Validating quantitative PCR assays for cfDNA detection without DNA extraction in exercising SLE patients.

Circulating cell-free DNA (cfDNA) has been investigated as a screening tool for many diseases. To avoid expensive and time-consuming DNA isolation, direct quantification PCR assays can be established. However, rigorous validation is required to provide reliable data in the clinical and non-clinical context. Considering the International Organization for Standardization, as well as bioanalytical method validation guidelines, we provide a comprehensive procedure to validate assays for cfDNA quantification from blood plasma without DNA isolation. A 90 and 222 bp assay was validated to study the kinetics of cfDNA after exercise in patients with systemic lupus erythematosus (SLE). The assays showed ultra-low limit of quantification (LOQ) with 0.47 and 0.69 ng/ml, repeatability ≤ 11.6% (95% CI 8.1-20.3), and intermediate precision ≤ 12.1% (95% CI 9.2-17.7). Incurred sample reanalysis confirmed the precision of the procedure. The additional consideration of pre-analytical factors shows that centrifugation speed and temperature do not change cfDNA concentrations. In SLE patients cfDNA increases ~ twofold after a walking exercise, normalizing after 60 min of rest. The established assays allow reliable and cost-efficient quantification of cfDNA in minute amounts of plasma in the clinical setting. Additionally, the assay can be used as a tool to determine the impact of pre-analytical factors and validate cfDNA quantity and quality of isolated samples.

Multiparameter flow cytometry in the differential diagnosis of aberrant T-cell clones of unclear significance.

Immunophenotypic distinction between neoplastic and reactive T-cell clones can be challenging, as peripheral T-cell lymphomas (PTCLs) lack an immunophenotypic marker of clonality. Systematic screening of 10,510 cases analyzed by immunophenotyping at our institution between 2006 and 2012 resulted in 49 cases with aberrant T-cell populations of unclear significance. Review of patient charts allowed us to assign these cases to three categories. In 21 cases, PTCL could later be confirmed by complementary diagnostics (PTCL group). In 20 cases, follow-up confirmed the reactive nature of the aberrant T-cells (non-PTCL group). Eight cases remained of unclear significance. Neither the population size nor the number of aberrant markers differed significantly between the PTCL and non-PTCL groups. Only loss of CD7 was found significantly more often in patients with PTCL than in patients with non-PTCL (p = 0.037). Our data show that aberrant T-cell populations need to be interpreted in the clinicopathological context, as reactive and neoplastic phenotypes largely overlap.