J Korean Med Sci.  2012 Oct;27(10):1137-1142. 10.3346/jkms.2012.27.10.1137.

Quantitative Determination of Plasmodium Parasitemia by Flow Cytometry and Microscopy

• Jun G
• Lee JS
• Jung YJ
• Park JW

• KMID: 1778817
• DOI: http://doi.org/10.3346/jkms.2012.27.10.1137

Abstract

The traditional light microscopy has limitations for precise growth assays of malaria parasites in culture or for assessment of new compounds for antimalarial activity; the speed and high reproducibility of flow cytometry can overcome these limitations. A flow cytometric method using PicoGreen, a DNA-binding fluorochrome, was developed with optimal precision suitable for performing growth assays of low-parasitemia field isolates. In addition, intra- and inter-person reproducibility of the flow cytometric and the microscopic method were compared in order to quantitatively demonstrate the improved precision. RNase treatment contributed to the precision of the flow cytometric measurements by enhancing the signal-to-noise ratios. Coefficients of variation of the method were smaller than 10% for 0.1% or higher parasitemia samples. The intra- and inter-person coefficients of variation of the flow cytometric method were three to six times smaller than those of the microscopic method. The flow cytometric method developed in this study yielded substantially more precise results than the microscopic method, allowing determination of parasitemia levels of 0.1% or higher, with coefficients of variation smaller than 10%. Thus, the PicoGreen method could be a reliable high sensitivity assay for analysis of low parasitemia samples and might be applied to a high throughput system testing antimalarial drug activity.

Keyword

Malaria; Flow Cytometry; Microscopy; Parasitemia; PicoGreen

MeSH Terms

*Flow Cytometry
Fluorescent Dyes/chemistry
Humans
*Microscopy
Organic Chemicals/chemistry
Parasitemia/*diagnosis
Plasmodium falciparum/*isolation & purification
Reproducibility of Results
Ribonucleases/metabolism
Signal-To-Noise Ratio
Fluorescent Dyes
Organic Chemicals
Ribonucleases

Figure

• 

  Fig. 1 Effect of RNase treatment. A 4.44% parasitemia sample (measured by microscopy) was diluted to prepare 1% and 0.5% parasitemia samples. Each of these three samples and an uninfected control sample were divided into two portions, and the two sets were incubated in a 37℃ water bath for 30 min: the first set without RNase and the second with RNase.

• 

  Fig. 2 Effect of PicoGreen concentration (1 × is the concentration suggested by the manufacturer).

• 

  Fig. 3 Precision of parasitemia determination by flow cytometry. An in vitro culture of P. falciparum was diluted to prepare samples of the desired parasitemia levels. (A) Each of the low range samples was measured 10 times. After deleting the largest and smallest values, the average and standard deviation were calculated for the remaining eight samples. (B) The data for each of the high range samples were obtained from five repeated measurements without deletion. (C) The coefficient of variation for each parasitemia was calculated by dividing the standard deviation by the true parasitemia, assuming perfectly accurate serial dilution. (D) 1.5% agarose gel showing PCR product obtained by nested multiplex PCR for 205 bp segment. DNA was extracted from the P. falciparum infected red blood cells. The % values of parasitemia, denoted in the table, were calculated by microscopic observation, and SD in the table means standard deviation.

• 

  Fig. 4 Reproducibility of parasitemia determination by flow cytometry and microscopy. Each of three examiners measured each sample five times. Each of the intra-person CVs is the average of three intra-person CVs obtained by the three examiners. The above experiment was repeated twice, and the graphs represent the average of the two experiments.

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