PLUS Assay Research Articles

Are the latest point-of-care D-dimer devices ready for use in general practice? A prospective clinical evaluation of five test systems with a capillary blood feature for suspected venous thromboembolism

IntroductionWe evaluated clinical performance of five novel point-of-care (POC) D-dimer devices with a capillary finger stick feature for predicting venous thromboembolism (VTE) in general practice: Exdia TRF Plus (E), AFIAS-1® (A), Standard F200® (S), LumiraDx™ (L) and Hipro AFS/1® (H). Materials and methodsPrimary care patients with a low suspicion of a VTE were asked to consent to (i) draw additional venous blood samples, (ii) perform a capillary POC D-dimer test, (iii) approach their general practitioner afterwards for clinical outcomes. Venous plasma samples were processed on all POC devices and a laboratory-based assay (STA-Liatest®D-Di PLUS assay). Results were compared with clinical outcomes to generate performance characteristics. Capillary and venous blood results were used for a matrix comparison. ResultsVenous plasma samples from 511 participants, of whom 57 had VTE, were used for clinical performance analyses. Areas under Receiving Operating Characteristic Curves ranged from 0.90 (95 % CI: 0.86–0.94) (H) to 0.93 (0.90–0.96) (E). All false-negative rates were below 1.4 % (95 % CI: 0.5 %–3.4 %). Matrix comparison demonstrated correlation coefficients ranging from r = 0.11 (95 % CI: −0.15–0.36) (H) to r = 0.94 (0.90–0.97) (A) with concordance percentages ranging from 71.4 % (applying a D-dimer cutoff of 500 ng/mL) (H) to 100 % (applying an age-dependent D-dimer cutoff) (A). ConclusionsClinical performance of the POC D-dimer devices for predicting a VTE in low-risk patients was comparable to that of a laboratory-based assay. However, our results indicate that the finger stick feature of certain devices should be further improved. (NL71809.028.19.)

Comparison of the Standard E TB-Feron ELISA and QuantiFERON-TB Gold PLUS assays: the advantageous use of whole recombinant protein antigens for latent tuberculosis diagnosis.

The laboratory diagnosis of latent tuberculosis is often performed using interferon-gamma release assays. Here, we compared two enzyme-linked immunosorbent assay-based interferon-gamma release assays, namely, the newly developed Standard E TB-Feron enzyme-linked immunosorbent assay (STFE) and the QuantiFERON-TB Gold PLUS assay (QFT-GP), using samples from 155 participants. The STFE is based on using whole EAST6 and CFP10 recombinant antigens for latent tuberculosis diagnosis. The participants were classified into four groups and screened using both assays per the manufacturers' instructions. Thereafter, two statistical analyses were conducted to compare the obtained results. First, the STFE results were compared with the QTF-GP results (used as the gold standard) to calculate the total concordance, sensitivity, and specificity of STFE. Second, positivity and negativity concordances were calculated to differentiate healthy participants from participants with tuberculosis. The STFE showed 97% and 94% sensitivity and specificity, respectively. Furthermore, its positivity and negativity concordances were 91% and 98%, respectively. These results indicate the coordinated clinical performance of STFE in detecting latent tuberculosis and its improved performance in targeting tuberculosis-infected participants. Based on the comparison of the latent tuberculosis diagnostic abilities of STFE and QFT-GP, we establish the suitability and superior performance of STFE as a diagnostic tool.

33O PRIMA/ENGOT-OV26/GOG-3012 study: Long-term conditional PFS

Niraparib (nir) showed a blinded independent central review–assessed PFS benefit as a first-line (1L) maintenance therapy (MT) in the primary analysis of PRIMA (data cut 17 May 2019) across biomarker subgroups, including a substantial benefit in patients (pts) with homologous recombination–deficient (HRd) tumours. These results were the basis for approval of nir as MT after response to 1L platinum-based chemotherapy (CT). Here we report investigator-assessed (IA) cPFS (the probability of remaining alive and progression free beyond a specified landmark) in PRIMA. This double-blind, placebo (PBO)-controlled phase 3 trial evaluated nir in pts with newly diagnosed, advanced high-grade serous or endometrioid ovarian, primary peritoneal, or fallopian tube cancer (OC) at high risk for relapse after a complete or partial response (CR or PR) to 1L platinum-based CT. Stratification factors were best response to 1L CT regimen (CR/PR), receipt of neoadjuvant CT (yes/no), and homologous recombination deficiency status (HRd or HRp/HRnd) per the Myriad myChoice® CDx PLUS assay. Pts received nir or PBO once daily (2:1 ratio). IA cPFS was analysed for the HRd and intention-to-treat (ITT) populations, using the 17 Nov 2021 data cut. The median follow-up time was 3.5 y. The estimated PFS rate at 4 y was 38% for nir-treated pts and 17% for PBO-treated pts in the HRd population and 24% (nir) vs 14% (PBO) in the ITT population. The 2-y cPFS probabilities beyond the 1- and 2-y landmarks were higher in the nir arm than in the PBO arm (HRd: 1 y: 62% vs 50%, 2 y: 74% vs 60%; ITT: 1 y: 54% vs 46%, 2 y: 67% vs 64%). Safety was previously reported (González-Martín, et al. Ann Oncol. 2022;33[suppl 7]:S789). A durable PFS benefit (nir vs PBO) was observed up to 4 y after randomisation in the ITT and HRd populations, as determined by IA. Pts free from disease progression or death at the 2-y landmark had a high probability of remaining free from progression or death at 4 y, supporting the use of nir as a 1L MT.Table: 33OHRdNirPBOLandmark time from randomizationEvents/total pts2-y probability from landmark, % (95% CI)Events/total pts2-y probability from landmark, % (95% CI)0137/24751 (44–57)98/12629 (21–37)1 y69/15962 (54–70)33/5750 (36–62)2 y26/11074 (64–82)11/3460aITTLandmark time from randomizationNirPBOEvents/total pts2-y probability from landmark, % (95% CI)Events/total pts2-y probability from landmark, % (95% CI)0332/48736 (31–40)199/24622 (17–28)1 y124/24454 (47–60)54/9246 (36–56)2 y42/15267 (57–76)15/5164aa95% CI were not calculated at time points with <10 pts. Open table in a new tab

Clinical Evaluation of the GeneXpert® Xpert® Xpress SARS-CoV-2/Flu/RSV PLUS Combination Test.

The GeneXpert® Xpert® Xpress SARS-CoV-2/Flu/RSV PLUS combination test (PLUS Assay) received Health Canada approval in January 2022. The PLUS Assay is similar to the SARS-CoV-2/Flu/RSV combination test, with modifications to improve assay robustness against circulating and emerging variants. The performance characteristics of the PLUS Assay were assessed at the Lakeridge Health Oshawa Hospital Centre and the National Microbiology Laboratory of Canada. The PLUS Assay was directly compared to the SARS-CoV-2/Flu/RSV combination test using SARS-CoV-2 culture from five variants and remnant clinical specimens collected across the COVID-19 pandemic. This included 50 clinical specimens negative for all pathogens, 110 clinical specimens positive for SARS-CoV-2, Influenza A, Influenza B, RSVA, and/or RSVB and an additional 11 mixed samples to screen for target interactions. The PLUS Assay showed a high percent agreement with the widely used SARS-CoV-2/Flu/RSV combination test. Based on these findings, the PLUS Assay and the Xpert SARS-CoV-2/Flu/RSV combination test results are largely consistent with no observed difference in sensitivity, specificity, or time to result when challenged with various SARS-CoV-2 variants. The reported Ct values provided by the new PLUS Assay was also unchanged, with the exception of a possible 1-2 cycle decrease reported Ct for RSVA across a limited sample size.

Assessing homologous recombination deficiency (HRD) in ovarian cancer: Optimizing concordance of the regulatory-approved companion diagnostic and a next-generation sequencing (NGS) assay kit.

e17571 Background: Olaparib + bevacizumab is approved as first-line maintenance treatment of ovarian cancer that is HRD positive, defined as the presence of a deleterious/suspected deleterious BRCA mutation (BRCAm) and/or genomic instability. HRD genomic instability score (GIS) has been assessed in clinical trials using a regulatory-approved companion diagnostic, Myriad myChoice® CDx. We evaluated the performance of an in-development NGS assay kit that identifies BRCA variants based on genomic content from Illumina’s TruSight™ Oncology 500 research assay, and, with additional genomic content, measures HRD GIS in tumor tissue (Illumina test) in parallel. Analytic concordance of the Illumina test versus the Myriad myChoice® PLUS assay (Myriad test) under improved algorithms for calculating GIS scores is reported. Methods: Ovarian cancer tissue samples were analyzed with the Illumina (40 ng DNA; N = 227) and Myriad (200 ng DNA; N = 254) tests. Agreement rates for BRCA analysis, HRD GIS, and overall HRD status (includes BRCAm and HRD GIS [cutoff, 42]) were analyzed. For the overall and the non-BRCAm analysis cohorts, Pearson correlation between the continuous HRD GIS of the Illumina and Myriad tests was determined. Analytic sensitivity and specificity of the Illumina-derived HRD GIS to classify HRD GIS (cutoff, 42) were evaluated using AUROC. After initial concordance calculations, modifications were made to increase the quality of the Illumina preprocessing data (ie, reducing allele dosage noise and allele bias) upstream of the GIS calling algorithm. Reprocessing of the existing raw data led to further improvement in the point estimates of positive and negative percent agreement. Results: Prevalence using the Illumina and Myriad tests was 51.2% and 49.2% (overall HRD positive) and 27.6% and 25.5% (BRCAm), respectively. Agreement rates are reported in the Table. Correlation of the Illumina HRD GIS with Myriad HRD GIS was 0.980 (all samples) and 0.976 (non-BRCAm cohort); AUROCs were 0.995 and 0.992, respectively. Conclusions: Comparison between the Illumina and Myriad tests showed that overall HRD status, BRCA analysis, and HRD GIS detection results were &gt; 90% concordant; HRD GIS was highly correlated (r &gt; 0.98); and prevalence estimates were similar. These data suggest that a distributable kit solution, such as the Illumina test under development, will approach the performance of the Myriad myChoice HRD assay.[Table: see text]

Dual staining for p16/Ki-67 to detect high-grade cervical lesions: Results from the Screening Triage Ascertaining Intraepithelial Neoplasia by Immunostain Testing study.

We compared clinical performance of p16/Ki-67 dual-stained cytology and human papillomavirus (HPV) genotyping, via different algorithms-alone, or in combination with cytology-to identify cervical intraepithelial neoplasia grade 2 or worse (CIN2+) and grade 3 or worse (CIN3+) in women referred to as colposcopy. We included 492 cervical specimens (134 normal, 130 CIN1, 99 CIN2, 121 CIN3, 8 cancers) randomly selected from 1158 specimens with valid conventional cytology, HPV (cobas 4800 HPV test) and biopsy results. Dual-stained cytology was retrospectively performed (CINtec PLUS assay) on PreservCyt material; slides were read by a cytologist and confirmed by two pathologists, blinded to cytology, biopsy and genotyping results. Sensitivity and specificity (95% confidence intervals in parentheses) of dual-stained cytology to detect CIN2+ and CIN3+ were compared to other screening tests available for the same women. Positivity rate for dual-stained cytology increased with histological severity: 30.6% in normal, 41.5% in CIN1, 72.7% in CIN2, 86.8% in CIN3 and 87.5% in cancer. Dual-stained cytology alone had lower sensitivity than HPV testing for CIN2+ [80.7% (75.0-85.6) vs 89.9% (85.3-93.5)] and CIN3+ [86.8% (79.7-92.1) vs 92.3% (86.2-96.2)]. However, corresponding specificity values were higher [64.0% (57.9-69.8) vs 56.1% (49.8-62.1) for CIN2+; 54.0% (48.7-59.2) vs 44.4% (39.2-49.6) for CIN3+]. Combining dual-stained cytology with an ASC-US abnormality threshold decreased specificity to 31.4% (25.9-37.4) for CIN2+ and 24.2% (19.9-29.0) for CIN3+. The corresponding values considering low squamous intraepithelial lesion threshold values were 42.8% (36.8-49.0) and 35.0% (30.1-40.1). Dual-stained cytology and HPV testing exhibited similar performance, although the former improved the specificity by 7.9% and 9.6% for CIN2+ and CIN3+, respectively.

High prevalence of genotype 6 hepatitis C virus infection in Southern Taiwan using Abbott genotype assays

Abbott RealTime Genotype II assay can effectively identify hepatitis C virus (HCV) genotypes (GTs), but some GT 6 subtypes might not be differentiated from GT 1. Abbott RealTime Genotype II PLUS and sequencing might be needed to resolve these ambiguous results. Unlike the high prevalence of GT 6 in Southeast Asia, GT 6 had rarely been reported in Taiwan except in intravenous drug abusers (IDU). But the prevalence of GT 6 in Taiwan might be underestimated. We conducted this study to determine the GTs in a HCV endemic area in Southern Taiwan. A total of 1147 patients with hepatitis C viremia for direct acting antivirals (DAA) treatment at the Chi Mei medical system in Tainan were enrolled. Genotype was determined using a working flow consisted of Abbott GT II, PLUS assays and 5' untranslated region (5' UTR)/core sequencing. Among the 1147 patients, 883 (77.0%) obtained GT results by GT II, 264 (23.0%) samples with ambiguous results by GT II assay received further tests, including 194 (73.5%) with PLUS assay and 70 (26.5%) with 5'UTR/core sequencing. Nearly three-quarters (73.5%) of ambiguous results by GT II assay were GT 6. Overall, 18.3% of samples were GT 6. Phylogenetic study of 11 samples of GT 6 subtypes showed 7 (63.6%) were 6g. GT 6 is the major factor for high ambiguous rate by GT II. Unexpected high prevalence of GT 6 (18.3%) in Southern Taiwan, especially subtype 6g, closely related to Indonesian strains, is first reported.

To Evaluate the Role of H. pylori in Patients with Chronic Recurrent Tonsillitis.

The usual indication for surgical resection of tonsils is chronic recurrent tonsillitis. Literature also does not indicate the reason behind the fact that only part of the population suffers from recurrent chronic tonsillitis in spite being exposed to similar conditions. This was a prospective study, in which 50 tonsil biopsy samples obtained from chronic tonsillitis patients. Specimens were analysed with rapid urease broth test, HelicotecUT PLUS assay and Toluidine blue staining for presence of Helicobacter pylori. The age ranged from 4 to 34years. The median age for patients with chronic recurrent tonsillitis was 9.5, 23 (46%) patients were male while 27 (54%) were female, presence of H. pylori by rapid urease broth test, HelicotecUT PLUS assay and Histopathology was 4%. Our analysis revealed that H. pylori did not significantly colonize the tonsils and does not play a role in the pathogenesis or development of chronic tonsillitis. The heterogeneity in study population and methodology may have contributed to the non significant results.

Evaluation of the Abbott RealTime HCV genotype II plus RUO (PLUS) assay with reference to core and NS5B sequencing

BackgroundHCV genotyping remains a critical tool for guiding initiation of therapy and selecting the most appropriate treatment regimen. Current commercial genotyping assays may have difficulty identifying 1a, 1b and genotype 6. ObjectiveTo evaluate the concordance for identifying 1a, 1b, and genotype 6 between two methods: the PLUS assay and core/NS5B sequencing. Study designThis study included 236 plasma and serum samples previously genotyped by core/NS5B sequencing. Of these, 25 samples were also previously tested by the Abbott RealTime HCV GT II Research Use Only (RUO) assay and yielded ambiguous results. The remaining 211 samples were routine genotype 1 (n=169) and genotype 6 (n=42). Genotypes obtained from sequence data were determined using a laboratory-developed HCV sequence analysis tool and the NCBI non-redundant database. ResultsAgreement between the PLUS assay and core/NS5B sequencing for genotype 1 samples was 95.8% (162/169), with 96% (127/132) and 95% (35/37) agreement for 1a and 1b samples respectively. PLUS results agreed with core/NS5B sequencing for 83% (35/42) of unselected genotype 6 samples, with the remaining seven “not detected” by the PLUS assay. Among the 25 samples with ambiguous GT II results, 15 were concordant by PLUS and core/NS5B sequencing, nine were not detected by PLUS, and one sample had an internal control failure. ConclusionsThe PLUS assay is an automated method that identifies 1a, 1b and genotype 6 with good agreement with gold-standard core/NS5B sequencing and can aid in the resolution of certain genotype samples with ambiguous GT II results.

Assessing the progression of chronic periodontitis using subgingival pathogen levels: a 24-month prospective multicenter cohort study

BackgroundThe diagnosis of the progression of periodontitis presently depends on the use of clinical symptoms (such as attachment loss) and radiographic imaging. The aim of the multicenter study described here was to evaluate the diagnostic use of the bacterial content of subgingival plaque recovered from the deepest pockets in assessing disease progression in chronic periodontitis patients.MethodsThis study consisted of a 24-month investigation of a total of 163 patients with chronic periodontitis who received trimonthly follow-up care. Subgingival plaque from the deepest pockets was recovered and assessed for bacterial content of Porphyromonas gingivalis, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans using the modified Invader PLUS assay. The corresponding serum IgG titers were measured using ELISA. Changes in clinical parameters were evaluated over the course of 24 months. The sensitivity, specificity, and prediction values were calculated and used to determine cutoff points for prediction of the progression of chronic periodontitis.ResultsOf the 124 individuals who completed the 24-month monitoring phase, 62 exhibited progression of periodontitis, whereas 62 demonstrated stable disease. The P. gingivalis counts of subgingival plaque from the deepest pockets was significantly associated with the progression of periodontitis (p < 0.001, positive predictive value = 0.708).ConclusionsThe P. gingivalis counts of subgingival plaque from the deepest pockets may be associated with the progression of periodontitis.

Increased Extracellular Nucleosome Levels and Microparticles in Atrial Fibrillation Patients Compared to the Age-Matched Normal Population

Introduction: Atrial fibrillation (AF) is currently the most common cardiac arrhythmia encountered in clinical practice and a major cause of morbidity and mortality among adults. It is estimated that atrial fibrillation affects approximately 2.7 million people in the United States. Extracellular plasma nucleosomes (PNs) are complexes of DNA and histones that are released during cell death. Histones have been shown to function as DAMPs when they are translocated from the nucleus to the extracellular space. It is hypothesized that these extracellular nucleosomes contribute to the observed hypercoagulable state and embolic complications in AF.Methods: The concentration of PNs in 48 plasma samples of atrial fibrillation patients and 38 aged-matched controls were measured using the Cell Death Detection ELISA PLUS assay (Roche Diagnostics, Mannheim, Germany) and functional microparticle levels were measured using an annexin binding method (Hyphen Biomedical, Paris, France).Results: When comparing the concentration of nucleosomes in plasma, the atrial fibrillation patients (306.1 ± 328.86 μg/mL) had much higher levels than the aged-matched controls (177.9 ± 146.7 μg/mL), (p = 0.0015). Functional microparticles were also markedly elevated in the AF patients (18.6 ± 4.1 nM) in comparison to the aged-matched controls (4.6 ± 3.1 nM), (p=0.001). A significant correlation was observed between the PN’s and microparticles, (p < 0.05).Conclusion: Both the microparticles and PNs were elevated in atrial fibrillation when compared to aged-matched controls, suggesting increased cell death resulting in the generation of these biomarkers. Extracellular nucleosomes function as DAMPs by binding to receptors and triggering the activation of multiple signal pathways which may contribute to the elevation of microparticles. Due to the involvement of inflammation and thrombosis in the pathology of atrial fibrillation, the simultaneously increased circulating nucleosome and microparticle levels in patients with AF implies their role in the pathogenesis of this syndrome. DisclosuresNo relevant conflicts of interest to declare.

Platelet Activation in End-Stage Renal Disease Is Mediated By Extracellular Nucleosomes That Originate from White Blood Cells

IntroductionExtracellular nucleosomes in plasma (PNs) are complexes of DNA and histones that are released during cell death and inflammatory responses. End-stage renal disease (ESRD) represents a complex syndrome where inflammation, endothelial dysfunction, and hemostatic aberrations contribute to the observed vascular manifestations. The purpose of this investigation is to profile PNs in patients with ESRD and to demonstrate their relevance to blood cells and platelet activation products.MethodsPre-dialysis plasma samples from patients undergoing maintenance hemodialysis (n = 90) at Loyola University outpatient dialysis unit were collected under an approved IRB protocol. Plasma samples from healthy individuals (n = 50) were purchased from a biobank as a control (George King Biomedical, Overland Park, Kansas). Complete blood count profiles, including white blood cells (WBCs), red blood cells (RBCs), and platelets were obtained from the patients’ medical records. The levels of PNs in ESRD patients and healthy volunteers were measured using the Cell Death Detection ELISA PLUS assay (Roche Diagnostics, Mannheim, Germany). MP-TF levels were measured using the Zymuphen MP-TF kit (Hyphen BioMed, Neuville-sur-Oise, France). PDGF levels were measured using the Human PDGF-BB Quantikine ELISA Kit (R&D Systems, Minneapolis, Minnesota). Human PF4 levels were measured using the Zymutest PF4 ELISA Kit (Hyphen BioMed, Neuville-sur-Oise, France). All individual results were tabulated and analyzed using the statistical software GraphPad Prism 7. The Mann-Whitney test for non-parametric data was utilized in comparing ESRD to control groups. PN levels were correlated with cell counts and platelet activation factors using the non-parametric Spearman correlation. Individual cell counts were also correlated with platelet activating factors using the same method.ResultsIn the ESRD patients, the average hematocrit was 31.7 ± 4.4 %, the average WBC count was 6.5 ± 4.0 K/uL, and the average platelet count was 179.4 ± 66.3 K/uL. The levels of PNs in the ESRD patients (15.5 ± 14.1 Arbitrary Units (AU)) were markedly higher in comparison to that of the controls (6.74 ± 13.7 AU; p < 0.0001). Similarly, MP-TF levels were significantly elevated in ESRD patients (3.00 ± 1.42 pg/mL) compared to normal (0.363 ± 0.263 pg/mL; p < 0.0001). PF4 levels were also significantly elevated in ESRD patients (95.3 ± 35.3 ng/mL) compared to normal (27.4 ± 19.8 ng/mL; p < 0.0001). While PDGF levels were higher in the ESRD group (116.0 ± 172.5 pg/mL) in comparison to the controls (82.7 ± 113.5 pg/mL), this increase was not statistically significant (p = 0.405). A positive correlation was observed between PNs and WBCs (p = 0.024; r = 0.244). PN levels did not show a correlation with RBC (p = 0.448; r = 0.083) and platelet levels (p = 0.545; r = 0.066). Furthermore, there was no correlation between PNs and MP-TF (p = 0.501; r = 0.077), PDGF (p = 0.314; r = 0.110) and PF4 (p = 0.524; r = -0.070) in the ESRD patients. However, the platelet count showed a positive correlation with PDGF (p = 0.044; r = 0.218) and MP-TF (p = 0.042; r = 0.237). Similarly, the WBC count showed a positive correlation with the platelet count (p < 0.0001; r = 0.476) and PDGF (p = 0.016; r = 0.260).ConclusionThis study clearly demonstrates that extracellular nucleosomes are elevated in the plasma of patients with ESRD. The fact that the PN levels correlated with the number of circulating white blood cells suggest that the PNs originate from these cells. Since the ESRD patients exhibited platelet activation, as evident by the observed increase in PDGF, MP-TF and PF4, it is plausible that this activation is mediated by PNs originating from the WBCs. As observed by the positive correlation between WBCs, PDGF, and platelet count, this study underscores the potential role of nucleosomes originating from WBCs in the activation of platelets. These results are consistent with previously reported observations that extracellular histones can induce platelet activation in a TLR2 and TLR4 dependent manner (http://www.ncbi.nlm.nih.gov/pubmed/21673343). DisclosuresNo relevant conflicts of interest to declare.

Using Targeted Sequencing of Paralogous Sequences for Noninvasive Detection of Selected Fetal Aneuploidies.

Current methods for noninvasive prenatal testing (NIPT) ascertain fetal aneuploidies using either direct counting measures of DNA fragments from specific genomic regions or relative measures of single nucleotide polymorphism frequencies. Alternatively, the ratios of paralogous sequence pairs were predicted to reflect fetal aneuploidy. We developed a NIPT assay that uses paralog sequences to enable noninvasive detection of fetal trisomy 21 (T21) and trisomy 18 (T18) using cell-free DNA (cfDNA) from maternal plasma. A total of 1060 primer pairs were designed to determine fetal aneuploidy status, fetal sex, and fetal fraction. Each library was prepared from cfDNA by coamplifying all 1060 target pairs together in a single reaction well. Products were measured using massively parallel sequencing and deviations from expected paralog ratios were determined based on the read depth from each paralog. We evaluated this assay in a blinded set of 480 cfDNA samples with fetal aneuploidy status determined by the MaterniT21® PLUS assay. Samples were sequenced (mean = 2.3 million reads) with 432 samples returning a result. Using the MaterniT21 PLUS assay for paired plasma aliquots from the same individuals as a reference, all 385 euploid samples, all 31 T21 samples, and 14 of 16 T18 samples were detected with no false positive results observed. This study introduces a novel NIPT aneuploidy detection approach using targeted sequencing of paralog motifs and establishes proof-of-concept for a potentially low-cost, highly scalable method for the identification of selected fetal aneuploidies with performance and nonreportable rate similar to other published methods.

Salivary pathogen and serum antibody to assess the progression of chronic periodontitis: a 24-mo prospective multicenter cohort study.

A diagnosis of periodontitis progression is presently limited to clinical parameters such as attachment loss and radiographic imaging. The aim of this multicenter study was to monitor disease progression in patients with chronic periodontitis during a 24-mo follow-up program and to evaluate the amount of bacteria in saliva and corresponding IgG titers in serum for determining the diagnostic usefulness of each in indicating disease progression and stability. A total of 163 patients with chronic periodontitis who received trimonthly follow-up care were observed for 24 mo. The clinical parameters and salivary content of Porphyromonas gingivalis, Prevotella intermedia and Aggregatibacter actinomycetemcomitans were assessed using the modified Invader PLUS assay, and the corresponding serum IgG titers were measured using ELISA. The changes through 24 mo were analyzed using cut-off values calculated for each factor. One-way ANOVA or Fisher's exact test was used to perform between-group comparison for the data collected. Diagnostic values were calculated using Fisher's exact test. Of the 124 individuals who completed the 24-mo monitoring phase, 62 exhibited periodontitis progression, whereas 62 demonstrated stable disease. Seven patients withdrew because of acute periodontal abscess. The ratio of P. gingivalis to total bacteria and the combination of P. gingivalis counts and IgG titers against P. gingivalis were significantly related to the progression of periodontitis. The combination of P. gingivalis ratio and P. gingivalis IgG titers was significantly associated with the progression of periodontitis (p = 0.001, sensitivity = 0.339, specificity = 0.790). It is suggested that the combination of P. gingivalis ratio in saliva and serum IgG titers against P. gingivalis may be associated with the progression of periodontitis.

FIBTEM PLUS Provides an Improved Thromboelastometry Test for Measurement of Fibrin-Based Clot Quality in Cardiac Surgery Patients

The viscoelastic functional fibrinogen (FF) and FIBTEM assays measure the contribution of fibrin to clot strength. Inhibition of platelet function is a necessary precondition for these tests to work. We investigated a novel test for measuring fibrin-based clotting, FIBTEM PLUS, in cardiac surgery and compared it with FF and FIBTEM. A prospective, observational study was performed which included 30 patients undergoing cardiac surgery with cardiopulmonary bypass (CPB). Blood samples were drawn at the beginning of surgery (pre-CPB), approximately 20 minutes before weaning from CPB and 5 minutes after heparin neutralization. FF, FIBTEM, and FIBTEM PLUS tests were performed in duplicate for all blood samples. Additional coagulation parameters, including platelet count, plasma fibrinogen levels, factor XIII activity, and heparin concentration, were also recorded for each sample. At all time points, the lowest mean maximum clot firmness (MCF) was observed with FIBTEM PLUS, although a statistically significant difference between FIBTEM and FIBTEM PLUS was observed only at baseline (mean values 22 vs 19 mm, P = 0.01; FF value for comparison: 27.7 mm). FF maximum amplitude (MA) values were significantly higher than FIBTEM MCF and FIBTEM PLUS MCF pre-CPB, during CPB and after heparin neutralization (P ≤ 0.001 for FF MA versus FIBTEM MCF and for FF MA versus FIBTEM PLUS MCF at all time points). The difference between FIBTEM MCF and FIBTEM PLUS MCF correlated with platelet count (r = 0.46;P < 0.001), whereas differences between FF MA and FIBTEM MCF, or FF MA and FIBTEM PLUS MCF did not (r = -0.07, P = 0.51; r = 0.16, P = 0.12, respectively). Differences between the assays were unrelated to heparin levels, which decreased considerably after protamine administration compared with heparin levels recorded before weaning from CPB (decrease from 2.1 to 0.1 U/mL and from 2.8 to 0.2 U/mL for anti-factor IIa and anti-factor Xa, respectively). Agreement between duplicate measurements was similar with FIBTEM and FIBTEM PLUS assays and lower with FF. Significant positive correlations were found between MCF or MA and fibrinogen concentration (all P < 0.001); the highest correlation was with FIBTEM PLUS MCF (r = 0.70). The FIBTEM PLUS assay produces precise results. At baseline, it provides greater inhibition of platelets than FIBTEM, but there is no meaningful difference between FIBTEM PLUS and FIBTEM in patients with low platelet counts.

Method comparison between INNOVANCE D-Dimer and PLUS D-Dimer

Objective To evaluate the clinical performance of INNOVANCE D-Dimer,and provide information for clinical application. Methods 402 cases of sodium citrate anticoagulant blood were tested with INNOVANCE assay and PLUS assay on CA7000 analyzer to measure plasma DDimer levels.VIDAS-30 immunology analyzer was also used to validate the two assays.4 patients with elevated D-Dimer were monitored continuously during 5 days using INNOVANCE assay and PLUS assay respectively,then the consistency of trend between 2 assays was analyzed.Plasma specimens added with hemoglobin,bilirubin and triglyceride were used to verify the anti-interference capability of INNOVANCE D-Dimer assay. Results In 402 specimens,the result ranges of INNOVANCE DDimer and PLUS D-Dimer were[2.15 (0.33,8.63)]mg/L FEU and[325.50 (123.75,974.00)]μg/L DDU,respectively.The consistency between two assays was poor (Z=-17.375,P=0.000),especially the results in the range of PLUS D-Dimer (201-300) μg/L DDU and (301-400) μg/L DDU,the coincidence rates were only 25% and 15%,respectively; the coincidence rate was up to 85% during PLUS D-Dimer (500-600) μg/L DDU; the coincidence rate was close to 100% when PLUS D-Dimer over 700μg/L DDU.Totally 47 of 402 cases were unmatched between two assays.Verified by VIDAS 30,83.0% (39/47) was false negative for PLUS assay,4.3% (2/47) was false negative for INNOVANCE assay,12.7% (6/47) was false positive for PLUS assay.There were 5 false positives and 39 false negative for PLUS assay,totally 45 cases; Two false negative for INNOVANCE assay.Four patients with elevated D-Dimer were monitored and the results showed similar trend between 2 assays.For INNOVANCE assay,the capacity of anti-interference to free bilirubin,unconjugated bilirubin,hemoglobin,and triglyceride was up to 217μmol/L,337μmol/L,41.04 g/L,18.35 mmol/L,respectively. Conclusions INNOVANCE assay can markedly reduce false negative results of D-Dimer compared with PLUS assay.INNOVANCE D-Dimer has good performance on anti-interference to jaundice,hemolysis and lipemia samples.(Chin J Lab Med,2013,36:638-642) Key words: D-Dimer; Laboratory techniques and procedures; Reagent kits; diagnostic

Microbiological Effect of Essential Oils in Combination with Subgingival Ultrasonic Instrumentation and Mouth Rinsing in Chronic Periodontitis Patients

Thirty chronic periodontitis patients were randomly assigned to 3 groups: control, saline, and essential oil-containing antiseptic (EO). Subgingival plaque was collected from a total of 90 pockets across all subjects. Subsequently, subgingival ultrasonic instrumentation (SUI) was performed by using EO or saline as the irrigation agent. After continuous mouth rinsing at home with EO or saline for 7 days, subgingival plaques were sampled again. Periodontopathic bacteria were quantified using the modified Invader PLUS assay. The total bacterial count in shallow pockets (probing pocket depth (PPD) = 4-5 mm) was significantly reduced in both saline (P < 0.05) and EO groups (P < 0.01). The total bacterial count (P < 0.05) and Porphyromonas gingivalis (P < 0.01) and Tannerella forsythia (P < 0.05) count in deep pockets (PPD ≥6 mm) were significantly reduced only in the EO group. In comparisons of the change ratio relative to baseline value of total bacteria counts across categories, both the saline and EO groups for PPD 4-5 mm and the EO group for PPD 6 mm showed a significantly low ratio (P < 0.05). The adjunctive use of EO may be effective in reducing subgingival bacterial counts in both shallow and deep pockets. This trial is registered with UMIN Clinical Trials Registry UMIN000007484.

D-Dimer levels at different stages of pregnancy in Australian women: A single centre study using two different immunoturbidimetric assays

BackgroundTo date there is minimal data available on D-Dimer levels at different stages of pregnancy. Patients and methodsWe prospectively measured D-Dimer levels in 632 consecutive pregnant women from March 2007 to January 2009. The median age of the participants was 31years (range; 18–42) with a median weight of 78 kilograms (range; 46–137). All subjects were investigated during each trimester with two different immunoturbidimetric assays; D-Dimer PLUS and INNOVANCE D-Dimer. D-Dimer levels were determined using a Sysmex® CA 1500 analyser. ResultsOur data demonstrate that D-Dimer levels in pregnancy show different patterns of rise within the first trimester, depending on the assay used; D-Dimer PLUS=0.88 (SD: mean ratio), INNOVANCE D-Dimer=0.72 (SD: mean ratio). Furthermore, the rise in mean results was greater for the INNOVANCE D-Dimer assay compared to the D-Dimer PLUS assay as shown by the ratio of third to first trimester results of 3.68 and 1.96 respectively. Both D-Dimer assays demonstrated moderate levels of intra-subject variability, with overall mean CVs of 16.5% (D-Dimer PLUS) and 16.9% (INNOVANCE D-Dimer). Furthermore, we studied the association between D-Dimer levels and occurrence of diseases of pregnancy. For both assays, there was no consistently interpretable evidence of an association between raised mean D-Dimer levels or rising D-Dimer levels and any of the diseases or conditions associated with pregnancy. ConclusionOur data suggest that the INNOVANCE D-Dimer assay increases significantly with the advancement of pregnancy, and is more sensitive than D-Dimer PLUS assay in the pregnant population.

P16/Ki-67 dual staining in cervico-vaginal cytology: Correlation with histology, Human Papillomavirus detection and genotyping in women undergoing colposcopy

ObjectivesTo evaluate the CINtec® PLUS assay (mtm laboratories), a new immunocytochemical method for the simultaneous detection of p16INK4a and Ki-67, in liquid-based cervico-vaginal cytology, investigating the association of the dual staining with HPV infection and genotyping as well as cytological and histological abnormalities. Methods140 women with a cervico-vaginal sample obtained immediately before the colposcopy were enrolled. This cytological sample was used for HPV testing with the Linear Array HPV Genotyping Test, the dual staining with the CINtec® PLUS kit and the morphology assessment. ResultsCytology results were 38 NILM, 16 ASC-US, 32L-SIL, 54H-SIL or worse. 113 patients also had a colposcopy-guided biopsy, classified as 14 negative, 35 CIN1, 24 CIN2, 37 CIN3, 3 invasive SCC. A strong association between p16/Ki-67 and HR-HPV infection was found (COR=6.86, 95% CI: 1.84–31.14). Importantly, the association between p16/Ki-67 positivity and HPV16 and/or 18 infection was 2-fold stronger compared to that with the infection by other HR-HPV types (COR=9.92, 95% CI: 2.39–47.77 vs COR=4.20, 95% CI: 0.99–20.87). In addition, p16/Ki-67 positivity rate significantly increased with the severity of the cytological and histological abnormalities (p<0.05 in both cases). p16/Ki-67 positivity resulted strongly associated with a CIN2+ diagnosis (COR=10.86 95% CI: 4.16–29.12). ConclusionsThis preliminary study evidenced that p16/Ki-67 immunostaining might have a relevant clinical role, since the dual staining was significantly associated with HR-HPV infection, particularly with HPV 16 and 18, and the increasing grade of the cervical lesions, the positivity for this biomarker being strongly related to the presence of a CIN2+ lesion.

PVI-1 Performance evaluation of the access HCV Ab PLUS assay on the UniCel DxI 800 in clinical routine

PVI-1 Performance evaluation of the access HCV Ab PLUS assay on the UniCel DxI 800 in clinical routine