Development and Validation of Flow Cytometry-Based Method to Quantify CD34/CD45+ Cells as a Release Criterion of...

We completed an international, multicenter, randomized, open-label Phase I/IIb trial assessing the safety and preliminary efficacy of transendocardial injection of autologous expanded CD34+ cells (ProtheraCytes) in patients after acute myocardial infarction (AMI; NCT02669810). A multicenter, randomized, controlled Phase III study is now being initiated. To support release of ProtheraCytes clinical batches, we validated two flow cytometry methods for accurate quantification of CD34/CD45⁺ cells (stem cell enumeration-SCE method) and characterization of accessory leukocyte subsets (monocytes, granulocytes, and B, T, NK lymphocytes-accessory populations immunophenotyping method). All the recovery rates for both methods, with calculations derived from QC materials specifications, met the acceptance criteria, based on precision assessment according to ICH Q2(R2), European Pharmacopeia (Ph. Eur. 2.7.23 and Ph. Eur. 2.7.24), and ISHAGE guidelines. In addition, the precision results (repeatability and intermediate precision) were lower than 28.3% (≤ 30% for accessory populations immunophenotyping method) and lower than 13.5% (≤ 25% for SCE method). Finally, a perfect linearity was demonstrated for SCE method across 1.7-2622.5 cells/μL with coefficient of determination (R²) of linear regression above 0.99 and matrix effects nearly negligible for both methods. The specificity, precision and accuracy of these methods were proven in the analysis of six determinations per operator in three different series. Altogether, these results indicate a good accuracy and precision of the proposed methods determining absolute counts, viability, and proportions of live CD34/CD45+ cells and accessory populations. This validated flow cytometry assay will be implemented for release testing in the forthcoming Phase III clinical trial of ProtheraCytes in post-AMI patients.