Flow cytometry is well established in research as well as in routine diagnostics. Starting with cellular counting and analysis, additional applications of particle-based counting rapidly increased the number of cytometric methods. Today, detection of cells, their functions, and a huge variety of bead-based assays allow sophisticated investigation of highly specific questions raised in clinical diagnostics and basic research.

In parallel, improvement of the technical equipment, software solutions, cell biological and molecular knowledge and more and more specific questions accelerated the necessity to make these methods reproducible if not standardized. Standardization is the key to make clinical diagnostics comparable between different laboratories [1] and is central for multicentric studies [2, 3]. Consequently, several national institutions such as the National Institute of Standards and Technology, NIST, or the German Physikalisch Technische Bundesanstalt, PTB, have begun to address the issue of harmonizing quantitative single cell analysis [4]. With Optimized Multiparametric Immunophenotype Panels, OMIPs, [5] and MIFlowCyt[6], general rules for flow cytometric experiments have been developed and their standardized form of publication established. For clinical cytometry, Good Laboratory Practices (GLP), guidelines and accreditation rules give clear guidance regarding how to manage flow cytometric approaches [7]. Here, three publications expand our knowledge in highly reproducible flow cytometric experiments and the technical knowledge concerning recent routine instrumentation.

Julfa Begum and colleagues from London and Swansea, both from the United Kingdom, and from Cambridge, MA, USA, describe a method for evaluating the use of fluorescent dyes that are required to track proliferation in cell lines by dye dilution [this issue, page 1085]. This article substantially contributes to understanding performance and limitations of this well established method [8] and add valuable information to an earlier critical comment by Roederer [9]. The authors labeled two different cell lines (Jurkat and A549) with several fluorescent dyes (including CFSE, Cell Tracker Violet, Dye Cycle Ruby) and compared them investigating and quantitating cell proliferation. By using uniform spectrally matched fluorescent particles, they could differentiate between intrinsic and extrinsic errors. Results allow selective and critical decision making when choosing cells and dyes for such investigations. Furthermore, general limitations of such approaches are described in detail.

Françoise Solly and co-authors from St Etienne, Lyon, Dijon, Limoges, Mulhouse, Bordeaux and Nantes, France, publish data from the GEIL (Groupe d'Etude Immunologique des Leucemies within the European Leukemia Net study) and thereby present a remarkable comparison between Canto II and Navios clinical flow cytometers [this issue, page 1066]. In multi-site studies, classical on-site setting of flow cytometers causes relevant differences in analysis (see also comments in 1–3). Reproducible analysis with multiparameter flow cytometers depends on electronic algorithms making adequate instrument settings manageable. The manuscript gives great advice how to compare the two different platforms to get identical information – and the results are convincing.

Even seven bench-top flow cytometers have been compared and evaluated by Arpad Czeh and coauthors from Pecs and Szeged, Hungary, and San Juan, Puerto Rico, with a modified mycotoxin kit [this issue, page 1073]. (Mycotoxins are secondary metabolites produced by microfungi that are capable of causing disease and death in humans. They commonly are taken up by infected crops or food.) Application of bead-based assays to detect soluble analytes is a well-established alternative to ELISA testing. Multiplex capabilities, sensitivity, expanded linear range and easy handling are in opposition to calibration challenges and platform dependency. Nevertheless, except for one out of the six analytes, results are comparable for all platforms.

These editorial-highlighted contributions underline the high level of precision in recent flow cytometers when following correct quality assurance and standardization. We are curious about the next applications that can be measured by the flow cytometer in a really reproducible way.

Literature Cited

  1. Top of page
  2. Literature Cited
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