(M1030-01-06) Comparison of Novel Whole Blood Preservation Methods as Alternatives to Cryopreserved PBMC for Immunophenotyping in Multicenter Clinical Trials

Purpose: A considerable challenge encountered when utilizing flow cytometric methods in multicenter clinical trials is specimen stability. Cryopreserved PBMC are a commonly used matrix, however, many clinical sites lack the expertise and/or infrastructure required for PBMC isolation, cryopreservation and shipment. Therefore, a feasibility study was conducted for the evaluation of novel whole blood preservation products as alternatives to PBMC. Three different whole blood preservation products were evaluated: Cryostor CS10 (CS), Cytodelics (CT), Smart Tube Proteomic Stabilization Buffer (ST).

Methods: Peripheral whole blood was collected from three healthy donors and processed at 3- and 30-hours post-collection according to the manufacturer’s recommendations. PBMC and CS samples were stored in LN2, CT tubes were stored at -20°C (CT-20) and -80°C (CT-80), and ST at -80°C. After a minimum of one week of storage, samples collected in each Test Matrix were evaluated in a 26-color spectral flow cytometric method previously optimized for PBMC. A total of 22 T- cell subsets were assessed. The variance (%CV) of the results generated with each Test Matrix was compared to the results from the PBMC sample processed at 30 hours post-collection.  In addition, a visual inspection of the gated data was conducted, and the resolution of each subset was scored as comparable, improved, or lower.

Results: The PBMC samples processed at 30 hours post-collection served as the Comparator Matrix for the three Test Matrices processed at 3 hours post-collection. CD3, CD4, and CD8 T cells, as well as Tregs were sufficiently resolved in all matrices. For CD4 and CD8 naïve and memory T cells, CS had similar resolution to PBMC isolation, but the resolutions of CT and ST were reduced. For CD4+ stem cell memory (TSCM), CS, CT, and ST resolution was comparable to PBMC across all donors.  Results were similar for CD8⁺ TSCM with the exception of one donor. The results for CD4⁺ exhaustion markers (PD-1, TIGIT, and KLRG1), were comparable in CS and CT-20 samples. For the CD8+ exhaustion markers, CT-20 and CT-80 provided comparable resolution to the PBMC group.

Conclusion: In terms of comparability of the test matrix to the comparator matrix, CS would be the preferred matrix. However, in terms of ease of collection, CT and ST are superior. Although, CS requires fewer manipulations than required for PBMC, the processing steps are still more complex compared with CT and ST. Between CT and ST, CT provides a logistical benefit in that it can be stored at -20°C, which has an advantage for investigative sites that do not have -80°C freezers. A key difference between CS and CT or ST, is that CT and ST contain fixatives which results in preservation of all the white blood cells including the granulocytes. As a result, the following modifications are warranted if these matrices would be used for the optimized 26-color spectral panel: 1) Antibody re-titration to improve resolution due to the possibility of altered epitopes or insufficient antibody concentration due to the presence of granulocytes. 2) Addition of a granulocyte marker to improve the specificity of the lymphocyte gate. The results for one donor were often different than the other two donors in terms of phenotype resolution, which highlights the importance of evaluating multiple donors for this type of feasibility study. Taken together, these results demonstrate that the commercially available whole blood preservation products provide viable alternatives to PBMC for immunophenotyping specimen preservation.