Hua-Jun He

Human CD4+ lymphocytes for antigen quantification: Characterization using conventional flow cytometry and mass cytometry

To transform the linear fluorescence intensity scale obtained with fluorescent microspheres to an antibody bound per cell (ABC) scale, a biological cell reference material is needed. Optimally, this material should have a reproducible and tight ABC value for the expression of a known clinical reference biomarker. In this study, we characterized commercially available cryopreserved peripheral blood mononuclear cells (PBMCs) and two lyophilized PBMC preparations, Cyto‐Trol and PBMC–National Institute for Biological Standard and Control (NIBSC) relative to freshly prepared PBMC and whole blood samples. It was found that the ABC values for CD4 expression on cryopreserved PBMC were consistent with those of freshly obtained PBMC and whole blood samples. By comparison, the ABC value for CD4 expression on Cyto‐Trol is lower and the value on PBMC–NIBSC is much lower than those of freshly prepared cell samples using both conventional flow cytometry and CyTOF™ mass cytometry. By performing simultaneous surface and intracellular staining measurements on these two cell samples, we found that both cell membranes are mostly intact. Moreover, CD4+ cell diameters from both lyophilized cell preparations are smaller than those of PBMC and whole blood. This could result in steric interference in antibody binding to the lyophilized cells. Further investigation of the fixation effect on the detected CD4 expression suggests that the very low ABC value obtained for CD4+ cells from lyophilized PBMC–NIBSC is largely due to paraformaldehyde fixation; this significantly decreases available antibody binding sites. This study provides confirmation that the results obtained from the newly developed mass cytometry are directly comparable to the results from conventional flow cytometry when both methods are standardized using the same ABC approach. Published 2012 Wiley Periodicals, Inc.

Breast cancer biomarker measurements and standards.

Cancer is a heterogeneous disease characterized by changes in the levels and activities of important cellular proteins, including oncogenes and tumor suppressors. Genetic mutations cause changes in protein activity and protein expression levels that result in the altered metabolism, proliferation, and metastasis seen in cancer cells. The identification of the critical biochemical changes in cancer has led to advances in its detection and treatment. An important example of this is the measurement of human epidermal growth factor receptor 2 (HER2), where increased expression occurs in approximately 20–30% of breast cancer tumors. HER2 is a member of the epidermal growth factor receptor family and is an important biomarker expressed on the cell surface. Measurement of the HER2 levels in tumor cells provides diagnostic, prognostic, and treatment information, because a targeted therapeutic is available. The most common methods to measure HER2 levels are immunohistochemistry and in situ hybridization assays. The accurate and reliable measurements of the specific changes in protein biomarkers for detection and treatment of cancer are important challenges. This review is focused on efforts to improve the quantitation and reliability of cancer biomarkers by using standards and reference materials.

Fluorescence resonance energy transfer-based method for detection of DNA binding activities on nuclear factor κB

The DNA binding protein nuclear factor κB (NF-κB) and the cellular signaling pathways in which it participates are the central coordinators of many biological processes, including innate and adapti...

Development of a candidate secondary reference procedure (immunoassay based measurement procedure of higher metrological order) for cardiac troponin I: I. Antibody characterization and preliminary validation

Abstract In this study, the first steps in the development of a secondary reference measurement procedure (RMP) ‘higher metrological order measurement procedure’ to support the cardiac troponin I (cTnI) standardization initiative is described. The RMP should be used to assign values to serum-based secondary reference materials (RMs) without analytical artifacts causing bias. A multiplexed bead-based assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to identify the optimum monoclonal antibody pair (clones 560 and 19C7) for the RMP. Using these antibodies, an ELISA-based procedure was developed to accurately measure the main cTnI forms present in blood. The proposed RMP appears to show no bias when tested on samples containing various troponin complexes, phosphorylated and dephosphorylated forms, and heparin. The candidate assay displayed suitable linearity and sensitivity (limit of detection, 0.052 μg/L) for the measurement of the proposed cTnI secondary RMs. Preliminary comparison data on patient samples with a commercial cTnI assay are also provided to support the suitability of RMP for value assignment to RMs. Full validation and final assessment of the RMP will be performed through transferability and inter-comparison studies. Clin Chem Lab Med 2010;48:1603–10.

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

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.

Quantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometry

BackgroundIn our previous study that characterized different human CD4+ lymphocyte preparations, it was found that both commercially available cryopreserved peripheral blood mononuclear cells (PBMC) and a commercially available lyophilized PBMC (Cyto-Trol™) preparation fulfilled a set of criteria for serving as biological calibrators for quantitative flow cytometry. However, the biomarker CD4 protein expression level measured for T helper cells from Cyto-Trol was about 16% lower than those for cryopreserved PBMC and fresh whole blood using flow cytometry and mass cytometry. A primary reason was hypothesized to be due to steric interference in anti- CD4 antibody binding to the smaller sized lyophilized control cells.MethodTargeted multiple reaction monitoring (MRM) mass spectrometry (MS) is used to quantify the copy number of CD4 receptor protein per CD4+ lymphocyte. Scanning electron microscopy (SEM) is utilized to assist searching the underlying reasons for the observed difference in CD4 receptor copy number per cell determined by MRM MS and CD4 expression measured previously by flow cytometry.ResultsThe copy number of CD4 receptor proteins on the surface of the CD4+ lymphocyte in cryopreserved PBMCs and in lyophilized control cells is determined to be (1.45 ± 0.09) × 105 and (0.85 ± 0.11) × 105, respectively, averaged over four signature peptides using MRM MS. In comparison with cryopreserved PBMCs, there are more variations in the CD4 copy number in lyophilized control cells determined based on each signature peptide. SEM images of CD4+ lymphocytes from lyophilized control cells are very different when compared to the CD4+ T cells from whole blood and cryopreserved PBMC.ConclusionBecause of the lyophilization process applied to Cyto-Trol control cells, a lower CD4 density value, defined as the copy number of CD4 receptors per CD4+ lymphocyte, averaged over three different production lots is most likely explained by the loss of the CD4 receptors on damaged and/or broken microvilli where CD4 receptors reside. Steric hindrance of antibody binding and the association of CD4 receptors with other biomolecules likely contribute significantly to the nearly 50% lower CD4 receptor density value for cryopreserved PBMC determined from flow cytometry compared to the value obtained from MRM MS.

Quantifying the Cluster of Differentiation 4 Receptor Density on Human T Lymphocytes Using Multiple Reaction Monitoring Mass Spectrometry

Cluster of differentiation 4 (CD4) is an important glycoprotein containing four extracellular domains, a transmembrane portion and a short intracellular tail. It locates on the surface of various types of immune cells and performs a critical role in multiple cellular functions such as signal amplification and activation of T cells. It is well-known as a clinical cell surface protein marker for study of HIV progression and for defining the T helper cell population in immunological applications. Moreover, CD4 protein has been used as a biological calibrator for quantification of other surface and intracellular proteins. However, flow cytometry, the conventional method of quantification of the CD4 density on the T cell surface depends on antibodies and has suffered from variables such as antibody clones, the fluorophore and conjugation chemistries, the fixation conditions, and the flow cytometric quantification methods used. In this study, we report the development of a highly reproducible nano liquid chromatography-multiple reaction monitoring mass spectrometry-based quantitative method to quantify the CD4 receptor density in units of copy number per cell on human CD4+ T cells. The method utilizes stable isotope-labeled full-length standard CD4 as an internal standard to measure endogenous CD4 directly, without the use of antibodies. The development of the mass spectrometry-based approach of CD4 protein quantification is important as a complementary strategy to validate the analysis from the cytometry-based conventional method. It also provides new support for quantitative understanding and advanced characterization of CD4 on CD4+ T cells.

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

NIST standard reference material (SRM) 2373 was developed to improve the measurements of the HER2 gene amplification in DNA samples. SRM 2373 consists of genomic DNA extracted from five breast cancer cell lines with different amounts of amplification of the HER2 gene. The five components are derived from the human cell lines SK-BR-3, MDA-MB-231, MDA-MB-361, MDA-MB-453, and BT-474. The certified values are the ratios of the HER2 gene copy numbers to the copy numbers of selected reference genes DCK, EIF5B, RPS27A, and PMM1. The ratios were measured using quantitative polymerase chain reaction and digital PCR, methods that gave similar ratios. The five components of SRM 2373 have certified HER2 amplification ratios that range from 1.3 to 17.7. The stability and homogeneity of the reference materials were shown by repeated measurements over a period of several years. SRM 2373 is a well characterized genomic DNA reference material that can be used to improve the confidence of the measurements of HER2 gene copy number.

Removal of Inhibitory Effects in a Serum Cardiac Troponin I Immunoassay

The measurement of cardiac troponin I (cTnI)1 has become the gold standard for the clinical diagnosis of myocardial infarction (1). Varieties of commercial sandwich-type immunoassays are used for the measurement of cTnI concentration in human serum or plasma. Several of these assays use pairs of monoclonal antihuman cTnI antibodies with high specificities for the invariant part of the cTnI molecule (amino acid residues 30–110), in accordance with the recommended guidelines (2). These assays are often based on magnetic beads or latex particles to take advantage of the ease of bead/particle washing, which can minimize assay interference by matrix proteins. Signal amplification by means of chemiluminescence or electrochemiluminescence allows the detection of serum cTnI at concentrations <0.05 μg/L. As a part of the recent effort of the IFCC Working Group for Standardization of Troponin I to develop a cTnI reference measurement system (1), we have used various measurement techniques to investigate binding affinities between 6 monoclonal antibodies (mAbs) obtained from HyTest (each serving as either the capture or the detection antibody) and cTnI, in the form of either a reference material (NIST SRM 2921) or a cTnI-positive serum pool (PS). One of the techniques is the multiplexed bead array (3), which resembles the commercial bead- or particle-based immunoassays. The capture mAb molecules are covalently immobilized on different bead populations to capture cTnI. The bound cTnI …

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.