Ring Trials Lymphocyte Immunophenotyping

External Quality Control Survey of Immunophenotyping of the
Central Reference Institution in Germany (1993-1995)

Issues of Specimen and Interlaboratory Variability

< Consensus

Organizers:

Working group of quality assessment in flow cytometry of the German Society of Clinical Chemistry

Members:

W.Prohaska, R.Kruse, W.-J.Geilenkeuser, T.Nebe, A.Radbruch, H.Diem, A.von Rücker

Address:

Deutsche Gesellschaft für Klinische Chemie e.V. Zentrale Referenzinstitution, P.O.B 150 139, D-53040 Bonn, Germany, Fax: +49/228/211529

Aim of the Survey:

Assessment and improvement of the interlaboratory standardization for the determination of lymphocyte subpopulations as defined by the markers CD3+, CD4+/CD3+, CD8+/CD3+, CD19+, CD16,56+/CD3- and CD3+/HLA-DR+

Specimens:

In the first two surveys washed and purified buffy coat, afterwards CDP (citrate/phosphate/dextrose) whole blood was used. The buffy coat was magnetically immunodepleted (Milteny Biotec immuno magnetic beads) to obtain pathologically low subpopulations. All specimens were shipped on the day of blood drawing and cell preparation.

Summary of Results:

On average, 109 laboratories participated in the surveys. After changing from immunodepleted buffy coat to CDP whole blood. there was a distinct decrease of the coefficient of variation (CV) in the determination of percentages of lymphocyte subpopulations: CV's improved from 4.5-12% to 3-7.5% (CD3+ cells) and CV's of CD4+/CD3+ cells moved from 5.8-14.4% to 3.2-6.6%. The CV's are lower than those reported by the College of American Pathologists Flow Cytometry Survey (1) and by surveys from other countries.
Clinically, the absolute cell count is more important than the percentages which show relatively small CV's. For the absolute cell counts we calculated CV's of 14-22.4% and 14.5-21% for CD3+ cells and CD4+/CD3+ cells, respectively. These values are considerably worse than those for the corresponding percentage counts.
As the leukocyte and lymphocyte count is the basis for calculating of the absolute cell counts of subpopulations, the calculation of these entities is important and can raise CV's two- to threefold. Subpopulations with low percentages and/or inhomogenic expression of marker epitopes (for instance HLA-DR) also yielded high CV's in relative counting.

Conclusion:

Fresh CPD whole blood is a natural, sufficiently stable and easy to handle specimen material for quality surveys in immunophenotyping. Aims to improve the results of quantitative immunophenotyping should address white cell and lymphocyte counting. Further standardisation of flow cytometry methods should improve quantitative immunophenotyping.

References:

1. Homburger, H.A., Rosenstock, W., Paxton, H., Paton, M.L., Landay, A.L.: Assessment of Interlaboratory ariability of Immunophenotyping; Ann.NY Acad. Sci.677:43-49(1993)