: A Sex-Specific Chimeric RNA and Its Role in Immune Sexual Dimorphism.

RNA processing mechanisms, such as alternative splicing and RNA editing, have been recognized as critical means to expand the transcriptome. Chimeric RNAs formed by intergenic splicing provide another potential layer of RNA diversification. By analyzing a large set of RNA-Seq data and validating results in over 1,200 blood samples, we identified , a female-specific chimeric transcript.

Optimization of novel loop-mediated isothermal amplification with colorimetric image analysis for forensic body fluid identification.

Accurate presumptive and confirmatory test use for forensic body fluid identification is essential for gaining contextual information for crime scene investigators. Loop-mediated isothermal amplification (LAMP) is an ideal method for forensic body fluid identification because it is highly specific and generates multi-sized amplicon DNA, and successful amplification results can be read out colorimetrically. Here, we show preliminary data on a LAMP method that rapidly identifies body fluids including venous blood, semen, and saliva, based on colorimetric response and image analysis.

A novel loop-mediated isothermal amplification method for identification of four body fluids with smartphone detection.

Messenger RNA profiling for body fluid identification (bfID) is a useful approach to collect contextual information associated with a crime. Current methods require costly fluorescent probes, lengthy amplification protocols and/or time-consuming sample preparation. To simplify this process, we developed a bfID method that has the potential to be rapid in analysis time, inexpensive and fluorescence-free, combining a universal operating procedure with a high-throughout (microwell plate) platform for simultaneous detection of mRNA markers from whole blood, semen, saliva, and vaginal fluid.

Use of Dual-Force Aggregation as a Multiplexed, Rapid Point-of-Care Screening Method for White Blood Cell Counting from Whole Blood Samples.

Background: Enumeration of blood cells is an integral metric for evaluating patient health and can be used to screen for a wide range of diseases and conditions. Conventional methods rely on large, expensive, and complicated instrumentation that requires trained technicians and is not amenable to point-of-care analysis. This work demonstrates the use of a multiplexed, bead-based assay for both rapid white blood cell (WBC) count screening and accurate, multiplexed WBC counts for point-of-care analysis.

Simple, Reagentless Quantification of Total Bilirubin in Blood Via Microfluidic Phototreatment and Image Analysis.

Total bilirubin (T-Bil) is an important clinical diagnostic marker that is measured frequently by physicians to assist in the diagnosis, treatment, and monitoring of multiple medical conditions. The work demonstrated here utilizes the 48-year-old mechanism of phototherapy that is commonly implemented in the treatment of infants with exaggerated physiologic and pathologic jaundice but adapts it to the microfluidic level for the ultimate purpose of total bilirubin quantitation. After acquisition of a small volume of blood (<10 μL) and through subsequent separation (plasma + red blood cells), a 3 μL plasma sample was imaged by a portable scanner and analyzed through a custom algorithm for color intensity.

Rotation-Driven Microfluidic Disc for White Blood Cell Enumeration Using Magnetic Bead Aggregation.

We recently defined a magnetic bead-based assay that exploited an agglutination-like response for DNA and applied it to DNA-containing cell enumeration using inexpensive benchtop hardware [ J. Am. Chem.

Optimization of Quantitative PCR Methods for Enteropathogen Detection.

Detection and quantification of enteropathogens in stool specimens is useful for diagnosing the cause of diarrhea but is technically challenging. Here we evaluate several important determinants of quantification: specimen collection, nucleic acid extraction, and extraction and amplification efficiency. First, we evaluate the molecular detection and quantification of pathogens in rectal swabs versus stool, using paired flocked rectal swabs and whole stool collected from 129 children hospitalized with diarrhea in Tanzania.

Hematocrit analysis through the use of an inexpensive centrifugal polyester-toner device with finger-to-chip blood loading capability.

Hematocrit (HCT) measurements are important clinical diagnostic variables that help physicians diagnose and treat various medical conditions, ailments, and diseases. In this work, we present the HCT Disc, a centrifugal microdevice fabricated by a Print, Cut and Laminate (PCL) method to generate a 12-sample HCT device from materials costing <0.5 USD (polyester and toner or PeT).

Sonorheometry assessment of platelet function in cardiopulmonary bypass patients: Correlation of blood clot stiffness with platelet integrin αIIbβ3 activity, aspirin usage, and transfusion risk.

Background: Impaired platelet function may underlie bleeding associated with cardiopulmonary bypass (CPB) and at present is incompletely evaluated with existing diagnostic technologies. Sonorheometry (SR) is a recently developed ultrasound-based technology that quantifies hemostasis and platelet activity from a blood sample by measuring ex vivo clot stiffness (S). We hypothesized that impaired platelet-fibrin interactions as assessed by SR would correlate with transfusion during CPB and history of prior aspirin therapy.

The ARTμS: a novel microfluidic CD4+ T-cell enumeration system for monitoring antiretroviral therapy in HIV patients.

We report on a novel and cost-effective microfluidic platform that integrates immunomagnetic separation and cell enumeration via DNA-induced bead aggregation. Using a two-stage immunocapture microdevice, 10 μL of whole blood was processed to isolate CD4+ T-cells. The first stage involved the immuno-subtraction of monocytes by anti-CD14 magnetic beads, followed by CD4+ T-cell capture with anti-CD4 magnetic beads.

Multilevel fluidic flow control in a rotationally-driven polyester film microdevice created using laser print, cut and laminate.

This paper presents a simple and cost-effective polyester toner microchip fabricated with laser print and cut lithography (PCL) to use with a battery-powered centrifugal platform for fluid handling. The combination of the PCL microfluidic disc and centrifugal platform: (1) allows parallel aliquoting of two different reagents of four different volumes ranging from nL to μL with an accuracy comparable to a piston-driven air pipette; (2) incorporates a reciprocating mixing unit driven by a surface-tension pump for further dilution of reagents, and (3) is amenable to larger scale integration of assay multiplexing (including all valves and mixers) without substantially increasing fabrication cost and time. For a proof of principle, a 10 min colorimetric assay for the quantitation of the protein level in the human blood plasma samples is demonstrated on chip with a limit of detection of ∼5 mg mL(-1) and coefficient of variance of ∼7%.

Evaluation of the initiation of urine drug screens intended for use in transfer patients.

Objective: The objective of this study was to determine if signs of clinical intoxication were present in patients who had transfer urine drug screens (UDS) performed and to determine the proportion of patients with UDS orders who were actually transferred to another facility.

Methods: Of all emergency department (ED) patient visits who had a transfer UDS ordered from November 19, 2011, to December 31, 2012, 54% of the population was randomly selected for review by 1 of 3 study investigators. For quality assurance, a random sample of 100 patient charts was independently reviewed by all 3 investigators to assure consistency in interpreting data.

Development and assessment of molecular diagnostic tests for 15 enteropathogens causing childhood diarrhoea: a multicentre study.

Background: Childhood diarrhoea can be caused by many pathogens that are difficult to assay in the laboratory. Molecular diagnostic techniques provide a uniform method to detect and quantify candidate enteropathogens. We aimed to develop and assess molecular tests for identification of enteropathogens and their association with disease.

Falsely increased chloride and missed anion gap elevation during treatment with sodium thiosulfate.

Background: Sodium thiosulfate (STS) is used to treat calciphylaxis and cyanide poisoning, but can lead to a serious anion-gap acidosis. We suspected that the calculated anion gap in a patient treated with STS for calciphylaxis was decreased to normal by a falsely increased chloride, and we hypothesized that STS directly interfered with chloride measurements.

Methods: Plasma pools were prepared with 12 concentrations of STS from 0 to 20 mmol/l.

From slide sets to sound bites: teaching and learning pathology in the digital age.

Educational evolution is particularly important in pathology, particularly cytopathology, due to the vast amounts of independent learning required to master this field. In this study, learning challenges faced by pathology residents were addressed through a variety of educational modalities including 24 short (∼10 minute) online tutorials (dubbed "Sound Bites") covering selected topics in cytopathology as well as other areas of anatomic and clinical pathology. Additionally, residents were provided with an annotated glass slide set covering pediatric pathology with an associated multiple choice self-assessment as well as multiheaded microscope slide review sessions.

Label-free method for cell counting in crude biological samples via paramagnetic bead aggregation.

Under chaotropic conditions, DNA released from lysed cells causes the aggregation of paramagnetic beads in a rotating magnetic field in a manner that is independent of the presence of other cellular components. The extent of aggregation correlates with the mass of DNA in a quantitative manner (Leslie, D. C.

Analytic evaluation of a new glucose meter system in 15 different critical care settings.

Background: Maintaining appropriate glycemic control in critically ill patients reduces morbidity and mortality. The use of point-of-care (POC) glucose devices is necessary to obtain rapid results at the patient's bedside. However, the devices should be thoroughly tested in the intended population before implementation.

Circulating microRNA as a biomarker of human growth hormone administration to patients.

Circulating microRNAs (miRNAs) in plasma are being studied for use as biomarkers of specific diseases and as markers of administration of pharmaceutical agents. Administration of recombinant human growth hormone (rhGH) is prohibited by sporting authorities, but it continues to be used by athletes attempting to gain an unfair advantage in athletic competition. Current methods for detection of rhGH use rely on immunoassay technology and are limited by a short time-frame in which detection of rhGH is possible.

From sample to PCR product in under 45 minutes: a polymeric integrated microdevice for clinical and forensic DNA analysis.

The extraction and amplification of DNA from biological samples is laborious and time-consuming, requiring numerous instruments and sample handling steps. An integrated, single-use, poly(methyl methacrylate) (PMMA) microdevice for DNA extraction and amplification would benefit clinical and forensic communities, providing a completely closed system with rapid sample-in-PCR-product-out capability. Here, we show the design and simple flow control required for enzyme-based DNA preparation and PCR from buccal swabs or liquid whole blood samples with an ~5-fold reduction in time.

A laboratory-developed TaqMan Array Card for simultaneous detection of 19 enteropathogens.

The TaqMan Array Card (TAC) system is a 384-well singleplex real-time PCR format that has been used to detect multiple infection targets. Here we developed an enteric TaqMan Array Card to detect 19 enteropathogens, including viruses (adenovirus, astrovirus, norovirus GII, rotavirus, and sapovirus), bacteria (Campylobacter jejuni/C. coli, Clostridium difficile, Salmonella, Vibrio cholerae, diarrheagenic Escherichia coli strains including enteroaggregative E.

Quantification of growth hormone mRNA in blood.

Background: Growth hormone (GH) is present in human lymphocytes and the relative concentration of GH mRNA in circulating cells is reportedly increased in patients with acromegaly. No quantitative assay has been described to measure GH in circulating cells.

Methods: To measure GH mRNA, we used real-time reverse-transcriptase (RT) quantitative PCR (qPCR) with nested primers, a synthetic nucleotide standard and SYBR Green I detection.

An enzyme-based DNA preparation method for application to forensic biological samples and degraded stains.

Extraction of DNA from forensic samples typically uses either an organic extraction protocol or solid phase extraction (SPE) and these methods generally involve numerous sample transfer, wash and centrifugation steps. Although SPE has been successfully adapted to the microdevice, it can be problematic because of lengthy load times and uneven packing of the solid phase. A closed-tube enzyme-based DNA preparation method has recently been developed which uses a neutral proteinase to lyse cells and degrade proteins and nucleases [14].

New detection modality for label-free quantification of DNA in biological samples via superparamagnetic bead aggregation.

Combining DNA and superparamagnetic beads in a rotating magnetic field produces multiparticle aggregates that are visually striking, enabling label-free optical detection and quantification of DNA at levels in the picogram per microliter range. DNA in biological samples can be quantified directly by simple analysis of optical images of microfluidic wells placed on a magnetic stirrer without prior DNA purification. Aggregation results from DNA/bead interactions driven either by the presence of a chaotrope (a nonspecific trigger for aggregation) or by hybridization with oligonucleotides on functionalized beads (sequence-specific).

Warfarin genotyping in a single PCR reaction for microchip electrophoresis.

Background: Warfarin is the most commonly prescribed oral anticoagulant medication but also is the second leading cause of emergency room visits for adverse drug reactions. Genetic testing for warfarin sensitivity may reduce hospitalization rates, but prospective genotyping is impeded in part by the turnaround time and costs of genotyping. Microfluidics-based assays can reduce reagent consumption and analysis time; however, no current assay has integrated multiplexed allele-specific PCR for warfarin genotyping with electrophoretic microfluidics hardware.