While numerous challenges exist in exploring the biology of disease identification and classification via multiplexing approaches, improvements in high-throughput technology bode well for new clinical offerings. This was one of the main take-home messages at two conferences that looked at multiplexing for research in areas such as cancer biomarkers, HLA typing, and genotyping for human papilloma virus (HPV).
Maurits de Koning, Ph.D., a scientist at DDL Diagnostic Laboratory discussed his company’s efforts at validating a human papilloma virus genotyping assay. The work, which he described at the BEBPA “Biological Assays” conference in Barcelona, is supported by GlaxoSmithKline Biologicals.
HPV is the causative agent of cervical cancer, for which two prophylactic vaccines are now available: Cervarix™ (containing HPV 16 and 18 VLPs) and Gardasil™, encompassing HPV 6, 11, 16, and 18 VLPs. The company’s assay is a PCR-based method, in which cervical biopsy material is obtained from patients, amplified with several primer sets covering 16 HPV types, and then measured using a Luminex-based procedure.
The DDL group constructed a prototype of the assay for proof of concept. Then, during the development phase, the team optimized and designed controls using this platform for evaluation of clinical samples. The next step was the validation of the system according to ICH guidelines.
The elements of validation, from the analytical side, include specificity, sensitivity, accuracy, precision, and robustness. Specificity takes into account the homology of various primers and probes for the many different HPV genomes, given that there may be numerous species that share some of these sequences. Dr. de Koning’s research group evaluated panels of samples and observed that the accuracy was quite high, 98%, when the samples were tested in a comparison with a reference assay.
The precision of the assay, i.e., the degree to which repeat tests of the same material were in agreement, was also quite high, 97%. Another property, that of robustness, or the stability of the assay in the face of minor variations in conditions, was found to be excellent at 95%, with both cervical swabs and formalin-fixed tissues.