Bridging the Gap between Digital Assays and Point-of-Care Testing: Automated, Low Cost, and Ultrasensitive Detection of Thyroid Stimulating Hormone.

Medical diagnostics is moving toward disease-related target detection at very low concentrations because of the (1) quest for early-stage diagnosis, at a point where only limited target amounts are present, (2) trend toward minimally invasive sample extraction, yielding samples containing low concentrations of target, and (3) need for straightforward sample collection, usually resulting in limited volume collected. Hence, diagnostic tools allowing ultrasensitive target detection at the point-of-care (POC) are crucial for simplified and timely diagnosis of many illnesses. Therefore, we developed an innovative, fully integrated, semi-automated, and economically viable platform based on (1) digital microfluidics (DMF), enabling automated manipulation and analysis of very low sample volumes and (2) low-cost disposable DMF chips with microwell arrays, fabricated via roll-to-roll processes and allowing digital target counting. Thyroid stimulating hormone detection was chosen as a relevant application to show the potential of the system. The assay buffer was selected using design of experiments, and the assay was optimized in terms of reagent concentration and incubation time toward maximum sensitivity. The hydrophobic-in-hydrophobic microwells showed an unparalleled seeding efficiency of 97.6% ± 0.6%. A calculated LOD of 0.0013 µIU/mL was obtained, showing the great potential of the platform, especially taking into account the very low sample volume analyzed (1.1 µL). Although validation (in biological matrix) and industrialization (full automation) steps still need to be taken, it is clear that the combination of DMF, low-cost DMF chips, and digital analyte counting in microwell arrays enables the implementation of ultrasensitive and reliable target detection at the POC.

Risk Stratification of Slide Tracheoplasty for Pediatric Airway Stenosis.

BACKGROUND: Slide tracheoplasty (STP) is the procedure of choice for treatment of long segmental congenital tracheal stenosis (LSCTS). Few studies predict factors leading to reintervention or mortality after STP. We analyzed a pediatric population to identify such factors and compared the outcome between 2 eras (1995-2012 and 2013-2017). METHODS: We analyzed 150 consecutive children who underwent STP from February 1995 to December 2017 in our hospital. RESULTS: Median age and weight were 6.9 months and 6.1 kg. Average tracheal diameter of LSCTS was 2.3 mm. Tracheal stenosis extended into bronchus in 36 patients and distal malacia in 38. Median follow-up was 67 months; mortality was 12.7%. Balloon dilatation was required in 81 patients (54%), stents in 29 (19%), and reoperation in 4 (3%). The presence of malacia, preoperative extracorporeal membrane oxygenation, congenital anomalies, and single lung anatomy increased the risk for reintervention. Cox regression analysis revealed preoperative ventilation to be an independent factor predicting reintervention and single lung tracheal anatomy for mortality. In the current era (after 2013), survival improved from 88% to 97% and stent requirement was reduced from 25% to 11%. CONCLUSIONS: Slide tracheoplasty can be applied to various airway configurations seen in LSCTS. The requirement for reintervention such as balloon dilatation and stenting is high in the group requiring preoperative ventilation. Mortality is highest in the single lung anatomy group. Centralization of care allowed us to develop the multidisciplinary team expertise to manage this and other rare airway conditions with acceptable outcomes.

Clinical, pathologic, cytogenetic, and molecular profiling in self-identified black women with uterine leiomyomata.

Black women are disproportionately affected by uterine leiomyomata (UL), or fibroids, compared to other racial groups, having a greater lifetime risk of developing UL and an earlier age of diagnosis. In order to elucidate molecular and genetic mechanisms responsible for the increased prevalence and morbidity associated with UL in black women, clinical, pathologic, cytogenetic, and select molecular profiling (MED12 mutation analysis) of 75 self-reported black women undergoing surgical treatment for UL was performed. Our observations are broadly representative of previous cytogenetic studies of UL: karyotypically abnormal tumors were detected in 30.7% of women and 17.4% of analyzed tumors. No notable association was observed between race and increased occurrence of cytogenetic abnormalities that might contribute to any population-specific morbidity or prevalence rate. Our data on MED12 mutation analyses (73.2% of tumors harbored a MED12 mutation) provide additional support for a significant role of MED12 in tumorigenesis. Although the effect of MED12-mediated tumorigenesis appears significant irrespective of race, other genetic events such as the distribution of karyotypic abnormalities appear differently in black women. This case series indicates that presently recognized genetic and molecular characteristics of UL do not appear to explain the increased prevalence and morbidity of UL in black women.

Structural Chromosomal Rearrangements Require Nucleotide-Level Resolution: Lessons from Next-Generation Sequencing in Prenatal Diagnosis.

In this exciting era of "next-gen cytogenetics," integrating genomic sequencing into the prenatal diagnostic setting is possible within an actionable time frame and can provide precise delineation of balanced chromosomal rearrangements at the nucleotide level. Given the increased risk of congenital abnormalities in newborns with de novo balanced chromosomal rearrangements, comprehensive interpretation of breakpoints could substantially improve prediction of phenotypic outcomes and support perinatal medical care. Herein, we present and evaluate sequencing results of balanced chromosomal rearrangements in ten prenatal subjects with respect to the location of regulatory chromatin domains (topologically associated domains [TADs]). The genomic material from all subjects was interpreted to be "normal" by microarray analyses, and their rearrangements would not have been detected by cell-free DNA (cfDNA) screening. The findings of our systematic approach correlate with phenotypes of both pregnancies with untoward outcomes (5/10) and with healthy newborns (3/10). Two pregnancies, one with a chromosomal aberration predicted to be of unknown clinical significance and another one predicted to be likely benign, were terminated prior to phenotype-genotype correlation (2/10). We demonstrate that the clinical interpretation of structural rearrangements should not be limited to interruption, deletion, or duplication of specific genes and should also incorporate regulatory domains of the human genome with critical ramifications for the control of gene expression. As detailed in this study, our molecular approach to both detecting and interpreting the breakpoints of structural rearrangements yields unparalleled information in comparison to other commonly used first-tier diagnostic methods, such as non-invasive cfDNA screening and microarray analysis, to provide improved genetic counseling for phenotypic outcome in the prenatal setting.

Immiscible phase nucleic acid purification eliminates PCR inhibitors with a single pass of paramagnetic particles through a hydrophobic liquid.

Extraction and purification of nucleic acids from complex biological samples for PCR are critical steps because inhibitors must be removed that can affect reaction efficiency and the accuracy of results. This preanalytical processing generally involves capturing nucleic acids on microparticles that are then washed with a series of buffers to desorb and dilute out interfering substances. We have developed a novel purification method that replaces multiple wash steps with a single pass of paramagnetic particles (PMPs) though an immiscible hydrophobic liquid. Only two aqueous solutions are required: a lysis buffer, in which nucleic acids are captured on PMPs, and an elution buffer, in which they are released for amplification. The PMPs containing the nucleic acids are magnetically transported through a channel containing liquid wax that connects the lysis chamber to the elution chamber in a specially designed cartridge. Transporting PMPs through the immiscible phase yielded DNA and RNA as pure as that obtained after extensive wash steps required by comparable purification methods. Our immiscible-phase process has been applied to targets in whole blood, plasma, and urine and will enable the development of faster and simpler purification systems.

Crash-resistant fuel system effectiveness in civil helicopter crashes.

BACKGROUND: Crash-resistant fuel systems (CRFS) have demonstrated close to 100% effectiveness in survivable crashes of Army helicopters, but the technology has been slow to transfer into the civil helicopter arena. Federal standards for civil helicopter CRFS are less stringent than those for military helicopters. A reduction in standards for CRFS in military helicopters is being considered. OBJECTIVE: The goal of this study was to determine whether crashes of civil helicopters with CRFS are less likely to result in post-crash fire than crashes of those without. METHOD: Crashes of civil helicopters during 1982-2004 were analyzed, comparing Bell 206 helicopters manufactured with CRFS with Aerospatial 350 helicopters manufactured during the same period (post-1981), but lacking CRFS. Bell 206 helicopters with CRFS were also compared with earlier models without CRFS. RESULTS: The highest proportion of crashes with post-crash fires (11.3%) was in AS-350s manufactured after 1981 (non-CRFS), and the lowest (3.7%) was in Bell 206s (with CRFS) [unadjusted risk ratio (RR) = 3.3, 95% confidence interval (CI) = 1.04, 10.50; adjusted for light and weather, RR = 2.81, Cl = 0.82, 9.69]. Earlier models of Bell 206s without CRFS had higher risk of post-crash fire than post-1981 models with CRFS (7.4% vs. 3.7%; adjusted RR = 2.11, Cl = 0.82, 5.45). CONCLUSIONS: The results of this study suggest a better performance, in terms of post-crash fire prevention, of CRFS-equipped civil helicopters as compared with those without CRFS. It is possible that CRFS in civil helicopters have not achieved the same degree of effectiveness as CRFS in military helicopters. CRFS should be used more widely in civil helicopters. The more stringent CRFS requirements for military helicopters should not be reduced without further research.

Identification and immunohistochemical characterization of a mucin-like glycoprotein expressed in early stage breast carcinoma.

In this report we describe a cDNA sequence, BS106, identified from Incyte Genomics LifeSeq Expressed Sequence Tag database. A multi-tissue mRNA expression array, northern blots, and RT-PCR assays demonstrate the expression of BS106 in mammary, salivary and prostate glands, but not in other tissue types. BS106 mRNA was detected in 90% of the breast tissues examined. The cDNA encodes a 90-amino acid protein characterized as a small, mucin-like protein based on amino acid composition, extensive O-linked glycosylation, and expression profile. BS106 protein was recombinantly expressed in human embryonic kidney 293 cells and the secreted product was purified from the culture media. Monoclonal antibodies were prepared and used for immunohistochemical analysis of early stage breast cancer. BS106 protein was detected in the vast majority of carcinomas (70-100%) and overexpressed in approximately 30% of the 22 specimens analyzed. BS106 protein was not detected in other solid tumor types including bladder carcinoma, colon carcinoma, endometrial carcinoma, gastric carcinoma, squamous cell lung carcinoma, adenocarcinoma of the lung, ovarian carcinoma, pancreatic and prostatic carcinoma.