Development and Validation of Measurement Traceability for In Situ Immunoassays

Background Immunoassays for protein analytes measured in situ support a $2 billion laboratory testing industry that suffers from significant interlaboratory disparities, affecting patient treatment. The root cause is that immunohistochemical testing lacks the generally accepted tools for analytic standardization, including reference standards and traceable units of measure. Until now, the creation of these tools has represented an insoluble technical hurdle. Methods We address the need with a new concept in metrology—that is, linked traceability. Rather than calculating analyte concentration directly, which has proven too variable, we calculate concentration by measuring an attached fluorescein, traceable to NIST Standard Reference Material 1934, a fluorescein standard. Results For validation, newly developed estrogen receptor (ER) calibrators were deployed in tandem with an array of 80 breast cancer tissue sections in a national external quality assessment program. Laboratory performance was assessed using both the ER standards and the tissue array. Similar to previous studies, the tissue array revealed substantial discrepancies in ER test results among the participating laboratories. The new ER calibrators revealed a broad range of analytic sensitivity, with the lower limits of detection ranging from 7310 to 74 790 molecules of ER. The data demonstrate, for the first time, that the variable test results correlate with analytic sensitivity, which can now be measured quantitatively. Conclusions The reference standard enables precise interlaboratory alignment of immunohistochemistry test sensitivity for measuring cellular proteins in situ. The introduction of a reference standard and traceable units of measure for protein expression marks an important milestone.

Comparison of the Fatty Acid Composition of Peanut Butter by Applying Different Fat Extraction Procedures

The aim of this work was to evaluate the influence of different fat extraction procedures on the fatty acids composition (FAs) of a NIST standard reference material, peanut butter (SRM�2387). Extraction of fat was made with petroleum ether by applying six extraction procedures: Soxhlet automatic with B�chi B-811 unit: Soxhlet standard (SS); Soxhlet warm (SW); Hot extraction (HE) and Continuous flow (CF), repeated extraction with solvent (SR) and Soxhlet traditional extraction (ST), by using a Soxhlet extractor. Identification of FAMEs was based on retention time (RT) and the ratio of characteristic ions (m/z) of the reference standards F.A.M.E. Mix C4-C24 and SRM�2377. FAMEs determination was realized by using internal standards (IS) and applying relative response factors (RRFi), and without IS by applying correction factors (Fi). Determination of FAMEs was performed on a GC-MS. Triglyceride of pentadecanoic acid (TAG-IS C15:0) was used as an IS to assess the analyte losses during FAMEs preparation steps, and tricosanoic acid methyl ester (FAME-IS C23:0) was used to evaluate the analyte losses on GC injection. Values of fat content and fatty acids composition determined by the 6 extraction procedures were compared with the NIST certified values of SRM�2387. All 4 procedures of extraction made with B�chi unit B-811 were effective in fat extraction and analysis of fatty acids composition compared to standard methods. Between the experimentally determined values and the certified values there were no significant differences.

Glassy carbon, NIST Standard Reference Material (SRM 3600): hydrogen content, neutron vibrational density of states and heat capacity

Commercial glassy carbon plates being used as absolute intensity calibration standards in small-angle X-ray scattering applications (NIST SRM 3600) have been characterized in several recent publications. This contribution adds to the characterization by measuring the hydrogen content of a plate to be (4.8 ± 0.2) × 10−4 (mol H)/(mol C), and by measuring the vibrational spectrum by neutron inelastic scattering. The spectrum bears a strong resemblance to published measurements on graphite, allowing the identification of several spectral features. The measured spectrum is used to calculate the heat capacity of low-hydrogen-content glassy carbon for comparison with measurements reported here from 20 to 295 K.

Development of NIST Standard Reference Material 2082, a Pathlength Standard for Measurements in the Ultraviolet Spectrum

New spectrophotometers and cuvettes have been designed to allow the measurement of absorbance values from samples using microliter volume sizes. These measurements are done using short pathlengths to decrease the sample volumes required. The major applications for these spectrophotometers and cuvettes are samples that are difficult to obtain in large amounts, such as proteins and nucleic acids that absorb light in the ultraviolet range. Existing ultraviolet absorbance standards have been designed for longer pathlength measurements. Standard Reference Material (SRM) 2082 was developed to validate the pathlengths of short-pathlength cuvettes and instruments using materials with absorbance spectra that are similar to the most commonly used samples. SRM 2082 consists of three individual components: a blank buffer solution, a solution of the amino acid tryptophan in the buffer, and a solution of the nucleobase uracil in the buffer. The tryptophan solution has an absorbance spectrum (peak at 280 nm) similar to proteins, and the uracil has an absorbance spectrum (peak at 260 nm) similar to nucleic acids. The absorbance values of these solutions were determined using a series of cuvettes with pathlengths from 0.1 mm to 2 mm. The pathlengths of the cuvettes used for the absorbance measurements were determined at the National Institute of Standards and Technology by physical and optical measurements. The effects of temperature and spectral bandwidth variations on the absorbance values of SRM 2082 were also investigated.

NIST Standard Reference Material 3600: Absolute Intensity Calibration Standard for Small-Angle X-ray Scattering

The certification of a new standard reference material for small-angle scattering [NIST Standard Reference Material (SRM) 3600: Absolute Intensity Calibration Standard for Small-Angle X-ray Scattering (SAXS)], based on glassy carbon, is presented. Creation of this SRM relies on the intrinsic primary calibration capabilities of the ultra-small-angle X-ray scattering technique. This article describes how the intensity calibration has been achieved and validated in the certifiedQrange,Q= 0.008–0.25 Å−1, together with the purpose, use and availability of the SRM. The intensity calibration afforded by this robust and stable SRM should be applicable universally to all SAXS instruments that employ a transmission measurement geometry, working with a wide range of X-ray energies or wavelengths. The validation of the SRM SAXS intensity calibration using small-angle neutron scattering (SANS) is discussed, together with the prospects for including SANS in a future renewal certification.