This study evaluated the ability of laboratories in the Chinese mainland to conduct molecular detection of seasonal A(H1N1), A(H1N1)pdm09, A(H3N2), A(H5N1), A(H7N9), A(H9N2), B(Victoria), and B(Yamagata). Based on a genetically engineered system of virus-like particles (VLPs), the National Center for Clinical Laboratories of China (NCCLs) developed an external quality assessment (EQA) panel. The panel was distributed to 35 laboratories in mainland China to investigate the proficiency of the 16 assays for influenza molecular detection. Using genetic engineering technology, VLPs encapsulating the 37 target genes of 8 influenza viruses were generated. After verification and quantification, 26 influenza virus surrogates with different concentrations were prepared for EQA. Among the 35 participating laboratories, 319 datasets were returned to the NCCLs. Overall, 95.6% (305/319) of datasets correctly reported all 30 samples, while 2.2% (7/319) of datasets with more than one incorrect result were considered as "improvable". A total of 16 misdiagnosed and 18 undiagnosed results were reported. The data analyzed in this study showed good reproducibility in China, but improvements are needed to decrease misdiagnosed and undiagnosed cases, particularly for the A(H9N2) NA gene. Moreover, VLPs are a good alternative specimen type for assay training and proficiency testing purposes.

Recent circulating tumor DNA (ctDNA) research has demonstrated its potential as a non-invasive biomarker for cancer. However, the deployment of ctDNA assays in routine clinical practice remains challenging owing to variability in analytical approaches and the assessment of clinical significance. A well-developed, analytically valid ctDNA assay is a prerequisite for integrating ctDNA into cancer management, and an appropriate analytical technology is crucial for the development of a ctDNA assay. Other determinants including pre-analytical procedures, test validation, internal quality control (IQC), and continual proficiency testing (PT) are also important for the accuracy of ctDNA assays. In the present review, we will focus on the most widely used ctDNA detection technologies and the key quality management measures used to assure the accuracy of ctDNA assays. The aim of this review is to provide useful information for technology selection during ctDNA assay development and assure a reliable test result in clinical practice.

Next-generation sequencing (NGS) is commonly used in a clinical setting for diagnostic and prognostic testing of genetic mutations to select optimal targeted therapies. Herein, we describe the development of a custom NGS assay for detecting single-nucleotide variants (SNVs) and copy number variations (CNVs) in a panel of 51 genes related to breast cancer. We designed and implemented a validation strategy in accordance with principles and guidelines developed by the Next-Generation Sequencing: Standardization of Clinical Testing work group using artificial, cell-free DNA (cfDNA) with mutant fragments prepared in a simple, rapid, and cost-effective manner. For SNV detection, our test had 96.30% sensitivity at mutant allele frequency ≥0.5% with high specificity (99.9997%) and accuracy (99.9996%). For CNV detection, the approach had 95.83% sensitivity for copy numbers at 1.25× (25.6% extra copies) with high specificity (99.77%) and accuracy (99.76%). In addition, our NGS-based assay demonstrated high intrarun and interrun reproducibility, high consistency compared to digital PCR, and a low cross-contamination rate. An overall assessment using cfDNA and plasma cfDNA samples demonstrated our custom NGS assay yields a reliable and robust detection sensitivity with a mutant allele frequency as low as 0.5% for SNVs and copy number of 1.25× for CNVs.

Irinotecan is widely used in the treatment of solid tumors, especially in colorectal cancer and lung cancer. Molecular testing for UGT1A1 genotyping is increasingly required in China for optimum irinotecan administration. In order to determine the performance of laboratories with regard to the whole testing process for UGT1A1 to ensure the consistency and accuracy of the test results, the National Center for Clinical Laboratories conducted an external quality assessment program for UGT1A1*28 genotyping in 2015. The panel, which comprised of four known mutational samples and six wild-type samples, was distributed to 45 laboratories that test for the presence of UGT1A1*28 polymorphisms. Participating laboratories were allowed to perform polymorphism analysis by using their routine methods. The accuracy of the genotyping and reporting of results was analyzed. Other information from the individual laboratories, including the number of samples tested each month, accreditation/certification status, and test methodology, was reviewed. Forty-four of the 45 participants reported the correct results for all samples. There was only one genotyping error, with a corresponding analytical sensitivity of 99.44% (179/180 challenges; 95% confidence interval: 96.94-99.99%) and an analytical specificity of 100% (270/270 challenges; 95% confidence interval: 98.64-100%). Both commercial kits and laboratory development tests were commonly used by the laboratories, and pyrosequencing was the main methodology used (n = 26, 57.8%). The style of the written reports showed large variation, and many reports showed a shortage of information. In summary, the first UGT1A1 genotyping external quality assessment result demonstrated that UGT1A1 genotype analysis of good quality was performed in the majority of pharmacogenetic testing centers that were investigated. However, greater education on the reporting of UGT1A1 genetic testing results is needed.

Vitiligo is a dermatological disorder with an autoimmune mechanism characterized by production of a variety of autoantibodies. Different levels of immunoglobulins can indicate the presence and the stage of some autoimmune diseases. We aimed to investigate serum IgA, IgM and IgG subclass levels and melanocyte-reactive antibodies in 65 vitiligo patients by the immunonephelometric assay (35 healthy people as controls). Compared with normal controls, a significant increase in total IgG, IgG1 and IgG2 (p = .005, p = .003 and p = .043, respectively) was observed in progressive nonsegmental vitiligo patients. Also, we found a significant decrease in IgG3 (p = .000 and p = .023) in progressive nonsegmental vitiligo patients and segmental patients. Moreover, we found the serum levels of IgG4 subclass in stable nonsegmental patients were significantly higher than those in normal controls (p = .018). Compared with controls, the positive rates of melanocyte-reactive antibodies were higher in progressive nonsegmental patients and stable nonsegmental patients (p = .032 and p = .046, respectively). Furthermore, we found higher level of IgG4 and lower level of IgM in male than those in female. Higher IgG1 level was also observed in patients with a family history than in those without a family history. In addition, there was a significant inverse correlation between the concentrations of IgG4 and disease duration. Our evaluation about the level of immunoglobulins might provide a useful insight into the pathological process of vitiligo.

Tamoxifen has been successfully used for treating breast cancer and preventing cancer recurrence. Cytochrome P450 2D6 (CYP2D6) plays a key role in the process of metabolizing tamoxifen to its active moiety, endoxifen. Patients with variants of the CYP2D6 gene may not receive the full benefit of tamoxifen treatment. The CYP2D6*10 variant (the most common variant in Asians) was analyzed to optimize the prescription of tamoxifen in China. To ensure referring clinicians have accurate information for genotype-guided tamoxifen treatment, the Chinese National Center for Clinical Laboratories (NCCL) organized a national proficiency testing (PT) to evaluate the performance of laboratories providing CYP2D6*10 genotyping. Ten genomic DNA samples with CYP2D6 wild-type or CYP2D6*10 variants were validated by PCR-sequencing and sent to 28 participant laboratories. The genotyping results and pharmacogenomic test reports were submitted and evaluated by NCCL experts. Additional information regarding the number of samples tested, the accreditation/certification status, and detecting technology was also requested. Thirty-one data sets were received, with a corresponding analytical sensitivity of 98.2% (548/558 challenges; 95% confidence interval: 96.7-99.1%) and an analytic specificity of 96.5% (675/682; 95% confidence interval: 97.9-99.5%). Overall, 25/28 participants correctly identified CYP2D6*10 status in 10 samples; however, two laboratories made serious genotyping errors. Most of the essential information was included in the 20 submitted CYP2D6*10 test reports. The majority of Chinese laboratories are reliable for detecting the CYP2D6*10 variant; however, several issues revealed in this study underline the importance of PT schemes in continued external assessment and provision of guidelines.

Cancer is characterized by multiple genetic and epigenetic alterations that drive malignant cell proliferation and confer chemoresistance. The ability to correct or ablate such mutations holds immense promise for combating cancer. Recently, because of its high efficiency and accuracy, the CRISPR-Cas9 genome editing technique has been widely used in cancer therapeutic explorations. Several studies used CRISPR-Cas9 to directly target cancer cell genomic DNA in cellular and animal cancer models which have shown therapeutic potential in expanding our anticancer protocols. Moreover, CRISPR-Cas9 can also be employed to fight oncogenic infections, explore anticancer drugs, and engineer immune cells and oncolytic viruses for cancer immunotherapeutic applications. Here, we summarize these preclinical CRISPR-Cas9-based therapeutic strategies against cancer, and discuss the challenges and improvements in translating therapeutic CRISPR-Cas9 into clinical use, which will facilitate better application of this technique in cancer research. Further, we propose potential directions of the CRISPR-Cas9 system in cancer therapy.

The κ/λ hybrid antibodies in normal human serum were reported recently, but their clinical relevance has not yet been explored. Rheumatoid arthritis (RA) is one of the major joint diseases, and the early diagnosis and treatment of RA remain a challenge. Here, we developed a double-sandwich enzyme-linked immunosorbent assay system to quantify relative serum κ/λ hybrid antibody levels in RA patients, osteoarthritis (OA) patients, and healthy controls (HC) in order to assess their potential use as a serological biomarker of early disease and clinical activity and to preliminarily investigate their immunomodulatory roles in RA. Surprisingly, we found that κ/λ hybrid antibody was markedly increased in both early and established RA. Serum κ/λ hybrid antibody levels were significantly correlated with clinical indexes and inflammatory markers in RA. Further analysis showed a positive correlation between κ/λ hybrid antibody levels and the 28-joint disease activity score (DAS28). In conclusion, serum κ/λ hybrid antibodies in RA were identified for the first time. High levels of κ/λ hybrid antibody may be a useful tool in distinguishing early RA from OA and HC. We suggest κ/λ hybrid antibody as a marker for disease activity. The increased κ/λ hybrid antibodies were associated with inflammatory conditions in RA.

To evaluate the proficiencies of laboratories utilizing next-generation sequencing (NGS) to detect somatic mutations in cancer-related genes, an external quality assessment (EQA) was implemented by the National Center for Clinical Laboratories of China in 2015. We prepared a panel of samples that comprised eight samples made by mixing synthetic mutated DNA fragments with normal human genomic DNA and one reference sample containing only genomic DNA. We validated our sample panel, and then distributed it to laboratories across China. We received complete results from 64 laboratories. The performances of 51.6 % (33/64) respondent labs were acceptable and 26.6 % (17/64) of the labs returned perfect results. In total, 449 mistakes were reported, including 201 false-negatives (201/449, 44.8 %) and 222 false-positives (222/449, 49.4 %) and 26 slightly discordant results (26/449, 5.8 %). We believe these unsatisfactory results and varied performances are mainly due to the enrichment methods used, the diverse sequencing chemistries of the different NGS platforms, and other errors within the sequencing process. The results indicate that our sample panel is suitable for use in EQA studies, and that further laboratory training in targeted NGS testing is urgently required. To address this, we propose a targeted NGS workflow with details on quality assurance procedures according to the current guidelines.

The only generally accepted serological marker currently used for the diagnosis of autoimmune pancreatitis (AIP) is IgG4. Our aim was mainly to determine whether hybrid κ\λ antibody can help to diagnose AIP and to differentiate it from pancreatic cancer. We established an enzyme-linked immunosorbent assay (ELISA) system to measure the levels of hybrid κ\λ antibodies in human sera. Sera were obtained from 338 patients, including 61 with AIP, 74 with pancreatic cancer, 50 with acute pancreatitis, 40 with ordinary chronic pancreatitis, 15 with miscellaneous pancreatic diseases, and 98 with normal pancreas. Our study showed levels of hybrid κ\λ antibodies in the AIP group were significantly higher than in the non-AIP group ( P < 0.001). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for the diagnosis of AIP were 80.3%, 91%, 66.2% and 95.5% respectively. Furthermore, the combined measurement of serum hybrid κ\λ antibody and IgG4 tended to increase the sensitivity although the difference was not statistically significant (90.2% vs. 78.7%, P = 0.08), compared to measurement of IgG4 alone. Our findings suggest that hybrid κ\λ antibody could be a new serological marker to diagnose AIP and differentiate it from pancreatic cancer.

Laboratories are increasingly requested to perform CYP2C19 genetic testing when managing clopidogrel therapy, especially in patients with acute coronary syndrome undergoing percutaneous coronary intervention. To ensure high quality molecular testing and ascertain that the referring clinician has the correct information for CYP2C19 genotype-directed antiplatelet therapy, a proficiency testing scheme was set up to evaluate the laboratory performance for the entire testing process. Proficiency panels of 10 cell samples encompassing the common CYP2C19 genetic polymorphisms were distributed to 62 participating laboratories for routine molecular testing and the responses were analyzed for accuracy of genotyping and the reporting of results. Data including the number of samples tested, the accreditation/certification status, and test methodology of each individual laboratory were also reviewed. Fifty-seven of the 62 participants correctly identified the CYP2C19 variants in all samples. There were six genotyping errors, with a corresponding analytical sensitivity of 98.5% (333/338 challenges; 95% confidence interval: 96.5-99.5%) and an analytic specificity of 99.6% (281/282; 95% confidence interval: 98.0-99.9%). Reports of the CYP2C19 genotyping results often lacked essential information. In conclusion, clinical laboratories demonstrated good analytical sensitivity and specificity; however, the pharmacogenetic testing community requires additional education regarding the correct reporting of CYP2C19 genetic test results.

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.

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.