Development and validation of flow cytometric measurement for parasitemia in cultures of P. falciparum vitally stained with YOYO-1

Authors


  • This article is a US government work and, as such, is in the public domain in the United States of America.

  • Material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the author and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense.

Abstract

Background:

The need for improved malaria diagnostics has long been recognized.

Methods:

Human parasitized erythrocytes based on the principles of flow cytometry (FCM) method is described for the determination of parasitemia in Plasmodium falciparum cultures using the fluorescence activated cell sorter and DNA-binding fluorescent dye, YOYO-1. The same assay samples can be also evaluated both microscopically and by scintillation counting after use of 3H-hypoxanthine-labeled parasites.

Results:

The counts of uninfected, infected, and nucleated cells occurred with high precision. The cells were categorized into different populations according to their physical or chemical properties such as RNase treatment and compensation required optimization. The detection and quantitation limits in the assay were 0.003% and 0.008% parasitemia, respectively. Overall, the parasite counts by FCM measurement correlated highly (r2 = 0.923–0.968) with the parasitemia measured by 3H-hypoxanthine incorporation assay when parasites variants incubated with various antimalarial drugs. In addition, the low levels of parasitemia (7.9%–21.3%) detected by microscopy than by FCM may be related to a number of tiny schizonts externally attached to the erythrocyte membranes but were not definitely inside the erythrocyte that normally would never be included in microscopy counting.

Conclusion:

On the basis of data reported herein, a rapid, high sensitivity, lower sampling error and reliable identification of human parasitized erythrocytes by the principles of FCM have been established. Published 2007 Wiley-Liss, Inc.

Microscopic analysis of patient blood smears is a potential problem in all affected areas. A published audit, the National External Quality Assessment Scheme (NEQAS) data and the observations of reference centers on slides submitted to them for confirmation of diagnosis have revealed shortcomings in the diagnosis of malaria in the United Kingdom (1) and in Thailand (2, 3). There was significant misdiagnosis with regard to false positives (7–36%), false negatives (5–18%), and false species (13–15%). A high frequency of technical errors (e.g. wrong pH or a poor quality film) was also noted. Therefore, microscopy is an imperfect “Gold Standard” diagnostic device (4).

Several new methods of malaria diagnosis have been developed in the past two decades (e.g. 3H-hypoxanthine incorporation assay), but these all rely on clinical suspicion and, consequently, an explicit clinical request. Although some methods lend themselves to automation (e.g. flow cytometry), no technique can yet be used for routine clinical automated screening. Such work has taken advantage of the absence of DNA in erythrocytes. Thus, if the parasite is inside the cell, its DNA can be stained with specific nucleic acid-binding dyes and detected by flow cytometry (FCM). Different dyes, such as Hoechst 33258 (5), acridine orange (6), thiazole orange (7), or hydroethidine (8), have been considered for the determination of parasitemia in cultures of P. falciparum by FCM. Jacobberger et al. (9) used DiOC1, a membrane potential responsive dye and Hoechst 33342 to evaluate parasitemia levels in mice (7). Recently, YOYO-1, a dimeric cyanine nucleic acid dye, is among the highest sensitivity fluorescent probes available for nucleic acid staining and has been added to this list (10, 11). YOYO-1 has an extremely high affinity for DNA, and it can be excited at 488 nm, which is the excitation wavelength available from most lasers employed in FCM. Its bright fluorescence signal and low background make it ideal for flow cytometric analysis of stained malaria nucleic acids (11, 12). Compared to our previous experience, the parasitized erythrocytes infected with P. berghei labeled with YOYO-1 can be completely separated from uninfected erythrocytes because of the increased relative fluorescence intensity. These features make YOYO-1 an extremely sensitive nucleic acid dye compared with many of the aforementioned dyes, a property that can be very useful in FCM (13).

The FCM analysis in cultured P. falciparum models of malaria is impeded by significant reduction of reticulocytes and normocytes containing detectable amounts of nucleic acids after blood treated. Therefore, the absence of reticulocytes and normocytes may reduce the interference in measurement of parasitemia (14). Present study proposes here use of a flow cytometer (Cell Lab QuantaTM, Beckman Coulter Inc., Fullerton, CA) with a 488 nm laser combined with and YOYO-1 nucleic acid dye. We compared our quick and reliable FCM method to traditional microscopic analysis of blood smears and the microdilution radioisotope method for the evaluation of parasitemia in parasite culture with P. falciparum. This report describes a dual-parameter procedure using autofluorescence to make a distinction of infected erythrocytes from uninfected erythrocytes and normocytes. This method is particularly well suited for measuring low and high parasitemias and significantly increased the sensitivity.

MATERIALS AND METHODS

Drugs and Chemicals

All of the artemisinin derivatives (artemisinin, dihydroartemisinin, artemether, arteether, artesunate, and artelinate) and positive control agents (mefloquine) tested were obtained through the Walter Reed Army Institute of Research chemical inventory system, except for chloroquine, which was purchased from Sigma. Stock solutions [8 × 10−3 to 40 × 10−3 M in dimethyl sulfoxide (DMSO)] were prepared, and aliquots were frozen at −20°C. Sodium citrate, heparin, D-glucose, glycerol and methanol were purchased from Sigma Chemical. Hema 3 stains were obtained from Fisher Scientific. YOYO-1 (oxazole yellow homodimer) was purchased from Invitrogen Corporation (Carlsbad, CA). Glutaraldehyde, Triton X-100, and ribounclease A (RNase A) were purchased from Sigma-Aldrich (St. Louis, MO). The test compounds used in this study are artemisinin, dihydroartemisinin, artemether, arteether, artesunate, artelinate, chloroquine, and mefloquine which are inventoried in the Walter Reed Army Institute of Research, Silver Spring, MD.

Sample Preparation for FCM

The progress of parasitemia in cultures of P. falciparum was assessed by flow cytometric analysis, as well as by conventional microscopic examination and 3H-hypoxanthine incorporation assay. A 300 μl of incubation medium (contain 1% washed human A-erythrocytes) in each vial in the incubation plate (96 vials) was used to perform 3H-hypoxanthine incorporation assay (see later). In parallel, a second plate (same contains with first plate) was centrifuged at 450g for 5 min and removed supernatant. The each vial in the plate was added directly with 0.3 ml of 0.04% glutaraldehyde, which concentration was selected from our optimization study. In the literature, 0.025–0.25% (v/v) glutaraldehyde has been used for fixation of malaria infected mouse and human blood in various FCM analyses. In this study, 1 ml of glutaraldehyde (0.00625–0.8%) in phosphate buffered saline (PBS) was tested. The cells were mixed with glutaraldehyde solution in various concentrations and stored at 4°C for 60 min. The fixation is preferred in case later treatment with RNase is required for digesting reticulocytes.

The plates were centrifuged at 450g for 5 min. The supernatant was removed by aspiration, and the cells resuspended in 0.3 ml PBS buffer supplemented with 0.25% Triton X-100 and left in this solution for 10 min at room temperature. The best concentration (0.25%) of Triton X-100 was selected from our previous study (14). After centrifugation, the permeabilized cells were re-suspended in 0.3 ml of RNase at 0.5 mg/ml and incubated for at least 2 h at 37°C for a complete digestion of reticulocyte RNA. Then DNA dye of each 20 μl of YOYO-1 solution was added into the 0.3 ml of sample to a final dye concentration of 500 ng/ml. The appropriate concentration of YOYO-1 was found to be optimal to discriminate infected erythrocytes from low to high counts (14).

All flow cytometric analyses were carried out with a Cell Lab Quanta™ (Beckman Coulter, Fullerton, CA) equipped with a 2–22-mW 488-nm air-cooled laser diode. Infected erythrocytes, uninfected erythrocytes, and leukocytes were gated on in logarithmic forward/side dot plots. Cells of 100,000 in each sample were analyzed at an average rate of 2,500 erythrocytes/s. Filters were placed before the green (FL-1) and red (FL-2) photomultiplier tubes (PMTs) such that the green PMT registered fluorescence emission between 520 and 555 nm, and the red PMT measured emission greater than 580 nm. When the samples were acquired by FCM, the erythrocytes infected with P. falciparum were used to model conditions without reticulocytes after treated with RNase and were critical in daily instrument setup and calibration. The control blood without parasite was stained in parallel with the test samples, so that adjustments to flow cytometer settings could be made to compensate for subtle changes in cell staining on a daily basis. While analyzing malaria infected blood samples, photomultiplier tube voltages and laser power were adjusted to maximize resolution between cells with and without malaria parasites. The parasites in the reticulocytes population should exhibit the same YOYO-1 associated fluorescence intensity as the parasites in the normal RBC. Compensation of YOYO-1 emission in FL-2 is an essential step whose only objective is to set up accurately the region of infected cell events. The region must be empirically determined by comparison of blood samples from uninfected and malaria infected rats by increasing compensation of YOYO-1 emission in FL-2 until a defined region for infected cell events is obtained.

Light Microscopy

Microscopic analysis of samples of incubated blood from P. falciparum was performed in blood smears (thin and thick) stained with diluted Giemsa according to a procedure described previously (15). The thick blood film provides enhanced sensitivity of the blood film technique and is much better than the thin film for detection of low levels of parasitemia and reappearance of circulating parasites during infection, recrudescence, or relapse.

Validation of FCM

Calibration curves were prepared in the percentage parasitemia range of 0.0002–10.0% (n = 4, at each level) for malaria infected blood from humans. The serial dilutions (vol/vol) of blood from 4 P. falciparum-infected human bloods were made with blood from blank control humans. The initial parasitemia of the infected human was 12.4% (measured by both microscopy and FCM) and the infected blood was serially diluted down to a theoretical 0.0002% of parasitemia. The lower calibration curve extended from 0.004 to 0.07% of parasitemia with standard values at 0.004%, 0.009%, 0.018%, 0.035%, and 0.07%, and the higher calibration curve extended from 0.28% to 9.0% with standard values at 0.56%, 1.12%, 2.3%, 4.5%, and 9%. The calibration curve was linearly fitted and weighted by 1/concentration with examination of the correlation coefficient. Percentages of parasites in subsequent test samples were calculated from the calibration curve of the human parasitemia.

Analytical accuracy in hematopathology compares the test result to a gold standard, morphology (16). The flow cytometric assessment of accuracy must therefore compare to microscopy. With rare events, however, the morphologic diagnosis is complex phenotypes in abnormal populations such as malaria infected rats with multi-parameter data such as single, multiple (invasion of single cell by multiple schizonts), and even early stage infections in adult erythrocytes and reticulocytes. Therefore, the microscopic diagnosis should consider the single, multiple, and even early stage infections in adult erythrocytes and reticulocytes. Descriptions of dim, moderate, and bright staining pattern can be diagnostic or prognostic and should be well characterized and documented for infected, uninfected erythrocytes, and nucleated cells.

Precision is a standard analytical parameter that measures the reproducibility of date points from a single sample stained and analyzed in duplicate at least 10 times. To validate the FCM for parasitemia, intra-day accuracy and precision were evaluated by analysis at various percentage levels on the same day. Six different parasitemia (1, 2, 4, 6, 8, and 12%) levels were selected to cover the entire span of the calibration curve. The concentrations of parasites were calculated from the calibration curves, and the lower limits of detection and quantitation were given by the highest level of the calibration curve. To assess the inter-day accuracy and precision, the intra-day assay was repeated on three different days.

Parasitemia sensitivity describes the ability to detect the minimum staining intensity above nonspecific or negative staining (17). The detection limit (DL) of an individual analytical procedure is the lowest amount of analyte in a sample that can be detected but not necessarily quantitated as an exact value. The quantitation limit (QL) is the lowest amount of analyte in a sample that can be quantitatively determined with suitable precision and accuracy. The QL is a parameter of quantitative assays for low levels of parasitemia in sample matrices, and is used particularly for the determination of impurities and/or degradation products.

The QL for this method is equivalent to the low point of the low range standard curve. The inter-day limit of quantitation was evaluated by taking the five back-calculated lowest standard calibration concentrations that were obtained in the inter-day precision study and presenting the mean, SD, CV, and RE as the inter-day results. The intra-day limit of quantitation was evaluated by analysis of five lowest standard calibrators and a complete set of standard curve calibrator samples run together within one day (or one run) and presenting the mean, SD, CV, and RE as the intra-day result.

Antimalarial Evaluation with 3H-hypoxanthine Incorporation Assay and FCM

The susceptibility of different malaria strains to the artemisinin derivatives and other control agents were determined using the tritiated hypoxanthine incorporation assay of Desjardins et al. (18) and Chulay et al. (19), as modified by Milhous et al. (20), except that the drug exposure period was 48 h. Four P. falciparum malaria parasites including clones and isolates were utilized for the drug testing. W2 is a clone of the Indochina I/UNC isolate and is resistant to chloroquine and pyrimethamine, but susceptible to mefloquine. D6 is a clone from the Sierra I/UNC isolate and is susceptible to chloroquine and pyrimethamine, but has reduced susceptibilities to mefloquine. The clones were derived by direct visualization and micromanipulation from patient isolates (21). TM90C2A and TM91C235 are P. falciparum isolates from Southeast Asia and highly resistant to mefloquine and a number of other antimalarials.

All parasites are maintained in continuous long term cultures in RPMI-1640 medium supplemented with 6% washed human A+ erythrocytes, 25 mM Hepes, 32 nM NaHCO3, and 10% heat inactivated A+ human plasma. Test compounds were initially dissolved in DMSO and diluted 400-fold in RPMI 1640 culture medium (Gibco BRL, Grand Island, NY). These solutions were subsequently serially diluted 2-fold, 11 times, to give a concentration range of 1,048-fold with a Biomek 2000 apparatus (Beckman, Fullerton, CA). The parasites were added to the diluted drugs plates for 24 h and incubated at 37°C with 5% O2, 5% CO2, and 90% N2. The addition of [3H] hypoxanthine was done after 24 h of incubation. After a further incubation of 18 h, parasite DNA was harvested from each microtiter well using a Packard Filter mate 196 harvester (Meriden, CT) onto glass filter mats. The uptake of [3H] hypoxanthine was measured with a Packard TopCount scintillation counter. Concentration-response data were analyzed by a nonlinear regression logistic dose-response model, and the IC50 values for each compound were calculated.

To validate this FCM method, the main artemisinin drugs (artemisinin, dihydroartemisinin, artemether, arteether, artesunate, and artelinate) and positive control agents (chloroquine and mefloquine) were used in the incubation with P. falciparum. The progress of parasitemia in cultures of P. falciparum was assessed by 3H-hypoxanthine incorporation assay and the flow cytometric analysis. A 300 μl of incubation medium (contain 1% washed human A-erythrocytes) in each vial in the incubation plate was used to perform 3H-hypoxanthine incorporation assay. In parallel, a second plate (300 μl each vial) was performed FCM analysis as described earlier

Data Analysis

The primary determination was the traditional microscopy method. Thin smears were prepared from tail blood, air dried, fixed with methanol, stained with Hema 3 according to the manufacture s instructions, and then examined under oil at 1000× magnification. One thousand red blood cells were examined. Parasitemia was calculated using the following formula: Parasitemia (%) = No. of infected red blood cells/(number of infected red blood cells + number of uninfected red blood cells) × 100. If the percentage of parasites is lower than 0.1%, 10,000 red blood cells will be counted following the same procedures. Using this approach, a negative smear will be one where parasitemia will be < 0.01%. IC50s of the drugs were determined using a nonlinear logistic dose response program.

RESULTS

Optimizations of Sample Preparation

Various options of the fixation, permeabilisation, RNA digestion, and YOYO-1 staining were investigated. The most important parameters to be optimized from the original staining procedures (11–14) were the cell fixation and digestion of reticulocyte RNA in the malaria infected rodent model. The optimal concentration of glutaraldehyde to obtain maximum resolution between uninfected and P. falciparum infected human erythrocytes was assessed. This was done to increase the difference in relative fluorescence unit for staining malaria nucleic acid versus uninfected events, as well as to reduce hemolysis or cell lysis induced by glutaraldehyde. Figure 1 shows that lowering the concentration of glutaraldehyde resulted in a significant cell lysis with a decrease of RBC and a statistically significant increase of the intensity in units between the cell populations from region 1 (uninfected RBC, green in Fig. 2, A1) to region 2 (infected RBC, orange in Fig. 2, A2). At 0.0125% glutaraldehyde, the intensity between the two region peaks (green and orange peaks) is 55.14 (from 6.5 to 59.4) units (Fig. 2, A3), and the hemolysis is 67.9% (Fig. 1). The two populations (green and orange) were clearly separated (Fig. 2, A2 and A3). When increased the concentration of glutaraldehyde to 0.05%, the interval was reduced to 44.85 units of intensities and the hemolysis is 8.1% (Fig. 1) at the middle dose. The two regions were still in a good separation. However, at high dose of glutaraldehyde (0.2%), short interval (32.8 units) in fluorescence intensity was found, and the hemolysis was not affected (0.01%). The two populations were not separated. Therefore, the maximum resolution to minimize the hemolysis and to obtain the highest interval or separation in intensity as possible was attained at a glutaraldehyde concentration of 0.04% (Fig. 1).

Figure 1.

Assessment of the optimum concentrations of glutaraldehyde as a fixative agent for FCM using YOYO-1 staining was shown for RBC. Sample blood from P. falciparum infected human RBCs was fixed with increasing concentrations of glutaraldehyde. The figure demonstrates that hemolysis of RBC is increased (empty circle and solid-line) at low glutaraldehyde concentrations. The YOYO-1 fluorescence intensity in intervals between the cell peaks from region 1 (uninfected RBC in green color) to region 2 (infected RBC in orange color) populations (triangle and dashed-line) is increased and decreased at low and high, respectively, glutaraldehyde concentrations. The sample shown was fixed according to the optimal concentration (0.04% glutaraldehyde, vertical dashed line), which points corresponding to keep minimum hemolysis and keep as wide as possible distance (intensity) between the two regions in present study.

Figure 2.

Flow cytometry analysis of erythrocytes and nucleated blood cells of YOYO-1 stained with bivariate graphs (YOYO-1/FL2) from uninfected (A1) and P. falciparum infected (A2) RBC with RNase showed a clear separation between the populations (A3, by a unidimensional analysis of YOYO-1 fluorescent intensity), and uninfected (B1) and infected (B2) RBC without RNase exhibited cells intermixture between the regions (B3, by a unidimensional analysis of YOYO-1 fluorescent intensity).

The background of incubated RBCs, which was washed with 0.9% saline, is clear with very few reticulocytes (0.005–0.142%) and nucleated cells without treatment with RNase (Fig. 2, B1). However, the background signal after the treatment of RNase (0.5 mg/ml) the reticulocytes, which is selected from our optimization test, was significantly reduced to 0.002% and the intensities of uninfected RBCs were also decreased (Fig. 2, A1). It is obvious that the glutaraldehyde and RNase can make a well separation of the uninfected RBC from the infected erythrocytes by a unidimensional analysis of YOYO-1 fluorescent intensity (Fig. 2, A3), when compared to those cells without treatment of RNase (Fig. 2, B3). Therefore, the maximum resolution to minimize the background signal and to obtain clear separation as possible was attained at treatments with appropriate glutaraldehyde and RNase.

Determination of the Regions in Bidimensional Analysis

The infected RBC showed multiple characteristic patterns (parasites in single ring, multiple rings, trophozoites, and schizonts) of staining different from that of uninfected and nucleated cells in the bidimensional analysis (Fig. 2, A1) with an appropriate compensation (2.4% YOYO-1/FL2). Undercompensation can result in the loss of infected RBC from the nucleated cells (WBC region in purple color). Overcompensation reduces fluorescent resolution between infected erythrocytes and uninfected RBC cells, and also with difference between parasitemias. Results obtained from P. falciparum-infected RBC (Fig. 2, A2) displays a histogram of cell counts of increasing fluorescence intensities in which the infected cells (orange region) are clearly separated from uninfected cells (green region) and also nucleated cells (purple and black regions). The parasitized, unparasitized, and nucleated populations were completely isolated in the own regions, respectively (Fig. 2, A2). The specificities of the three regions were calculated in the range of 98.8–99.2% on validations of the infected, uninfected, and nucleated populations in the present study. In addition, this compensation exhibits a chromatograph of cell peaks (Fig. 2, A3) of increasing fluorescence intensities in which the single ring (first orange peak at 65.7 units), multiple ring or trophozoites (second orange peak at 91.2 units), and schizonts (third orange peak at 107.8 units) are separated from each other (12, 22).

Sensitivity and Linearity

The increased specificity of YOYO-1/FL2 analysis could significantly improve the sensitivity of the measurements of parasitemia in terms of reproducibility and lower quantitative limitation. Calibration curve parameters for parasitemia in the incubated human erythrocytes infected with P. falciparum are shown in Figure 3. Results were calculated using various levels of parasitemia from four individual incubations. Calibration curves for parasitemia were linear using weighted (1/concentration) linear regression in the concentration range of 0.004–10.0% on four incubations with a mean correlation coefficient (r2) greater than or equal to 0.972 for all curves. Different numbers of erythrocytes were acquired to establish the limits of sensitivity of the techniques. In the present assay, the data analyzed were found to be linear from 0.02% to 10.0% and the DL of the YOYO-1 method was 0.003%, measured as the percentage of parasitaemia detectable as background concentration due to the similarity with uninfected control samples. The quantitative limit (QL) was calculated as 0.00828% (Fig. 3), which measured as the percentage of parasitaemia to be detected 3-fold over the DL (0.00267%) in according with FDA Guidance (23).

Figure 3.

Sensitivity of detection from calibration curves of P. falciparum infected erythrocytes from four separate incubations. Parasites counted by flow cytometry were compared with the parasitemia calculated from the initial parasitemia and the dilution factor. The DL and quantitative limit (QL) was determined with 0.00267% and 0.00828%, respectively, from the background using the bidimensional FL-1 (YOYO-1)/FL-2 method with the decimal logarithm of the mean of duplicate samples for each dilution tested (n = 4).

The lower and higher parasitemia calibrations were also analyzed in this study, and the parameters for parasitemia are listed in Table 1. Calibration curves for lower and higher parasitemias were linear with the correlation coefficient (r2) greater than or equal from 0.958 to 0.992 for all curves (Table 1), suggesting that the method is well suited for detection of low and high parasitemia in the malaria-rat model.

Table 1. Assay Linearity of the Flow Cytometry Method on Lower Parasitemia (0.004–0.07%) and Higher Parasitemia (0.28–9.0%) Samples in W2 Strain and D6 Strain by Calibration Curvesa (n = 3)
CalibrationSlopey-interceptCoefficient of correlation (r2)
  • a

    The calibration curve was linearly fitted and weighted by 1/concentration with examination of the correlation coefficient.

W2
 Lower parasitemia calibration1.743 ± 0.3690.004 ± 0.0020.962 ± 0.032
 Higher parasitemia calibration0.998 ± 0.0570.199 ± 0.0670.992 ± 0.005
D6
 Lower parasitemia calibration1.548 ± 0.6080.004 ± 0.0020.958 ± 0.044
 Higher parasitemia calibration0.987 ± 0.0360.203 ± 0.0560.991 ± 0.008

Accuracy and Precision

The accuracy and precision measured the reproducibility of individual parasitemia (with various clones and isolates of P. falciparum) stained and analyzed in duplicate at least 10 times compared with FCM measurement, and the data are shown in Table 2. The inter-day coefficients of variation (precision) for parasitemia samples (uninfected control, 0.97%, 2.31%, 3.53%, 4.28%, 6.445%, 7.34%, 8.42%, and 12.41%) measured by FCM were 31.34%, 6.11%, 4.51%, 4.30%, 2.73%, 4.01%, 3.45%, 2.15%, and 2.72%, respectively. The accuracy for parasitemia analyzed by FCM was in a range of 97.34–104.39%. The data obtained for parasitemia obtained by FCM were well within the acceptable limits to meet FDA guidelines for bioanalytical methods guideline for bioanalytical validation (24).

Table 2. Accuracy and Precision Assessments of Parasitemias From individual P. falciparum (Clones and Isolates) Infected Human RBCs Using Validation Analysis Via the Reproducibility of a Single Sample Stained and Analyzed in Duplicate at Least 10 samples
Parasite clonesFCM measured parasitemia (%)Measured parasitemia (%)FCM accuracy (%)FCM precision (%)FCM vs microscopy accuracy (%)
  1. Accuracy: 100% measured parasitemia/nominal parasitemia from flow cytometry or microscopy methods. FCM, flow cytometry; W-2, Indochina clone, resistant to chloroquine, sensitive to mefloquine; D-6, Sierra Leone clone, sensitive to chloroquine, resistant to mefloquine; TM90C2A and TM91C235 are P. falciparum isolates from Southeast Asia and highly resistant to mefloquine and a number of other antimalarials.

Uninfected samples0.0020109.0935.14
 0.004092.5031.34
Infected samples3.533.0297.874.30114.07
 W26.445.68104.394.01118.36
 8.427.2298.572.15114.96
 12.4111.2097.342.72107.86
 D64.283.66103.762.73121.34
 7.346.1599.283.45118.49
 TM90C2A2.312.0698.874.51110.87
 TM91C2350.970.9299.196.11109.01
Mean5.714.9999.913.75114.37
S.D.  2.661.274.86
Variability  7.081.6123.62

Accuracy of FCM Compared to Microscopy

The parasitemia measured by FCM was in a range from 107.86% to 121.34% (Table 2) when calculated relatively to microscopy counts as 100% in same sample. After having analyzed 242 samples from infected rats in the experiments during our methodological evaluation, we found that most of the parasitemias read by FCM were, on average, 14% higher than the counts of microscopy. The differences observed may be explained by the fact that microscopy is a subjective technique and many cells that do not show a healthy morphology may be discarded during counting, whereas FCM detects parasites irrespective of their health status (12). In fact, we found some schizont-like cells attached to the membrane of normal RBCs (Fig. 4, A1) under light microscopic observation. The same slides were also shown to have tiny stains of Giemsa dye in the schizonts after adjusting the focus (Fig. 4, A2). After staining with YOYO-1 and analysis by fluorescent microscopy, the slides showed that the schizonts were attached to the RBC membranes (Figs. 4, B1–B4). If these particles are real schizonts, the parasites would never be counted by microscopy in our experience before. Although the schizonts will still be confirmed via further investigation, the finding may be a main explanation for the high parasitemia on FCM.

Figure 4.

Photomicrographs of infected rat erythrocytes with P. falciparum show at day 5 after infection. A1 and A2: light microscopy with Giemsa-stained blood smears. Arrows identify schizonts on the membrane surface and penetrating or reinvading the RBC just like vacuoles attached (A1), and a Giemsa stained nucleus shows in the vacuoles (A2). B1–B4: fluorescent microscopy. Arrows are used to identify the schizonts to penetrate or reinvade RBC on the membrane surface stained with YOYO-1. Such as a preinfection phase of parasite is in attachment to the RBC membrane, which parasites were not definitely inside the erythrocyte would never be included in parasitaemia counts by light microscopy in our laboratory but could be measured by flow cytometry.

Application of Parasitemia Measurement by FCM

The staining and FCM procedure utilized for this method has been used to determine the antimalarial effects of six artemisinins (artemisinin, dihydroartemisinin, artemether, arteether, artesunate, and artelinate) and two positive control drugs (chloroquine and mefloquine) in incubated RBCs infected with two clones of P. Falciparum (W2 and D6). Once parameters were established with the malaria-infected blood, experimental samples were analyzed to determine the correlation data between FCM- and with 3H-hypoxanthine incorporation assay. In these experiments, the RBC infected with W2 and D6 parasites were incubated with eight different drugs at various concentrations and the same blood samples were measured for both the flow cytometric analysis and the 3H-hypoxanthine incorporation assay. The summary of the 50% inhibitory concentration (IC50) of the eight compounds is shown in Table 3. The two data are very similar between the two methods without significant difference.

Table 3. In Vitro Sensitivity Profile with 50% Inhibitory Concentration (IC50) of Artemisinin Derivatives and Control Drugs (Chloroquine and Mefloquine) Against Chloroquine-Resistant Clone of P. falciparum from Indochina (W2), Mefloquine-Resistant Clone of P. falciparum from Sierra Leone (D6) by 3H-Hypoxanthine Incorporation Assay (3H-HIA), and Flow Cytometry (FCM)
CompoundsW2 clone of P. falciparumD6 clone of P. falciparum
FCM (IC50) (ng/ml)3H-HIA (IC50) (ng/ml)P valueFCM (IC50) (ng/ml)3H-HIA (IC50) (ng/ml)P value
  1. Values are mean ± SD; W-2: Indochina clone, resistant to chloroquine, sensitive to mefloquine. D-6: Sierra Leone clone, sensitive to chloroquine, resistant to mefloquine.

Artemisinin0.89 ± 0.090.83 ± 0.100.2821.32 ± 0.441.49 ± 0.040.945
Dihydroartemisinin0.31 ± 0.100.34 ± 0.120.1040.26 ± 0.080.29 ± 0.090.568
Artemether0.45 ± 0.060.48 ± 0.070.0780.70 ± 0.210.73 ± 0.240.804
Arteether0.38 ± 0.050.37 ± 0.090.4640.57 ± 0.180.48 ± 0.140.179
Artesunate0.20 ± 0.030.25 ± 0.060.2670.30 ± 0.140.35 ± 0.050.971
Artelinic acid1.28 ± 0.301.24 ± 0.150.3382.21 ± 1.692.78 ± 0.760.752
Chloroquine183 ± 19.4203 ± 38.10.4194.94 ± 1.345.75 ± 1.230.554
Mefloquine2.98 ± 0.563.08 ± 0.900.9659.91 ± 1.347.24 ± 2.400.067

The 3H-hypoxanthine incorporation assay determined parasitemia frequencies were then compared with the corresponding FCM counts. Regression analysis was performed to determine the correspondence between methods. The resulting r2 values of 1.000 indicated a high degree of linear correlation between these two methodologies. Results obtained from P. falciparum-infected rats are exhibited in Figure 5. Thirty-six measurements per drug and per each method were done on samples taken from three individual incubations, and showed a direct correlation (r2 = 0.927–0.968) between flow cytometric and 3H-hypoxanthine incorporation determinations with the eight drugs (Fig. 5).

Figure 5.

Correlations (r2 = 0.927–0.968) are shown between parasitemias by using 3H-hypoxanthine incorporation assay and flow cytometry method. Samples (markers) were taken from incubation RBC infected with P. falciparum (W2) with various concentrations of artemisinin (QHS), dihydroartemisinin (DHA), artemether (AM), arteether (AE), artesunate (AS), artelinate (AL), chloroquine (CQ), and mefloquine (MFQ) (n = 3).

DISCUSSION

The need for improved malaria diagnostics has long been recognized. Recognizing these needs, many flow cytometric assays using different DNA-specific dyes have been evaluated. Several reports show YOYO-1 is better than Hoechst 33258 to easily differentiate between uninfected and infected RBC when parasitaemia is low (6, 11). Our published data by using FCM indicated that the infected rat erythrocytes with YOYO-1 can be completely separated from uninfected erythrocytes because of its high relative fluorescence intensity of the dye. These characteristics were also shown in the present study because YOYO-1 can be excited with 488 nm blue lasers. It shows excellent properties in terms of brightness and is quite stable upon binding to DNA, therefore, allowing samples to be diluted before acquisition without significant loss of signal intensity. With YOYO-1, the total fluorescence intensity in a parasite is 100–200 times less than that of nucleated blood cells. Therefore, the nucleated blood cells can easily be distinguished from infected cells on the basis of the difference in the fluorescence intensity (25). These features make YOYO-1 an extremely sensitive nucleic acid dye compared with many of the aforementioned dyes, a property that can be very useful in FCM (12, 13).

To avoid hemolysis due to lysis by glutaraldehyde (26, 27), the use of glutaraldehyde as fixative agent should be at the lowest concentration possible. The concentration of glutaraldehyde (0.04%) selected in our assay format was different from a lower concentration (0.025%) reported by Barkan et al. (11) and Jimenez-Diaz et al. (12) or a higher concentration (0.25%) described by Janse and Van Vianen (25) and Barkan et al. (11). The concentration recommended in these studies did not allow for optimal discrimination between infected and uninfected erythrocytes in our analysis conditions. Low glutaraldehyde concentration could increase the difference in fluorescence intensity to keep the separation of the infected region from the uninfected region. However, the difference in concentration of glutaraldehyde has significance in cell lysis for the testing samples under test. The best concentration obtained in our laboratory is 0.04%, which could minimize the cell lysis and also maximize the distance between the two regions.

The results obtained in our experiments determined that the background signal obtained using unidimensional YOYO-1 determination was low (< 0.004% in all cases) compared with high and highly variable (0.02% and up to 0.07%) in our previous studies with rodent parasites (14). The stable and clear background presented in this assay was highly related to that (1) the RBCs have been washed with 0.9% saline before the culture; (2) the appropriate concentration of a potential activity of RNase. Some infections of human parasites are accompanied with substantial reticulocytosis (most of the red blood cells might be reticulocytes). Under such conditions, large amounts of RNA are present which could contribute to the parasite-derived signal, thus producing some false positive readings or high noise background. Therefore, treatment of permeabilized infected and uninfected cells with active RNase has been added for reduction or clearance of the background signal. This RNase-specific differential value of fluorescence (differential fluorescence) was plotted against the cell fluorescence measured after RNase treatment. However, appropriate compensation of YOYO-1 emission in FL-2 could be set up to accurately delineate the regions of infected and uninfected events. In this study, typical values of compensation in FL-1 (YOYO-1) were obtained for optimal resolution between infected and uninfected populations after RNase treatment.

In this assay, the clear background levels produced a successful DL at 0.00267% parasitemia. The high sensitivity obtained allowed for the flow cytometric approach to be used to routinely evaluate the efficacy of novel antimalarial drugs in our laboratory. The expected sensitivity that can be achieved by an experienced microscopist for the examination of thick blood films is about 50 parasites/μl of blood, which is equivalent to 0.001% of infected RBCs (28). Milne et al. (1) found that most routine diagnostic laboratories generally achieved a lower sensitivity of detection (average, 0.01% RBC infected, 500 parasites/μl) in an examination of results from British laboratories submitted to the Malaria Reference Laboratory. The sensitivity in QL by FCM (0.00828%) in present study seems to be same as that by microscopy (0.01%). Therefore, this FCM approach was an acceptable practical limit of sensitivity. Since its improvement, the FCM with YOYO-1 has been in use for eight drugs incubated with W2 and D6 parasites in our laboratory. After having analyzed 384 samples from culture RBC infected with P. falciparum in experiments of antimalarial activity using this technique, the results demonstrated that this procedure is sensitive and reproducible. Therefore, the correlation found between 3H-hypoxanthine incorporation assay and FCM assays was strong (r2 = 0.927–0.968).

The mean parasitemia determined by the FCM approach was slightly higher (14%) than those determined microscopically. It is not surprising that the FCM method gave higher values (7–21%), as this is the case in almost all publications when compared with microscopic data (11, 12, 25, 29–33). We carefully checked many facts including using multiple microscopists, but the values were the same. However, we did find many tiny particles attached to normal RBC membranes in the infected blood slides. These particles look like vacuoles around the RBC. After adjusting the focus on the light microscopy, the Giemsa stained nucleus could be seen within the vacuoles. We thought that maybe schizonts from blood induced infection starting to penetrate or reinvade RBC. After staining with YOYO-1, the same samples were viewed by fluorescence microscopy. As on the Giemsa stained slides, the same particles with fluorescence were attached on the membranes of normal RBCs (Fig. 3). These parasites were not definitely inside the erythrocyte would never be included in parasitaemia counts by light microscopy in our laboratory, but could be measured by FCM. Of course, further investigation on the schizonts will be necessary for the final conclusion. However, this finding may be one possible explanation for the differences observed.

The FCM assay has been validated, and the results of validation show the method is reproducible and accurate within the acceptable limits to meet FDA guidelines for validation of bioanalytical methods (23). The sensitivity is also suggested that the high QL (0.008% parasitemia) produced from the FCM method was comparable to microscopy (0.01%). Therefore, based on data reported herein, a rapid, lower sampling error, and reliable identification of rodent erythrocyte parasites based on the principles of the FCM have replaced the traditional blood smear in our laboratory.

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