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Biotherapies, Cellular Therapies, and Immunotherapies

(P-BT-23) Qualification of Flow Cytometry Assays against CD45, CD3, CD4, CD8, CD14, CD19 and CD56 for Leukopak Donor Screening

Sunday, October 20, 2024
12:00 PM - 1:00 PM  
Background/Case Studies: Leukopaks are blood products produced through apheresis and which contain enriched fractions of mononuclear leukocytes. In addition to serving a significant role in immunological research, leukopaks are an indispensable source material for the production of advanced therapy products. Downstream applications often require a level of consistency between leukopaks or a particular immunological profile. To meet these requirements in light of inherent donor variability, it is essential to characterize leukopak composition through immunophenotyping. The aim of this project was to qualify flow cytometry panels to accomplish leukopak characterization through immunophenotyping.


Study

Design/Methods:

Flow cytometry assay panels were designed using fluorescent-tagged antibodies in separate panels containing seven immunophenotyping markers: CD45, CD3, CD3/CD4, CD3/CD8, CD14, CD19 and CD3/CD56. Commercially available leukopak samples were used for assay qualification. Inter-assay precision for each panel was assessed through six measurements across six days and two operators. Intra-assay precision was performed in six replicates during a single run. Accuracy was assessed by dilutions of leukopak sample in a nonreactive background at predetermined ratios prior to staining. Measurements were taken on two separate days by two operators. Each instance consisted of triplicate measurements at two dilution ratios. Sensitivity was determined using Fc-fusion recombinant proteins for the relevant CD marker, immobilized on Protein G polystyrene beads. Eight two-fold dilutions of CD target-coated beads were generated within a nonreactive, human IgG-coated Protein G background prior to staining. Triplicate measurements were taken for each dilution point between neat and 0.8% target. Dilution points producing signal values with a CV of ≤ 20% between triplicates were plotted for linearity. Sensitivity was defined as the highest dilution which met a CV of ≤ 20%.

Results/Findings:

Inter-assay and intra-assay precision of the assays were acceptable, meeting a CV of ≤ 20%. Accuracy measurements for each marker met a percent recovery of 70%-130% of the predetermined value for each of two dilutions. Linearity studies  produced an R2 value of ≥ 0.90 for each panel and established sensitivities between 0.8% and 6.4%.

Conclusions:

The data presented shows the successful qualification of flow cytometry panels for leukopak samples against CD45, CD3, CD3/CD4, CD3/CD8, CD14, CD19 and CD3/CD56. Each assay displays excellent precision, accuracy, linearity and sensitivity This characterization of leukopaks could provide invaluable insight, especially in the selection of the best starting materials for downstream manufacturing into advanced therapy products.