WP2: Enumeration methods for nucleic acids

The aim of this work package is to establish procedures suitable for primary reference measurements of amount of nucleic acid based on single-molecule detection and counting (enumeration) and assignment of SI traceability to control samples, calibrators and reference materials. This work package also aims at defining uncertainty for the developed procedures. The principal experimental factors and sample characteristics that affect quantitative measurement using digital PCR (dPCR) will be investigated and the resulting information used to prepare general guidance for validation of digital PCR measurement systems and the evaluation of uncertainty arising from these experimental factors.

Characterisation of the influence quantities and perturbing factors needed to establish dPCR as a primary method will be undertaken in line with the following ISO standards:

  • ISO 15193: In vitro diagnostic medical devices – Measurement of quantities in samples of biological origin- presentation of reference measurement procedures
  •  ISO 15194: In vitro diagnostic medical devices – Measurement of quantities in samples of biological origin – requirements for certified reference materials and the content of supporting documentation
  • ISO 17511: In vitro diagnostic medical devices – Measurement of quantities in biological samples – Metrological traceability of values assigned to calibrators and control materials

The work package will characterise enumeration approaches using a range of materials including synthetic DNA and genomic DNA. A minimal residual disease (MRD) clinical model system (e.g. hematologic malignancies) and/or circulating tumour cell model will be chosen in line with the model chosen for WP4 (enumeration of macroscopic entities – rare cell detection) allowing comparison of results across at least two measurement methods. Methods for assessing the purity of the test materials will be covered in WP5 and methods for value assignment of the materials in terms of copy numbers will be covered here in WP2.

MRD refers to small numbers of malignant cells that remain in a patient during treatment or after treatment when the patient is in remission. It is the major cause of relapse in cancer and leukaemia. MRD testing has several important roles: determining whether treatment has eradicated the cancer or whether traces remain, comparing the efficacy of different treatments, monitoring patient remission status and recurrence of the leukaemia or cancer and choosing the treatment that will best meet those needs.

The two methods currently used in the clinic for MRD monitoring are flow cytometry for aberrant cell phenotype counting and quantitative polymerase chain reaction (qPCR) for quantification of molecular biomarkers. The molecular targets can include clonal gene rearrangements, recurrent gene mutations, or aberrant gene expression of fusion gene transcripts. The most studied biomarker to date being the BCR-ABL1 fusion transcript.

For both flow cytometry and molecular approaches the need for more sensitive detection methods, (one malignant cell or mutation in a background of thousands or millions of normal cells), more accurate quantification of malignant cells or mutations and improved standardisation of measurements has been highlighted as essential for improving determination of disease status. The molecular enumeration approaches proposed in this WP have the potential to improve both the sensitivity and accuracy of disease monitoring over qPCR approaches and provide improved routes to SI traceability.

Test materials produced for evaluation will be characterised by multiple enumeration approaches and will also form the basis of the material used later on in the project for assessing approaches for measuring nucleic acid purity.