Flow cytometric analysis of blood cells stained with the cyanine dye DiOC₁[3]: Reticulocyte quantification

Abstract

The fluorescent dye 3,3'-dimethyloxacarbocyanine (DiOC1[3]) is taken up by all cells in mammalian blood which then fluoresce as follows: mature erythrocytes < immature erythrocytes = platelets < leukocytes. A continuous fluorescence distribution can be generated for the red blood cells by flow cytometry and deconvolved into two arbitrary populations, mature and immature erythrocytes (mRBC and imRBC). This analysis mimics the established method of counting imRBC stained with the supravital dyes, newWe have described the use of the cyanine dye 3,3'-dimethyloxacarbocyanine (DiOCJ31) for flow cytometric analysis of blood cells from mice infected with the rodent malaria parasites P vinckei (8) and P chabaudi (9). It was shown that using measurements of low angle forward light scatter (LALS) correlated with those of fluorescence intensity @I), the parasitized (pRBC), immature (imRBC), and mature (mRBC) components of the red blood cell (RBC) population could be quantified. However, because of the high degree of fluorescence overlap between the populations, the analysis was imprecise.In order to improve the analysis of parasitized blood, a protocol using a combination of DiOC1[3] and the DNA binding dye Hoechst 33342 (Ho) has been devised. In this manner, the DNA containing pRBC can be quantified precisely by measurement of Ho fluorescence. The uninfected RBC population, which is composed of RBC in various stages of maturation, can then be analyzed with respect to DiOC1[3] fluorescence after gating on Ho fluorescence. The quantification of the immature RBC population then becomes the same problem as encountered in any normal or anemic blood sample from an uninfected host. This paper describes the flow cytometric analysis of RBC maturity using DiOC1 [3]. Although we have primarily developed the assay in order to pursue goals in malaria research, the procedure will work well for studies in which quantification of an immature subpopulation of mouse RBC is necessary or desirable. Further,

Methodology and application of flow cytometry for investigation of human malaria parasites

“…In 1977 (Jackson et al, 1977) the first experiments were performed to investigate malaria parasites using flow cytometry by staining cells with ethidium bromide and acridine orange. Other early experiments were carried out on parasites which infect other mammals such as Plasmodium vinckei with (DiOC 1- 3 + Hoechst 33342) (Jacobberger et al, 1984) and Plasmodium berghei ( P. berghei , DRAQ5)(Billker et al, 2004). Neither of these stain/stain combinations have been used to investigate human malaria, thus far.…”

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

“…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 respon-sive 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).…”

Clinical Flow Cytometric Reticulocyte Analysis

“…However, pyronin Y, as well as ethidium bromide and propidium iodide, are impermeable to the cell membrane of 'viable' cells and require a fixation or permeabilization step in the stain ing procedure. Jacobberger et al [10] have successfully employed the cyanine dye dimethyloxycarbocyanine (DiOCi [3]) for FCM reticulocyte analysis, but this dye has potential shortcomings in its low quantum yield after binding to RNA and fluorescent sensitivity to membrane potential changes. All the fluorescent dyes used for FCM reticulocyte counting share the practical disadvantage of staining the sample lines by the instrument, thus requir ing a period of thorough detergent and/or bleach treat ment prior to the next flow cytometric application.…”

“…There are two basic approaches to the analysis of reticulocyte FCM histograms. Either mathe matical curve-fitting algorithms [10,20,21,26] or deter mination of a threshold level of fluorescent intensity for defining reticulocytes based upon unstained control samples [3,13,17] have been employed. Either approach is satisfactory when comparisons are made to manual techniques of reticulocyte counting, but additional stud ies are required to determine the most accurate and reproducible technique of data analysis.…”