Assessment of serum total 25-hydroxyvitamin D assays for Vitamin D External Quality Assessment Scheme (DEQAS) materials distributed at ambient and frozen conditions.

The Vitamin D External Quality Assessment Scheme (DEQAS) distributes human serum samples four times per year to over 1000 participants worldwide for the determination of total serum 25-hydroxyvitamin D [25(OH)D)]. These samples are stored at -40 °C prior to distribution and the participants are instructed to store the samples frozen at -20 °C or lower after receipt; however, the samples are shipped to participants at ambient conditions (i.e., no temperature control). To address the question of whether shipment at ambient conditions is sufficient for reliable performance of various 25(OH)D assays, the equivalence of DEQAS human serum samples shipped under frozen and ambient conditions was assessed. As part of a Vitamin D Standardization Program (VDSP) commutability study, two sets of the same nine DEQAS samples were shipped to participants at ambient temperature and frozen on dry ice. Twenty-eight laboratories participated in this study and provided 34 sets of results for the measurement of 25(OH)D using 20 ligand binding assays and 14 liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. Equivalence of the assay response for the frozen versus ambient DEQAS samples for each assay was evaluated using multi-level modeling, paired t-tests including a false discovery rate (FDR) approach, and ordinary least squares linear regression analysis of frozen versus ambient results. Using the paired t-test and confirmed by FDR testing, differences in the results for the ambient and frozen samples were found to be statistically significant at p < 0.05 for four assays (DiaSorin, DIAsource, Siemens, and SNIBE prototype). For all 14 LC-MS/MS assays, the differences in the results for the ambient- and frozen-shipped samples were not found to be significant at p < 0.05 indicating that these analytes were stable during shipment at ambient conditions. Even though assay results have been shown to vary considerably among different 25(OH)D assays in other studies, the results of this study also indicate that sample handling/transport conditions may influence 25(OH)D assay response for several assays.

An Interlaboratory Comparison on the Characterization of a Sub-micrometer Polydisperse Particle Dispersion.

The measurement of polydisperse protein aggregates and particles in biotherapeutics remains a challenge, especially for particles with diameters of ≈ 1 µm and below (sub-micrometer). This paper describes an interlaboratory comparison with the goal of assessing the measurement variability for the characterization of a sub-micrometer polydisperse particle dispersion composed of five sub-populations of poly(methyl methacrylate) (PMMA) and silica beads. The study included 20 participating laboratories from industry, academia, and government, and a variety of state-of-the-art particle-counting instruments. The received datasets were organized by instrument class to enable comparison of intralaboratory and interlaboratory performance. The main findings included high variability between datasets from different laboratories, with coefficients of variation from 13 % to 189 %. Intralaboratory variability was, on average, 37 % of the interlaboratory variability for an instrument class and particle sub-population. Drop-offs at either end of the size range and poor agreement on maximum counts of particle sub-populations were noted. The mean distributions from an instrument class, however, showed the size-coverage range for that class. The study shows that a polydisperse sample can be used to assess performance capabilities of an instrument set-up (including hardware, software, and user settings) and provides guidance for the development of polydisperse reference materials.

Lessons from the NIST micronutrients quality assurance program for vitamin C, 1993 to 2015: sample stability, assay reproducibility, and use of controls to improve comparability.

Vitamin C is a necessary micronutrient that is involved in many biological processes. In preserved human plasma and serum, vitamin C is most meaningfully analyzed as total ascorbic acid (TAA). From 1993 through 2015, the National Institute of Standards and Technology (NIST) coordinated 40 interlaboratory studies (ILS) devoted to improving the between-participant comparability of TAA measurements. The results from these ILS demonstrate that the concentration of TAA ([TAA]) is stable for at least 20 years in serum diluted 1 + 1 (volume fraction) with 10% mass concentration aqueous metaphosphoric acid (MPA) and stored at -80 °C. The between-participant relative reproducibility precision, expressed as a coefficient of variation (CV), improved from over 16% to under 9% over the course of the studies. Normalization of test samples (i.e., ex post-facto recalibrating the as-submitted results) using participant-prepared serum-free calibration solutions did not improve reproducibility. Normalization to one control sample having a similar serum-based matrix as the test samples improved the CV to 7%; normalization to two such controls reduced the CV to 4%. Multicenter studies that require the highest degree of measurement comparability among the participants should consider calibrating with materials that have a serum-based matrix as similar as possible to that of the samples of interest.

Evaluating digital PCR for the quantification of human nuclear DNA: determining target strandedness.

Digital polymerase chain reaction (dPCR) methodology has been asserted to be a "potentially primary" analytical approach for assigning DNA concentration. The essence of dPCR measurements is the independent dispersal of fragments into multiple reaction partitions, amplifying fragments containing a target nucleotide sequence until the signal from all partitions containing at least one such fragment rises above threshold, and then determining the proportion of partitions with an above-threshold signal. Should originally double-stranded DNA (dsDNA) fragments be converted into two single strands (ssDNA) prior to dispersal, the dPCR measurements could be biased high by as much as a factor of two. Realizing dPCR's metrological potential therefore requires analytical methods for determining the proportion of ssDNA in nominally dsDNA samples. To meet this need, we have investigated several approaches to this determination: A260 ratio, dPCR ratio, cdPCR staircase, and ddPCR enzyme. In our hands, only the endonuclease-based approach provides adequately accurate estimates for relatively small ssDNA proportions. We present four (enzyme, assay) pairs that provide self-consistent results for human nuclear DNA extracts. However, the proportion of ssDNA differs by as much as 50% between assays, apparently related to the guanine-cytosine (GC) content of the fragment near the assay's target sequence. While materials extracted by us have no more than 6% ssDNA content even after long storage, a commercially obtained PCR assay calibrant contains ≈18% ssDNA. Graphical abstract.

NIST Reference Materials: Utility and Future.

The National Institute of Standards and Technology (NIST), formerly the National Bureau of Standards, was established by the US Congress in 1901 and charged with establishing a measurement foundation to facilitate US and international commerce. This broad language provides NIST with the ability to establish and implement its programs in response to changes in national needs and priorities. This review traces some of the changes in NIST's reference material programs over time and presents the NIST Material Measurement Laboratory's current approach to promoting accuracy and metrological traceability of chemical measurements and validation of chemical measurement processes.

NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods.

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.

The role of the CCQM OAWG in providing SI traceable calibrators for organic chemical measurements.

Metrological traceability for organic chemical measurements is a documented unbroken chain of calibrations with stated uncertainties that ideally link the measurement result for a sample to a primary calibrator in appropriate SI units (e.g., mass fraction). A comprehensive chemical purity determination of the organic calibrator is required to ensure a true assessment of this result. We explore the evolution of chemical purity capabilities across metrology institute members of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology's Organic Analysis Working Group (OAWG). The OAWG work program has promoted the development of robust measurement capabilities, using indirect "mass balance" determinations via rigorous assessment of impurities and direct determination using quantitative nuclear magnetic resonance spectroscopy methods. A combination of mass balance and qNMR has been shown to provide a best practice approach. Awareness of the importance of the traceability of organic calibrators continues to grow across stakeholder groups, particularly in key areas such as clinical chemistry where activities related to the Joint Committee for Traceability in Laboratory Medicine have raised the profile of traceable calibrators.

Evaluating droplet digital PCR for the quantification of human genomic DNA: converting copies per nanoliter to nanograms nuclear DNA per microliter.

The highly multiplexed polymerase chain reaction (PCR) assays used for forensic human identification perform best when used with an accurately determined quantity of input DNA. To help ensure the reliable performance of these assays, we are developing a certified reference material (CRM) for calibrating human genomic DNA working standards. To enable sharing information over time and place, CRMs must provide accurate and stable values that are metrologically traceable to a common reference. We have shown that droplet digital PCR (ddPCR) limiting dilution end-point measurements of the concentration of DNA copies per volume of sample can be traceably linked to the International System of Units (SI). Unlike values assigned using conventional relationships between ultraviolet absorbance and DNA mass concentration, entity-based ddPCR measurements are expected to be stable over time. However, the forensic community expects DNA quantity to be stated in terms of mass concentration rather than entity concentration. The transformation can be accomplished given SI-traceable values and uncertainties for the number of nucleotide bases per human haploid genome equivalent (HHGE) and the average molar mass of a nucleotide monomer in the DNA polymer. This report presents the considerations required to establish the metrological traceability of ddPCR-based mass concentration estimates of human nuclear DNA. Graphical abstract The roots of metrological traceability for human nuclear DNA mass concentration results. Values for the factors in blue must be established experimentally. Values for the factors in red have been established from authoritative source materials. HHGE stands for "haploid human genome equivalent"; there are two HHGE per diploid human genome.

Steps to achieve quantitative measurements of microRNA using two step droplet digital PCR.

Droplet digital PCR (ddPCR) is being advocated as a reference method to measure rare genomic targets. It has consistently been proven to be more sensitive and direct at discerning copy numbers of DNA than other quantitative methods. However, one of the largest obstacles to measuring microRNA (miRNA) using ddPCR is that reverse transcription efficiency depends upon the target, meaning small RNA nucleotide composition directly effects primer specificity in a manner that prevents traditional quantitation optimization strategies. Additionally, the use of reagents that are optimized for miRNA measurements using quantitative real-time PCR (qRT-PCR) appear to either cause false positive or false negative detection of certain targets when used with traditional ddPCR quantification methods. False readings are often related to using inadequate enzymes, primers and probes. Given that two-step miRNA quantification using ddPCR relies solely on reverse transcription and uses proprietary reagents previously optimized only for qRT-PCR, these barriers are substantial. Therefore, here we outline essential controls, optimization techniques, and an efficacy model to improve the quality of ddPCR miRNA measurements. We have applied two-step principles used for miRNA qRT-PCR measurements and leveraged the use of synthetic miRNA targets to evaluate ddPCR following cDNA synthesis with four different commercial kits. We have identified inefficiencies and limitations as well as proposed ways to circumvent identified obstacles. Lastly, we show that we can apply these criteria to a model system to confidently quantify miRNA copy number. Our measurement technique is a novel way to quantify specific miRNA copy number in a single sample, without using standard curves for individual experiments. Our methodology can be used for validation and control measurements, as well as a diagnostic technique that allows scientists, technicians, clinicians, and regulators to base miRNA measures on a single unit of measurement rather than a ratio of values.

Evaluating Droplet Digital Polymerase Chain Reaction for the Quantification of Human Genomic DNA: Lifting the Traceability Fog.

Digital polymerase chain reaction (dPCR) end point platforms directly estimate the number of DNA target copies per reaction partition, λ, where the partitions are fixed-location chambers (cdPCR) or aqueous droplets floating in oil (ddPCR). For use in the certification of target concentration in primary calibrant certified reference materials (CRMs), both λ and the partition volume, V, must be metrologically traceable to some accessible reference system, ideally, the International System of Units (SI). The fixed spatial distribution of cdPCR chambers enables real-time monitoring of PCR amplification. Analysis of the resulting reaction curves enables validation of the critical dPCR assumptions that are essential for establishing the SI traceability of λ. We know of no direct method for validating these assumptions for ddPCR platforms. The manufacturers of the cdPCR and ddPCR systems available to us do not provide traceable partition volume specifications. Our colleagues at the National Institute of Standards and Technology (NIST) have developed a reliable method for determining ddPCR droplet volume and have demonstrated that different ddPCR reagents yield droplets of somewhat different size. Thus, neither dPCR platform by itself provides metrologically traceable estimates of target concentration. We show here that evaluating split samples with both cdPCR and ddPCR platforms can transfer the λ traceability characteristics of a cdPCR assay to its ddPCR analogue, establishing fully traceable ddPCR estimates of CRM target concentration.

Evaluating Digital PCR for the Quantification of Human Genomic DNA: Accessible Amplifiable Targets.

Polymerase chain reaction (PCR) multiplexed assays perform best when the input quantity of template DNA is controlled to within about a factor of √2. To help ensure that PCR assays yield consistent results over time and place, results from methods used to determine DNA quantity need to be metrologically traceable to a common reference. Many DNA quantitation systems can be accurately calibrated with solutions of DNA in aqueous buffer. Since they do not require external calibration, end-point limiting dilution technologies, collectively termed "digital PCR (dPCR)", have been proposed as suitable for value assigning such DNA calibrants. The performance characteristics of several commercially available dPCR systems have recently been documented using plasmid, viral, or fragmented genomic DNA; dPCR performance with more complex materials, such as human genomic DNA, has been less studied. With the goal of providing a human genomic reference material traceably certified for mass concentration, we are investigating the measurement characteristics of several dPCR systems. We here report results of measurements from multiple PCR assays, on four human genomic DNAs treated with four endonuclease restriction enzymes using both chamber and droplet dPCR platforms. We conclude that dPCR does not estimate the absolute number of PCR targets in a given volume but rather the number of accessible and amplifiable targets. While enzymatic restriction of human genomic DNA increases accessibility for some assays, in well-optimized PCR assays it can reduce the number of amplifiable targets and increase assay variability relative to uncut sample.

The evaluation of the scoring systems: the fixed effects model under known variances.

A number of scoring systems for proficiency testing and interlaboratory comparison are in use by the metrology community. The choice of scoring system for a given study is often based on the study coordinator's experience and anecdotal knowledge, perhaps attributable to a historic lack of detailed and formal explanation about the foundation of these systems. This has influenced the development of new scoring systems, some of them departing from the well-established hypothesis testing theory. Often, different scoring systems give different results not because one may be better than the others but because, as they are documented, the user cannot control the confidence level of the test. We present a formal evaluation of seven of these systems under the fixed effects model assuming known variances. Under these sound assumptions, the systems analyzed all have the same statistical properties. Furthermore, these systems are all members of a family of systems based on strictly increasing functions in which the statistical decision problem is invariant. Under the fixed effects model with known variances, no unbiased scoring system can provide greater statistical power than the members of this family of systems. We apply these results to the lead content of water example provided in International Standard ISO 13528:2015 "Statistical methods for use in proficiency testing by interlaboratory comparisons."

Real-time cdPCR opens a window into events occurring in the first few PCR amplification cycles.

Polymerase chain reaction (PCR) end-point limiting dilution techniques, collectively termed "digital PCR (dPCR)", have been proposed as providing a potentially primary method for DNA quantification. We are evaluating several commercially available dPCR systems for use in certifying mass concentration in human genomic DNA reference materials. To better understand observed anomalies among results from chamber- and droplet-dPCR (cdPCR and ddPCR) systems, we have developed a graphical tool for evaluating and documenting the performance of PCR assays in real-time cdPCR systems: the ogive plot, the cumulative distribution of crossing threshold values. The ogive structure appears to embed information about early amplification events. We have successfully simulated ogives observed with different assays and reaction conditions using a four-stage amplification model parameterized by the probability of creating an intact 1) first generation "long" amplicon of indeterminate length from an original DNA target, 2) second generation defined-length amplicon from a long amplicon, and 3) defined-length amplicon from another defined-length amplicon. We are using insights from this model to optimize dPCR assay design and reaction conditions and to help validate assays proposed for use in value-assigning DNA reference materials.

Developmental validation of the PowerPlex(®) ESI 16/17 Fast and PowerPlex(®) ESX 16/17 Fast Systems.

The PowerPlex(®) ESI 16 Fast, ESI 17 Fast, ESX 16 Fast, and ESX 17 Fast Systems represent faster cycling versions (50min or less) of the PowerPlex(®) ESI and ESX Systems released by Promega in 2009 to accommodate the ENFSI and EDNAP groups' call for new STR multiplexes for Europe. In addition to amplification of purified DNA samples, these new faster cycling systems allow for direct amplification from single-source blood and buccal samples deposited on FTA(®) and nonFTA paper as well as from SwabSolution™ extracts of buccal swabs without the need for purification and quantitation. There are no changes to the autosomal primer pair sequences in the PowerPlex(®) ESI Fast and ESX Fast Systems compared to the original multiplexes, and full concordance at all autosomal loci and amelogenin was observed with data generated previously with the original PowerPlex(®) ESI and ESX Systems. This paper describes the developmental validation study performed on these new fast systems following guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM) and those of the DNA Advisory Board (DAB). Validation data demonstrate that these systems are sensitive for detecting low levels of DNA while also being capable of generating robust profiles from the high amount of input DNA present in direct-amplification samples. These systems are also tolerant to both high concentrations of PCR inhibitors as well as to slight variations in the final concentration of master mix and primer pair present in the amplification reaction that might be encountered due to pipetting error. The results of this validation study demonstrate that these systems may be used on multiple thermal cyclers and capillary electrophoresis platforms.

International comparison of a hydrocarbon gas standard at the picomol per mol level.

Studies of climate change increasingly recognize the diverse influences of hydrocarbons in the atmosphere, including roles in particulates and ozone formation. Measurements of key nonmethane hydrocarbons (NMHCs) suggest atmospheric mole fractions ranging from low picomoles per mol (ppt) to nanomoles per mol (ppb), depending on location and compound. To accurately establish mole fraction trends and to relate measurement records from many laboratories and researchers, it is essential to have accurate, stable, calibration standards. In February of 2008, the National Institute of Standards and Technology (NIST) developed and reported on picomoles per mol standards containing 18 nonmethane hydrocarbon compounds covering the mole fraction range of 60 picomoles per mol to 230 picomoles per mol. The stability of these gas mixtures was only characterized over a short time period (2 to 3 months). NIST recently prepared a suite of primary standard gas mixtures by gravimetric dilution to ascertain the stability of the 2008 picomoles per mol NMHC standards suite. The data from this recent chromatographic intercomparison of the 2008 to the 2011 suites confirm a much longer stability of almost 5 years for 15 of the 18 hydrocarbons; the double-bonded alkenes of propene, isobutene, and 1-pentene showed instability, in line with previous publications. The agreement between the gravimetric values from preparation and the analytical mole fractions determined from regression illustrate the internal consistency of the suite within ±2 pmol/mol. However, results for several of the compounds reflect stability problems for the three double-bonded hydrocarbons. An international intercomparison on one of the 2008 standards has also been completed. Participants included National Metrology Institutes, United States government laboratories, and academic laboratories. In general, results for this intercomparison agree to within about ±5% with the gravimetric mole fractions of the hydrocarbons.

An international assessment of the metrological equivalence of higher-order measurement services for creatinine in serum.

The Consultative committee for amount of substance-metrology in chemistry (CCQM)-K80 Key Comparison directly assessed the equivalence of many of the world's higher-order value-assigned materials (HOVAMs) for creatinine in human serum. This 2009 international study compared the certified values and uncertainties of the materials using measurements made under repeatability conditions. The study evaluated 17 materials submitted by 6 national metrology institutes (NMIs). The creatinine quantity in these materials ranged from 3 mg/kg to 57 mg/kg (about 0.3 mg/dL to 6 mg/dL or 30 nmol/L to 500 nmol/L). All materials were stored and prepared according the specifications provided by the participating NMIs. Samples were processed and analyzed under repeatability conditions by one analyst using isotope-dilution liquid chromatography-mass spectrometry in two measurement campaigns. The certified values and repeatability measurements were compared using uncertainty-weighted generalized distance regression. The instrumental repeatability relative standard deviation was 1.2%. The measurement design required assessment of within-unit and between-campaign variability in addition to measurement repeatability. At a 95% level of confidence, the certified values for all 17 materials agreed to within their assigned uncertainties. CCQM-K80 demonstrated the metrological equivalence of the currently available HOVAMs for creatinine in human serum and of the creatinine measurement services provided by the participating NMIs.

A Bayesian approach to the evaluation of comparisons of individually value-assigned reference materials.

Several recent international comparison studies used a relatively novel experimental design to evaluate the measurement capabilities of participating organizations. These studies compared the values assigned by each participant to one or more qualitatively similar materials with measurements made on all of the materials by one laboratory under repeatability conditions. A statistical model was then established relating the values to the repeatability measurements; the extent of agreement between the assigned value(s) and the consensus model reflected the participants' measurement capabilities. Since each participant used their own supplies, equipment, and methods to produce and value-assign their material(s), the agreement between the assigned value(s) and the model was a fairer reflection of their intrinsic capabilities than provided by studies that directly compared time- and material-constrained measurements on unknown samples prepared elsewhere. A new statistical procedure is presented for the analysis of such data. The procedure incorporates several novel concepts, most importantly a leave-one-out strategy for the estimation of the consensus value of the measurand, model fitting via Bayesian posterior probabilities, and posterior coverage probability calculation for the assigned 95% uncertainty intervals. The benefits of the new procedure are illustrated using data from the CCQM-K54 comparison of eight cylinders of n-hexane in methane.

Preparation and value assignment of standard reference material 968e fat-soluble vitamins, carotenoids, and cholesterol in human serum.

Standard Reference Material 968e Fat-Soluble Vitamins, Carotenoids, and Cholesterol in Human Serum provides certified values for total retinol, γ- and α-tocopherol, total lutein, total zeaxanthin, total ß-cryptoxanthin, total ß-carotene, 25-hydroxyvitamin D(3), and cholesterol. Reference and information values are also reported for nine additional compounds including total α-cryptoxanthin, trans- and total lycopene, total α-carotene, trans-ß-carotene, and coenzyme Q(10). The certified values for the fat-soluble vitamins and carotenoids in SRM 968e were based on the agreement of results from the means of two liquid chromatographic methods used at the National Institute of Standards and Technology (NIST) and from the median of results of an interlaboratory comparison exercise among institutions that participate in the NIST Micronutrients Measurement Quality Assurance Program. The assigned values for cholesterol and 25-hydroxyvitamin D(3) in the SRM are the means of results obtained using the NIST reference method based upon gas chromatography-isotope dilution mass spectrometry and liquid chromatography-isotope dilution tandem mass spectrometry, respectively. SRM 968e is currently one of two available health-related NIST reference materials with concentration values assigned for selected fat-soluble vitamins, carotenoids, and cholesterol in human serum matrix. This SRM is used extensively by laboratories worldwide primarily to validate methods for determining these analytes in human serum and plasma and for assigning values to in-house control materials. The value assignment of the analytes in this SRM will help support measurement accuracy and traceability for laboratories performing health-related measurements in the clinical and nutritional communities.

Dietary supplement laboratory quality assurance program: the first five exercises.

The National Institute of Standards and Technology (NIST) has established a Dietary Supplement Laboratory Quality Assurance Program (DSQAP) in collaboration with the National Institutes of Health Office of Dietary Supplements. Program participants measure concentrations of active and/or marker compounds as well as nutritional and toxic elements in food and dietary supplements distributed by NIST. Data are compiled at NIST, where they are analyzed for accuracy relative to reference values and concordance among the participants. Performance reports and certificates of completion are provided to participants, which can be used to demonstrate compliance with current Good Manufacturing Practices as promulgated by the U.S. Food and Drug Administration. The DSQAP has conducted five exercises to date, with total participation including more than 75 different laboratories and many more individual analysts.