Development of NIST standard reference material 2373: Genomic DNA standards for HER2 measurements

He, Hua‐Jun; Almeida, Jamie L.; Lund, Steven P.; Steffen, Carolyn R.; Choquette, Steven J.; Cole, Kenneth D.

doi:10.1016/j.bdq.2016.02.001

Cited by 24 publications

(28 citation statements)

References 31 publications

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“…These assays were developed for the certification of NIST cancer biomarker standards (23,24). The calculation of copy number measurements from droplet digital PCR requires an accurate measurement of the droplet size.…”

Section: Resultsmentioning

confidence: 99%

Limitations of methods for measuring the concentration of human genomic DNA and oligonucleotide samples

Stein

DeRose

et al. 2018

BioTechniques

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We compared different methods (absorbance, fluorescent dye-binding, and digital PCR) for measuring the concentrations of human genomic DNA from cultured cells and absorbance measurements of a synthetic DNA oligonucleotide. NIST Standard Reference Material (SRM) 2082, a pathlength absorbance standard, was used to benchmark the absorbance measurements done with microvolume spectrophotometers and a microvolume plate reader. Control absorbance values were measured on a high accuracy spectrophotometer and a NIST calibrated pathlength cuvette. Measurements of the human genomic DNA sample were done with several types of fluorescent dye binding assays using different DNA calibrators. The fluorescent dye binding methods gave different results for genomic DNA depending upon the type of DNA calibrator and the fluorescent dye that was used. The human genomic DNA sample was also characterized by using six different droplet digital PCR assays (amplicons located on different chromosomes) to measure the average copy number. Conversion of the digital PCR data to copy numbers was sensitive to the droplet size used for calculations and conversion to mass concentration was dependent upon the molecular weight of the human genome used for the calculations. The results from the different methods were compared and the caveats for each measurement method were discussed.

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Section: Resultsmentioning

confidence: 99%

Limitations of methods for measuring the concentration of human genomic DNA and oligonucleotide samples

Stein

DeRose

et al. 2018

BioTechniques

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“…Details regarding the certification of NIST SRM 2373 are available from the certificate of analysis and an article describing the development and characterization of SRM 2373. 23 The cell lines were obtained from the American Type Culture Collection (ATCC) (Manassas, VA) and grown in the NIST laboratories using the recommended culture conditions. The breast cancer cell lines have different sources, hormone sensitivity, 24 and karyotypes 25e28 ( Table 1).…”

Section: Preparation Of Cell Linesmentioning

confidence: 99%

“…The DNA samples were treated with bovine pancreatic ribonuclease A before purification to remove the enzyme. 23 All purified genomic DNA samples were prepared in buffer (10 mmol/L Tris, 0.1 mmol/L EDTA, pH 8.0) and stored at 4 C. The cell line DNA was authenticated using the AmpFLSTR Identifiler Plus PCR Amplification Kit (catalog number 4427368; Life Technologies, Carlsbad, CA) on a 3500xl Genetic Analyzer with a 36-cm capillary array and POP-4 polymer (Life Technologies). The DNA samples were analyzed both when received from the repository and when the cells were expanded to produce the genomic DNA used for the production of SRM 2373.…”

Section: Dna Extraction Identity Authentication and Reference Matermentioning

confidence: 99%

Certified DNA Reference Materials to Compare HER2 Gene Amplification Measurements Using Next-Generation Sequencing Methods

Lih

Das

et al. 2016

The Journal of Molecular Diagnostics

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The National Institute of Standards and Technology (NIST) Standard Reference Materials 2373 is a set of genomic DNA samples prepared from five breast cancer cell lines with certified values for the ratio of the HER2 gene copy number to the copy numbers of reference genes determined by real-time quantitative PCR and digital PCR. Targeted-amplicon, whole-exome, and whole-genome sequencing measurements were used with the reference material to compare the performance of both the laboratory steps and the bioinformatic approaches of the different methods using a range of amplification ratios. Although good reproducibility was observed in each next-generation sequencing method, slightly different HER2 copy numbers associated with platform-specific biases were obtained. This study clearly demonstrates the value of Standard Reference Materials 2373 as reference material and as a calibrator for evaluating assay performance as well as for increasing confidence in reporting HER2 amplification for clinical applications.

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“…A call for the development of a cell line HER-2 measurement standard in 2003 19 led to a HER-2 genomic DNA standard this year (2016) 20,21 but, at least so far, no HER-2 protein standard. Other descriptions regarding the use of cell lines in IHC testing, sometimes in the context of proficiency testing, often do not characterize cell lines in terms of the number of HER-2 molecules per cell.…”

Section: Discussionmentioning

confidence: 99%

Analytic Response Curves of Clinical Breast Cancer IHC Tests

Vani

Sompuram

Schaedle

et al. 2017

J Histochem Cytochem.

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An important limitation in the field of immunohistochemistry (IHC) is the inability to correlate stain intensity with specific analyte concentrations. Clinical immunohistochemical tests are not described in terms of analytic response curves, namely, the analyte concentrations in a tissue sample at which an immunohistochemical stain (1) is first visible, (2) increases in proportion to the analyte concentration, and (3) ultimately approaches a maximum color intensity. Using a new immunostaining tool ( IHControls), we measured the analytic response curves of the major clinical immunohistochemical tests for human epidermal growth factor receptor type II (HER-2), estrogen receptor (ER), and progesterone receptor (PR). The IHControls comprise the analytes HER-2, ER, and PR at approximately log concentration intervals across the range of biological expression, from 100 to 1,000,000 molecules per test microbead. We stained IHControls of various concentrations using instruments, reagents, and protocols from three major IHC vendors. Stain intensity at each analyte concentration was measured, thereby generating an analytic response curve. We learned that for HER-2 and PR, there is significant variability in test results between clinical kits for samples with analyte concentrations of approximately 10 molecules/microbead. We propose that the characterization of immunostains is an important step toward standardization.

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Development of NIST standard reference material 2373: Genomic DNA standards for HER2 measurements

Cited by 24 publications

References 31 publications

Limitations of methods for measuring the concentration of human genomic DNA and oligonucleotide samples

Limitations of methods for measuring the concentration of human genomic DNA and oligonucleotide samples

Certified DNA Reference Materials to Compare HER2 Gene Amplification Measurements Using Next-Generation Sequencing Methods

Analytic Response Curves of Clinical Breast Cancer IHC Tests

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