Impact of blood storage and sample handling on quality of high dimensional flow cytometric data in multicenter clinical research.

Obtaining reliable and reproducible high quality data in multicenter clinical research settings requires design of optimal standard operating procedures. While the need for standardization in sample processing and data analysis is well-recognized, the impact of sample handling in the pre-analytical phase remains underestimated. We evaluated the impact of sample storage time (≈transport time) and temperature, type of anticoagulant, and limited blood volume on reproducibility of flow cytometric studies. EDTA and Na-Heparin samples processed with the EuroFlow bulk lysis protocol, stained and stored at 4 °C showed fairly stable expression of cell surface markers and distribution of the major leukocyte populations for up to 72 h. Additional sample fixation (1% PFA, Fix & Perm) did not have any beneficial effects. Blood samples stored for <24 h at room temperature before processing and staining seemed suitable for reliable immunophenotyping, although losses in absolute cell numbers were observed. The major losses were observed in myeloid cells and monocytes, while lymphocytes seemed less affected. Expression of cell surface markers and population distribution were more stable in Na-Heparin blood than in EDTA blood. However, storage of Na-Heparin samples was associated with faster decrease in leukocyte counts over time. Whole blood fixation strategies (Cyto-Chex, TransFix) improved long-term population distribution, but were detrimental for expression of cellular markers. The main conclusions from this study on healthy donor blood samples were successfully confirmed in EDTA clinical (patient) blood samples with different time delays until processing. Finally, we recognized the need for adjustments in bulk lysis in case of insufficient blood volumes. Despite clear overall conclusions, individual markers and cell populations had different preferred conditions. Therefore, specific guidelines for sample handling should always be adjusted to the clinical application and the main target leukocyte population.

Prediction of the anti-RhD donor population size for managerial decision-making.

BACKGROUND: Rhesus D (RhD)-negative women pregnant with a RhD-positive child receive prophylactic injections to prevent haemolytic disease of the newborn. Because of the success of the prophylaxis, the number of naturally immunized women has decreased, thereby also decreasing the number of potential donors who provide the plasma from which the prophylaxis is made. As the current donor pool is ageing, the availability of the prophylaxis is threatened. OBJECTIVES: Objectives are to investigate whether the anti-D population and the changes therein can be described by a relatively simple model, to determine the impact of ageing of the anti-D donors on the decline of the population and how many new donors should be recruited to meet future supply demand. METHODS: Data on Dutch anti-D donors in 1994-2013 were used to simulate the donor population size and age composition for various donor recruitment scenarios. RESULTS: With a continuous influx of 27 new donors per year and a donor stopping rate of 10% per year, the population size will stabilize at 195 donors, with 2·3% of donors stopping annually due to reaching the donor age limit. A formula is derived to estimate which donor recruitment and retention efforts are required to maintain a prespecified donor pool. CONCLUSION: A relatively simple model can already describe and predict the size of the anti-D donor population and the impact of ageing accurately.