Direct bisulphite conversion of cervical samples for DNA methylation analysis 119 5 screening settings associated with high-throughput. Of note, an automated solution for preparation of purified bisulphite-converted DNA from direct conversion of plasma and urine has been reported 17. Our findings provide the basis for development of streamlined and automated workflows for methylation analysis. The direct conversion protocol with 1/40th of the original sample volume as input used basically similar input amount in qMSP, i.e., 1/800th of original sample volume, as the protocol with prior DNA isolation, when considering a typical DNA concentration of about 20 ng/μl as seen in this study. Three of the six initially invalid samples with the direct protocol had ACTB Cq values close to the threshold for valid test results (i.e., <0.5 Cq difference). Three corresponded with a low DNA concentration (<5 ng/μl) in the reference protocol suggesting a low amount of cells in the original PreservCyt sample. Using four times more input of these samples in the direct cell conversion protocol generated a valid result. Overall, our data support congruence between the two different bisulphite protocols. We found a particularly strong correlation of ΔΔCq values of FAM19A4 and miR124-2 between both protocols in samples associated with CIN2+. The divergence between results was slightly larger at lower levels of methylation, corresponding to higher Cq values in PCR. A higher variation seen with higher Cq values is inherent to the PCR process, which becomes more stochastic in the presence of less template. Of interest, when we exploratively applied the assay’s cut-off to score a sample hypermethylation-positive or -negative 14, the direct cell conversion protocol also showed a high agreement (108/119, 90.8%, Cohen’s kappa 0.711) with the reference protocol. The percent agreement and kappa value are well in line with an earlier study reporting on the intra- and inter-laboratory agreement of the FAM19A4/miR124-2 methylation test 18. In that study, an inter-laboratory workflow (i.e., bisulphite conversion and assay combined) agreement of 90.0% and kappa score of 0.76 were found. The data presented herein support further studies to validate the clinical performance of the new protocol 19. Various bisulphite conversion kits, including those with direct cell input, have been evaluated before 20, but this is the first study comparing a direct cell protocol with a protocol including DNA isolation and normalisation, on cervical samples. Preliminary findings also support the direct conversion protocol for use with self-collected cervicovaginal samples. Self-sampling is seen increasingly as a credible approach to improve cervical screening coverage, and particularly gaining attention in view of the new challenges imposed by the COVID-19 pandemic 21.
RkJQdWJsaXNoZXIy MjY0ODMw