Flow Cytometric Analysis of Specimens by a Central Reference Laboratory in a Multi-Center Study: Factors Affecting Data Quality.

Central reference flow cytometry laboratories are used in multicenter trials to eliminate inter laboratory variation in testing. Yet, factors that materially affect the quality of data such as graft processing, sample composition, packaging, and shipment have been little studied. The Baylor Center for Cell and Gene Therapy (BCGT) was the central reference laboratory for the NMDP IRB-2002-0068 multicenter study on the correlation of graft composition on clinical outcome. Samples of processed or non-processed grafts of 489 bone marrow (BM) and 696 peripheral blood progenitor cell (PBPC) grafts from July 2003-March 2004 were shipped to BCGT using 2 types of shipping containers with frozen gel packs to keep samples cool during transit. On arrival samples were assessed for temperature of gel packs, sample integrity (clumps, clots requiring filtration), viability by flow cytometry using 7AAD, and immunophenotype (CD34+, CD3+, and CD3+ T-cell subsets, CD25, CD69, HLA-DR). The FDA “standard” of 70% viability was used to define acceptable sample viability. Integrity and viability of BM and PBPC samples were not influenced by the method of cell processing: non-processed, plasma depletion, mononuclear cell concentration or T-cell depletion. BM samples were not affected by either temperature or time to evaluation. Viability of PBPC samples was adversely affected by both gel pack temperature and time from collection to evaluation. A 55% mean viability was observed with gel packs arriving thawed-warm vs 89% with frozen gel packs. Additionally, the samples that arrived >48hr after collection also had mean viability less than the 70% standard value. Viability of samples was inversely correlated with gel pack temperature on arrival and time from collection to evaluation. Therefore, the quality of FACS data and by implication, the graft utility was largely influenced by cell type, temperature in transit, and the time from collection to evaluation. Thus, while the use of a central reference laboratory can produce reliable data on cells following a variety of processing methods, certain factors must be optimized in order to collect data that accurately reflects the samples under study. Shipping standards to maintain graft viability and reliability of laboratory assessments of graft composition must be established and validated against the success of transplantation outcomes.