PB0900 - Developing standards to support platelets and platelet components

Background: Platelet biology is notoriously difficult to standardise due to high susceptibility to misinterpretation and differences in platelet responses from person to person. Platelets are analysed using a plethora of methods; however, the high sensitivity, shape variation and small size of platelets can lead to subjective data between laboratories and users. There are currently no reference materials that can aid in the training/education of operators, validation of tests, quality control and characterisation of platelets. Therefore, there is an urgent need for physical platelet standards to ensure the quality and consistency of experiments.

Aims: Develop a lyophilised viable material for assessment by flow cytometry. The main parameters to be evaluated by flow cytometry are size and granularity, viability, expression of CD41a+/CD42b+, and contamination with nucleated cells.

Methods: Washed platelets were pre-stained with a viability dye and fixed with formaldehyde. A fixed unstained platelet control was prepared in parallel. Fixed platelets were lyophilised with a trehalose buffer containing 2% BSA. Platelets were analysed by flow cytometry pre- and post-lyophilisation to assess platelet viability and expression of CD41a+/CD42b+.

Results: Lyophilisation was successful, resulting in robust pellet formation. There was no loss in percentage of viable cells following lyophilisation (98.6%, Figure 2B) in comparison to pre-lyophilisation (98.2%, Figure 1B). Additionally, 9 months post-lyophilisation with storage at -20⁰C, minimal loss of viable cells (96%) was observed. Post-lyophilisation ~88% of viable cells stained positive for CD41a+/CD42b expression.

Conclusion(s): An additional trial fill will be conducted to include contaminating nucleated cells, followed by a final manufacturing fill. The manufacturing fill will be used in a collaborative study where participants will be asked to estimate percentages of CD41a+/CD42b+, viable platelets and contaminating cells. A globally recognised reference material will aid researchers and the clinical community to characterise platelet populations and provide meaningful comparable results between laboratories and equipment.