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Noninvasive prenatal diagnosis (NIPD) aims to detect fetal-related genetic disorders before birth by detecting markers in the peripheral blood of pregnant women, holding the potential in reducing the risk of fetal birth defects. Fetal-nucleated red blood cells (fNRBCs) can be used as biomarkers for NIPD, given their remarkable nature of carrying the entire genetic information of the fetus. Here, we review recent advances in NIPD technologies based on the isolation and analysis of fNRBCs. Conventional cell separation methods rely primarily on physical properties and surface antigens of fNRBCs, such as density gradient centrifugation, fluorescence-activated cell sorting, and magnetic-activated cell sorting. Due to the limitations of sensitivity and purity in Conventional methods, separation techniques based on micro-/nanomaterials have been developed as novel methods for isolating and enriching fNRBCs. We also discuss emerging methods based on microfluidic chips and nanostructured substrates for static and dynamic isolation of fNRBCs. Additionally, we introduce the identification techniques of fNRBCs and address the potential clinical diagnostic values of fNRBCs. Finally, we highlight the challenges and the future directions of fNRBCs as treatment guidelines in NIPD. Graphical Abstract

Background Relative haplotype dosage (RHDO) approach has been applied in noninvasive prenatal diagnosis (NIPD) of Duchenne muscular dystrophy (DMD). However, the RHDO procedure is relatively complicated and the parental haplotypes need to be constructed. Furthermore, it is not suitable for the diagnosis of de novo mutations or mosaicism in germ cells. Here, we investigated NIPD of DMD using a relative mutation dosage (RMD)-based approach—cell-free DNA Barcode-Enabled Single-Molecule Test (cfBEST), which has not previously been applied in the diagnosis of exon deletion. Methods Five DMD families caused by DMD gene point mutations or exon deletion were recruited for this study. After the breakpoints of exon deletion were precisely mapped with multiple PCR, the genotypes of the fetuses from the five DMD families were inferred using cfBEST, and were further validated by invasive prenatal diagnosis. Results The cfBEST results of the five families indicated that one fetus was female and did not carry the familial molecular alteration, three fetuses were carriers and one was male without the familial mutation. The invasive prenatal diagnosis results were consistent with those of the cfBEST procedure. Conclusion This is the first report of NIPD of DMD using the RMD-based approach. We extended the application of cfBEST from point mutation to exon deletion mutation. The results showed that cfBEST would be suitable for NIPD of DMD caused by different kinds of mutation types.

Hepatitis C virus (HCV) is a major health problem in hemodialysis patients, which leads to significant morbidity and mortality through progressive hepatic fibrosis or cirrhosis. Percutaneous liver biopsy is the gold standard to stage hepatic fibrosis. However, it is an invasive procedure with postbiopsy complications. Because uremia may significantly increase the risk of fatal and nonfatal bleeding events, the use of noninvasive means to assess the severity of hepatic fibrosis is particularly appealing to hemodialysis patients. To date, researchers have evaluated the performance of various biochemical, serological, and radiological indices for hepatic fibrosis in hemodialysis patients with HCV infection. In this review, we will summarize the progress of noninvasive indices for assessing hepatic fibrosis and propose a pragmatic recommendation to diagnose the stage of hepatic fibrosis with a noninvasive index, in hemodialysis patients with HCV infection.

Background Though massively parallel sequencing has been widely applied to noninvasive prenatal screen for common trisomy, the clinical use of massively parallel sequencing to noninvasive prenatal diagnose monogenic disorders is limited. This study was to develop a method for directly determining paternal haplotypes for noninvasive prenatal diagnosis of monogenic disorders without requiring proband’s samples. Methods The study recruited 40 families at high risk for autosomal recessive diseases. The targeted linked-read sequencing was performed on high molecular weight (HMW) DNA of parents using customized probes designed to capture targeted genes and single-nucleotide polymorphisms (SNPs) distributed within 1Mb flanking region of targeted genes. Plasma DNA from pregnant mothers also underwent targeted sequencing using the same probes to determine fetal haplotypes according to parental haplotypes. The results were further confirmed by invasive prenatal diagnosis. Results Seventy-eight parental haplotypes of targeted gene were successfully determined by targeted linked-read sequencing. The predicted fetal inheritance of variant was correctly deduced in 38 families in which the variants had been confirmed by invasive prenatal diagnosis. Two families were determined to be no-call. Conclusions Targeted linked-read sequencing method demonstrated to be an effective means to phase personal haplotype for noninvasive prenatal diagnosis of monogenic disorders.

Non-alcoholic fatty liver disease (NAFLD) is a potentially serious liver disease that affects approximately one-quarter of the global adult population, causing a substantial burden of ill health with wide-ranging social and economic implications. It is a multisystem disease and is considered the hepatic component of metabolic syndrome. Unlike other highly prevalent conditions, NAFLD has received little attention from the global public health community. Health system and public health responses to NAFLD have been weak and fragmented, and, despite its pervasiveness, NAFLD is largely unknown outside hepatology and gastroenterology. There is only a nascent global public health movement addressing NAFLD, and the disease is absent from nearly all national and international strategies and policies for non-communicable diseases, including obesity. In this global Delphi study, a multidisciplinary group of experts developed consensus statements and recommendations, which a larger group of collaborators reviewed over three rounds until consensus was achieved. The resulting consensus statements and recommendations address a broad range of topics — from epidemiology, awareness, care and treatment to public health policies and leadership — that have general relevance for policy-makers, health-care practitioners, civil society groups, research institutions and affected populations. These recommendations should provide a strong foundation for a comprehensive public health response to NAFLD.Non-alcoholic fatty liver disease (NAFLD) is a potentially serious liver disease with a substantial burden worldwide. In this Consensus Statement, a global multidisciplinary group of experts develop consensus statements and recommendations addressing a broad range of topics on NAFLD to raise awareness and spur action.

BACKGROUNDCurrent clinical management of patients with pulmonary nodules involves either repeated low-dose CT (LDCT)/CT scans or invasive procedures, yet causes significant patient misclassification. An accurate noninvasive test is needed to identify malignant nodules and reduce unnecessary invasive tests.METHODWe developed a diagnostic model based on targeted DNA methylation sequencing of 389 pulmonary nodule patients' plasma samples and then validation in 140 plasma samples independently. We tested the model in different stages and subtypes of pulmonary nodules.RESULTSA 100-feature model was developed and validated for pulmonary nodule diagnosis; the model achieved a receiver operating characteristic curve-AUC (ROC-AUC) of 0.843 on 140 independent validation samples, with an accuracy of 0.800. The performance was well maintained in (a) a 6 to 20 mm size subgroup (n = 100), with a sensitivity of 1.000 and adjusted negative predictive value (NPV) of 1.000 at 10% prevalence; (b) stage I malignancy (n = 90), with a sensitivity of 0.971; (c) different nodule types: solid nodules (n = 78) with a sensitivity of 1.000 and adjusted NPV of 1.000, part-solid nodules (n = 75) with a sensitivity of 0.947 and adjusted NPV of 0.983, and ground-glass nodules (n = 67) with a sensitivity of 0.964 and adjusted NPV of 0.989 at 10% prevalence. This methylation test, called PulmoSeek, outperformed PET-CT and 2 clinical prediction models (Mayo Clinic and Veterans Affairs) in discriminating malignant pulmonary nodules from benign ones.CONCLUSIONThis study suggests that the blood-based DNA methylation model may provide a better test for classifying pulmonary nodules, which could help facilitate the accurate diagnosis of early stage lung cancer from pulmonary nodule patients and guide clinical decisions.FUNDINGThe National Key Research and Development Program of China; Science and Technology Planning Project of Guangdong Province; The National Natural Science Foundation of China National.

To study the detection limits of chromosomal microaberrations in non-invasive prenatal testing with aim for five target microdeletion syndromes, including DiGeorge, Prader-Willi/Angelman, 1p36, Cri-Du-Chat, and Wolf-Hirschhorn syndromes. We used known cases of pathogenic deletions from ISCA database to specifically define regions critical for the target syndromes. Our approach to detect microdeletions, from whole genome sequencing data, is based on sample normalization and read counting for individual bins. We performed both an in-silico study using artificially created data sets and a laboratory test on mixed DNA samples, with known microdeletions, to assess the sensitivity of prediction for varying fetal fractions, deletion lengths, and sequencing read counts. The in-silico study showed sensitivity of 79.3% for 10% fetal fraction with 20M read count, which further increased to 98.4% if we searched only for deletions longer than 3Mb. The test on laboratory-prepared mixed samples was in agreement with in-silico results, while we were able to correctly detect 24 out of 29 control samples. Our results suggest that it is possible to incorporate microaberration detection into basic NIPT as part of the offered screening/diagnostics procedure, however, accuracy and reliability depends on several specific factors.

Saliva is a noninvasive biofluid that can contain metabolite signatures of oral squamous cell carcinoma (OSCC). Conductive polymer spray ionization mass spectrometry (CPSI-MS) is employed to record a wide range of metabolite species within a few seconds, making this technique appealing as a point-of-care method for the early detection of OSCC. Saliva samples from 373 volunteers, 124 who are healthy, 124 who have premalignant lesions, and 125 who are OSCC patients, were collected for discovering and validating dysregulated metabolites and determining altered metabolic pathways. Metabolite markers were reconfirmed at the primary tissue level by desorption electrospray ionization MS imaging (DESI-MSI), demonstrating the reliability of diagnoses based on saliva metabolomics. With the aid of machine learning (ML), OSCC and premalignant lesions can be distinguished from the normal physical condition in real time with an accuracy of 86.7%, on a person by person basis. These results suggest that the combination of CPSI-MS and ML is a feasible tool for accurate, automated diagnosis of OSCC in clinical practice.

Physical therapists routinely make diagnostic and prognostic decisions in the course of patient care. The purpose of this clinical perspective is to illustrate what we believe is the optimal method for interpreting the results of studies that describe the diagnostic or prognostic accuracy of examination procedures. To illustrate our points, we chose the Berg Balance Test as an exemplar measure. We combined the data from 2 previously published research reports designed to determine the validity of the Berg Balance Test for predicting risk of falls among elderly people. We calculated the most common validity indexes, including sensitivity, specificity, predictive values, and likelihood ratios for the combined data. Clinical scenarios were used to demonstrate how we believe these validity indexes should be used to guide clinical decisions. We believe therapists should use validity indexes to decrease the uncertainty associated with diagnostic and prognostic decisions. More studies of the accuracy of diagnostic and prognostic tests used by physical therapists are urgently needed.