Jiehong Xie

Multicolor flow cytometry analysis of the proliferations of T-lymphocyte subsets in vitro by EdU incorporation.

EdU (5‐ethynyl‐2′‐deoxyuridine) incorporation has proved advantageous in the studies of cell kinetics, DNA synthesis, and cellular proliferation in vitro and in vivo compared to [3H]thymidine incorporation and BrdU (5‐bromo‐2′‐deoxyuridine) incorporation. Here, we describe a method that combines EdU incorporation and immunostaining with flow cytometric analysis to detect the proliferations of T lymphocyte subsets in vitro and optimized the assays conditions. We found that the number of EdU+ cells were associated with EdU concentration, incubation time, and the volume of Click reaction solution, the best EdU concentration 10–50 μM, the optimal incubation time 8–12 h and the proper volume of Click volume 100 μl for labeling 1 × 106 lymphocytes. Fixation was better to be performed before permeabilization, not together with. Furthermore, the permeabilization detergent reagent, PBS with 0.05% saponin was better than Tris buffer saline (TBS) with 0.1% Triton X‐100. In addition, sufficient wash with PBS with 0.05% saponin has no influence on the staining of EdU+ cells. Also, the lymphocytes incorporating EdU could be stored at 4°C, −80°C, and in liquid nitrogen up to 21 days. The present study will aid in optimization of flow cytometry assay to detect the proliferations of T cell subsets by EdU incorporation and the labeling of cell surface antigens.

Using a novel microRNA delivery system to inhibit osteoclastogenesis.

Previously, we developed a novel microRNA (miRNA) delivery system based on bacteriophage MS2 virus-like particles (MS2 VLPs). In this current study, we used this system to transport miR-146a into human peripheral blood mononuclear cells (PBMCs), and demonstrated the inhibition of osteoclastogenesis in precursors. Two cytokines, receptor activator of NF-κB ligand (RANKL), and macrophage-colony stimulating factor (M-CSF) were used to induce osteoclastogenesis. MS2 VLPs were transfected into PBMCs. qRT-PCR was applied to measure expression levels of miR-146a and osteoclast (OC)-specific genes. Western blot (WB) was conducted to evaluate miR-146a downstream target proteins: epidermal growth factor receptor (EGFR) and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6). The formation and activity of OCs were assessed by cytochemical staining and bone resorption assay, respectively. In PBMCs treated with MS2-miR146a VLPs, qRT-PCR assays showed increased expression of miR-146a (p < 0.01) and decreased expression of all four OC-specific genes (p < 0.05). WB results indicated decreased expression of EGFR (p < 0.01) and TRAF6 (p < 0.05). The number of OCs decreased markedly and bone resorption assay demonstrated inhibited activity. This miR-146a delivery system could be applied to induce overexpression of miR-146a and to inhibit the differentiation and function of OCs.

Evaluation of an individual-donation nucleic acid amplification testing algorithm for detecting hepatitis B virus infection in Chinese blood donors

This multicenter study was performed to evaluate the efficiency of a multiplex individual‐donation nucleic acid amplification technology (ID‐NAT) and discriminatory testing algorithm for detecting hepatitis B virus (HBV) infection in Chinese blood donors.

A New Type of Natural Bispecific Antibody With Potential Protective Effect in Hashimoto Thyroiditis

CONTEXT As a new antibody concept, natural bispecific antibodies (nBsAbs) have been detected in long-term passive immunization and some diseases, but their potential immunomodulatory role remains unclear. Hashimoto thyroiditis (HT) appears to fulfill the condition for nBsAb production but has not yet been characterized. OBJECTIVE The objective of the study was to identify a new nBsAb against thyroid peroxidase (TPO) and thyroglobulin (Tg) in HT patients and to preliminarily explore its immunomodulatory role. DESIGN, SETTING, AND PATIENTS Serum samples were obtained from 136 HT patients, 92 diseased controls, and 99 healthy controls for anti-TPO/Tg nBsAb detection. The relationship between anti-TPO/Tg nBsAb and other clinical parameters was also analyzed. MAIN OUTCOME MEASURES The anti-TPO/Tg nBsAb was detected using a double-antigen sandwich ELISA. Higher nBsAb levels were found to be associated with decreased inflammation in HT patients. RESULTS The prevalence of anti-TPO/Tg nBsAb in HT was 44.9% (61 of 136), significantly higher than that of diseased controls (2.2%, 2 of 92) (P < .0001) and healthy controls (0%, 0 of 99) (P < .0001). HT patients who were nBsAb positive were prone to have significantly lower levels of serum C-reactive protein and TNF-α compared with the nBsAb-negative individuals (P < .05). The serum amyloid A and interferon-γ levels also showed a similar trend in the two groups. The IgG subclass of anti-TPO/Tg nBsAb was IgG4. Further analysis showed a negative correlation between anti-TPO/Tg nBsAb and serum total IgG4 (r = -0.697, P = .025) in IgG4 thyroiditis patients. CONCLUSIONS A new type of nBsAb against TPO and Tg in HT patients is identified. Our data also indicate a protective effect of anti-TPO/Tg nBsAb in the pathogenesis of HT and extend prior knowledge about nBsAb in diseases.

External Quality Assessment for the Detection of Measles Virus by Reverse Transcription-PCR Using Armored RNA

In recent years, nucleic acid tests for detection of measles virus RNA have been widely applied in laboratories belonging to the measles surveillance system of China. An external quality assessment program was established by the National Center for Clinical Laboratories to evaluate the performance of nucleic acid tests for measles virus. The external quality assessment panel, which consisted of 10 specimens, was prepared using armored RNAs, complex of noninfectious MS2 bacteriophage coat proteins encapsulated RNA of measles virus, as measles virus surrogate controls. Conserved sequences amplified from a circulating measles virus strain or from a vaccine strain were encapsulated into these armored RNAs. Forty-one participating laboratories from 15 provinces, municipalities, or autonomous regions that currently conduct molecular detection of measles virus enrolled in the external quality assessment program, including 40 measles surveillance system laboratories and one diagnostic reagent manufacturer. Forty laboratories used commercial reverse transcription-quantitative PCR kits, with only one laboratory applying a conventional PCR method developed in-house. The results indicated that most of the participants (38/41, 92.7%) were able to accurately detect the panel with 100% sensitivity and 100% specificity. Although a wide range of commercially available kits for nucleic acid extraction and reverse transcription polymerase chain reaction were used by the participants, only two false-negative results and one false-positive result were generated; these were generated by three separate laboratories. Both false-negative results were obtained with tests performed on specimens with the lowest concentration (1.2 × 104 genomic equivalents/mL). In addition, all 18 participants from Beijing achieved 100% sensitivity and 100% specificity. Overall, we conclude that the majority of the laboratories evaluated have reliable diagnostic capacities for the detection of measles virus.

Improvements in CYP2C9 Genotyping Accuracy Are Needed: A Report of the First Proficiency Testing for Warfarin-related CYP2C9 and VKORC1 Genotyping in China.

Abstract: Warfarin is the most commonly used oral anticoagulant in clinical practice. The cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex 1 (VKORC1) genotypes have been confirmed to be associated with warfarin dose requirements. Accurate genotyping results are of particular importance for obtaining reliable genotype-guided warfarin dosing information. This study aims to determine analytic performance of laboratories offering CYP2C9 and VKORC1 testing in China. A proficiency panel of 15 validated cell samples covering common CYP2C9 and VKORC1 genetic polymorphisms was provided to 31 participating laboratories, and their genotyping results were evaluated. Fourteen data sets (45.2%) performed well with the entire panel of samples, and 17 data sets (54.8%) reported at least one genotyping error. For VKORC1 (−1639G>A), participating laboratories were 100% successful in detecting genotypes of GG, GA, and AA. For CYP2C9, participants were greater than 90% successful in detecting genotypes of *1/*1, *1/*2, *1/*3, *2/*3, and *3/*3. However, 15 laboratories failed to detect rarely encountered variant genotype *2/*2. The poor performance of CYP2C9 genotyping may be because of the limitation of methodologies used for detecting CYP2C9*2 allele. The proficiency testing survey highlighted the need for improving genotyping accuracy for some laboratories in this field.

External quality assessment for the molecular detection of MERS-CoV in China

Abstract Background In May 2015, an imported case of Middle East respiratory syndrome coronavirus (MERS-CoV) infection occurred in China, so rapid and reliable diagnosis of suspected cases was necessary. Objectives An external quality assessment (EQA) program for the molecular detection of MERS-CoV was organized by the National Center for Clinical Laboratories (NCCL). Study design MS2 virus-like particles (VLPs) encapsulating specific RNA sequences of MERS-CoV were prepared as positive specimens. The assessment panel, which comprised of three negative and seven positive samples with different concentrations of VLPs, was distributed to 56 laboratories from 16 provinces, municipalities, or autonomous regions for molecular detection. Results Among the received data sets, three employed an in-house-developed real-time reverse-transcription polymerase chain reaction (rRT-PCR) assay and the others applied various commercial rRT-PCR kits. Overall, the majority of laboratories (46/56, 82.1%) could achieve 100% accuracy for MERS-CoV detection, but three laboratories (5.4%) still had room for improvement. Consequently, all negative samples were identified correctly, reaching 100% specificity. The false-negative rate was 3.1%, and most of the false-negative results were obtained from samples with relatively low concentration, indicating an urgent need to improve detection in weak-positive specimens. Conclusions The majority of participants possessed reliable diagnostic capacity for the detection of MERS-CoV. Moreover, EQA is indispensable because it can help enhance the diagnostic capability for the surveillance of MERS-CoV infections and allow comparison of the results among different laboratories.

What is the meaning of a nonresolved viral nucleic acid test-reactive minipool?

This study aimed at analyzing the prevalence of hepatitis B virus (HBV) DNA among hepatitis B surface antigen (HBsAg)–negative donations by cobas TaqScreen MPX test (Roche Molecular Systems) and discussing the meaning of a reactive minipool (MP) that does not resolve to an individual donation (ID)–reactive result.

Reduced vitamin D levels are associated with autoimmune response in tuberculosis patients

A recent report found that vitamin D deficiency was associated with an increased autoimmune response in healthy individuals and systemic lupus erythematosus patients.In addition, a potential mechanism about the role of vitamin D in B cell hyperactivity and autoantibody production had been proposed.1 However, increased autoimmune response and autoantibody production can also be observed in several other diseases such as tuberculosis (TB). Accumulating evidences demonstrate that TB patients have high titres of a variety of autoantibodies.2,–,4 Our research sought to investigate whether a …

Synthetic Circulating Cell-free DNA as Quality Control Materials for Somatic Mutation Detection in Liquid Biopsy for Cancer

BACKGROUND Detection of somatic genomic alterations in tumor-derived cell-free DNA (cfDNA) in the plasma is challenging owing to the low concentrations of cfDNA, variable detection methods, and complex workflows. Moreover, no proper quality control materials are available currently. METHODS We developed a set of synthetic cfDNA quality control materials (SCQCMs) containing spike-in cfDNA on the basis of micrococcal nuclease digestion carrying somatic mutations as simulated cfDNA and matched genomic DNA as genetic background to emulate paired tumor-normal samples in real clinical tests. Site-directed mutagenesis DNA that contained 1500-2000 bases with single-nucleotide variants or indels and genomic DNA from CRISPR/Cas9 edited cells with EML4-ALK rearrangements was fragmented, quantified, and added into micrococcal nuclease-digested DNA derived from HEK293T cells. To prove their suitability, the SCQCMs were compared with patient-derived plasma samples and validated in a collaborative study that encompassed 11 laboratories. RESULTS The results of SCQCM analysis by next-generation sequencing showed strong agreement with those of patient-derived plasma samples, including the size profile of cfDNA and the quality control metrics of the sequencing data. More than 95% of laboratories correctly detected the SCQCMs with EGFR T790M, L858R, KRAS G12D, and a deletion in exon 19, as well as with EML4-ALK variant 2. CONCLUSIONS The SCQCMs were successfully applied in a broad range of settings, methodologies, and informatics techniques. We conclude that SCQCMs can be used as optimal quality controls in test performance assessments for circulating tumor DNA somatic mutation detection.