In the realm for standardization in immunophenotyping


  • Attila Tárnok

    Corresponding author
    1. Department of Pediatric Cardiology, Cardiac Centre, University of Leipzig, Leipzig, Germany
    2. Translational Centre for Regenerative Medicine (TRM), Universität Leipzig, Germany
    • Dept. Pediatric Cardiology, Cardiac Centre, University Leipzig, Strümpellstr. 39, 04289 Leipzig, Germany
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  • The work presented in this editorial was made possible by funding from the German Federal Ministry of Education and Research (BMBF, PtJ-Bio, 0315883).

Standardization and quality control is the keystone of reliable and trustworthy diagnosis. This is particularly true for cellular diagnostics where the preanalytics, instrumentation, and reagents are very diverse and prone to innumerable variations affecting the results (1). Furthermore, as critical are the objects of interest—the cells from biological sources—that are fine-tuned in their composition and behavior in the organism and may be sensitive to various modifications of environmental changes once outside the body. So defining the best and most reproducible assays for their detailed analysis is of utmost relevance for trustworthy diagnosis.

Roussel and colleagues from Rennes, France, Bangor, Maine and Seattle, Washington USA (this issue, page 973) scrutinized on behalf of the International Council for Standardization in Hematology (ICSH) three different flow cytometric methods in order to find the reference method for leukocyte differential counts in human blood. In clinical laboratories, nowadays hematology analyzers are routinely used to obtain whole blood differential leukocyte counts. Unfortunately, each of the systems has its own bias as has recently been shown (1) when it comes to rare or unusual cells. In this study, the ICSH study group compared in a multicenter endeavor (three countries), different instruments including automated analyzers, manual counting and in addition two different standard flow cytometers were used. The immunophenotype panels and whole blood, no wash staining and analysis protocols were adapted to each of the flow cytometers from different providers. There are several conclusions from this thorough study. First of all, the bias of hematological automates is that up to 50% of the analysis has to be confirmed by tedious and time consuming manual counting on a microscope. This is not necessary when using flow cytometry analysis. The use of staining of the cell nucleus by DNA specific dyes is useful in order to detect all nucleated cells including premature red blood cells but is difficult to standardize. Finally, the protocols need to be extended to quantify the clinically relevant immature leukocytes and blasts by extending the antibody panel. Also, the characterization of other specific clinically relevant subsets like resident and activated monocytes may be of relevance (2). So in summary, new higher level consensus protocols need evaluation in a clinical setting to approach clinical cytomics in diagnosis.

In this respect, various research groups took the effort to increase the depth of analysis by combining a multitude of differentially colorized monoclonal antibodies in a painful and tedious development of optimized multicolor immunofluorescence panels (OMIPs). As examples, recently a 10-color OMIP for the differentiation of hematopoietic cell samples (3) or for memory B-cells (4) were introduced. Now, Mahnke and colleagues from Bethesda, Maryland, USA (this issue, page 935) present a new 13-color 12-antibody panel in order to detail the differentiation of human T-cells. This panel has the advantage of combining phenotypic markers for the unequivocal identification of specific cell types with differentiation and activation markers for their further characterization. Many of the published OMIPs have the potential of giving invaluable input for future standardized clinical panels and many more are to come.

Next to the identification of the right instrument and the staining panel, the correct way to isolate and store cells until the analysis is essential. Scheible and colleagues from Rochester, New York, USA (this issue, page 937) used umbilical cord blood leukocytes and tested framework conditions for their appropriate phenotypic and functional analysis. Umbilical cord blood cells are of relevance in identifying and quantifying stem and progenitor cells (5) and also congenital immune defects. Unfortunately, the time and circumstances of sampling, storage, and transport until their preparation and analysis are often difficult to follow and record in the day-to-day clinical routine. Scheible and colleagues take a closer look at different variables that may influence results obtained from isolated umbilical cord mononuclear cells including influence of cryopreservation and time of preparation after isolation. By analyzing the effects on cell viability, percentage of cell subsets and expression of a plethora of activation markers, they performed T-lymphocyte activation assays and looked at the production of various intracellular cytokines. The authors report that storage up to 24 hours after harvesting has very minor effects. However, they add that preterm neonates or those with infectious problems were not included and may lead to differing results requiring additional studies and more neonates to be enrolled.

Summarizing, several important consensus efforts and standardization studies are presently already at hand for high quality clinical immune diagnostics. However, more such important studies are needed to reach a fully standardized sampling storage, additional preanalytical procedures as well as the final cellular analysis.