Patients who receive cancer immunotherapy treatments in clinical trials must have their peripheral blood mononuclear cell (PBMC) samples collected at baseline and at later time points. Immune monitoring facilities provide laboratory testing of these PBMC samples in order to gauge the response of the patient’s immune system to the test treatment.
A primary focus of immune monitoring facilities is therefore to develop cutting-edge technologies whilst also standardizing and validating immune assays with rigorous quality-control standards to ensure data reliability. After all, the weight of clinical trial results depends on the accuracy, precision, and reliability of data generated from these PBMC samples.
The fragile nature of biological samples makes standardization of laboratory procedures an especially important focus. Parameters that can affect data include the anticoagulant used for preserving PBMC samples, the time frame between sampling and processing, storage/shipping temperature en route to the central processing lab, the cryopreservation and thawing process, as well as the cell culture media.
A team of scientists from Torrey Pines Institute for Molecular Studies and from ImmunoCellular Therapeutics, Inc. tackled one assay important for cancer immunotherapy. The ELISPOT, or enzyme-linked-immunosorbant-spot assay, monitors PBMC-mediated immunity by detecting T cells in PBMC populations that are specifically targeted to fighting cancer in the patient.
HemaCare provided PBMC samples from 4 donors positive for HLA-A*0201. These donors had the serotype for human leukocyte antigen (HLA) class I molecules of the A*02 allele; this is analogous to having HLA-A*02 flagpoles for flying protein flags of intracellular proteins that fit this flagpole. There are 456+ different HLA-A*02 molecules, and HLA-A*0201 is another level of specificity.
The research team was interested in investigating whether overnight resting of PBMC could counter the effects of delayed processing and long-term cryopreservation. PBMC samples were thawed and assayed by ELISPOT immediately and were compared with resting the PBMC samples overnight in an incubator.
Since PBMC samples were obtained from “normal” donors, the T cells to be detected by ELISPOT were not cancer-fighting PBMC but rather T cells against what donors were likely to have been exposed to, such as common viruses. Virus antigens known to be presentable on HLA-A*0201 molecules were used in this assay, and T cells specific to these were enumerated using ELISPOT.
Using statistical analyses, controlled studies, and reporting their assays in compliance with the Minimal Information about T-cell Assays (MIATA) guidelines, this research team demonstrated that overnight resting of PBMC samples significantly improved the magnitude of response of T cells as well as the statistical significance.
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- Santos R, Buying A, Sabri N, Yu J, Gringeri A, Bender J, Janetzki S, Pinilla C, Judkowski VA. Improvement of IFN-gamma ELISPOT Performance Following Overnight Resting of Frozen PBMC Samples Confirmed Through Rigorous Statistical Analysis. Cells. 2014 Dec 24;4(1):1-18.