The development and implementation of quality assurance programs to support nutritional measurements.

The National Institute of Standards and Technology administers quality assurance programs devoted to improving measurements of nutrients and related metabolites in foods, dietary supplements, and serum and plasma samples. These programs have been developed in collaboration with the National Institutes of Health to assist measurement communities in their efforts to achieve accurate results that are comparable among different laboratories and over time. Targeted analytes include micronutrients, botanical markers, nutritional elements, contaminants, fatty acids, and vitamin D metabolites.

Development and certification of green tea-containing standard reference materials.

A suite of three green tea-containing Standard Reference Materials (SRMs) has been issued by the National Institute of Standards and Technology (NIST): SRM 3254 Camellia sinensis (Green Tea) Leaves, SRM 3255 Camellia sinensis (Green Tea) Extract, and SRM 3256 Green Tea-Containing Solid Oral Dosage Form. The materials are characterized for catechins, xanthine alkaloids, theanine, and toxic elements. As many as five methods were used in assigning certified and reference values to the constituents, with measurements carried out at NIST and at collaborating laboratories. The materials are intended for use in the development and validation of new analytical methods, and for use as control materials as a component in the support of claims of metrological traceability.

Certification of vitamins and carotenoids in SRM 3280 multivitamin/multielement tablets.

A new multivitamin/multielement dietary supplement Standard Reference Material (SRM) has been issued by the National Institute of Standards and Technology (NIST), with certified and reference concentration values for 13 vitamins, 24 elements, and 2 carotenoids. The constituents have been measured by multiple analytical methods with data contributed by NIST and by collaborating laboratories. This effort included the first use of isotope dilution mass spectrometry for value assignment of both fat-soluble vitamins (FSVs) and water-soluble vitamins (WSVs). Excellent agreement was obtained among the methods, with relative expanded uncertainties for the certified concentration values typically ranging from <2% to 15% for vitamins.

NIST mixed stain studies #1 and #2: interlaboratory comparison of DNA quantification practice and short tandem repeat multiplex performance with multiple-source samples.

The Mixed Stain Study 1 (MSS1, Apr.-Nov. 1997) and Mixed Stain Study 2 (MSS2, Jan.-May 1999) evaluated multiplexed short-tandem repeat (STR) DNA typing systems with samples containing DNA from more than one source. These interlaboratory challenge studies evaluated forensic STR measurement, interpretation, and reporting practice using well-characterized samples of very different analytical difficulty. None of the relatively few errors reported in either exercise resulted in a false identification of a reference source; several errors in evaluating the unknown source in three-source samples would hinder matching the profile in any archival database. None of the measurement anomalies reported is associated with any particular STR multiplex; all DNA amplification anomalies are associated with inefficient DNA extraction, inaccurate DNA quantitation, and/or analytical threshold policies.

Preparation and value assignment of Standard Reference Material 968c Fat-Soluble Vitamins, Carotenoids, and Cholesterol in Human Serum.

Standard Reference Material 968c Fat-Soluble Vitamins, Carotenoids, and Cholesterol in Human Serum provides certified values for retinal, delta-, gamma-, and alpha-tocopherol, trans- and total beta-carotene, and cholesterol in human serum. Values are also reported for 16 additional compounds including lutein, zeaxanthin, alpha- and beta-cryptoxanthin, lycopene, alpha-carotene, retinyl palmitate, and 25-hydroxyvitamin D. The certified values for the fat-soluble vitamins and carotenoids in SRM 968c were based on the agreement of results from the means of at least two liquid chromatographic methods used at the National Institute of Standards and Technology (NIST) and from the medians from an interlaboratory comparison study among institutions that participate in the NIST Micronutrients Measurement Quality Assurance Program. The assigned values for cholesterol in the SRM are the means of results obtained using the NIST definitive method, gas chromatography-isotope dilution mass spectrometry.

Investigating the effect of changing ammunition on the composition of organic additives in gunshot residue (OGSR).

The measurement of the organic additives in smokeless gunpowder is an attractive approach for the detection of handgun use because it provides compositional information that can help associate residues and unfired gunpowder. We investigate several factors that will be required to advance the characterization of organic gunshot residue (OGSR) as a useful forensic tool, including evaluating residue contamination from previously fired ammunition, particle-to-particle compositional variability, and compositional features resulting from the type of firing primer used. Using ammunition loaded with known smokeless powders containing different stabilizers, a sequence of shots was fired from a .357 magnum revolver, and the muzzle exit residues were collected. Compositional analysis of the residues, both in bulk and as single particles, showed only a trace of the previously fired powder in the first shot and none in subsequent shots. In an additional experiment testing conventional leaded and the new lead-free firing primers, the OGSR composition was found not to depend on the primer type.

RFLP band size standards: NIST standard reference material 2390.

The procedural standard for DNA profiling developed by the U.S. advisory board on DNA quality assurance methods mandates annual confirmation of forensic DNA measurement systems against an appropriate reference material supplied by or traceable to the National Institute of Standards and Technology (NIST). NIST Standard Reference Material (SRM) 2390 is a suitable and appropriate standard for HaeIII restriction enzyme-based restriction fragment length polymorphism (RFLP) profiling systems. Originally issued in 1992, an among-laboratory SRM 2390 recertification study was initiated in 1997. Using data provided by the 20 state, local, or commercial forensic laboratory participants, quantitative band sizes values (expected mean values and associated bivariate tolerance intervals) are established for two different-source DNAs (female cell line K562 and healthy male "TAW") for genetic loci D1S7, D2S44, D4S139, D5S110, D1OS28, and D17S79. Methods for validating an RFLP measurement system, validating a control material or other secondary standard, and for tracing a particular set of RFLP measurements to NIST SRM 2390 are described in detail.

RFLP band size standards: cell line K562 values from 1991 to 1997 proficiency studies.

Cell line K562 is the defacto forensic control material for forensic restriction fragment length polymorphism (RFLP) DNA profiling in the U.S. Fifty-one proficiency tests conducted from 1991 through 1997 enable a detailed description of RFLP measurement performance during this period. Sufficient data are available to define reference distributions for all commonly utilized and many less commonly reported genetic loci, for both HaeIII- and HinfI-based RFLP systems. The average measured size of HaeIII locus D1S7 and D5S110 bands has varied slightly over time; while relatively small, these temporal changes add to the overall interlaboratory measurement uncertainty. The characteristic standard deviation for HinfI RFLP system measurements has a nearly identical dependence on expected band size as does the standard deviation for HaeIII measurements. The ellipsoidal distance, K, is suggested for use as an RFLP data quality metric; the critical threshold value that on average excludes 1% of plausibly valid proficiency data for a given polymorphic locus is K1% = 14.2.

NIST micronutrients measurement quality assurance program: characterizing the measurement community's performance over time.

The Micronutrients Measurement Quality Assurance Program (M2QAP) at the National Institute of Standards and Technology was created in 1984 with the goal of improving among-participant measurement comparability for fat-soluble vitamin-related compounds in human serum. We recently described improved tools for evaluating comparison exercise data; we here extend and apply these tools to the evaluation of the measurement community's performance over the entire 15-year history of the M2QAP. We here display measurement performance characteristics for the 14 measurands most commonly reported by the M2QAP community. We confirm that among-participant comparability for total beta-carotene cannot be much improved without improving average long-term within-participant measurement stability. We demonstrate that improved measurand definition and/or identification of interferences may help participants improve comparability for many of the M2QAP's other commonly reported measurands. The reported measurement performance characteristics may be of interest to clinical, nutritional, and epidemiological studies involving any of these measurands. The data analysis techniques utilized may be applicable to other programs.

NIST micronutrients measurement quality assurance program: characterizing individual participant measurement performance over time.

The mission of the Micronutrients Measurement Quality Assurance Program (M2QAP) at the National Institute of Standards and Technology is enhanced interlaboratory measurement comparability for fat-soluble vitamin-related measurands in human serum. We recently described improved tools for evaluating individual participant measurement performance in single interlaboratory comparison exercises; we here apply and extend these tools to the evaluation of participant performance over the entire 15-year history of the M2QAP. We describe and illustrate a set of interconnected graphical reporting tools for identifying long-term trends and single-exercise events. We document and discuss recurrent patterns we observe in the measurement performance characteristics for M2QAP participants. The graphical analysis techniques utilized may be applicable to other interlaboratory comparison programs.

Graphical tools for RFLP measurement quality assurance: laboratory performance charts.

Forensic restriction fragment length polymorphism analyses typically provide two band size results at each genetic locus for each sample. In collaboration with the member laboratories of the Technical Working Group for DNA Analysis Methods, we have developed graphical techniques that compactly summarize even large numbers of such paired measurements. This paper provides a detailed description of the basic tool, a modified bivariate control chart for data from multiple samples and/or multiple loci. We demonstrate how various modifications and combinations of these "laboratory performance charts" can be used for quality control, quality assurance, and quality demonstration.

Micronutrients Measurement Quality Assurance Program: helping participants use interlaboratory comparison exercise results to improve their long-term measurement performance.

Over the past decade, the Micronutrients Measurement Quality Assurance Program (M2QAP) at the National Institute of Standards and Technology (NIST) has administered nearly 40 interlaboratory comparison exercises devoted to fat-soluble vitamin-related analytes in human serum. While M2QAP studies have been used to help certify reference materials and to document the performance of analytical systems, the primary focus of the M2QAP has been, and remains, the improvement of among-participant measurement comparability for target analytes. Recent analysis of historical measurement performance indicated the most efficient mechanism for further improving measurement comparability among participants is the improvement of long-term (months to years) comparability within each laboratory. The summary reports for the M2QAP studies are being redesigned to provide more chemist-friendly analyses of participant performance, dissecting systematic and random components of measurement incomparability as functions of analyte level and time. This report documents the semantic and graphical tools developed to help interlaboratory-comparison-exercise participants interpret their own measurement performance.

The stability of retinol, alpha-tocopherol, trans-lycopene, and trans-beta-carotene in liquid-frozen and lyophilized serum.

The concentrations of retinol, alpha-tocopherol, and trans-beta-carotene in lyophilized serum stored at -25 degrees C and -80 degrees C have been monitored for 10 years. There was no evidence of degradation of any of these compounds over the 10-year period. Retinol, alpha-tocopherol, and trans-beta-carotene were less stable at -25 degrees C in liquid-frozen serum than they were in lyophilized serum. At -80 degrees C, trans-beta-carotene levels were stable for up to 3 years of storage in liquid-frozen serum. Both retinol and alpha-tocopherol appeared stable in liquid-frozen serum for at least 5 years at -80 degrees C. The effect of repeated freeze/thaw cycles on retinol, alpha-tocopherol, trans-lycopene, and trans-beta-carotene in liquid-frozen and reconstituted lyophilized serum both stored at -20 degrees C was also studied. Retinol, alpha-tocopherol, trans-lycopene, and trans-beta-carotene in reconstituted lyophilized serum stored at -20 degrees C were stable for at least 3 days with minimal (< 5) freeze/thaw cycles.

Interlaboratory comparison of autoradiographic DNA profiling measurements: precision and concordance.

Knowledge of the expected uncertainty in restriction fragment length polymorphism (RFLP) measurements is required for confident exchange of such data among different laboratories. The total measurement uncertainty among all Technical Working Group for DNA Analysis Methods laboratories has previously been characterized and found to be acceptably small. Casework cell line control measurements provided by six Royal Canadian Mounted Police (RCMP) and 30 U.S. commercial, local, state, and Federal forensic laboratories enable quantitative determination of the within-laboratory precision and among-laboratory concordance components of measurement uncertainty typical of both sets of laboratories. Measurement precision is the same in the two countries for DNA fragments of size 1000 base pairs (bp) to 10,000 bp. However, the measurement concordance among the RCMP laboratories is clearly superior to that within the U.S. forensic community. This result is attributable to the use of a single analytical protocol in all RCMP laboratories. Concordance among U.S. laboratories cannot be improved through simple mathematical adjustments. Community-wide efforts focused on improved concordance may be the most efficient mechanism for further reduction of among-laboratory RFLP measurement uncertainty, should the resources required to fully evaluate potential cross-jurisdictional matches become burdensome as the number of RFLP profiles on record increases.

Products of partial digestion with Hae III. Part 1. Characterization, casework experience and confirmation of the theory of three-, four- and five-banded RFLP pattern origins using partial digestion.

The sizes of Hae III partial digestion products at D1S7, D2S44, D4S139, D5S110, D10S28, and D17S26 were evaluated in experimentally generated partial digestions of liquid blood DNA. The partial digestion products were highly predictable, suggesting a very high level of sequence conservation in regions flanking variable number tandem repeat (VNTR) blocks. Partial digestion bands associated with three-or-more-banded patterns were also characterized. Partial digestion of three-banded patterns can be used to determine whether the extra bands arise due to internal Hae III sites in the VNTR block and to identify hidden three-banded patterns. Partial digestion products from forensic casework also conformed to size expectations. Presumed partial digestion bands from 27 forensic samples were compared to the experimentally generated data. The causes of partial digestion are examined and recommendations for interpreting forensic DNA evidence exhibiting partial digestion products are given.

Products of partial digestion with Hae III. Part 2. Quantification.

The base pair size of the excess DNA in the smallest three partial digestion bands for the variable number of tandem repeat loci D1S7, D2S44, D4S139, D5S110, D10S28, and D17S26 has been quantitatively evaluated using data obtained from intentional partial digestion of liquid blood DNA. Restriction fragment length polymorphism (RFLP) measurement characteristics specific to the performing laboratory were evaluated from that laboratory's historical K562 cell line control data. The expected size of the excess DNA is estimated as the weighted mean of the differences between the measured size of the partially digested bands and the fully digested band, with the weights predicted using knowledge of RFLP measurement characteristics. Confidence limits are developed for evaluating whether the size differences among a set of RFLP band multiplets observed in pristine samples are consistent with those expected from partial digestion. The base pair size of excess DNA for partials observed in evidentiary samples appears to be somewhat less than that from pristine samples.

Interlaboratory evaluation of short tandem repeat triplex CTT.

An interlaboratory comparison of typing results for Short Tandem Repeats (STRs) at the GenBank loci HUMCSF1PO, HUMTPOX, HUMTH01, and HUMVWFA31 using the "CTT triplex" and "CTTv quadruplex" has been evaluated. These STRs all have a nominal four basepair (bp) repeat. Seven different samples were distributed to 41 laboratories. The 34 laboratories that returned results used a wide variety of analytical systems. Comparable results were obtained for all samples at all loci when results were reported as an allelic name. Raw sizing results obtained from internal-lane sizing standards differed by nearly five bp at some loci. Many different factors contribute to this observed sizing variability, including choice of sizing standards and matrix composition. Although sizing results can be made more comparable by locus-specific offsets or calibration to a comprehensive set of alleles at each locus, samples typed to the allelic name can now be validly compared regardless of analytical method. Interlaboratory comparison of raw allelic size remains problematic.

Interlaboratory comparison of autoradiographic DNA profiling measurements. 4. Protocol effects.

The observed total interlaboratory uncertainty in restriction fragment length polymorphism (RFLP) measurements is sufficiently small to be of little significance given current forensic needs. However, as the number of RFLP data increase, further reduction in the total uncertainty could help minimize the resources required to evaluate potential profile matches. The large number of data available enable quantitative estimation of the within-laboratory imprecision and among-laboratory bias contributions to the total uncertainty. Some small but consistent among-laboratory measurement biases can be attributed to specific procedural or materials differences. The bias direction is often fragment-specific and thus unpredictable for unknown samples. Actions that would minimize currently recognized sources of interlaboratory bias include the following: (1) all laboratories should use the same algorithm for data interpolation, (2) all laboratories should use the same sizing ladders, (3) each laboratory should prepare control DNA and sample DNA in the same manner and with the identical reagents, (4) all laboratories should adopt a uniform policy on ethidium bromide use, and (5) all laboratories should adopt the same control DNA sizing acceptability criteria.

NIST/NCI Micronutrients Measurement Quality Assurance Program: measurement repeatabilities and reproducibilities for fat-soluble vitamin-related compounds in human sera.

The NIST/NCI Micronutrient Measurement Quality Assurance Program has conducted 33 interlaboratory comparison exercises for fat-soluble vitamin-related compounds in human sera over the past 12 years. Periodic reanalysis of lyophilized serum samples prepared from more than 70 different sera has enabled estimation of the short- and long-term measurement characteristics. Median- and interquartile-range-based statistics adequately estimate the distribution of results from laboratories that are in analytical control from total distributions that include a significant minority of outlier data. Short-term interlaboratory reproducibility standard deviations (SDs) are predictable functions of analyte concentration, with an asymptotic limit at low analyte concentration and a linear relationship at high concentrations. Long-term trends in the interlaboratory reproducibility can be estimated by standardizing the short-term SD at the observed analyte concentration to an expected SD at a given physiologically significant analyte concentration. The "average" laboratory's same-day analytical repeatability SD is about one-third of the estimated interlaboratory reproducibility; repeatability for longer periods between analyses is, on average, on better than the reproducibility. While a few exceptional laboratories have maintained excellent repeatability over the entire decade, long-term study measurements generated within a single laboratory are not generally more internally consistent than results from multiple laboratories. Enhanced and more consistently implemented intralaboratory quality control and quality assurance methods are required to further improve and maintain interlaboratory measurement comparability.

Certification of fat-soluble vitamins, carotenoids, and cholesterol in human serum: Standard Reference Material 968b.

In Standard Reference Material 968b, fat-soluble vitamins and cholesterol in human serum, certified values are provided for cholesterol, retinol, retinyl palmitate, alpha-tocopherol, trans-beta-carotene, total beta-carotene ( trans plus cis isomers), total alpha-carotene, and lutein. Non-certified values are also reported for gamma-tocopherol (includes beta-tocopherol), delta-tocopherol, zeaxanthin, beta-cryptoxanthin, trans-lycopene, trans-lycopene, trans-alpha-carotene, total lycopene, 9- cis-betacarotene, 13- plus 15- cis-beta-carotene, and 15- cis-beta-carotene. Both certified and non-certified values are based on the agreement among results from three different liquid chromatographic analytical procedures developed at NIST and from an interlaboratory comparison exercise among institutions that participate in a NIST-managed Micronutrients Measurement Quality Assurance Program. Cholesterol is certified in this material using the NIST isotope dilution/mass spectrometric definitive method.