Validation of ctDNA Quality Control Materials Through a Precompetitive Collaboration of the Foundation for the National Institutes of Health

PURPOSE We report the results from a Foundation for the National Institutes of Health Biomarkers Consortium project to address the absence of well-validated quality control materials (QCMs) for circulating tumor DNA (ctDNA) testing. This absence is considered a cause of variance and inconsistencies in translating ctDNA results into clinical actions. METHODS In this phase I study, QCMs with 14 clinically relevant mutations representing single nucleotide variants, insertions or deletions (indels), translocations, and copy number variants were sourced from three commercial manufacturers with variant allele frequencies (VAFs) of 5%, 2.5%, 1%, 0.1%, and 0%. Four laboratories tested samples in quadruplicate using two allele-specific droplet digital polymerase chain reaction and three (amplicon and hybrid capture) next-generation sequencing (NGS) panels. RESULTS The two droplet digital polymerase chain reaction assays reported VAF values very close to the manufacturers’ claimed concentrations for all QCMs. NGS assays reported most single nucleotide variants and indels, but not translocations, close to the expected VAF values. Notably, two NGS assays reported lower VAF than expected for all translocations in all QCM mixtures, possibly related to technical challenges detecting these variants. The ability to call ERBB2 copy number amplifications varied across assays. All three QCMs provided valuable insight into assay precision. Each assay across all variant types demonstrated dropouts at 0.1%, suggesting that the QCM can serve for testing of an assay’s limit of detection with confidence claims for specific variants. CONCLUSION These results support the utility of the QCM in testing ctDNA assay analytical performance. However, unique designs and manufacturing methods for the QCM, and variations in a laboratory’s testing configuration, may require testing of multiple QCMs to find the best reagents for accurate result interpretation.

Evaluation of the performance of sysmex XN-3100 automated hematology analyzer regarding the sysmex XE-2100 and microscopic examination

ObjectivesWe performed a verification study of the Sysmex XN-3100 hematology analyzer in comparison with the XE-2100 according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) and the International Council for Standardization in Hematology (ICSH).Materials and methodsBlood samples and quality control materials were used for precision. For comparison, we used the current XE-2100 as the comparative method and analyzed 540 blood samples. The Passing-Bablok and Bland-Altman tests were performed according to the CLSI EP09-A3 and a carryover study was performed according to the CLSI H26-A2 guidelines. The flagging performance of the two analyzers was compared, using two experienced laboratory technicians as the reference method.ResultsThe Sysmex XN-3100 demonstrated high levels of precision for most parameters. For the comparison analysis, all parameters, except for MCHC, monocytes and basophils were within the systematic error limits of desirable biological variability criterion (SeDBV). The carryover was less than 0.4% for all parameters. The flagging performance of the XN-3100 was satisfactory and the overall efficiency was high.ConclusionsThe XN-3100 not only showed a strong correlation and agreement with the XE-2100 but also displayed a comparable analytical sensitivity, and increased specificity, which may result in an improved turnaround time and throughpu.

Performance evaluation of Siemens Atellica enhanced estradiol assay

BackgroundSerum estradiol (E2) levels may be used in the diagnostic and/or monitoring of a broad variety of clinical conditions. The aims of this study were to evaluate the Siemens enhanced estradiol assay (eE2) on Atellica IM 1600 (Siemens Healthineers) and to perform a sample comparison with the Siemens ADVIA Centaur XP (Siemens Healthineers).MethodsWithin-day and between-day coefficient of variation (CV) were determined using serum sample pools and quality control materials. Six serum samples with decreasing concentrations of E2 were used to assess the limit of quantification. Linearity was evaluated using two different serum samples. Accuracy was evaluated by measuring three certified reference materials. Passing–Bablok regression and Bland–Altman plot were used for comparing Atellica and Centaur XP in 119 serum samples ranging from 45 to 10,059 pmol/L.ResultsWithin-day and between-day imprecision was <6.6%. Accuracy was <6.0% for all values except for 114 pmol/L (22%). The study of limit of quantification resulted in an interday imprecision <20%. High correlation between measured and expected estradiol dilution results was observed (R = 0.99), with recoveries ranging from 77 to 93%. Comparison study showed good agreement and no significant bias.ConclusionsThe study shows that Atellica eE2 assay presents acceptable imprecision and accuracy and correlates well with Centaur XP.

An efficient method for immortalization of amniocytes with chromosome abnormalities for renewable application of rare disease sources

Objective: We describe a new method of immortalizing amniocytes with chromosome abnormalities to generate renewable resources for studying rare diseases. Methods: Three methods were investigated by randomly dividing 180 cases of adherent amniocytes into groups A, B, and C. Group A cells were digested with 0.25% trypsin for 10, 20, 30, 60, 90, and 120 mins. Group B and C cells were digested with 0.25% trypsin for 3–5 minutes. Group A and B cells were then transfected with PT67 cell-produced SV40LT. Group C digested cells underwent liposome-mediated SV40LT-transfection. Results: The percentage of clones immortalized by SV40LT transfection in groups A, B, and C were 18.3, 5.0, and 16.7%, respectively, after G418 screening. Group A produced a higher percentage of immortalized clones than did groups B and C, but only significantly differed from group B. The karyotypes and chromosome 13, 18, 21, X, and Y fluorescence signals in transfected cells of all groups were identical to those of the primary cells after passaging for 10–15 generations. Conclusions: The trypsin-mediated SV40LT transfection used in group A can be applied for routine establishment of amniocyte lines to obtain a renewable resource for the study of rare diseases and as quality control materials for prenatal diagnosis.