Many hematological laboratories use flow cytometry to detect and characterize malignant cells in lymphoma or leukemia because it can help identify cell classes and maturation stages. Unfortunately, application of the technique for diagnosis, which would require classification of normal cells in blood and bone marrow, has been limited by a variety of factors, including reagent cost, sample preparation, acquisition time and postacquisition analysis.Traditionally, labs have determined leukocyte differential counts – the proportion of various types of leukocytes (or other cells), expressed in percentages – through microscopical classification of a stained smear of a sample, with a skilled technician identifying at least 14 cell classes in blood or bone marrow. Besides being labor-intensive, it also is a somewhat subjective technique.Using a flow cytometer to perform these counts automatically could advance the labs’ work considerably. “Cellular classification in blood and bone marrow is fundamental in diagnostic hematology,” said Sven Björnsson, a researcher at University Hospital MAS in Malmö, Sweden, “and there is an urgent need to replace the subjective morphological classification system presently in use.”In a Cytometry Part B paper published earlier this year, Björnsson and colleagues at the hospital reported on a study in which they took advantage of the seven parameters available in their flow cytometer to come up with a reliable differential count using only one test tube. The one-tube immunophenotyping panel for leukocyte and platelet classification that they developed could contribute significantly to the diagnosis of disease and the monitoring of treatment. The researchers demonstrated the technique using an FC500 flow cytometer made by Beckman Coulter Inc. of Fullerton, Calif. Excitation was provided by a single 488-nm argon laser or by that laser in conjunction with a collinear 635-nm red diode laser. Emission was detected in five channels. A lyse-no-wash method ensured minimal loss of the fragile cells, and they employed live gating on DNA stain to obtain nucleated cells only.For the latter, the researchers chose the DNA stain DRAQ5 from Biostatus Ltd. of Leicestershire, UK. Published fluorescence images of DRAQ 5 indicated that its DNA selectivity, membrane permeability properties and Stokes shift made it a unique DNA stain, Björnsson said. Certain cell-permeant Syto green stains might have served as alternatives for the study, but they were reported to bind differently to different cell types, “which is a major drawback,” Björnsson said. Researchers have reported a new method for cell cycle analysis in antibody-gated populations using the DNA stain DRAQ5. The figure shown demonstrates how the stain can be used for gating on live and apoptic cells in bone marrow. If investigators gate on high mean fluorescence intensity (MFI) DRAQ (left), they will see all live cells classified into different populations that are differently colored. If they gate on low MFI DRAQ (right), they will see the apoptotic cells only and will note that these are mainly erythroblasts (light brown) and mature granulocytes (blue).It’s beneficialCompany spokesman Roy Edward noted some benefits of using the stain. “Clearly, using a far-red-emitting DNA dye has some advantage in terms of experimental design. Most of the common fluors you would choose first for your antibodies are in the visible range; PE Texas Red, for instance. And none of these exhibit any problematic spectral overlap with DRAQ5.”Because it is a stoichiometric dye, he added, it provides a molecule-by-molecule measure of DNA, which means that the signal from DRAQ5 is equivalent to the DNA in the sample. “Binding to DNA causes no difference other than a slight redshift. So the dye gives you a linear response, which is much easier to quantify,” Edward said. The disadvantage, he noted, is that DRAQ5 is a relatively dull dye. As a result, Björnsson said, high concentrations are needed for stoichiometric staining, causing some quenching of other fluorescence emissions. The dye also exhibits some cell-dependent binding, he added, although this can be overcome by selecting staining conditions, which are discussed in the Cytometry Part B paper and which will be addressed further in an upcoming publication.The experiments suggested that classification of total nucleated cells in blood or bone marrow can be achieved reliably using the seven parameters available in a single tube in the researchers’ cytometer. In addition, he said, the study yielded two novel findings: First, the method used for red cell lysis affects the degree of staining. Also, DRAQ5 works well in the FL-5 channel on the Beckman Coulter flow cytometer, leaving four channels available for antibody staining. In contrast, he said, the FACSCalibur from BD Biosciences of San Diego is not well suited for DRAQ5. “The [company’s] FACSCanto 5+2 should be very similar to the FC500 if you skip the red laser channels. I will try to work out a method for the FACSCanto 4+2 in order to be eligible for the ISLH trial of blood cell classification by flow.”Björnsson plans to use the DNA stain in future studies. For example, he hopes to contribute to the diagnosis of myelodysplastic syndrome by exploiting the ploidy of megakaryocytes. “Unfortunately, I’m not yet able to identify megakaryocytes in our sample. I’ve just begun to stain epithelial tumor cells with DRAQ5 for ploidy investigation. I need better methods to disperse these cells, though.”Contact: Sven Björnsson, University Hospital MAS; e-mail: sven.bjornsson@skane.se; Roy Edward, Biostatus Ltd.; e-mail: roy@biostatus.com.