Mechanism and interpretation of two-for-one response to premature atrial complexes during atrioventricular node re-entry tachycardia.

AIMS: The response to premature atrial complexes (PACs) during tachycardia has been shown to differentiate atrioventricular nodal re-entrant tachycardia (AVNRT) from focal junctional tachycardia (JT). His refractory PAC (HrPACs) perturbing the next His (resetting with fusion) is diagnostic of AVNRT and such a late PAC fusing with the native beat cannot reset the focal source of JT. Early PAC advancing the immediate His with continuation of tachycardia suggests JT but can also occur in AVNRT due to simultaneous conduction through the AV nodal fast and slow pathways [two-for-one response (TFOR)]. The objective of this study was to evaluate the incidence and mechanism of TFOR after early premature atrial complexes (ePACs) during AVNRT and to differentiate it from the known response to ePACs during JT. METHODS AND RESULTS: Typical AVNRT cases were diagnosed using standard criteria. We evaluated the responses to scanning PACs delivered during tachycardia in 100 patients undergoing AV node slow pathway modification for AVNRT. The responses to HrPACs and ePACs delivered from coronary sinus os or high right atrium were retrospectively reviewed. In 10 patients, ePACs advanced the immediate His with continuation of tachycardia. In all 10 cases, HrPACs advanced the next His, confirming AVNRT as the mechanism, and indicating a TFOR. CONCLUSION: A TFOR can occur in a small number of patients during AVNRT and is therefore not diagnostic of JT. However, HrPACs always perturbed the next His in these cases, confirming the diagnosis of AVNRT and allowing for differentiation from JT.

Single-center experience of the FIRM technique to ablate paroxysmal and persistent atrial fibrillation.

INTRODUCTION: Focal impulse and rotor modulation (FIRM)-guided ablation has had mixed results of published success, and most studies have had a follow-up for a year or less. We aimed to study a consecutive group of patients followed for at least 1.5 years, subgrouped into those with an initial FIRM ablation and those with a previous, failed ablation who now received a FIRM guided one, to evaluate for success in each group and factors that might affect success. METHODS: Of 181 patients, 167 were available for analysis. Group 1 (n = 122) had a first or primary ablation (paroxysmal atrial fibrillation [PAF] 51; persistent atrial fibrillation [PeAF] 71) and group 2 (n = 45) had a redo ablation (PAF 18; PeAF 27). All patients were done under general anesthesia. FIRM mapping was done in the right atrium first and then the left, and only rotors consistently seen on multiple epochs were ablated, using 15 to 30 W. Rotor ablation was discontinued when remapping showed elimination of rotational activity at the site. Wide area catheter ablation was done for pulmonary vein isolation (PVI). Routine follow-up was at 3, 6, and 12 months of the first year, with a Holter monitor at 6 months, and then every 6 months thereafter. Event recorders were given to patients with potential arrhythmic symptoms. RESULTS: Mean follow-up was 16 months. Nearly 40% of patients had obstructive sleep apnea; mean body mass index was 32; and average left atrial size was 39.7 mm and 46.2 mm for PAF and PeAF patients, respectively. Freedom from atrial arrhythmia recurrence was: in group 1 patients, 82.4% for PAF and 67.6% for PeAF patients; in group 2 patients, 83.3% for PAF, but only 40.7% for PeAF patients. Comparing outcomes for the first 10 patients studied to the next 20 or more done by three operators showed no difference, suggesting no learning curve affecting the ablation results. Furthermore, the univariate analysis did not show any demographic factor to have an independent significance for ablation success or failure. Spontaneous termination during rotor ablation occurred in 76.8% of PAF and 27.6% of PeAF patients but did not affect the long-term outcomes for maintenance of sinus rhythm. CONCLUSIONS: FIRM-guided atrial ablation plus PVI in our patient population resulted in good success from a recurrence of atrial arrhythmias in patients undergoing an initial ablation procedure. For those with persistent AF undergoing a second procedure now using FIRM guidance plus PVI, the results are lower. Further research is needed to define better the appropriate population for FIRM-guided ablation and the degree of ablation needed for success in these patients.

Application of microdroplet PCR for large-scale targeted bisulfite sequencing.

Cytosine methylation of DNA CpG dinucleotides in gene promoters is an epigenetic modification that regulates gene transcription. While many methods exist to interrogate methylation states, few current methods offer large-scale, targeted, single CpG resolution. We report an approach combining bisulfite treatment followed by microdroplet PCR with next-generation sequencing to assay the methylation state of 50 genes in the regions 1 kb upstream of and downstream from their transcription start sites. This method yielded 96% coverage of the targeted CpGs and demonstrated high correlation between CpG island (CGI) DNA methylation and transcriptional regulation. The method was scaled to interrogate the methylation status of 77,674 CpGs in the promoter regions of 2100 genes in primary CD4 T cells. The 2100 gene library yielded 97% coverage of all targeted CpGs and 99% of the target amplicons.

Dynamic regulation of HIV-1 mRNA populations analyzed by single-molecule enrichment and long-read sequencing.

Alternative RNA splicing greatly expands the repertoire of proteins encoded by genomes. Next-generation sequencing (NGS) is attractive for studying alternative splicing because of the efficiency and low cost per base, but short reads typical of NGS only report mRNA fragments containing one or few splice junctions. Here, we used single-molecule amplification and long-read sequencing to study the HIV-1 provirus, which is only 9700 bp in length, but encodes nine major proteins via alternative splicing. Our data showed that the clinical isolate HIV-1(89.6) produces at least 109 different spliced RNAs, including a previously unappreciated ∼1 kb class of messages, two of which encode new proteins. HIV-1 message populations differed between cell types, longitudinally during infection, and among T cells from different human donors. These findings open a new window on a little studied aspect of HIV-1 replication, suggest therapeutic opportunities and provide advanced tools for the study of alternative splicing.

Multiplex picoliter-droplet digital PCR for quantitative assessment of DNA integrity in clinical samples.

BACKGROUND: Assessment of DNA integrity and quantity remains a bottleneck for high-throughput molecular genotyping technologies, including next-generation sequencing. In particular, DNA extracted from paraffin-embedded tissues, a major potential source of tumor DNA, varies widely in quality, leading to unpredictable sequencing data. We describe a picoliter droplet-based digital PCR method that enables simultaneous detection of DNA integrity and the quantity of amplifiable DNA. METHODS: Using a multiplex assay, we detected 4 different target lengths (78, 159, 197, and 550 bp). Assays were validated with human genomic DNA fragmented to sizes of 170 bp to 3000 bp. The technique was validated with DNA quantities as low as 1 ng. We evaluated 12 DNA samples extracted from paraffin-embedded lung adenocarcinoma tissues. RESULTS: One sample contained no amplifiable DNA. The fractions of amplifiable DNA for the 11 other samples were between 0.05% and 10.1% for 78-bp fragments and ≤1% for longer fragments. Four samples were chosen for enrichment and next-generation sequencing. The quality of the sequencing data was in agreement with the results of the DNA-integrity test. Specifically, DNA with low integrity yielded sequencing results with lower levels of coverage and uniformity and had higher levels of false-positive variants. CONCLUSIONS: The development of DNA-quality assays will enable researchers to downselect samples or process more DNA to achieve reliable genome sequencing with the highest possible efficiency of cost and effort, as well as minimize the waste of precious samples.

Accurate variant detection across non-amplified and whole genome amplified DNA using targeted next generation sequencing.

BACKGROUND: Many hypothesis-driven genetic studies require the ability to comprehensively and efficiently target specific regions of the genome to detect sequence variations. Often, sample availability is limited requiring the use of whole genome amplification (WGA). We evaluated a high-throughput microdroplet-based PCR approach in combination with next generation sequencing (NGS) to target 384 discrete exons from 373 genes involved in cancer. In our evaluation, we compared the performance of six non-amplified gDNA samples from two HapMap family trios. Three of these samples were also preamplified by WGA and evaluated. We tested sample pooling or multiplexing strategies at different stages of the tested targeted NGS (T-NGS) workflow. RESULTS: The results demonstrated comparable sequence performance between non-amplified and preamplified samples and between different indexing strategies [sequence specificity of 66.0% ± 3.4%, uniformity (coverage at 0.2× of the mean) of 85.6% ± 0.6%]. The average genotype concordance maintained across all the samples was 99.5% ± 0.4%, regardless of sample type or pooling strategy. We did not detect any errors in the Mendelian patterns of inheritance of genotypes between the parents and offspring within each trio. We also demonstrated the ability to detect minor allele frequencies within the pooled samples that conform to predicted models. CONCLUSION: Our described PCR-based sample multiplex approach and the ability to use WGA material for NGS may enable researchers to perform deep resequencing studies and explore variants at very low frequencies and cost.

The surface electrocardiogram predicts risk of heart block during right heart catheterization in patients with preexisting left bundle branch block: implications for the definition of complete left bundle branch block.

BACKGROUND: Patients with left bundle branch block (LBBB) undergoing right heart catheterization can develop complete heart block (CHB) or right bundle branch block (RBBB) in response to right bundle branch (RBB) trauma. We hypothesized that LBBB patients with an initial r wave (>or=1 mm) in lead V1 have intact left to right ventricular septal (VS) activation suggesting persistent conduction over the left bundle branch. Trauma to the RBB should result in RBBB pattern rather than CHB in such patients. METHODS: Between January 2002 and February 2007, we prospectively evaluated 27 consecutive patients with LBBB developing either CHB or RBBB during right heart catheterization. The prevalence of an r wave >or=1 mm in lead V1 was determined using 118 serial LBBB electrocardiographs (ECGs) from our hospital database. RESULTS: Catheter trauma to the RBB resulted in CHB in 18 patients and RBBB in 9 patients. All 6 patients with >or=1 mm r wave in V1 developed RBBB. Among these 6 patients q wave in lead I, V5, or V6 were present in 3. Four patients (3 in CHB group and 1 in RBBB group) developed spontaneous CHB during a median follow-up of 61 months. V1 q wave >or=1 mm was present in 28% of hospitalized complete LBBB patients. CONCLUSIONS: An initial r wave of >or=1 mm in lead V1 suggests intact left to right VS activation and identifies LBBB patients at low risk of CHB during right heart catheterization. These preliminary findings indicate that an initial r wave of >or=1 mm in lead V1, present in approximately 28% of ECGs with classically defined LBBB, may constitute a new exclusion criterion when defining complete LBBB.

Prevalence and impact of manic traits in depressed patients initiating interferon therapy for chronic hepatitis C infection.

GOALS: To evaluate the impact of manic traits on adverse events in depressed hepatitis C patients initiating interferon therapy. BACKGROUND: Interferon alpha therapy for hepatitis C can exacerbate preexisting depression. Bipolar disorder frequently presents as depressive symptoms that are indistinguishable from or misdiagnosed as major depressive disorder. The impact of bipolar disorder on adverse psychiatric events during therapy is unknown. STUDY: A retrospective study was performed on consecutive patients initiating interferon therapy in the Hepatology clinic at a tertiary-care center between December 2004 and October 2007. All patients completed the Physicians Health Questionnaire (PHQ-9), a validated survey for major depressive disorder. Patients with a positive PHQ screen completed the Mood Disorders Questionnaire (MDQ), a validated screening tool for manic traits. Patients with a negative PHQ served as controls. All adverse psychiatric events were documented through retrospective record review for 6 months after interferon initiation. RESULTS: A total of 165 patients were treated with interferon alpha. One hundred thirty-two (80%) had a negative PHQ (controls) and 33 had a positive PHQ. Forty-one (30%) of the control patients had adverse psychiatric events. Psychiatric events occurred in 8 of 22 (36%) patients with positive PHQ but negative MDQ; 8 of 11 (73%) with positive PHQ and positive MDQ had psychiatric adverse events. This finding was statistically significant compared with the control group (P=0.007). The overall sustained viral response rate was 58% and was not statistically significant among groups. CONCLUSIONS: Baseline manic traits, as detected by the MDQ, were associated with high rates of adverse psychiatric events among individuals receiving antiviral therapy.

Bilateral macular hole formation secondary to sclopetaria caused by shockwaves transmitted by a posterior vector: case report.

BACKGROUND: Sclopetaria is a rare ophthalmic finding in trauma CASE PRESENTATION: This is a report of a patient who developed macular holes from sclopetaria induced by indirect trauma. A 22-year-old male, suffered a gunshot wound that passed behind his eyes, resulting in bilateral macular hole formation CONCLUSION: To our knowledge, this is the first reported case in which trauma posterior to the globes caused bilateral macular hole formation.

How a State Measures Up: Ambulatory Care Pharmacists' Perception of Practice Management Systems for Comprehensive Medication Management in Utah.

Comprehensive medication management (CMM) is a patient-centered standard of care that ensures a patient's medications are optimized. The CMM Practice Management Assessment Tool (PMAT) is a tool to assess areas of CMM practice management. The purpose of this project was to assess the state of CMM practice management based on clinical pharmacist perception for two health systems in the state of Utah, and to identify areas of excellence and/or improvement utilizing a novel method for PMAT analysis. The PMAT was distributed to all primary care-focused ambulatory care pharmacists employed by University of Utah Health (U of U Health) and Intermountain Healthcare (Intermountain). Ordinal responses were assigned to three possible categories of CMM support (High, Indifferent, and Low). Ten surveys were completed from U of U Health, and nine were completed from Intermountain. Thirty-two of the 86 survey questions resulted in a high level of support, and 25 questions resulted in a low level of support from the majority of respondents. Statistically significant differences between the institutions were found for 18 questions. The utilization of the PMAT within two Utah health systems highlighted areas of excellence and improvement and demonstrates a unique method for analysis of PMAT results.

Rationale and design of the impact of anticoagulation therapy on the Cognitive Decline and Dementia in Patients with Nonvalvular Atrial Fibrillation (CAF) Trial: A Vanguard study.

Atrial fibrillation (AF) is associated with a risk for cognitive impairment and dementia, which is more pronounced in patients with a history of clinical stroke. Observational trials suggest that the implementation and quality of long-term anticoagulation impact dementia risk. Emerging evidence suggests that direct oral anticoagulants may improve long-term risk of dementia in AF patients. This manuscript describes the rational and trial design of the the Cognitive Decline and Dementia in Atrial Fibrillation Patients (CAF) Trial. CAF investigates if AF patients randomized to dabigatran etexilate will have long-term higher cognition scores and lower rates of dementia compared in the long term to dose-adjusted warfarin (International Normalized Ratio [INR]: 2.0-3.0). As of 27 February 2019, a total of 120 subjects will be enrolled at one investigational site in the United States and will be followed for 2 years after study enrollment. To date, 97 have been enrolled. The average age is 74.2 years, 53% are male, and 9% had a prior stroke. In this Vanguard study, patients will be followed for 2 years after study enrollment. These prospective, randomized data will inform the understanding of two anticoagulants in AF patients as it relates to risk of cognitive decline and dementia. Cranial imaging and biomarkers collected will assist in understanding mechanisms of brain injury.

"Pharming out" support: a promising approach to integrating clinical pharmacists into established primary care medical home practices.

Objective Embedding clinical pharmacists into ambulatory care settings needs to be assessed in the context of established medical home models. Methods A retrospective, observational study examined the effectiveness of the Intermountain Healthcare Collaborative Pharmacist Support Services (CPSS) program from 2012-2015 among adult patients diagnosed with diabetes mellitus (DM) and/or high blood pressure (HBP). Patients who attended this program were considered the intervention (CPSS) cohort. These patients were matched using propensity scores with a reference group (no-CPSS cohort) to determine the effect of achieving disease management goals and time to achievement. Results A total of 17,684 patients had an in-person office visit with their provider and 359 received CPSS (the matched no-CPSS cohort included 999 patients). CPSS patients were 93% more likely to achieve a blood pressure goal < 140/90 mmHg, 57% more likely to achieve HbA1c values < 8%, and 87% more likely to achieve both disease management goals compared with the reference group. Time to goal achievement demonstrated increasing separation between the study cohorts across the entire study period ( P < .001), and specifically, at 180 days post-intervention (HBP: 48% vs 27% P < .001 and DM: 39% vs 30%, P < .05). Conclusions CPSS participation is associated with significant improvement in achievement of disease management goals, time to achievement, and increased ambulatory encounters compared with the matched no-CPSS cohort.

Comparison of 2D-3D Measurements of Hallux and First Ray Sagittal Motion in Patients With and Without Hallux Valgus.

BACKGROUND: Clinicians base treatment decisions on measures of hallux and first ray motion in the management of first metatarsophalangeal joint disorders. Women account for a majority of the patients. This study assessed the reliability of a 2D approach for the measurements of sagittal motion, and compared the result to a Cardan (3D) angle criterion standard and evaluated how hallux valgus (bunion) deformity affected the comparisons. METHODS: Twenty-nine women (controls n = 10; bunion n = 19) were examined using a retrospective repeated measures design. Weightbearing magnetic resonance (MR) images were acquired to replicate the position of the foot during the stance phase of gait. The images were reconstructed into virtual bone models using computer processes, whereby measures of hallux and first ray motion were represented by 2D and 3D methods of measurement. An examiner measured 2D motion on the image data sets using a goniometer, and reliability was assessed. The 3D Cardan angle result was derived from a matrix calculation. The 2D-3D comparison of measurements was evaluated with an analysis of variance (ANOVA) model across gait conditions, run separate for groups. RESULTS: The 2D measurement was reliable (ICC ≥ 0.98, SEM ≤ 0.89 degrees). There was no method-by-condition interaction (F ≤ 1.37, P ≥ .25) between variables. No significant difference was detected between the 2D-3D measurements in the control group (F ≤ 1.24, P ≥ .30), but the measurements were statistically different (F ≥ 4.46, P ≤ .049) in the bunion group. CONCLUSION: This study described a reliable 2D approach for measuring hallux and first ray sagittal motion from weightbearing images. The 2D measurements were comparable to a Cardan angle component motion result in controls, but not in women with bunion. CLINICAL RELEVANCE: Joint motion measurements may augment clinical decision making. These results suggest that a 2D image-based approach may be adequate to estimate hallux and first ray sagittal motion, although bunion deformity creates out-of-plane motions that may require 3D methods to accurately quantify. Further clinical study is required to assess the differences in clinical outcomes between measurement techniques.

Continuous murmur caused by a ventricular septal defect post-myocardial infarction: visual display with a handheld computer.

Ventricular septal defects do not typically result in a continuous murmur. The only previously reported case involved a congenital ventricular septal defect. We report a case of an acquired ventricular septal defect following a large anterior myocardial infarction resulting in a continuous murmur. The continUous nature was confirmed both with 2D echocardiography and a specially equipped stethoscope in conjunction with a handheld computer to provide a visual display of the murmur. A proposed mechanism for the continuous murmur was discussed.

DNA stabilization is critical for maximizing performance of fecal DNA-based colorectal cancer tests.

We have developed a multitarget, fecal DNA screening assay that detects the presence of gene-specific mutations and long DNA fragments associated with colorectal cancer (CRC). We continue to investigate methods that may be used to optimize clinical sensitivity. The goals of this investigation are to establish how sample handling conditions affect the stability of DNA in stool, thereby potentially limiting clinical sensitivity, and to determine conditions to ameliorate DNA degradation. A study was run comparing paired sample aliquots. Quantitative PCR data for matched aliquots was used to determine first the effect of sample incubation on total recovery and integrity of DNA, then the effect of stabilization buffer addition to stool on recoverable DNA, and finally the impact of buffer addition on assay sensitivity. Comparison of quantitative PCR data for paired aliquots shows that the amount of recoverable human DNA is negatively affected by storing stool samples (N = 43) at room temperature for > or =36 hours (P = 0.0018). However, the addition of stabilization buffer leads to a significant increase in recovery of DNA (P = 0.010), compared with samples incubated without buffer. Whereas the DNA Integrity Assay (DIA) is found to be sensitive to DNA degradation (sensitivity was reduced by 82%; P = 0.0002), point mutation marker sensitivity is more refractory. Overall, DNA can be stabilized by addition of buffer to the sample, leading to increased assay sensitivity.

Computerized clinical decision support improves warfarin management and decreases recurrent venous thromboembolism.

BACKGROUND: An explicit approach to warfarin dose adjustment using computerized clinical decision support (CDS) improves warfarin management. We report metrics of quality for warfarin management before and after implementation of CDS in a large health care system. METHODS: A total of 2591 chronically anticoagulated patients were eligible for inclusion. We compared interpatient time in therapeutic range (TTR) and international normalized ratio (INR) variability before and after implementation of CDS. We report outcomes of major bleeding, thrombosis, and health care utilization. RESULTS: Implementation of CDS significantly improved TTR (from 63.99% to 65.13%; P = .04) and reduced out-of-range INRs (from 42.39% to 39.97%; P < .001). Venous thromboembolism (relative risk [RR] 0.41; P < .001) emergency department utilization (RR 0.62; P < .001), and hospitalization (RR 0.62; P < .001) were reduced after CDS implementation. Major hemorrhage was more frequent after CDS implementation (RR 1.42; P = .01). CONCLUSION: The CDS warfarin management was associated with improved TTR and decreased INR variability in a large cohort of chronically anticoagulated patients. Clinically relevant outcomes were broadly improved, although more bleeding events were observed.

Clinical relevance of KRAS-mutated subclones detected with picodroplet digital PCR in advanced colorectal cancer treated with anti-EGFR therapy.

PURPOSE: KRAS mutations are predictive of nonresponse to anti-EGFR therapies in metastatic colorectal cancer (mCRC). However, only 50% of nonmutated patients benefit from them. KRAS-mutated subclonal populations nondetectable by conventional methods have been suggested as the cause of early progression. Molecular analysis technology with high sensitivity and precision is required to test this hypothesis. EXPERIMENTAL DESIGN: From two cohorts of patients with mCRC, 136 KRAS, NRAS, and BRAF wild-type tumors with sufficient tumor material to perform highly sensitive picodroplet digital PCR (dPCR) and 41 KRAS-mutated tumors were selected. All these patients were treated by anti-EGFR therapy. dPCR was used for KRAS or BRAF mutation screening and compared with qPCR. Progression-free survival (PFS) and overall survival (OS) were analyzed according to the KRAS-mutated allele fraction. RESULTS: In addition to the confirmation of the 41 patients with KRAS-mutated tumors, dPCR also identified KRAS mutations in 22 samples considered as KRAS wild-type by qPCR. The fraction of KRAS-mutated allele quantified by dPCR was inversely correlated with anti-EGFR therapy response rate (P < 0.001). In a Cox model, the fraction of KRAS-mutated allele was associated with worse PFS and OS. Patients with less than 1% of mutant KRAS allele have similar PFS and OS than those with wild-type KRAS tumors. CONCLUSIONS: This study suggests that patients with mCRC with KRAS-mutated subclones (at least those with a KRAS-mutated subclones fraction lower or equal to 1%) had a benefit from anti-EGFR therapies.

Determining lower limits of detection of digital PCR assays for cancer-related gene mutations.

Digital PCR offers very high sensitivity compared to many other technologies for processing molecular detection assays. Herein, a process is outlined for determining the lower limit of detection (LoD) of two droplet-based digital PCR assays for point mutations of the epidermal growth factor receptor (EGFR) gene. Hydrolysis probe mutation-detection assays for EGFR p.L858R and p.T790M mutations were characterized in detail. Furthermore, sixteen additional cancer-related mutation assays were explored by the same approach. For the EGFR L8585R assay, the assay sensitivity is extremely good, and thus, the LoD is limited by the amount of amplifiable DNA that is analyzed. With 95% confidence limits, the LoD is one mutant in 180,000 wild-type molecules for the evaluation of 3.3 µg of genomic DNA, and detection of one mutant molecule in over 4 million wild-type molecules was achieved when 70 million copies of DNA were processed. The measured false-positive rate for the EGFR L8585R assay is one in 14 million, which indicates the theoretical LoD if an unlimited amount of DNA is evaluated. For the EFGR T790M assay, the LoD is one mutant in 13,000 for analysis of a 3.3 µg sample of genomic DNA, and the dPCR assay limit sensitivity approaches one mutant in 22,000 wild-type molecules.

Electrocardiographic features and prevalence of bilateral bundle-branch delay.

BACKGROUND: Definitive diagnosis of bilateral bundle-branch delay/block may be made when catheter-induced right bundle-branch block (RBBB) develops in patients with baseline left bundle-branch (LBB) block. We hypothesized that a RBBB pattern with absent S waves in leads I and aVL will identify bilateral bundle-branch delay/block. METHODS AND RESULTS: Fifty patients developing transient RBBB pattern in lead V1 during right heart catheterization were studied. Patients were grouped according to whether the baseline ECG demonstrated a normal QRS, left fascicular blocks, or LBB block pattern. The RBBB morphologies in each group were compared. The prevalence of bilateral bundle-branch delay/block pattern was examined in our hospital ECG database. All patients with baseline normal QRS complexes (n=30) or left fascicular blocks (4 anterior, 5 posterior) developed a typical RBBB pattern. Among the 11 patients with a baseline LBB block pattern, 7 developed an atypical RBBB pattern with absent S waves in leads I and aVL and the remaining 4 demonstrated a typical RBBB. The absence of S waves in leads I and aVL during RBBB was 100% specific and 64% sensitive for the presence of pre-existing LBB block. Among the consecutive 2253 hospitalized patients with RBBB, 34 (1.5%) had the bilateral bundle-branch delay/block pattern. CONCLUSIONS: An ECG pattern of RBBB in lead V1 with absent S wave in leads I and aVL indicates concomitant LBB delay. Pure RBBB and bifascicular blocks are associated with S waves in leads I and aVL.

Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR.

Quantitative polymerase chain reactions (qPCR) based on real-time PCR constitute a powerful and sensitive method for the analysis of nucleic acids. However, in qPCR, the ability to multiplex targets using differently colored fluorescent probes is typically limited to 4-fold by the spectral overlap of the fluorophores. Furthermore, multiplexing qPCR assays requires expensive instrumentation and most often lengthy assay development cycles. Digital PCR (dPCR), which is based on the amplification of single target DNA molecules in many separate reactions, is an attractive alternative to qPCR. Here we report a novel and easy method for multiplexing dPCR in picolitre droplets within emulsions-generated and read out in microfluidic devices-that takes advantage of both the very high numbers of reactions possible within emulsions (>10(6)) as well as the high likelihood that the amplification of only a single target DNA molecule will initiate within each droplet. By varying the concentration of different fluorogenic probes of the same color, it is possible to identify the different probes on the basis of fluorescence intensity. Adding multiple colors increases the number of possible reactions geometrically, rather than linearly as with qPCR. Accurate and precise copy numbers of up to sixteen per cell were measured using a model system. A 5-plex assay for spinal muscular atrophy was demonstrated with just two fluorophores to simultaneously measure the copy number of two genes (SMN1 and SMN2) and to genotype a single nucleotide polymorphism (c.815A>G, SMN1). Results of a pilot study with SMA patients are presented.