Table 2 Implementation of NGS test in the diagnostics.
From: ERIC recommendations for TP53 mutation analysis in chronic lymphocytic leukemia—2024 update
Commercial IVD/IVDR/FDA assaya | Laboratory-developed test (in-house/custom) | |||||
|---|---|---|---|---|---|---|
Process | Tools, material | Steps to be fulfilled | Steps to be fulfilled | Specifications and TP53-CLL-specific notes | ||
Test familiarization | Explore the clinical and analytical needs | Define turnaround time, costs, number of samples, instrumentation, personnel, etc. | Standalone or panel assay | |||
Test development | Study available technologies | Choose a commercial method | Test design | Regions to be sequenced Library preparation method Sequencing technology Bioinformatics | ||
Determine required test performance metrics and sequencing settings | Aim to detect low-VAF variants Consider: DNA input, coverage (each base of TP53 gene has to meet the minimum read depth) | |||||
Prepare SOP | ||||||
Optimization | Perform pilot run(s) Reference material Non-DNA controls | General assessment of method applicability for the purpose | Identify and solve errors and weaknesses | |||
Check if the required parameters are met | ||||||
Set the critical values (pass/fail criteria) | Output library quality and quantity Sequencing performance (level of background noise/overall error rate, coverage non-uniformity, low/high coverage, base call quality metrics etc.) | |||||
Verification/validation | Reference material (different from the material used during optimization) | Verification | Prepare verification protocol | Validation | Prepare validation protocol | |
Test the ranges provided by the manufacturer and technical variables in the laboratory | Include variables influencing assay performance | Amount of input DNA Number, type of samples in sequencing run Sequencing machine and other instruments Personnel | ||||
Verify the values provided by the manufacturer | Assess parameters describing the test performance | Limit of blank, limit of detection Repeatability, reproducibility Proportion of true/false positives/negatives, etc. (See Supplementary Table 3) | ||||
Quality control (QC) and continuous monitoring | Diagnostic samples Regularly analyzed positive and negative controls | Monitor of specimen parameters | Sample purity, DNA quality, etc. | |||
Check variant profile | Proportion of TP53-mut/del(17p) and low-burden TP53 mutations, variant profile (Figs. 1, 4, 6) | |||||
Samples supplied by EQA provider | External quality assessment (EQA) | |||||
Samples provided by independent laboratory | Interlaboratory comparison | |||||
