Analytical validation of digital cytometry (iSort) for leukocyte enumeration using stored blood.

Abstract

3542 Background: Blood leukocyte enumeration is a cornerstone for clinical diagnosis and immune monitoring of diverse cancers and immunotherapies. Existing methods rely on intact/living cells and can thus be limiting due to handling time constraints and need for predefined antibody panels. While indirect cytometry methods including digital cytometry can overcome this limitation on archival specimens, their clinical performance has not been extensively characterized. We developed iSort, a novel transcriptome deconvolution method based on CIBERSORTx. Here, we comprehensively evaluated iSort and validated it against established diagnostic standards. Methods: We recruited 36 healthy adult blood donors and characterized their blood leukocyte profiles. We used several established clinical cytometry methods requiring intact cells in a CLIA laboratory including Complete Blood Count [CBC] and 6-color TBNK [TBNK]. We also immunophenotyped leukocytes by a research flow cytometry panel (FACS) and by mass cytometry (CyTOF). We then used these techniques as standards for validating leukocyte populations enumerated by iSort. iSort was performed on whole blood through deconvolution of 22 subsets from RNA-Seq. We assessed iSort’s analytical detection performance by spiking purified lymphocyte subsets into lymphodepleted human blood and by simulating blood mixtures using defined leukocyte mixtures within latin square designs. We assessed iSort concordance with an FDA approved IVD assay (TBNK) comparing distinct RNA-Seq library preparation chemistries. Results: iSort was highly correlated with TBNK/CBC across CD4 T, CD8 T, B cells, NK cells, monocytes, and neutrophils (r≥0.95). When comparing correlations to TBNK/CBC, we found no significant differences between iSort, CyTOF, and FACS, nor between RNA-Seq library chemistries. iSort demonstrated high linearity at low abundance levels (0.1 - 1%, r = 0.99, B-cells spiked into lymphodepleted blood samples after Rituximab) and at higher abundance levels (0.5 - 90%, r > 0.99) across lymphoid and myeloid subsets. iSort also showed high reproducibility among triplicate blood tubes for each population (median CV = 11%). Conclusions: iSort digital cytometry achieves highly accurate and robust leukocyte enumeration for diverse hematopoietic subsets. Given its favorable performance against existing clinical standards that require intact/living cells, iSort is a promising approach for the development of immunotherapy biomarkers.