Pre-analytical processes in medical diagnostics: New regulatory requirements and standards.

In May 2017, the European In Vitro Diagnostic Regulation (IVDR) entered into force and will apply to in vitro diagnostics from May 26th, 2022. This will have a major impact on the in vitro diagnostics (IVD) industry as all devices falling under the scope of the IVDR will require new or re-certification. It will also affect health institutions developing and using in-house devices. The IVDR also has implications with respect to product performance validation and verification including the pre-analytics of biological samples used by IVD developers and diagnostic service providers. In parallel to the IVDR, a series of standards on pre-analytical sample processing has been published by the International Organization for Standardization (ISO) and the European Committee for Standardization (CEN). These standards describe pre-analytical requirements for various types of analyses in various types of biospecimens. They are of relevance for IVD product developers in the context of (re)certification under the IVDR and to some extent also to devices manufactured and used only within health institutions. This review highlights the background and the rational for the pre-analytical standards. It describes the procedure that leads to these standards, the major implications of the standards and the requirements on pre-analytical workflows. In addition, it discusses the relationship between the standards and the IVDR.

Assessment of Digital PCR as a Primary Reference Measurement Procedure to Support Advances in Precision Medicine.

BACKGROUND: Genetic testing of tumor tissue and circulating cell-free DNA for somatic variants guides patient treatment of many cancers. Such measurements will be fundamental in the future support of precision medicine. However, there are currently no primary reference measurement procedures available for nucleic acid quantification that would support translation of tests for circulating tumor DNA into routine use. METHODS: We assessed the accuracy of digital PCR (dPCR) for copy number quantification of a frequently occurring single-nucleotide variant in colorectal cancer (KRAS c.35G>A, p.Gly12Asp, from hereon termed G12D) by evaluating potential sources of uncertainty that influence dPCR measurement. RESULTS: Concentration values for samples of KRAS G12D and wild-type plasmid templates varied by <1.2-fold when measured using 5 different assays with varying detection chemistry (hydrolysis, scorpion probes, and intercalating dyes) and <1.3-fold with 4 commercial dPCR platforms. Measurement trueness of a selected dPCR assay and platform was validated by comparison with an orthogonal method (inductively coupled plasma mass spectrometry). The candidate dPCR reference measurement procedure showed linear quantification over a wide range of copies per reaction and high repeatability and interlaboratory reproducibility (CV, 2%-8% and 5%-10%, respectively). CONCLUSIONS: This work validates dPCR as an SI-traceable reference measurement procedure based on enumeration and demonstrates how it can be applied for assignment of copy number concentration and fractional abundance values to DNA reference materials in an aqueous solution. High-accuracy measurements using dPCR will support the implementation and traceable standardization of molecular diagnostic procedures needed for advancements in precision medicine.

International Interlaboratory Digital PCR Study Demonstrating High Reproducibility for the Measurement of a Rare Sequence Variant.

This study tested the claim that digital PCR (dPCR) can offer highly reproducible quantitative measurements in disparate laboratories. Twenty-one laboratories measured four blinded samples containing different quantities of a KRAS fragment encoding G12D, an important genetic marker for guiding therapy of certain cancers. This marker is challenging to quantify reproducibly using quantitative PCR (qPCR) or next generation sequencing (NGS) due to the presence of competing wild type sequences and the need for calibration. Using dPCR, 18 laboratories were able to quantify the G12D marker within 12% of each other in all samples. Three laboratories appeared to measure consistently outlying results; however, proper application of a follow-up analysis recommendation rectified their data. Our findings show that dPCR has demonstrable reproducibility across a large number of laboratories without calibration. This could enable the reproducible application of molecular stratification to guide therapy and, potentially, for molecular diagnostics.

An international comparability study on quantification of mRNA gene expression ratios: CCQM-P103.1.

Measurement of RNA can be used to study and monitor a range of infectious and non-communicable diseases, with profiling of multiple gene expression mRNA transcripts being increasingly applied to cancer stratification and prognosis. An international comparison study (Consultative Committee for Amount of Substance (CCQM)-P103.1) was performed in order to evaluate the comparability of measurements of RNA copy number ratio for multiple gene targets between two samples. Six exogenous synthetic targets comprising of External RNA Control Consortium (ERCC) standards were measured alongside transcripts for three endogenous gene targets present in the background of human cell line RNA. The study was carried out under the auspices of the Nucleic Acids (formerly Bioanalysis) Working Group of the CCQM. It was coordinated by LGC (United Kingdom) with the support of National Institute of Standards and Technology (USA) and results were submitted from thirteen National Metrology Institutes and Designated Institutes. The majority of laboratories performed RNA measurements using RT-qPCR, with datasets also being submitted by two laboratories based on reverse transcription digital polymerase chain reaction and one laboratory using a next-generation sequencing method. In RT-qPCR analysis, the RNA copy number ratios between the two samples were quantified using either a standard curve or a relative quantification approach. In general, good agreement was observed between the reported results of ERCC RNA copy number ratio measurements. Measurements of the RNA copy number ratios for endogenous genes between the two samples were also consistent between the majority of laboratories. Some differences in the reported values and confidence intervals ('measurement uncertainties') were noted which may be attributable to choice of measurement method or quantification approach. This highlights the need for standardised practices for the calculation of fold change ratios and uncertainties in the area of gene expression profiling.

International Comparison of Enumeration-Based Quantification of DNA Copy-Concentration Using Flow Cytometric Counting and Digital Polymerase Chain Reaction.

Enumeration-based determination of DNA copy-concentration was assessed through an international comparison among national metrology institutes (NMIs) and designated institutes (DIs). Enumeration-based quantification does not require a calibration standard thereby providing a route to "absolute quantification", which offers the potential for reliable value assignments of DNA reference materials, and International System of Units (SI) traceability to copy number 1 through accurate counting. In this study, 2 enumeration-based methods, flow cytometric (FCM) counting and the digital polymerase chain reaction (dPCR), were compared to quantify a solution of the pBR322 plasmid at a concentration of several thousand copies per microliter. In addition, 2 orthogonal chemical-analysis methods based on nucleotide quantification, isotope-dilution mass spectrometry (IDMS) and capillary electrophoresis (CE) were applied to quantify a more concentrated solution of the plasmid. Although 9 dPCR results from 8 laboratories showed some dispersion (relative standard deviation [RSD] = 11.8%), their means were closely aligned with those of the FCM-based counting method and the orthogonal chemical-analysis methods, corrected for gravimetric dilution factors. Using the means of dPCR results, the RSD of all 4 methods was 1.8%, which strongly supported the validity of the recent enumeration approaches. Despite a good overall agreement, the individual dPCR results were not sufficiently covered by the reported measurement uncertainties. These findings suggest that some laboratories may not have considered all factors contributing to the measurement uncertainty of dPCR, and further investigation of this possibility is warranted.

Standardization of Nucleic Acid Tests for Clinical Measurements of Bacteria and Viruses.

Nucleic acid-based tests for infectious diseases currently used in the clinical laboratory and in point-of-care devices are diverse. Measurement challenges associated with standardization of quantitative viral load testing are discussed in relation to human cytomegalovirus, BK virus, and Epstein-Barr virus, while the importance of defining the performance of qualitative methods is illustrated with Mycobacterium tuberculosis and influenza virus. The development of certified reference materials whose values are traceable to higher-order standards and reference measurement procedures, using, for instance, digital PCR, will further contribute to the understanding of analytical performance characteristics and promote clinical data comparability.

Fully traceable absolute protein quantification of somatropin that allows independent comparison of somatropin standards.

BACKGROUND: Measurement traceability in clinical chemistry is required to standardize clinical results irrespective of the measurement procedure and laboratory. The traceability of many protein substances is maintained by reference to the first standard produced, which may no longer exist, with values assigned by consensus. Independent methods that provide traceability to the Système d'Unité International for all relevant properties of a protein standard could remove reliance on the original standard preparations. METHODS: We developed a method based on the traceable quantification of tryptic peptides released from the protein by isotope dilution mass spectrometry to compare 2 standard preparations of somatropin (recombinant human growth hormone), WHO 98/574 and Ph.Eur.CRS S0947000. Relative quantification using isotope-coded affinity tagging, isobaric tagging for relative and absolute quantification, and standard additions were also performed to validate the digestion method used and to determine whether any modifications were present. RESULTS: The total somatropin content in both materials was determined and an uncertainty estimation undertaken [WHO 2.19 +/- 0.21) mg/vial, European Pharmacopeia 2.06 +/- 0.21 mg/vial]. Each uncertainty in this paper is a fully estimated uncertainty, with 95% CI (k = 2). Isotope coded affinity tag and standard addition results fully validated the robustness of the digestion method used. In addition, iTRAQ (isobaric tagging for relative and absolute quantification analysis) identified 2 modifications, neither of which impacted the quantification. CONCLUSIONS: An independent method that does not rely on a preexisting protein standard has been developed and validated for the traceable value-assignment of total somatropin. The methods reported here address the amount of substance (mass fraction) of the standard materials but address neither biological activity nor other characteristics that may be important in assessing suitability for use as a calibrator.

Primary ocular melioidosis due to a single genotype of Burkholderia pseudomallei in two cats from Arnhem Land in the Northern Territory of Australia.

Melioidosis was diagnosed in two domestic crossbred cats presented for unilateral ocular disease. One patient was born and bred in Nhulunbuy, Arnhem Land, while the other had moved there 6 months previously from Townsville, Queensland. Both patients were presented with sudden onset of a 'red eye' and blepharospasm, which progressed to an enlarged, painful, firm globe with loss of pupillary light reflexes and vision. An obvious primary focus of infection outside the eye was not detected in either cat. In both patients, the affected eye was surgically removed and vitreal culture revealed a pure growth of Burkholderia pseudomallei. In each instance, the infection had penetrated the sclera to produce retrobulbar cellulitis, and in one case frank retrobulbar abscessation. Histologically, there was a pyogranulomatous uveitis with extensive destruction of intraocular structures. The first case was still alive approximately 1 year following enucleation and limited antimicrobial therapy using amoxicillin clavulanate and doxycycline. The second was euthanased when a localised abscess developed on the same side of the face as the healed surgical incision, despite appropriate antimicrobial therapy. Both cases were caused by the same multilocus sequence type of B pseudomallei (ST 116), which had only been isolated previously from two human patients, both living in the same isolated geographical area as the cats of this report. Apart from the geographical clustering, no epidemiological links were evident between the two cats and/or the two people. The presumptive pathogenesis of these infections is discussed in relation to current knowledge about melioidosis in northern Australia.

The costs of using unauthenticated, over-passaged cell lines: how much more data do we need?

Increasing data demonstrate that cellular cross-contamination, misidentified cell lines, and the use of cultures at high-passage levels contribute to the generation of erroneous and misleading results as well as wasted research funds. Contamination of cell lines by other lines has been recognized and documented back to the 1950s. Based on submissions to major cell repositories in the last decade, it is estimated that between 18% and 36% of cell lines may be contaminated or misidentified. More recently, problems surrounding practices of over-subculturing cells are being identified. As a result of selective pressures and genetic drift, cell lines, when kept in culture too long, exhibit reduced or altered key functions and often no longer represent reliable models of their original source material. A review of papers showing significant experimental variances between low- and high-passage cell culture numbers, as well as contaminated lines, makes a strong case for using verified, tested cell lines at low- or defined passage numbers. In the absence of cell culture guidelines, mandates from the National Institutes of Health (NIH) and other funding agencies or journal requirements, it becomes the responsibility of the scientific community to perform due diligence to ensure the integrity of cell cultures used in research.

The External RNA Controls Consortium: a progress report.

Standard controls and best practice guidelines advance acceptance of data from research, preclinical and clinical laboratories by providing a means for evaluating data quality. The External RNA Controls Consortium (ERCC) is developing commonly agreed-upon and tested controls for use in expression assays, a true industry-wide standard control.

Real-time detection of genetically modified soya using Lightcycler and ABI 7700 platforms with TaqMan, Scorpion, and SYBR Green I chemistries.

A comparative cross platform evaluation of real-time polymerase chain reaction detection of DNA sequences present in Roundup Ready soya was undertaken using the ABI 7700 and Roche Lightcycler detection systems in combination with 3 different detection chemistries: TaqMan, Scorpion primers, and SYBR Green I fluorescent dye. Various copy numbers of a plasmid containing the soya lectin sequence were used to determine the sensitivity and reproducibility of the different technology combinations and to examine both inter and intra machine variability. To examine the relative accuracy of each technology, the genetically modified soya content of baked products containing known amounts of Roundup Ready soya was determined by detection of lectin and the EPSPS transgene. It was determined that the combination of TaqMan detection chemistry and the ABI 7700 platform represented the best method for quantitative detection of genetically modified organisms in terms of both precision and accuracy.

Detection of genetically modified crops and their derivatives: critical steps in sample preparation and extraction.

The detection of genetically modified crops in foodstuff relies on detection of transgenic DNA or protein material in the sample matrix. Purified DNA or proteins are used as analytical material for polymerase chain reaction technologies and immunodiagnostics. Successful sample preparation is critical to the validity of subsequent analysis. For routine analysis, a good sample preparation technique should be simple, safe, and inexpensive while reproducibly generating DNA/protein of sufficient quality and yield. The suitability of isolated DNA or protein as an analyte for a detection or characterization technique depends on amount or concentration, purity, and integrity, each of which may be influenced by sample matrix and the extraction technique, and, in turn, may impact the validity of analytical techniques. The key sample preparation steps of homogenization, pretreatment, extraction, and purification are discussed as well as typical analytical methods. Consideration is given to application of these steps for particular sample matrixes to maximize yield, reduce inhibition effects, and minimize contamination. The choice of the most appropriate and valid methods for sample preparation from particular foods is discussed with respect to DNA analysis. Attention is also given to ease of use, cost, and generic applicability of the procedures.