UBA1-CDK16 : 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 UBA1-CDK16 , a female-specific chimeric transcript. Intriguingly, both parental genes, are expressed in males and females. Mechanistically, UBA1-CDK16 is produced by cis-splicing between the two adjacent X-linked genes, originating from the inactive X chromosome. A female-specific chromatin loop, formed between the junction sites, facilitates the alternative splicing of its readthrough precursor. This unique chimeric transcript exhibits evolutionary conservation, evolving to be female-specific from non-human primates to humans. Furthermore, our investigation reveals that UBA1-CDK16 is enriched in the myeloid lineage and plays a regulatory role in myeloid differentiation. Notably, female COVID-19 patients who tested negative for this chimeric transcript displayed higher counts of neutrophils, highlighting its potential role in disease pathogenesis. These findings support the notion that chimeric RNAs represent a new repertoire of transcripts that can be regulated independently from the parental genes, and a new class of RNA variance with potential implications in sexual dimorphism and immune responses.

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. The method is designed for easy implementation into forensic casework protocols with minimal disruption to DNA analysis. LAMP naturally increases target specificity due to the use of multiple primers for one target and mRNA targets were used for tissue and human specificity. With colorimetric detection as an inherent part of LAMP, samples that are positive or negative for any of the body fluids are readily identified by image capture and analysis, thus eliminating subjectivity. Results show by using the 3D-printed imaging system specific color ranges can be set for easy determination of body fluids. The resulting color change can be seen in <30 min using a universal temperature and primer concentration for all body fluids. This simple method and imaging system allow for minimal hands-on time with objective image analysis and presents a pathway for creating a new potential method for forensic body fluid identification.

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. Full bfID sample preparation and analysis of 23 samples was completed in under 3 h using smart phone optical detection and analysis and show efficacy of the method in a validated blind study. The results provide an efficient, sensitive and specific approach to supplement the current biochemical tests in a forensic laboratory.

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. After blue light irradiation for 10 min at 470 nm, the sample was reimaged and analyzed. The resulting intensities obtained pre- and postimaging (clearly observed through a color change from yellow to clear) were then utilized to calculate the total bilirubin concentration. A total of 34 blood samples were analyzed with microfluidic photo treatment-image analysis (µPIA) and were found to have a Deming-regression slope of 0.97 (R2 = 0.960) when compared to the total bilirubin values determined in the clinical laboratory. We demonstrate the implementation of a centrifugal microdevice fabricated through the Print, Cut, and Laminate (PCL) method that accepts eight whole blood samples and provides the capabilities to not only quantitate total bilirubin (Deming-regression slope of 0.95, R2 = 0.990) but allow future integration with excess plasma sufficient for additional downstream clinical assays. This work will highlight the inexpensive nature of the analysis (absence of caustic, viscous, or additional reagents), the simplicity (does not require any chemical reactions), speed (sample-to-answer in <15 min), insusceptibility to biofouling (no protein matrix effects, hemoglobin interferences, and minimized turbidity), low volume plasma requirement (3 µL), and the ability for future downstream integration.

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. METHODS: Blood samples were lysed and diluted before being incubated with silica-coated magnetic particles under chaotropic conditions, a rotating magnetic field, and a source of agitation. The resulting bead aggregation was imaged and correlated to a known WBC count. After establishing standard curves, the WBC count for 18 whole blood samples were determined by this method and compared to values obtained conventionally. RESULTS: When the optimal dilution factor for lysis of whole blood samples was established, 17 of 18 samples (94.4%) were correctly screened and categorized as having high, typical, or low WBC count, while 14 of 18 samples were within 16% of the reported clinical values. The developed system provides analysis of 13 samples in <3 min with a total analysis time of approximately 10 min (including incubation and dilution) and represents comparable throughput to conventional instrumentation, while providing point-of-care capability with reduced size (14 × 21 × 14 cm) and simplicity. CONCLUSIONS: This work demonstrates the potential for a multiplexed, bead-based assay to be used as a rapid, point-of-care screening method for WBC counting from whole blood samples.

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. Soc. 2012 , 134 ( 12 ), 5689 - 96 ]. Although cost-efficient, the open-well format assay required numerous manual steps, and the magnetic field actuation scheme was not readily adaptable for integration. Here, we demonstrate a low-cost (<$2 in-lab), higher-throughput "pinwheel assay" platform that relies on a combination of a disposable rotation-driven microdisc (RDM), and a simple bidirectional rotating magnetic field (bi-RMF). The assay was transformed into an integrated microfluidic system using a multilayered polyester microfluidic disc created through laser print, cut and laminate fabrication, with fluid flow controlled by rotation speed without any mechanical valves. The RDM accepts four samples that undergo on-chip dilution to five different concentrations that cover the effective concentration range needed for downstream cell counting by pinwheel assay. We show that a bi-RMF is effective for the simultaneous actuation of pinwheel assays in 20 detection chambers. The optimization of the bi-RMF frequencies allows the RDM-based pinwheel assay detect human genomic DNA down to a mass of human genomic DNA (5.5 picograms) that is roughly equal to the mass in a single cell. For proof of principle, enumeration of the white blood cells in human blood samples on the RDM provided data correlating well (C.V. of 10%) with those obtained in a clinical lab. Fusing the cost-effective RDM with a simple bi-RMF provides a promising strategy for automation and multiplexing of magnetic particle-based agglutination assays.

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. Swabs generally yielded a higher quantification cycle (Cq) (average 29.7, standard deviation 3.5 vs. 25.3 ± 2.9 from stool, P<0.001) but were still able to detect 80% of pathogens with a Cq < 30 in stool. Second, a simplified total nucleic acid (TNA) extraction procedure was compared to separate DNA and RNA extractions and showed 92% (318/344) sensitivity and 98% (951/968) specificity, with no difference in Cq value for the positive results (ΔCq(DNA+RNA-TNA) = -0.01 ± 1.17, P = 0.972, N = 318). Third, we devised a quantification scheme that adjusts pathogen quantity to the specimen's extraction and amplification efficiency, and show that this better estimates the quantity of spiked specimens than the raw target Cq. In sum, these methods for enteropathogen quantification, stool sample collection, and nucleic acid extraction will be useful for laboratories studying enteric disease.

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). Following introduction from a drop of blood (finger stick), whole blood metering and cell sedimentation are controlled by centrifugal force, only requiring a CD player motor as external hardware and, ultimately, a cell phone for detection. The sedimented volume from patient blood in the HCT Disc was analyzed using a conventional scanner/custom algorithm for analysis of the image to determine a hematocrit value, and these were compared to values generated in a clinical laboratory, which correlated well. To enhance portability and assure simplicity of the HCT measurement, values from image analysis by a cell phone using a custom application was compared to the scanner. Fifteen samples were analyzed with cell phone image analysis system and were found to be within 4% of the HCT values determined in the clinical lab. We demonstrate the feasibility of the PeT device for HCT measurement, and highlight its uniquely low cost (<0.5 USD), speed (sample-to-answer <8 min), multiplexability (12 samples), low volume whole blood requirement (<3 µL), rotation speeds (<4000 rpm) needed for effective measurement as well as the direct finger-to-chip sample loading capability.

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%.

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. The super hydrophilic surface generated during polydimethylsiloxane (PDMS) plasma treatment allowed for accurate metering of the CD4+ T-cell lysate, which then interacted with silica-coated magnetic beads under chaotropic conditions to form aggregates. Images of the resulting aggregates were captured and processed to reveal the mass of DNA, which was used to back-calculate the CD4+ T-cell number. Studies with clinical samples revealed that the analysis of blood within 24 hours of phlebotomy yielded the best results. Under these conditions, an accurate cell count was achieved (R(2) = 0.98) when compared to cell enumeration via flow cytometry, and over a functional dynamic range from 106-2337 cells per µL.

Sonorheometry assessment of platelet function in cardiopulmonary bypass patients: Correlation of blood clot stiffness with platelet integrin αIIbß3 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. METHODS: Thirty-nine patients undergoing elective cardiopulmonary bypass (CPB) were enrolled following informed consent (University of Virginia IRB#14050) in a prospective observational pilot study to assess pre-operative platelet function and transfusion frequency. To assess platelet activity, abciximab was added to blood prior to SR and native S versus abciximab treated S created a differential test for platelet activity. Patient blood samples were activated with kaolin and SR was then used to measure clot stiffness. Patients were transfused with blood products as directed by clinical practice, with the surgical team blinded to SR results. RESULTS: Blood clot stiffness with and without abciximab, was compared in a ratio test (S/Sabciximab) named the Platelet Function Index (PFI). PFI was hypothesized to be positively correlated with platelet contributions through integrin αIIbß3 to clot stiffness. PFI for CPB subjects was lower for those receiving transfusions than those not receiving transfusions (p<0.006). A receiver-operator characteristics (ROC) analysis correlating the PFI with the blinded surgical team's decision on transfusions that included platelet concentrates generated an area under the curve (AUC) of 0.79 (p<0.001). Additionally, the mean value of PFI for subjects on aspirin therapy was lower than for those not on aspirin therapy (p<0.02) and correlated with a 1.73-fold enhanced risk of receiving a peri-operative transfusion. CONCLUSION: Evaluation of platelet function with SR may help in the specification of blood transfusion needs in cardiac surgery and in the assessment of aspirin effects on risk of surgical bleeding.

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. Demographics, clinical characteristics and history, disposition, and laboratory results were recorded. RESULTS: Of the 639 patients included in this study, only 18% were transferred to another psychiatric facility. Pediatric patients and those with presenting with suicidal ideation were more likely to be transferred to an outside facility. Thirty-six percent of the UDS were positive for at least one substance. Marijuana was the most common substance (23%), followed by cocaine (7%) and opiates (7%). There was no evidence that the UDS changed acute management decisions. CONCLUSIONS: Few (<6%) patients demonstrated any clinical characteristics that were consistent with an acute intoxication. Less than 20% of patients who had a transfer UDS were actually transferred to an outside facility corresponding with more than 80% not ordered appropriately according to the ED established guidelines. This number of inappropriate tests represented more than $152 000 of avoidable UDS cost during the study period.

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. METHODS: We developed and assessed molecular diagnostic tests for 15 enteropathogens across three platforms-PCR-Luminex, multiplex real-time PCR, and TaqMan array card-at five laboratories worldwide. We judged the analytical and clinical performance of these molecular techniques against comparator methods (bacterial culture, ELISA, and PCR) using 867 diarrhoeal and 619 non-diarrhoeal stool specimens. We also measured molecular quantities of pathogens to predict the association with diarrhoea, by univariate logistic regression analysis. FINDINGS: The molecular tests showed very good analytical and clinical performance at all five laboratories. Comparator methods had limited sensitivity compared with the molecular techniques (20-85% depending on the target) but good specificity (median 97·3%, IQR 96·5-98·9; mean 95·2%, SD 9·1). Positive samples by comparator methods usually had higher molecular quantities of pathogens than did negative samples, across almost all platforms and for most pathogens (p<0·05). The odds ratio for diarrhoea at a given quantity (measured by quantification cycle, Cq) showed that for most pathogens associated with diarrhoea-including Campylobacter jejuni and Campylobacter coli, Cryptosporidium spp, enteropathogenic Escherichia coli, heat-stable enterotoxigenic E coli, rotavirus, Shigella spp and enteroinvasive E coli, and Vibrio cholerae-the strength of association with diarrhoea increased at higher pathogen loads. For example, Shigella spp at a Cq range of 15-20 had an odds ratio of 8·0 (p<0·0001), but at a Cq range of 25-30 the odds ratio fell to 1·7 (p=0·043). INTERPRETATION: Molecular diagnostic tests can be implemented successfully and with fidelity across laboratories around the world. In the case of diarrhoea, these techniques can detect pathogens with high sensitivity and ascribe diarrhoeal associations based on quantification, including in mixed infections, providing rich and unprecedented measurements of infectious causes. FUNDING: Bill & Melinda Gates Foundation Next Generation Molecular Diagnostics Project.

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. Chloride was measured in each sample on 9 analyzers: Architect 16200, StatProfile pHOx Plus, RapidLab 1265®, Vitros 350®, Advia 1800, Roche Modular, iSTAT1, RAPIDpoint 500, and Radiometer ABL735. RESULTS: Statistically significant, dose-dependent increases in reported chloride concentrations were seen with all analyzers except the RAPIDpoint 500 and Vitros. The increases ranged from 5 to 75 mmol/l at the peak thiosulfate concentrations (33 mmol/l) expected in treated patients. The CLIA-allowable error of 5% was exceeded by 4 analyzers (Architect 16200, iSTAT1, StatProfile pHOx Plus, and Radiometer ABL735). The RAPIDpoint 500 showed a 3-mmol/l decrease in measured chloride over the tested range. The Vitros analyzer showed no interference. CONCLUSIONS: Interference of STS in chloride measurement in several common analyzers may lead to erroneous anion-gap calculations and confound the diagnosis of STS-induced anion-gap acidosis.

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. We hypothesized that administration of rhGH would alter expression of circulating miRNAs and that any changes could be detected. To identify potential miRNA targets, we utilized miRNA microarrays for screening. Confirmatory testing used real-time reverse-transcriptase (RT) quantitative PCR (qPCR) assays of selected miRNAs in 35 plasma samples obtained from (1) individuals with no known pituitary disorders, (2) patients with excess GH production, and (3) patients receiving therapeutic replacement doses of rhGH. We identified and confirmed four miRNAs that were differentially expressed in all individuals using therapeutic replacement doses of rhGH when compared to individuals with naturally high levels of GH and normal controls. This study further develops the hypothesis that circulating miRNAs may be used as biomarkers for detection of doping in sports.

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. Use of these modalities was tracked and residents surveyed about their experiences using them. All 20 residents (100%) reported using Sound Bites either from work computers, home computers, or mobile devices. Residents reported that easy accessibility, brevity, and opportunities for self-assessment were important variables contributing to this use, and that Sound Bite use would make them more likely to benefit from in-person teaching through lectures and/or slide sessions. Within 12 months of the release of the first Sound Bite, individual Sound Bites were accessed a total of 1169 times (mean: 49 times per Sound Bite). In contrast, slide sets were only accessed about once a month and were only employed by 30% of residents (6 of 20) for independent study; only 20% (4 of 20) completed the accompanying multiple choice self-assessment. All residents attended multiheaded microscope slide review sessions. Whereas traditional educational methods remain valuable tools in pathology education, these data suggest that short, web-based tutorials represent a valuable adjuvant teaching tool.

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. et al., J. Am. Chem. Soc. 2012, 134, 5689-96), and from this, the number of DNA-containing cells in the sample can be enumerated. Microbial growth testing is demonstrated by monitoring bead aggregation with E. coli in the presence of ampicillin. Without the need for fluorescent labeling or Coulter counting, the white blood cell count can be defined directly from a microliter of crude whole blood. Specificity is brought to the process by coupling bead-based immunocapture with DNA-bead aggregation allowing for the enumeration of CD4+ T cells from human blood samples. The results of DNA-induced bead aggregation had a 95% correlation with those generated by flow cytometry. With the process requiring only inexpensive, widely available benchtop laboratory hardware, a digital camera, and a simple algorithm, this provided a highly accessible alternative to more expensive cell-counting techniques.

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. The use of POC glucose meters in critically ill patients has been questioned both in the literature and by regulatory agencies. The aim of this study was to determine if the ACCU-CHEK® Inform II system (Roche Diagnostics) POC glucose meter demonstrated the desired accuracy and precision, as defined by Clinical and Laboratory Standards Institute guideline POCT12-A3, in a large number of critically ill patients from multiple intensive care settings at two academic medical centers. METHODS: A total of 1200 whole blood meter results from 600 patients were compared with central laboratory plasma values. Whole blood aliquots from venous samples were used to obtain duplicate meter results with the remaining sample being processed to obtain plasma for central laboratory testing within 5 min of meter testing. RESULTS: A total of 1185 (98.8%) of the new meter's glucose values were within ± 12.5% (± 12 mg/dl for values ≥ 100 mg/dl) of the comparative laboratory glucose values, and 1198 (99.8%) were within ± 20% (± 20 mg/dl for values <100 mg/dl). CONCLUSIONS: Considering the large number of patients from numerous critical care units examined, the new glucose meter system appears to have sufficient analytic accuracy for use in critically ill patients.

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 swab containing cells or DNA could be loaded into a novel receptacle together with the DNA liberation reagents, heated using an infrared heating system, mixed with PCR reagents for one of three different target sets under syringe-driven flow, and thermally-cycled in less than 45 min, an ~6-fold reduction in analysis time as compared to conventional methods. The 4 : 1 PCR reagents : DNA ratio required to provide the correct final concentration of all PCR components for effective amplification was verified using image analysis of colored dyes in the PCR chamber. Novel single-actuation, 'normally-open' adhesive valves were shown to effectively seal the PCR chamber during thermal cycling, preventing air bubble expansion. The effectiveness of the device was demonstrated using three target sets: the sex-typing gene Amelogenin, co-amplification of the ß-globin and gelsolin genes, and the amplification of 15 short tandem repeat (STR) loci plus Amelogenin. The use of the integrated microdevice was expanded to the analysis of liquid blood samples which, when incubated with the DNA liberation reagents, form a brown precipitate that inhibits PCR. A simple centrifugation of the integrated microchips (on a custom centrifuge), mobilized the precipitate away from the microchannel entrance, improving amplification of the ß-globin and gelsolin gene fragments by ~6-fold. This plastic integrated microdevice represents a microfluidic platform with potential for evolution into point-of-care prototypes for application to both clinical and forensic analyses, providing a 5-fold reduction from conventional analysis time.