axis). The data are the mean of tripli-cates � SEM pooled from two experiments. The bars for devia-

tions are only shown if bigger than the size of symbols.

ORIGINAL ARTICLE


species could be gated with the same region, although each

species produced characteristic patterns of staining that corre-

lated with their affinity for reticulocytes and degree of natural

synchronization (Fig. 2B). As expected, P. yoelii infection

reduced the percentage of uninfected high FL2585 reticulocytes

because of its preference for these cells and showed all erythro-

cytic stages (nonsynchronized growth) (Fig. 2B). Conversely,

neither P. chabaudi nor P. vinckei significantly diminished the

percentage of uninfected high FL2585 reticulocytes and showed

an apparent high degree of synchronization (2). Interestingly,

P. falciparum, which does not productively infect mE (30),

lacked high FL2585 reticulocytes, probably because of the inhi-

bition of murine erythropoiesis, and showed all erythrocytic

stages in peripheral blood (nonsynchronized growth). Apply-

ing the gate of acquisition defined previously, the background

signal in samples taken from uninfected CD1 or humanized

NODscid/b2m-/- mice was \0.05% in all cases. Importantly,

since the pattern of staining was similar in samples not neutra-

lized with glutaraldehyde, it was decided that the neutraliza-

tion step would be maintained for safety reasons.

The next step was to ascertain whether parasitemias

measured using SYTO-16530/SYTO-16585 were accurate. Accu-

racy is defined as the capability of the cytometric technique to

give true values of parasitemia by comparison with a reference

standard. YOYO-1530/YOYO-1585 was employed as the refer-

ence method, since it has been validated independently for

standard rodent models of malaria (16,31) and P. falciparum

cultures (32) and utilizes the same strategy of analysis (16).

Thus, we measured parasitemias in parallel using YOYO-1530/

YOYO-1585 and SYTO-16530/SYTO-16585 in 212 samples taken

at different times from peripheral blood of CD1 or humanized

NODscid/b2m-/- mice infected with rodent Plasmodium spp. or

Figure 2. Gates of infected erythrocytes in bidimensional SYTO-16530/SYTO-16585 dot plots for murine models of malaria. (A) Specificity of

detection of P. yoelii at low parasitemias using a one-dimensional pattern of staining SYTO-16530. We measured blood samples from con-

trol uninfected CD1 mice (left panels) or 24 h after infection with P. yoelii (i.v., 103 106 infected erythrocytes/mouse, n5 5 mice/group, rightpanels). Upper panel on the left shows the background signal in uninfected mice, whereas the upper panel on the right shows the percent-

age of infection measured as indicated above. Lower panels show the very same samples in bidimensional FL1530/FL2585 dot plots without

compensation. The arrows show the putative infected events. Data are from a representative individual of each experimental group. (B)

Effect of compensation of FL2585 on bidimensional SYTO-16530/SYTO-16585 dot plots and establishment of gates of infected events. We

took blood samples from control uninfected CD1 mice or 48 h after infection with P. yoelii, P. chabaudi or P. vinckei (i.v., 10 3 106 infected

erythrocytes/mouse, n 5 5 mice/group). We also took blood samples from control uninfected humanized NODscidb2m-/- or 6 days after infec-

tion with P. falciparum Pf3D70087/N9 (i.v., 203 106 infected erythrocytes/mouse, n 5 3 mice/group). The plots shown are from one represen-

tative mouse of each experimental group. The arrow indicates the population of erythrocytes stained with SYTO-16 (mostly reticulocytes),

which is present in blood from uninfected CD1 mice but not in P. yoelii-infected CD-1 and humanized NODscidb2m-/-mice.

ORIGINAL ARTICLE


P. falciparum, respectively. The rodent malaria samples were

taken between days 0 and 6 after infection and showed a range

of parasitemia between 0.02 and 92%.The samples from the

murine P. falciparum model were taken between days 0 and 10

after infection and showed a range of parasitemia between

0.02 and 10%. As shown in Figure 3A, there was a strong cor-

relation between both methods (Spearman’s coefficient of cor-

relation r 5 0.997, P \ 0.0001). Moreover, SYTO-16530/

SYTO-16585 measurements were deemed accurate (mean 91.9

� 1.1%, median 92.6%) in the range assayed (0.07–92% of

parasitemia), although a tendency for SYTO-16 to give slightly

lower values than YOYO-1 was detected (Fig. 3B, Wilcoxon-

signed rank test, P \ 0.0001 for departure of the theoretical

median 100%). The next step was to determine the range of

linearity and sensitivity, as well as to obtain an estimation of

the precision of measurements in the detection range. In order

to do this, triplicate 1/2 serial dilutions (vol/vol) of blood

from P. yoelii-infected CD1 mice with blood from uninfected

CD1 controls were tested. As expected, the data were found to

be linear (r2 5 0.95) for parasitemias above 0.07% (Fig. 3C).

This defined the suitable range of parasitemias over which

changes in murine parasitemia could be accurately measured.

However, the sensitivity of the method, that is, the lowest de-

tectable parasitemia, was 0.01% and the precision, expressed

as coefficient of variation, was 4.3 � 2.8. Therefore, the cell-

permeant nucleic acid dye SYTO-16 provided a fast SYTO-

16530/SYTO-16585 method of measuring Plasmodium spp.

parasitemias in murine models of malaria that did not require

complex sample processing and is as accurate, sensitive, and

precise as YOYO-1530/YOYO-1585 (16).

Simultaneous Detection of Erythrocyte

Subpopulations Using Monoclonal Antibodies

With the exception of some nonhuman primates, P. fal-

ciparum only productively infects hE. In order to evaluate the

therapeutic efficacy of antimalarial compounds in P. falcipa-

rum-infected humanized NODscid/b2m-/- mice, it is mandatory

to measure both parasitemia and the percentage of hE in pe-

ripheral blood of mice to discard the possibility that any drop

in the former is due to an enhanced clearance of hE, as found

in other analogous models (33). To assess this practical pro-

blem, we tested whether the nonhemolytic rat anti-mouse

mAb TER-119, which is specific for the murine erythroid line-

age (34), could be used for the simultaneous detection of mE

and parasites by the SYTO-16530/SYTO-16585 technique. First,

experiments were performed to determine the concentration

of antibody necessary to achieve minimum background, maxi-

mum discrimination of murine and hE while showing no he-

molytic activity and no significant erythrocyte aggregation

(35). Thus, 2 ll of blood from an uninfected humanized

NODscid/b2m-/- mouse with~50% hE in peripheral blood (�20

3 106 erythrocytes) was added to 100 ll saline containing

SYTO-16 at 5 lM and increasing concentrations of TER-119-

PE (from 0.1 to 50 lg/ml) in 96-well plate format. Each sam-

ple was then incubated, neutralized, and acquired in Trucount

tubes as described. At 5–10 lg/ml, TER-119-PE reached

saturation of staining without any hemolytic activity (Fig. 4A)

and negligible background staining with PE-conjugated isoty-

pic control (not shown). To ascertain whether staining with

TER-119-PE would render accurate percentages of erythro-

cytes, blood of known concentration of erythrocytes obtained

from NODscid/b2m-/- mice and human donors were mixed at

various proportions (vol/vol) and the relative percentage of

each cell type was measured. The results shown in Figure 4B

indicated that TER-119-PE measurements were accurate

(93.2% � 2.5%), although the values of percentage of mE

were slightly lower than the predicted ones (One sample t test,

P5 0.021) in the range assayed (0.7–100%). Next, the specificity

of TER-119-PE/SYTO-16530–585 for detection of P. falciparum-

Figure 3. Accuracy, linearity, and sensitivity for detection of

infected erythrocytes (A) Correlation between parasitemias meas-

ured using SYTO-16530/SYTO-16585 and YOYO-1530/YOYO-1585.

We randomly took blood samples from CD1 mice infected with P.yoelii (n 5 66 samples), P. berghei (n 5 18 samples), P. chabaudi(n 5 24 samples), P. vinckei (n 5 24 samples), or humanized

NODscidb2m-/- infected with P. falciparum Pf3D70087/N9 (n 5 105

samples) at different times after i.v. infection. The Spearman’s

coefficient of correlation was r 5 0.997 (P \ 0.0001). (B)

Bland�Altman plot to detect any bias in the measurements of par-asitemia using SYTO-16530/SYTO-16585 compared to YOYO-1530/

YOYO-1585. The plot shows the ratio between the % of parasitemia

measured by SYTO-16530/SYTO-16585 and YOYO-1530/YOYO-1585versus the average % of parasitemia measured by both methods.

Data plotted are the same used in correlation studies shown in

the previous section. (C) Sensitivity and linearity of detection of

SYTO-16530/SYTO-16585 Plasmodium yoelii-infected erythrocytesat low parasitemias. We serially diluted (1/2 vol/vol) erythrocytes

from a mouse having 0.5% of parasitemia with erythrocytes from

a control CD1 mouse. The theoretical parasitemias were calcu-

lated for the different number of erythrocytes in each type of

blood. The data shown are the mean � SEM of the decimal loga-

rithm of triplicate samples for each dilution tested, pooled from

three different experiments.

ORIGINAL ARTICLE


parasitized erythrocytes was determined. Most of the back-

ground signal detectable in peripheral blood of mice was due

to mE, as shown in Figure 4C. Accordingly, the use of TER-

119-PE enabled us to reduce background signal to\0.01% in

uninfected HM. Using serial dilutions of infected samples

with blood from uninfected NODscid/b2m-/- mice, in order to

increase the relative percentage of interfering reticulocytes, the

sensitivity of detection was observed to be \0.01% and the

measurements were linear (r2 5 0.9882), at least for parasite-

mias � 0.01% (Fig. 4D). Thus, in the humanized mouse

model of P. falciparum, TER-119-PE/SYTO-16530–585 staining

significantly increased the specificity, range of linearity, and

sensitivity of detection of parasitized erythrocytes by at least

one order of magnitude compared with SYTO-16530/SYTO-

16585 alone.

Quantitative Determination of Blood Populations in

HM Using Trucount Tubes

The use of Trucount tubes containing a defined number

of fluorescent beads has been validated in clinical practice for

measurement of cells and platelets in human blood (36–38).

Since the use of SYTO-16 involves no loss of erythrocytes due

to sample processing before acquisition in flow cytometry,

experiments were carried out to determine whether Trucount

tubes could be used to measure the concentration of erythro-

cytes in blood in our assay conditions. Erythrocytes were

counted in electronic hemocytometer Coulter AcT.5.Diff

(Beckman Coulter, Fullerton, CA) and in Neubauer hemocyt-

ometer as reference methods. In these experiments, blood

from uninfected CD1 was used, since the Coulter AcT.5.Diff

could not unambiguously resolve the erythrocyte populations

present in samples of HM. Hence, 2 ll of blood from an unin-

fected CD1 mouse was added to 100 ll saline containing

SYTO-16 at 2.5 lM in 96-well plate format (n 5 10 repli-

cates). The samples were incubated for 20 min at room tem-

perature in the dark, neutralized with glutaraldehyde for

5 min, and 30 ll of each sample was acquired in Trucount tubes

containing 270 ll of sterile saline. Trucount beads were distin-guished from erythrocytes by their fluorescence detected at

660 � 20 (FL4660) in SSC/FL4660 dot plots after excitation at

633 nm. Leukocytes were excluded on the basis of their high

fluorescence on FL1530. Although all three methods rendered

similar results in terms of accuracy and precision, it was found

that flow cytometry counting gave consistently lower values

than the hemocytometer (P \ 0.01, one-factor ANOVA fol-

lowed by Bonferroni posttest), as shown in Table 1. Next, these

results were confirmed by measuring in parallel erythrocyte

concentrations using microscopy (performed by different

Figure 4. Simultaneous staining with SYTO-16 and TER-119 mAb. (A) Titration of TER-119-PEmAb for staining 2 ll of blood from peripheral

blood of a humanized NODscidb2m-/-mice with about 50% of hE in peripheral blood. (B) Accuracy of measurement of mE in samples stained

with TER-119-PE mAb. We mixed known volumes of human blood (5.2 � 0.1 3 109 erythrocytes/ml) with blood from a control NODscidb2m-/-

mouse (6.1 � 0.43 109 erythrocytes/ml). Data are the mean � SEM of triplicate measurement per data point. (C) Flow cytometry pattern for

samples of human blood, NODscidb2m-/-mice, uninfected HM or Pf3D70087/N9-infected HM stained simultaneously with TER-119-PE mAb and

SYTO-16 (TER-119-PE/SYTO-16530�585). Data are representative of n5 44 infected mice. (D) Linearity and sensitivity of detection of P. falcip-arum-infected erythrocytes in samples stained simultaneously with TER-119-PE/SYTO-16530�585. We serially diluted (1/2 vol/vol) bloodfrom a humanized NODscidb2m-/-mouse having 0.5% of parasitemia (7.7 � 0.3 3 109 erythrocytes/ml) with erythrocytes from a control non-

chimeric NODscidb2m-/-mouse (5.2 � 0.23 109 erythrocytes/ml). The theoretical parasitemias were calculated for the different number of ery-throcytes in each type of blood. The data shown are the mean � SEM of the decimal logarithm of triplicate samples for each dilution tested.

ORIGINAL ARTICLE


technicians with different degrees of training), and TER-119-

PE/SYTO-16530–585 plus Trucount beads (TSC) in 26 samples

taken from peripheral blood of humanized NODscid/b2m-/-

mice having between 2.2 3 109 and 11.9 3 109 erythrocytes/

ml. As expected from previous results, both sets of data corre-

lated significantly (Spearman’s coefficient of correlation r 50.76, P\ 0.0001) and TSC measurements showed an accuracy

of 117% � 28% (median 114.7), indicating a tendency to give

slightly higher values than microscopy under routine labora-

tory conditions (Wilcoxon-signed rank test, P \ 0.0028 for

departure of the theoretical median 100%). Finally, the linear-

ity and sensitivity of counting by TSC was determined using a

LSRII digital flow cytometer in order to maximize the sensitiv-

ity of counting infected erythrocytes using 2 ll blood samples.

First, the concentration of erythrocytes was measured in sam-

ples obtained by 1/2 (vol/vol) triplicate serial dilutions of

blood from a P. falciparum-infected humanized NODscid/b2m-/-

mouse with human serum. The data were linear over the entire

range tested (2.6 3 109 down to 4.1 3 107 erythrocytes/ml,

which modeled an extreme anemia), as shown in Figure 5A.

The accuracy with respect to the theoretical values in the range

tested was 104 � 3.3% (one sample t test, P 5 0.26 for depar-

ture from a theoretical accuracy of 100%) and the limit of

sensitivity was 4.0 3 105 erythrocytes/ml due to the high

background found in human serum alone (3.7 � 0.1 3 105

erythrocytes/ml). Second, we measured the concentration of

infected hE (ihE) in samples obtained by 1/2 (vol/vol) tripli-

cate serial dilutions of blood from a P. falciparum-infected

humanized NODscid/b2m-/- mouse with blood from an unin-

fected NODscid/b2m-/- mouse to maximize the unspecific back-

ground due to murine reticulocytes. Since the measurement of

the concentration of cells depends on the concentration of

beads in the tube, we eliminated the uninfected erythrocytes

by establishing a threshold for acquisition in FL1530. This

strategy dramatically diminished the number of events to be

acquired, limiting this number, in practical terms, to the num-

ber of beads per Trucount tubes (Fig. 5B). As shown in Figure

5C, the measurements were linear over the whole range tested

(83.1 3 106 down to 1.6 3 103 ihE/ml, which encompassed a

range of parasitemias from 1.2% down to 0.0018%). The accu-

racy with respect to the theoretical values in the range assayed

was 111% � 7.1% (one sample t test, P 5 0.14 for departure

of the theoretical 100% of accuracy) and the limit of sensitiv-

ity was 7.0 3 102 erythrocytes/ml (background was 0.0006 �0.00005 3 106 erythrocytes/ml). In summary, these data indi-

cated that Trucount tubes can be used in combination with

SYTO-16 and TER-119-PE mAb to accurately measure the

concentration of P. falciparum-infected or uninfected erythro-

cytes in a range of five orders of magnitude while generating

small size listmode files.

DISCUSSION

In this article, we describe a simple, sensitive, and accurate

quantitative method to measure parasitemias in peripheral

Figure 5. Measurement of the concentration of erythrocytes in

HM by TSC. (A) Linearity and accuracy of measurement of con-

centration of hE using TSC. We serially diluted (1/2 vol/vol) blood

from a humanized NODscidb2m-/-mouse infected with P. falciparum(7.0 � 0.2 3 109 erythrocytes/ml) with decomplemented human

serum. The data shown are the mean � SEM of the decimal loga-

rithm of the concentration of hE from triplicate samples for each

dilution tested. (B) Flow cytometry patterns for samples of P. fal-ciparum-infected HM (upper and middle panels) or uninfected HM

(lower panel) mice, following acquisition of all SYTO-165301 and

FL46601 events. SYTO-16530

1 events encompass ihE and back-

ground cells, whereas FL46601 events are beads. (C) Linearity and

accuracy of measurement of the concentration of ihE using TSC.

We serially diluted (1/2 vol/vol) blood from a humanized

NODscidb2m-/- mouse infected with P. falciparum (1.1% parasite-

mia, 7.0 � 0.2 3 109 erythrocytes/ml) with blood from a control

nonchimeric NODscidb2m-/- mouse (8.8 � 0.4 3 109 erythrocytes/

ml). The data shown are the mean � SEM of the decimal loga-

rithm of the concentration of parasitized erythrocytes in blood in

triplicate samples for each dilution tested. All samples were

acquired in a Becton Dickinson LSRII flow cytometer and analyzed

using BD FACSDiva 5.0 software.

Table 1. Comparison of three methods for measurement of the

concentration of erythrocytes in murine blooda

NEUBAUER COULTER TRUCOUNTTM

Experiment 1 7.84 � 0.74 7.71 � 0.44 7.01 � 0.42b

Experiment 2 7.32 � 0.44 8.15 � 0.8b 7.89 � 0.26b

Experiment 3 8.08 � 0.52 8.67 � 0.41b 7.19 � 0.33b

a Concentration of erythrocytes in samples of blood from the

same CD1 mouse in each experiment. Data are the mean erythro-

cytes/ml 3 109 � SEM of 10 replicates for measurements

performed in Coulter AcT.5.Diff or using TrucountTM beads. For

Neubauer counting, four fields containing at least 100 erythro-

cytes were counted.b The difference of the mean compared to Neubauer counting

was significant (P\0.05, one-factor ANOVA, Dunnett’s posttest).

ORIGINAL ARTICLE


blood of mice infected with Plasmodium spp. using SYTO-

16530/SYTO-16585.

Any flow cytometry technique for murine models of

malaria should have adequate specificity, that is, the ability to

discriminate between infected and background erythrocytes in

the range of its intended use. In this respect, the enhanced

specificity of the YOYO-1530/YOYO-1585 method for detection

of parasitemias in mice allowed the unequivocal identification

of parasites in many circumstances in which the previously

described methods had failed (16). Despite the quality of this

technique, it had two major shortcomings. First, it did not

allow a quick determination of parasitemia since a minimum

of 2 h of fixation in glutaraldehyde was required before sample

processing. Second, measuring the concentration of infected

erythrocytes in blood samples was not possible, because of the

extensive manipulation of the blood samples. Therefore,

SYTO-16, a membrane-permeant nucleic acid dye with spec-

tral properties similar to YOYO-1, was tested because of its

potential to maintain the level of specificity achieved with

YOYO-1530/YOYO-1585 while allowing a fast and quantitative

measurement of infected erythrocytes in mice infected with

Plasmodium spp.

Before implementing the SYTO-16530/SYTO-16585 bidi-

mensional method, in which compensation is of critical im-

portance, we determined the concentration of SYTO-16 that

maximized the MFIinfected/MFIuninfected ratio at 530 nm in

samples showing a negligible percentage of uninfected reticu-

locytes (e.g., P. yoelii 48–72 h after i.v. infection with 107 para-

sites). SYTO-16 has been previously used to detect P. falcipa-

rum in vitro (25,26), and P. berghei (26,27) and Theileria

sergenti (28) in mice by measuring its emission around 530 nm

after excitation at 488 nm. The concentrations of dye used by

these authors ranged from 20 nM (26) to 3 lM (27). Unfortu-

nately, no information has been published on the criteria and

the titration curve results in the different conditions of stain-

ing with SYTO-16. Most of the studies used PBS as staining

buffer (26–28) although there seems to be evidence indicating

that phosphate salts may affect the binding of SYTO-16 to

DNA (24). Moreover, there is experimental evidence indicat-

ing that changing the staining buffer and/or the interactions

with other reagents may markedly reduce the MFI after stain-

ing with SYTO-16 (26). Therefore, it is very difficult to com-

pare the concentrations deployed in the aforementioned stu-

dies with the relatively high concentrations that were shown to

be required to maximize the MFIinfected/MFIuninfected ratio. In

summary, it is likely that the wide differences in the concen-

trations of SYTO-16 employed reflect not only different condi-

tions of staining but also different criteria of optimization.

Since SYTO-16 has a quantum yield that is higher than most

nucleic acids dyes, it is not surprising that sufficient resolution

for some uses was also obtained at lower concentrations of

SYTO-16. In fact, in our experimental conditions, the MFIin-

fected/MFIuninfected ratio for SYTO-16 at 0.5 lM (MFIinfected/

MFIuninfected ratio � 100) was actually higher than the highest

ratio obtained for YOYO-1 at 0.25 lM (optimal MFIinfected/

MFIuninfected ratio � 60) (16) and appeared to be very similar

to the resolution described by Huber et al. (27) using 3 lM in

PBS. Regardless of the differences between findings reported

in the present study and those reported by other authors,

SYTO-16 showed a MFIinfected/MFIuninfected that was five times

higher than YOYO-1 (at optimum concentration), hydroethi-

dine or thiazole orange and even higher when compared to

Hoechst, propidium iodide, or acridine orange, according to

data presented by other authors (20,23,28,39–41). This trait of

SYTO-16 was crucial to reproducibly measure very low con-

centrations of parasites (below\0.01%) since the background

signal was minimized.

The detection specificity of the SYTO-16530/SYTO-16585method is dependent on the differential distribution of values

of the bidimensional vector (SYTO-16530, SYTO-16585) for the

populations of uninfected erythrocytes containing residual

nucleic acids and infected erythrocytes containing Plasmodium

spp. Although SYTO-16 is not a DNA-selective dye, its peaks

of absorption (488 or 494 nm bound to DNA or RNA, respec-

tively) and emission (518 and 525 nm, bound to DNA or

RNA, respectively) were slightly shifted to longer wavelengths,

and its quantum yield was significantly lower (0.65 vs. 0.24,

bound to DNA or RNA, respectively) when bound to RNA

(24). Thus, the first component of the vector (SYTO530),

which is close to the emission maximum of SYTO-16, prob-

ably reflects the amount and type of predominant nucleic acid

inside erythrocytes, that is, DNA in infected erythrocytes and

RNA in uninfected reticulocytes. Consequently, as occurred in

one-dimensional methods, SYTO-16530 alone (the first com-

ponent of the bidimensional vector), this component may not

discriminate between infected and uninfected erythrocytes

containing similar amounts of nucleic acids. This discrimina-

tion was achieved by measuring the compensated SYTO-16585component. The relative contribution of SYTO-16 bound to

DNA, RNA, other undefined sources of fluorescence, and

autofluorescence to the emission in FL2585 is not currently

known, but it may differ significantly from YOYO-1. The

SYTO-16530/SYTO-16585 and YOYO-1530/YOYO-1585 methods

were equivalent in terms of measurement of the percentage of

parasitemia in blood from infected mice, but some experimen-

tal evidences indicate that the patterns of staining might be

qualitatively different. First, in contrast with the YOYO-1530/

YOYO-1585 method, the SYTO-16530/SYTO-16585 procedure

does not involve digestion of RNA. It is likely that SYTO-16

bound to RNA may be more relevant in the SYTO-16585 com-

ponent. This would explain why the SYTO-16530/SYTO-16585method did not permit a clear differentiation between infected

reticulocytes and infected normocytes, in contrast with the

pattern of infected erythrocytes observed with YOYO-1530/

YOYO-1585 (16). Consequently, the presence of significant

amounts of RNA in infected erythrocytes may mask detectable

differences in fluorescence between infected reticulocytes and

infected normocytes that are unveiled in YOYO-1530/YOYO-

1585 upon fixation with glutaraldehyde and RNA digestion.

Second, the SYTO-16530/SYTO-16585 procedure does not

require fixation with glutaraldehyde. Even though a short

5-min period of fixation with 0.25% glutaraldehyde may

induce significant autofluorescence in erythrocytes, our results

suggest that autofluorescence does not interfere in the pattern

ORIGINAL ARTICLE


of staining. Therefore, it is likely that the autofluorescence of

uninfected background events may be more important for dis-

crimination between infected and uninfected events in YOYO-

1530/YOYO-1585 than in the SYTO-16530/SYTO-16585 proce-

dure. This might explain why the RNA present in noninfected

erythrocytes could be compensated in FL2585 as the specific

signal in infected erythrocytes in SYTO-16530/SYTO-16585(Fig. 2), whereas the effect of compensation (up to 70%) on

background erythrocytes compared to infected erythrocytes

was negligible in YOYO-1530/YOYO-1585 (16). Third, since the

staining with SYTO-16 was performed using fresh erythro-

cytes, SYTO-16 may accumulate in mitochondria, the parasi-

tophore vacuole, or cytoplasmic structures of parasites, as

found in many eukaryotic cells (24,42). As a whole, the afore-

mentioned characteristics make the interpretation of SYTO-

16530/SYTO-16585 patterns of staining difficult in terms of

unequivocally identifying the different stages of the parasite in

vivo, as has been addressed recently for monitoring P. falcipa-

rum in vitro using Hoechst and thiazole orange (41). A poten-

tial way to overcome staining pattern interpretation problems

might be to test permeable DNA-selective dyes that can be

excited at 488 or at 532 nm (e.g., Vybrant Dye-CycleTM Green

or Orange stains), as has been suggested recently (43). These

reagents emit fluorescence when bound to double-stranded

DNA, and the intensity of emission is proportional to the

amount of DNA in the cell (24). Although a systematic com-

parison using a bidimensional method FL1530/FL2585 is still in

progress, our preliminary results indicate that SYTO-16530/

SYTO-16585 and Vybrant Dye-CycleTM Green530/Vybrant Dye-

CycleTM Green585 produce similar patterns of staining, SYTO-

16 being brighter than Vybrant Dye-Cycle Green while the lat-

ter dye renders lower levels of background.

A number of techniques aimed at simultaneously staining

membrane antigens and intraerythrocytic parasite’s nucleic

acids have been described. All of them were intended to char-

acterize specific subpopulations of infected erythrocytes (44–

46) or detect antibodies against membrane antigens found on

the surface of infected erythrocytes (47,48). In addition to this

approach, the present study demonstrates that mAb targeted

against erythrocytic populations responsible for background

signals can be used to significantly enhance the specificity and

sensitivity of cytometric measurements. This strategy seemed

more reliable than trying to select the target population (e.g.

P. falciparum-infected erythrocytes) using antibodies against

human glycophorin A, since this is a high-density antigen that

might cause artefacts due to agglutination of erythrocytes

(35), thereby reducing the quality of measurements of ihE in

HM at very low parasitemias. The sensitivity achieved using

TSC in HM was remarkable (�0.001%). Previously, the most

sensitive procedures for detection of parasites in peripheral

blood had required the lysis of erythrocytes and detection of

released parasites (22,49). With these procedures, the sensitiv-

ity in human samples was 0.005% and significantly lower (49),

or even unacceptably unspecific (22), in rodent malaria mod-

els. Compared with these methods, TSC does not require sam-

ple lysis, employs smaller quantities of blood [2 ll vs. 50 ll(49) or 25 ll (22), respectively] and gives accurate values of

concentration of ihE over the entire range tested. The main

disadvantage is the relatively long time of acquisition per sam-

ple required to achieve maximum sensitivity. Indeed, our

results suggest that there is still room for improvement in TSC

procedure, since platelets are a major source of background in

mice at very low parasitemias. Thus, mAbs against murine pla-

telets could be employed to enhance the sensitivity and speci-

ficity of measurements in expensive PK/PD studies in P. falcip-

arum HM models.

The technique described in the present article does not

require complex sample processing. As there are no changes in

sample volume, the measurement of the actual concentrations

of erythrocytes in blood was possible. The use of Trucount

tubes was strongly advocated because of its demonstrated

reliability in clinical practice to measure the concentration of

leukocytes (36), red cell vesicles in thalassemic patients (37),

and particularly, platelets or residual cells in plasma products

(38). However, given the relatively high cost of these tubes,

other approaches could be validated for each particular analyt-

ical purpose. Therefore, methods based on the stability of the

flow rate of bench cytometers (50) or, preferably, with stand-

ards prepared with biotin-labeled erythrocytes (51), might

provide cheaper alternatives.

Murine models of malaria may become key tools to

understand the in vivo PK/PD relationships that govern the

therapeutic efficacy of antimalarials (52). These studies

demand accurate quantitative measurements of parasite con-

centration over wide ranges of parasitemia (7,52) and, as in

the case of P. falciparum HM models, the capacity to resolve

complex cellular dynamics. The TER-119-PE/SYTO-16530–585technique described here was developed to measure ihE with

high specificity and sensitivity in HM used in PK/PD studies

of antimalarial therapeutic efficacy. This procedure provided in-

formation on the hematological status of HM (e.g. erythrocyte

concentration and degree of engraftment with hE), significantly

improved the specificity of detection of P. falciparum-ihE over

SYTO-16530/SYTO-16585 alone and enabled us to accurately

measure the concentration of ihE in blood over the entire

range of detection (five orders of magnitude). Moreover, this

technique can be modified to measure the reticulocytosis and

the degree of anemia induced by different strains of rodent

Plasmodium spp. in immunocompetent mice using anti-

mouse CD71 mAb instead of TER-119 (data not shown).

Hence, it may become a useful tool in any experimental design

in murine models of malaria requiring quantitative assessment

of erythrocytic subpopulations.

ACKNOWLEDGMENTS

This work was supported in part by Medicines for

Malaria Venture (Geneva, Switzerland) through GSK-Diseases

of the Developing World/MMV agreement. We acknowledge

the support of Antonio Martınez and all the staff at Labora-

tory Animal Science Department in GlaxoSmithKline-Diseases

of the Developing World in Tres Cantos (Spain) for providing

and maintaining the mice used in this study. The authors are

also indebted to Elena Jimenez and the staff from Experimen-

tal Pharmacophysiology at GSK DDW-Biology for their con-

ORIGINAL ARTICLE


tribution counting blood samples with the electronic hemo-

cytometer Coulter AcT.5.Diff. The authors are indebted to Jo-

seph J. Campo (CRESIB, Barcelona, Spain) for critical review

of the manuscript. The authors are also grateful to Nick

Guthrie for the thorough review of the English grammar and

syntax of the manuscript.

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Quantitative measurement of Plasmodium -infected erythrocytes in murine models of malaria by flow...

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