False-positive Samples Research Articles

Cascaded hybrid residual U-Net for glioma segmentation

Glioma segmentation is critical for making surgical plans. Recently, the traditional glioma segmentation method is less competitive with two deep learning segmentation strategies: the patch-based method which focuses more on the local feature for each pixel, and the image-based method which fully leverages the global feature and captures the overall shape, size and other characteristics of the lesion in a neighborhood of a pixel. In this study, we investigate and integrate the advantages of 2-D and 3-D image-based architectures, and propose a new convolutional neural network called the Cascaded Hybrid Residual U-Net (CHR-U-Net) for MRI glioma segmentation. The CHR-U-Net exploits both the 2D local features as well as the 3D global spatial contextual information simultaneously. In the first-level of CHR-U-Net, the R-2D-U-Net combines the 2D-U-Net and the residual unit for quick lesion area detecting without any miss. To prevent from missing false-positive pixels, the output of R-2D-U-Net is resampled by using the hard-mining to collect more possible false-positive samples. In the second-level of CHR-U-Net, the axial, coronal, and sagittal 3D-U-Nets are trained to predict whether pixels belong to the area of glioma. The results of three 3D-U-Nets are fused to improve the accuracy and reduce false positives. The database of 2017 BRATS challenge were used in our experiments for the verification. The Dices and Sensitivities of Enhancing, Whole, and Core areas were calculated. The Dices are 0.73, 0.90, and 0.83 and the Sensitivities are 0.83, 0.90, and 0.82, respectively, for the axial, coronal, and sagittal 3D-U-Nets. Experimental results show that the proposed model significantly improves the performance of glioma segmentation.

Diagnostic Accuracy of Pan-Degenerate Amplification and Adaptation Assay for HIV-1 Drug Resistance Mutation Analysis in Low- and Middle-Income Countries.

HIV drug resistance (HIVDR) is a barrier to sustained virologic suppression in low- and middle-income countries (LMICs). Point mutation assays targeting priority drug resistance mutations (DRMs) are being evaluated to improve access to HIVDR testing. In a cross-sectional study (June 2018 to September 2019), we evaluated the diagnostic accuracy of a simple and rapid HIVDR assay (the pan-degenerate amplification and adaptation [PANDAA] assay targeting the mutations K65R, K103NS, M184VI, Y181C, and G190A) compared to Sanger sequencing and next-generation sequencing (NGS). Plasma samples from adolescents and young adults (aged 10 to 24 years) failing antiretroviral therapy (viral load, >1,000 copies/ml on 2 consecutive occasions 1 month apart) were analyzed. Sensitivity and specificity of the PANDAA assay were determined by a proprietary application designed by Aldatu Biosciences. Agreement between genotyping methods was evaluated using Cohen's kappa coefficient. One hundred fifty samples previously characterized by Sanger sequencing were evaluated using PANDAA. For all DRMs detected, PANDAA showed a sensitivity and specificity of 98% and 94%, respectively. For nucleotide reverse transcriptase inhibitor DRMs, sensitivity and specificity were 98% (95% confidence interval [CI], 92% to 100%) and 100% (94% to 100%), respectively. For non-nucleotide reverse transcriptase inhibitor DRMs, sensitivity and specificity were 100% (97% to 100%) and 76% (61% to 87%), respectively. PANDAA showed strong agreement with Sanger sequencing for K65R, K103NS, M184VI, and G190A (kappa > 0.85) and substantial agreement for Y181C (kappa = 0.720). Of the 21 false-positive samples genotyped by PANDAA, only 6 (29%) were identified as low-abundance variants by NGS. With the high sensitivity and specificity to detect major DRMs, PANDAA could represent a simple and rapid alternative HIVDR assay in LMICs.

Validation of tumor DNA in bronchial lavage as a diagnostic tool in lung cancer.

9020 Background: Diagnosing lung cancer requires invasive procedures with risk of complications for the patient. The HOXA9 gene is highly methylated in lung cancer, and methylated tumor DNA (meth-tDNA) in bronchial lavage has previously shown potential as a diagnostic biomarker. The aim of the present study was to validate these preliminary results. Methods: Patients were referred by the general practitioner on suspicion of lung cancer. The Danish diagnostic package includes chest and abdominal CT scan, bronchoscopy, blood tests, and histopathological or cytological verification. Twelve ml lavage fluid was collected at bronchoscopy for analysis of meth-tDNA based on droplet digital PCR according to our published method. A positive test was defined as ≥ 4 droplets containing meth-tDNA and a ratio between HOXA9 and Albumin of > 0.15%. The analysis was performed blinded to clinical data and meth-tDNA status was compared with the final diagnosis. Results: The study population was 204 consecutively enrolled patients. The material consisted of a discovery cohort (n = 105, presented at ASCO 2019) used for establishing the cut-points, and a validation cohort (n = 99). Six were excluded from analysis due to malignancy other than lung cancer and one due to failed analysis. In the discovery cohort, the sensitivity was 68.7% (95% CI 56.2-79.4%), specificity 88.2% (95% CI 72.6-96.7%), and positive predictive value (PPV) 92.0% (95% CI 80.8-97.8%). In the validation cohort, the same values were 76.9% (95% CI 63.2-87.5%), 77.3% (95% CI 62.2-88.5%), and 80.0% (95% CI 66.3-90.0%), respectively. Analyzing the entire patient material (n = 197) the sensitivity, specificity, and PPV were 72.3% (95% CI 63.3-80.1%), 82.1% (95% CI 71.7-89.8%), and 86.0% (95% CI 77.6-92.1%), respectively. The false positive samples were equally distributed among patients with cryptogenic organizing pneumonia, granulomatous inflammation, and acute inflammatory disease. The false negative samples were mainly from patients with peripheral tumor, no radiologically detectable tumor, and mesothelioma. Conclusions: Meth-tDNA in bronchial lavage holds potential as a supplementary tool in the diagnosis of lung cancer with a clinically relevant sensitivity and specificity. Routine clinical application awaits further validation in a clinical trial. [Table: see text]

Analytical validation of a tissue agnostic ctDNA MRD assay using tumor specific methylation and somatic variant profiles in early-stage CRC.

e15549 Background: Liquid biopsy is a candidate for detection of minimal residual disease (MRD) in early colorectal cancer. Detection of circulating tumor (ct)DNA in early stage cancer is challenging given the low abundance of ctDNA molecules. We developed a tissue agnostic MRD Test to optimize sensitivity and specificity of ctDNA detection by integrating tumor-specific genomic mutation and DNA methylation signatures coupled to noise-reduction of non-tumor background cfDNA signals. Methods: This MRD Test was developed following CLIA, Nex-StoCT Working Group, and AMP/CAP guidance and validation principles. Using a single input plasma sample, data from somatic and epigenomic targeted capture panels are integrated to generate a final test result: ctDNA detected or ctDNA not detected. ctDNA detected is defined by the presence of mutation or epigenomic classifier inputs exceeding a defined threshold. Quality control (QC) measures were implemented in both process and final sequencing results. Analytical specificity, sensitivity, and accuracy were determined using 130 samples. Results: The MRD Test specificity was 95% (70/74) using colonoscopy screened negative samples.A retrospective review of the clinical histories of these false positive samples showed high risk clinical features such as smoking, family history of CRC, and sub-optimal bowel prep prior to the colonoscopy. Analytical sensitivity (LoD) was established by diluting late stage CRC samples at two clinically relevant cfDNA inputs at three dilutions (30ng or 10ng, ranging 0.1% to 0.75% tumor fraction). The 95% LoD was determined to be 0.3% for 10ng and < 0.1% for 30ng, the lowest level tested for each input. Accuracy was determined by diluting advanced CRC samples to 0.5-0.75% MAF tested at 30ng or 10ng cfDNA with a detection result of 100% (38/38). Conclusions: We present a CLIA validated assay with performance characteristics sufficient to detect residual disease in Stage II/III CRC post-curative intent therapy. This assay is currently being used in multiple interventional clinical trials.

Results from extended lymphadenectomies with [111In]PSMA-617 for intraoperative detection of PSMA-PET/CT-positive nodal metastatic prostate cancer

PurposeIdentification of suspicious PSMA-PET/CT-positive lymph node (LN) metastases (LNM) from prostate cancer (PCa) during lymphadenectomy (LA) is challenging. We evaluated an 111In-labelled PSMA ligand (DKFZ-617, referred to as [111In]PSMA-617) as a γ-emitting tracer for intraoperative γ-probe application for resected tissue samples in PCa patients. Forty-eight hours prior to LA, [111In]PSMA-617 was administered intravenously in 23 patients with suspected LNM on PSMA-PET/CT (n = 21 with biochemical relapse, n = 2 at primary therapy). Resected tissue samples (LN, LNM and fibrofatty tissue) were measured ex situ by a γ-probe expressed as counts per second (CPSnorm). [111In]PSMA-617 tissue sample uptake was measured by a germanium detector for verification and calculated as %IAlbm (percent injected activity per kilogram lean body mass at time of surgery). Based on a clinical requirement for a specificity > 95%, thresholds for both ex situ measurements were chosen accordingly. Correlation of the results from PET/CT, γ-probe and germanium detector with histopathology was done.ResultsEight hundred sixty-four LNs (197 LNM) were removed from 275 subregions in 23 patients, on average 8.6 ± 14.9 LNM per patient. One hundred four of 275 tissue samples showed cancer. Median γ-probe and germanium detector results were significantly different between tumour-affected (33.5 CPSnorm, 0.71 %IAlbm) and tumour-free subregions (3.0 CPSnorm, 0.03 %IAlbm) (each p value < 0.0001). For the chosen γ-probe cut-off (CPSnorm > 23) and germanium detector cut-off (%IAlbm > 0.27), 64 and 74 true-positive and 158 true-negative samples for both measurements were identified. Thirty-nine and 30 false-negative and 6 and 5 false-positive tissue samples were identified by γ-probe and germanium detector measurements.Conclusion[111In]PSMA-617 application for LA is feasible in terms of an intraoperative real-time measurement with a γ-probe for detection of tumour-affected tissue samples. γ-probe results can be confirmed by precise germanium detector measurements and were significantly different between tumour-affected and tumour-free samples.

A Bitcoin Transaction Network Analytic Method for Future Blockchain Forensic Investigation

Popular Blockchain-based cryptocurrencies, like Bitcoin, are increasingly being used maliciously for illegal trades. In order to trace and analyze suspected Bitcoin transactions and addresses, address clustering methods and Bitcoin flow analysis methods are gaining attention recently. However, existing methods only focus on Bitcoin addresses and flow, and neglect other important information, such as transaction structure and behavior features. In order to exploit all useful features of transactions, this paper proposes a Bitcoin transaction network analytic method for facilitating Blockchain forensic investigation based on an extended safe Petri Net. The structural features and dynamic semantics of Petri net are used in our proposed model to define the static and dynamic features of Bitcoin transactions. Nineteen features have been identified to define Bitcoin transaction patterns for analyzing and finding suspected addresses. Bitcoin gene has been embedded into the Petri net transitions to trace and analyze Bitcoin flow accurately. Finally, marginal distribution analysis of Bitcoin transaction features and data visualization techniques are used to eliminate some false positive samples further and to improve the accuracy of identifying suspected addresses. The proposed Bitcoin transaction network analytic method provides a reliable forensic investigation model along with a prototype platform which is beneficial for financial security. The efficiency of our proposed method is empirically verified based on a real-life case study analysis.

IdenPC-MIIP: identify protein complexes from weighted PPI networks using mutual important interacting partner relation.

Protein complexes are key units for studying a cell system. During the past decades, the genome-scale protein-protein interaction (PPI) data have been determined by high-throughput approaches, which enables the identification of protein complexes from PPI networks. However, the high-throughput approaches often produce considerable fraction of false positive and negative samples. In this study, we propose the mutual important interacting partner relation to reflect the co-complex relationship of two proteins based on their interaction neighborhoods. In addition, a new algorithm called idenPC-MIIP is developed to identify protein complexes from weighted PPI networks. The experimental results on two widely used datasets show that idenPC-MIIP outperforms 17 state-of-the-art methods, especially for identification of small protein complexes with only two or three proteins.

Development and evaluation of swine vesicular disease isotype-specific antibody ELISAs based on recombinant virus-like particles.

Swine vesicular disease (SVD) is a contagious viral disease of pigs. The clinical signs of SVD are indistinguishable from other vesicular diseases, such as senecavirus A infection (SVA) and foot-and-mouth disease (FMD). Rapid and accurate diagnostic tests of SVD are considered essential in countries free of vesicular diseases. Competitive ELISA (cELISA) is the serological test used routinely. However, although cELISA is the standard test for SVD antibody testing, this test produces a small number of false-positive results, which caused problems in international trade. The current project developed a SVD isotype antibody ELISA using recombinant SVD virus-like particles (VLP) and an SVD-specific monoclonal antibody (mAb) to reduce the percentage of false positives. The diagnostic specificities of SVD-VLP isotype ELISAs were 98.7% and 99.6% for IgM and IgG. The SVD isotype ELISAs were SVD-specific, without cross-reactivity to other vesicular diseases. A panel of 16 SVD-positive reference sera was evaluated using the SVD-VLP isotype ELISAs. All sera were correctly identified as positive by the two combined SVD-VLP isotype ELISAs. Comparison of the test results showed a high level of correlation between the SVDV antigen isotype ELISAs and SVD-VLP isotype ELISAs. 303 sera from animals lacking clinical signs and history of SVDV exposure were identified positive using SVD cELISA. These samples were examined using SVD-VLP isotype ELISAs. Of the 303 serum samples, five were positive for IgM, and five of 303 were positive for IgG. Comparable to virus neutralization test results, SVD isotype ELISAs significantly reduced the false-positive samples. Based on above test results, the combined use of cELISA and isotype ELISAs can reduce the number of false-positive samples and the use of time-consuming virus neutralization tests, with benefit for international trade in swine and related products.

A retrospective study of the added value of parallel titers compared with serial titers.

Prenatal antibody titers for alloimmunized patients are subject to multiple sources of variation. A parallel titer on the previous sample at the same time as the current sample is recommended. The purpose of this study was to determine the added value of parallel titers. This is a retrospective study of samples from consecutive prenatal patients with at least two prenatal antibody titers performed in the same pregnancy at a single institution between October 2010 and March 2017. Prenatal titers were performed using gel technology. Data were collected to determine the sensitivity and specificity of a clinically significant increase (twofold or greater) in serial titers compared with the gold standard of using parallel titers. There were 155 serial prenatal titers performed in 59 alloimmunized pregnant women. Nineteen samples (12%) had a serial titer increase of twofold or greater with eight false positive samples (increase less than twofold when using parallel titers). Thirty-six samples (23%) had a serial titer increase of onefold with two false negative samples (increase of twofold or greater using parallel titers). One hundred samples (65%) had no increase (or a decrease) in serial titer with none having an increase of twofold or greater using parallel titers. The sensitivity of a twofold or greater increase in serial titers was 84.6% (95% CI 55-98%) and the specificity was 94.4% (95% CI 89-98%) when compared with parallel titers. This study questions the value of parallel titers on every prenatal titer performed. When no increase in serial titers was observed, parallel titers added no new information.

Herpes simplex virus type 2 (HSV-2) IgG index values in two immunoassays in relation to HSV-2 IgG inhibition assay results

CDC guidelines recommend confirmatory testing of sera with low-positive indices (1.10–3.50) in the HerpeSelect® (HSLT) HSV-2 IgG screening assay. To determine if this recommendation is adequate for our patient population, we reviewed HSLT HSV-2 IgG screening indices for 262 screen-positive sera (index >1.10) tested in our confirmatory assay, which assesses inhibition of binding to recombinant gG2 by HSV-1- and HSV-2-infected cell lysates. To determine how the recommendation affects other screening assays, we tested these samples in the Liaison® HSV-2 IgG assay. Of 124 false-positive sera, 20% and 39% had an index >3.50 in the HSLT and Liaison screening assays, respectively. In both assays, 51% of 63 indeterminate sera (inhibition by HSV-1 lysate) had indices >3.50. Similarly, ≥75% of 75 true-positive samples exhibited indices >3.50 in both assays. Thus, confirmatory testing only of sera with low-positive HSV-2 IgG indices misses some false-positive and indeterminate samples, leading to misdiagnosis of HSV-2 infection.

GroEL gene typing and genetic diversity of Anaplasma bovis in ticks in Shaanxi, China

Anaplasma bovis, causative agent of bovine anaplasmosis, is usually identified by nested-PCR amplifying the rrs gene. However, it is difficult to determine the genetic relationship among different variants within A. bovis using this gene because of high conservation. In this study, two tick species, identified as Rhipicephalus microplus and Haemaphysalis longicornis based on morphological and molecular methods by analyzing COI gene, were collected from cattle, goat or sheep. Subsequently, A. bovis was initially detected by PCR amplifying the rrs gene in ticks in Shaanxi Province, China. The sequencing and Blast results showed that some false positive samples were found when only based on the amplification of partial rrs gene, presenting these sequences resembled those of other Alphaproteobacteria rather than A. bovis. Although major surface proteins genes were proposed and used successfully to identify members within Anaplasmataceae, these genes were unavailable for A. bovis. Hence, primers targeting the groEL gene were designed and a PCR assay was developed. The PCR products were sequenced and similarity and phylogenetic analysis suggested all these sequences are the groEL gene of A. bovis. In addition, phylogenetic analysis based on the groEL gene also revealed the genetic diversity of A. bovis worldwide, as well as in Shaanxi Province of China, which wasn't reflected by analyzing the rrs gene. In sum, groEL gene is important for molecular detection and phylogenetic analysis of A. bovis.

Improved Positive Predictive Performance of Listeria Indicator Broth: A Sensitive Environmental Screening Test to Identify Presumptively Positive Swab Samples.

PDX-LIB, Listeria Indicator Broth, was developed as a proprietary sensitive screening test to identify presumptively positive environmental swab samples for Listeria sp. The original formulation, while sensitive, initially proved to exhibit acceptable levels of false positive test results. Paradigm Diagnostics has been undertaken to modify the medium formulation to render it more selective while not sacrificing its sensitivity. After identification of a candidate formulation through laboratory studies, a field trial was conducted to validate the test performance parameters, including the true positive frequency and false positive frequency in several different food-processing facilities. Identical swab samples were enriched in both the original medium formulation and the new formulation. Presumptive positive samples were confirmed by plating on selective differential agar and qPCR analysis. The field trial data demonstrate that the new formulation significantly reduces the frequency of false positive samples compared to the original Listeria Indicator Broth formulation, without compromising the sensitivity of the original formulation. The new medium formulation resulted in no false positive samples compared to the 54% increased presumptive positive samples obtained with the original medium formulation.

Development of a progesterone-specific IgE assay for diagnosing patients with suspected progestogen hypersensitivity

BackgroundProgesterone hypersensitivity (PH) manifests as a spectrum of allergic symptoms during the luteal phase of the menstrual cycle. Confirming progesterone-specific immunoglobulin E (IgE; sIgE) by skin testing is unreliable because of irritant responses. ObjectiveTo develop a progesterone sIgE assay to assist in diagnosing PH. MethodsA progesterone–bovine serum albumin (BSA) conjugate was characterized and used to analyze sera collected from women in our center with suspected PH in a 1-batch enzyme-linked immunosorbent assay (ELISA) to establish high, low and negative cut-points. Sera collected from healthy nonatopic female subjects and from women with classical PH symptoms were included as negative and positive controls, respectively. Values exceeding the average negative control (OD + 3× the standard deviation) were considered positive. These cut-points were subsequently used to establish positive and negative results for serum from women with suspected PH received from other centers. A subset of high positive sera was used for ELISA-inhibition and in a beta-hexosaminidase mediator release assay to evaluate the specificity and functional relevance of progesterone-specific serum IgE, respectively. The numbers of true negative, false negative, true positive, and false positive samples were determined. ResultsThe direct progesterone sIgE ELISA results ranged from high positive to low positive and negative compared with healthy nonatopic control sera. Enzyme-linked immunosorbent assay inhibition and beta-hexosaminidase mediator release confirmed specificity and functional relevance of progesterone-sIgE, respectively. The sensitivity, specificity positive predictive values and negative predictive values were found to be 82%, 100%, 86%, and 100%, respectively, using the mediator release assay results as the gold standard. ConclusionThis assay has a good specificity and positive predictive value for screening women with suspected PH for progesterone sIgE.

Catalase-Containing Silica Particles as Ultrasound-Based Hydrogen Peroxide Sensors to Determine Infected From Noninfected Fluid Collections in Humans.

OBJECTIVE. Hydrogen peroxide (H2O2) plays a key role in neutrophil oxidative defense against infection. Catalase-containing silica nanoshells are nanoparticles that generate O2 microbubbles imaged with ultrasound in the presence of elevated H2O2. We aimed to determine whether ultrasound-detectable O2 microbubbles produced by catalase-containing silica nanoshells can determine whether fluid collections drained from patients are infected. SUBJECTS AND METHODS. During this HIPAA-compliant, institutional review board-approved study, 52 human fluid samples were collected from clinically required image-guided percutaneous drainage procedures. Catalase-containing silica nanoshells were added to the fluid samples during imaging in real time using a Sequoia-512 15L8-S linear transducer (Siemens Healthcare). Production of detectable microbubbles was graded subjectively as negative (noninfected) or positive (infected) with low, moderate, or high confidence by a single observer blinded to all clinical data. The truth standard was microbiology laboratory culture results. Performance characteristics including ROC curves were calculated. RESULTS. Microbubble detection to distinguish infected from noninfected fluids was 84% sensitive and 72% specific and offered negative and positive predictive values of 89% and 64%, respectively. The AUC was 0.79. Six of nine false-positive samples were peritoneal fluid collections that were all collected from patients with decompensated cirrhosis. CONCLUSION. The presence of elevated H2O2 indicated by microbubble formation in the presence of catalase-containing silica nanoshells is sensitive in distinguishing infected from noninfected fluids and offers a relatively high negative predictive value. False-positive cases may result from noninfectious oxidative stress. Catalase-containing silica nanoshells may constitute a novel point-of-care test performed at time of percutaneous drainage, potentially obviating placement of drains into otherwise sterile collections and minimizing risk of secondary infection or other complication.

A Novel Enzyme Immunoassay for the Detection of Buprenorphine, Norbuprenorphine and Their Glucuronides in Urine.

Buprenorphine is a commonly used opioid in pain therapy as well as in opiate maintenance therapy. Immunoassays are quick and cost-effective methods for the necessary toxicological urine analysis of maintenance therapy patients. In this study a novel enzymatic immunoassay, the Thermo Fisher Scientific CEDIA Buprenorphine II assay (Bup2) was evaluated for the detection of buprenorphine, norbuprenorphine and their conjugated metabolites in human urine samples. The Bup2 assay has a cut-off of 10 ng/mL with ±25% controls, whereas the existing CEDIA Buprenorphine assay (Bup1) has a cut-off of 5 ng/mL and ±40% controls. Both assays were analyzed on a Thermo Scientific Indiko Plus benchtop analyzer. Seven-day precision studies of Bup2 assay demonstrated excellent precision of 7.2-10.6%. No crossover between control samples and the cut-off level were observed. Urine samples of 120 patients undergoing opiate maintenance therapy were collected. Immunoassay results of Bup1 and Bup2 were confirmed by gas chromatography mass spectrometry (GC/MS) for buprenorphine and norbuprenorphine as well as for their glucuronides. Comparison showed a specificity of 0.99 between the Bup2 assay and GC/MS, whereas the Bup1 assay had a specificity 0.70 due to 21 false positive samples. The reason is a known cross-reactivity of the Bup1 assay to opiate compounds. The Bup2 assay revealed one false positive result close to the cut-off value; no specific candidate possibly causing a cross-reaction was detected by GC/MS and liquid chromatography tandem mass-spectrometry (LC/MS/MS) methods. The data presented demonstrate an excellent correlation of the Bup2 assay to GC/MS, showing improved specificity and sensitivity when compared to the Bup1 assay. Thus, the Bup2 assay is highly suitable for urine testing, even for opiate maintenance patients receiving high doses of morphine.

A comprehensive multiplex PCR based exome-sequencing assay for rapid bloodspot confirmation of inborn errors of metabolism

BackgroundTandem mass spectrometry (MS MS) and simple fluorometric assays are currently used in newborn screening programs to detect inborn errors of metabolism (IEM). The aim of the study was to evaluate the clinical utility of exome sequencing as a second tier screening method to assist clinical diagnosis of the newborn.MethodsA novel PCR-exome amplification and re-sequencing (PEARS) assay was designed and used to detect mutations in 122 genes associated with 101 IEM. Newborn bloodspots positive by biochemical testing were analysed by PEARS assay to detect pathogenic mutations relevant to the IEM.ResultsIn initial validation studies of genomic DNA samples, PEARS assay correctly detected 25 known mutations associated with 17 different IEM. Retrospective gene analysis of newborns with clinical phenylketonuria (PKU), identified compound heterozygote phenylalanine hydroxylase (PAH) gene mutations in eight of nine samples (89%). Prospective analysis of 211 bloodspots correctly identified the two true PKU samples, yielding positive and negative predictive values of 100%. Testing of 8 true positive MS MS samples correctly identified potentially pathogenic compound heterozygote genotypes in 2 cases of citrullinemia type 1 and one case each of methylmalonic acidemia, isobutyryl-CoA dehydrogenase deficiency, short chain acyl-CoA dehydrogenase deficiency and glutaric acid type II and heterozygous genotypes in 2 cases of autosomal dominant methioninemia. Analysis of 11 of 12 false positive MS MS samples for other IEM identified heterozygous carriers in 8 cases for the relevant genes associated with the suspected IEM. In the remaining 3 cases, the test revealed compound heterozygote mutations in other metabolic genes not associated with the suspected IEM, indicating a misinterpretation of the original MS MS data.ConclusionsThe PEARS assay has clinical utility as a rapid and cost effective second-tier test to assist the clinician to accurately diagnose newborns with a suspected IEM.

Evaluation of the Abbott m2000 system for dried blood spot detection of hepatitis C virus RNA

BackgroundHepatitis C virus RNA testing using dried blood spots (DBS) offers a method for detecting ongoing HCV infection in “hard to reach” populations. Abbott Molecular have developed a quantitative HCV RNA DBS protocol (currently for research use only) for extraction and real-time PCR amplification using them2000sp and m2000rt system. MethodsA panel of seventy “mock” DBS were made from patient whole blood; who were known to be either HCV RNA negative or positive. This panel compared the “mock” DBS and the plasma viral load results. A further dilution panel of “mock” DBS made from one HCV positive patient was used to estimate the detection limit of the assay. Abbott was then compared with an in-house real-time Taqman PCR using patient DBS samples. ResultsAll “mock” DBS samples with a viral load >1000IU/ml were detected by Abbott, with only 1/8 detected at <1000 IU/ml. The dilution panel suggested the limit of detection to be between 178 to 1779 IU/ml. There were two false positive samples detected at low level <282 IU/ml, both samples were from patients who had been previously positive. The overall sensitivity of the Abbott RUO DBS protocol when compared to plasma was 86% (95 CI 73.76%–74.18%) increasing to 100% (CI 91.59%–100%) when the viral load was >1000IU/ml. Abbott compared well with the in-house assay with sensitivity of 97.5% (95% CI 86.84%–99.94%) and specificity of 100% (95% CI 91.19%–100%). ConclusionsThe Abbott system is an automated platform which can be used for DBS HCV RNA extraction and amplification. The preliminary data presented here showed a high sensitivity and specificity for DBS with viral loads greater than 1000IU/ml and compared well with a published in-house method.

High false positive rate of white blood cells in urine samples of pregnant women may be caused by epithelial cells being misclassified by the sysmex UF-1000i urine flow cytometer.

BackgroundThe UF‐1000i has been widely used in screening urinary sediments. However, the interference factor of the UF‐1000i in the screening urinary sediments of pregnant women has not been reported. The aim of the study was to demonstrate that epithelial cells (ECs) cause a high false positive rate of white blood cells (WBCs) by the UF‐1000i in pregnant women.MethodsUrine samples were collected from 207 pregnant women. All samples were measured by the UF‐1000i and a microscopic method.ResultsThe areas under the curve (AUC) for WBC and EC counts were 0.837 (95% CI, 0.773–0.901) and 0.844 (95% CI, 0.785–0.903), respectively. The positive rates of the WBC and EC were 73.43% and 37.20%, respectively, by the UF‐1000i, and they were 19.32% and 72.95% by the microscopic method. The positive predictive value, negative predictive value, false positive rates, and false negative rates by the UF‐1000i were for WBC 25.66%, 98.18%, 74.34%, and 1.82%, respectively, and for EC they were 96.1%, 40.77%, 3.9%, and 59.23%, respectively. The coefficient of correlation R value was 0.503 (P < 0.01) between WBC by UF‐1000i and EC by the microscopic method in WBC false positive samples.ConclusionsEC could be an interference factor for the UF‐1000i in screening urinary WBC of pregnant women, and the high false positive rate for WBC may be caused by ECs being misclassified as WBCs by the UF‐1000i. © 2018 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.

Efficient multi-kernel multi-instance learning using weakly supervised and imbalanced data for diabetic retinopathy diagnosis

ObjectiveDiabetic retinopathy (DR) is one of the most serious complications of diabetes. Early detection and treatment of DR are key public health interventions that can significantly reduce the risk of vision loss. How to effectively screen and diagnose the retinal fundus image in order to identify retinopathy in time is a major challenge. In the traditional DR screening system, the accuracy of micro-aneurysm (MA) and hemorrhagic (H) lesion detection determines the final screening performance. The detection method produced a large number of false positive samples for guaranteeing high sensitivity, and the classification model was not effective in removing false positives since the suspicious lesions lack label information. MethodsIn order to solve the problem of supervised learning in the diagnosis of DR, we formulate weakly supervised multi-class DR grading as a multi-class multi-instance problem where each image (bag) is labeled as healthy or abnormal and consists of unlabeled candidate lesion regions (instances). Specifically, we proposed a multi-kernel multi-instance learning method based on graph kernel. Moreover, we develop an under-sampling from instance level and over-sampling from bag level to improve the performance of the multi-instance learning in the diagnosis of DR. ResultsThrough empirical evaluation and comparison with different baselinemethods and the state-of-the-art methods on data from Messidor, we illustrate that the proposed method reports favorable results, with an overall classification accuracy of 0.916 and an AUC of 0.957. ConclusionsThe experiments results demonstrate that the proposed multi-kernel multi-instance learning framework with bi-level re-sampling can solve the problem in the imbalanced and weakly supervised data for grading diabetic retinopathy, and it improves the diagnosis performance over several state-of-the-art competing methods.

False positive screen test for mucopolysaccharidoses in healthy female newborns

BackgroundIn total, 930 urine samples obtained on 2nd and 3rd day from birth have been analyzed for the early diagnosis of Mucopolysaccharidoses. MethodsDimethylmethylene blue (DMB) assay and one-dimensional electrophoresis were performed in all urine samples. Agarose gel electrophoresis, before and after treatment with chondroitinase ABC and heparinases, was used for a comprehensive characterization. ResultsOut of 930 urine samples 7 showed anomalous electrophoretic pattern; 5 of them had high GAG levels by DMB test. Atypical samples (n = 7) were analyzed by agarose gel electrophoresis. After enzymatic digestion, some slow bands were still visible. A second urine sample of the above 7 newborns was analyzed at the age of 1 month, demonstrating both a normal pattern and normal GAG levels. Additional urine and vaginal mucus samples from 10 term neonates with vaginal bleeding showed the same electrophoretic pattern observed in the 7 false positive samples. ConclusionsThe altered electrophoretic pattern may be due to the presence of glycoproteins and not to specific GAGs, due to high levels of maternal hormones exposure during pregnancy. To our knowledge, this is the first time maternal estrogen hormones are proposed as a likely cause of false-positive urinary glycosaminoglycan screen test in healthy newborns.