Reagent Lots Research Articles

The development of smart screening technology kit (SST-Kit) for sorting cassava cultivars based on cyanide analysis

Cyanides are deadly toxic substances commonly found in cassava root tuber. Higher content of cyanides causes bitter taste of the root tuber. Therefore, analysis of cyanide content has been used as one of criteria for bitterness in cassava. The substitute common method used for this purpose was organoleptic test that could lead to poisoning, or argentometry, which is complicated, time-consuming, and requires lots of reagents. Therefore, a fast, easy, and safe method is strongly demanded. In this work, a smart screening technology kit (SST-Kit) was developed using ninhydrin reagent. This simple, fast, and low-cost method can be applied in the field for the analysis of cyanide levels in cassava. This method was optimized to the concentration of sodium carbonate from 0.5 to 2.0% by measuring the red color intensity produced under various times of reading from 1 to 3 minutes and three sample preparation techniques. The optimum condition for SST-Kit to read the cyanides level was achieved under the condition of 0.5% of sodium carbonate with a time readout of 2 minutes, using grinding preparation technique followed by dilution. The SST-kit had been applied on cassava samples with the accuracy of more than 90%.

A five-year retrospective audit of anti-myeloperoxidase antibody titres measured using a commercial chemiluminescent immunoassay

Background: In 2015, ACT Pathology transitioned to a commercial chemiluminescent immunoassay (QUANTA-Flash MPO, Inova Diagnostics) to measure anti-MPO IgG antibodies. Monitoring of internal performance check sera identified discordant results following a change in reagent lot. To investigate this further, we conducted a five-year retrospective audit of all anti-MPO titres measured using this assay. Methods: Samples were stratified according to titre (negative <6 IU/mL, low-positive 6–23 IU/mL and high-positive ≥24 IU/mL), clinical information provided on request forms, ANCA pattern as detected by indirect immunofluorescence (IIF), and reagent lot number. Results: A total of 2005 anti-MPO titres were measured between 15 May 2015 and 13 August 2019. The mean number of tests performed each month doubled between 2015–2016 (25 tests/month) to 2017–2019 (50 tests/month). This was associated with increased testing of patients with known AAV. Some variation in the percentage of low-positive (5.8–16.4%) and high-positive (7.7–21.4%) results was observed between the 12 reagent lots used, however this may have been confounded by duration of use (range 2–296 days). The predicted probability of p-ANCA detection was significantly higher in patients with higher anti-MPO IgG titres (OR=17.66, 95% CI 13.72–21.13, p<0.0001). Conclusion: Anti-MPO IgG testing at ACT Pathology has increased over the last five years. While some variation between reagent lots has been observed, this has not significantly affected overall assay performance.

Online Novel Techniques for Selective Simultaneous Determination of Alkalinity/Bicarbonate and Hardness in Drinking Water by Skalar San++ Segmented Flow Analysis.

The procedures of the current novel automated technique for the determination of hardness, Carbonate, bicarbonate and alkalinity in drinking water have been modified from both classical batch titration and the method of the flow analysis Skalar San++ method (classical scalar current). The forms of standards in the current methods have been modified. The calcium chloride used previously in the current method for the determination of both calcium and total hardness was replaced by a mixed standard that contains calcium and magnesium carbonate in an aqueous solution. Likewise, calcium carbonate and sodium bicarbonate standards currently used in determination of alkalinity and bicarbonate respectively were replaced by using only one standard of calcium, sodium carbonate salt, and the required bicarbonate concentrations were derived from the same calcium carbonate formula mathematically in the stoichiometry equation. The new approach method was validated and applied successfully for analysis of drinking water and proficiency tests samples (PTS).The technique is used for an automatic online analysis of total, calcium hardness, total alkalinity and bicarbonate in drinking water covering a range of 10 - 100 ppm for total hardness and calcium hardness and a range of 5-100 ppm for alkalinity and bicarbonate mg/L as CaCO3. The method gave a distinct merit over the manual titration method and the current skalar method, with enhanced detection limit, high precision (RSD) which was found to be less than 1%) and accuracy of ( ± 1-2%), as well as a high spectral peak resolution and sample throughput.. Moreover, additional merits of the proposed method over the classical skalar one, other than being economic in use, as there is some chemical cut off, is the feasibility of the automatic determination of carbonate ions concentrations in the same run, which could not be achieved automatically by the classical skalarc method, as it always needs mathematical subtraction of bicarbonate concentration from alkalinity concentrations from multi runs. The latter approach is tedious, involved lots of reagents and procedures, time consuming, and could impact the overall method precision and accuracy. The methoddemonstrated selectivity as online automated approach for selective determination of total hardness and calcium hardness in one run and determination of total alkalinity and bicarbonate from the same standard in the second run, when keeping all of the four chemistry reagents aspirated automatically. Sole analysis for every parameter as in the current method was achieved demonstrating simultaneous analysis without discernment, the recovery was found to be comparable with the novel proposed method, but the latter gave a marked enhancement in precision and peak resolution.. Analysis for drinking water samples gave quantitative results with Z score of less than ± 1.2. Accuracy of the automated analysis was investigated by multiple techniques including recovery by standard addition method, run of CRM and t test statistical analysis. The merit of the accuracy for this proposed method has been demonstrated successfully through applications to real samples.

Alloseq HCT: A Wide Range Chimerism Testing Solution with High Sensitivity, Accuracy and Precision for Routine Post Hematopoietic Cell Transplant Monitoring

Genetic chimerism testing in hematopoietic cell transplant (HCT) recipients is typically done using methods based on Short Tandem Repeats (STR), with lower limit of detection around 2%. More sensitive chimerism detection methods may aid in engraftment monitoring and potentially predict early relapse of the disease. We have developed and analytically validated a highly sensitive chimerism test based on Next Generation Sequencing that utilizes single-nucleotide polymorphisms located throughout the genome. The test combines a clinical laboratory compatible protocol with streamlined workflow and a fully automated analysis software to accurately quantify the level of chimerism. Twenty-two samples designed to mimic genomic DNA (gDNA) derived from HCT recipient samples were generated using four distinct gDNA samples mixed at fractions ranging between 0.1-30%. These samples were used to validate the entire AlloSeq HCT™ workflow across multiple DNA input amounts, operators, reagent lots and sequencing runs. Sequencing was performed on the Illumina MiSeq and chimerism quantification was done using the AlloSeq HCT software. The data obtained by the AlloSeq HCT was compared to the expected mixtures and a correlation of 0.99 was obtained. The assay was sensitive down to 0.1%. We next evaluated within-run and across-run variability, which were on average 4.7% and 4.1%, respectively. The within-run and across-run variability was calculated over a range of input gDNA between 3 ng and 100 ng, showing the robustness of the method at different test conditions. AlloSeq HCT test accurately and reproducibly quantified the level of chimerism across a wide dynamic range with higher sensitivity than current standard of care. Combined with automated data analysis, 24-hour workflow and minimal hands-on time, AlloSeq HCT is an optimal method with high sensitivity and precision for routine chimerism testing.

A validated non-aqueous potentiometric titration method for quantitative determination of bromhexine hydrochloride from pharmaceutical preparation

A simple precise, rapid accurate and sensitive non-aqueous potentiometric titration method was developed for quantitative determination of bromhexine hydrochloride from pharmaceutical dosage form. The titration was carried out using standardized 0.1 N perchloric acid. The proposed method was found to be precise with % RSD 0.9999) between 50% to 250% of 0.100 mg of drug substance weight. The percentage recovery of bromhexine hydrochloride in the optimized method was between 99.38 to 99.76%. The method is also found to be rugged when checked by different analysts and using different lots of reagents and different makes of titrators.

A Validated Simple Titrimetric Method for the Quantitative determination of Isoxsuprine hydrochloride from Pharmaceutical dosages

A simple precise, rapid accurate and sensitive titration method was developed for quantitative determination of isoxsuprine hydrochloride in pharmaceutical dosage form. The titration was carried out using standardized 0.1 N sodium hydroxide solution. The proposed method was found to be precise with % RSD 0.99) between 10% to 50% of 500 mg of drug, substance weight. The percentage recovery of isoxsuprine hydrochloride in the optimized method was 99.02% to 99.84%. The method is also found to be rugged when checked by different analysts and using different lots of reagents and different laboratories.

Performance of Afinion HbA1c measurements in general practice as judged by external quality assurance data.

Background It has been debated whether point-of care (POC) glycated hemoglobin (HbA1c) measurements methods can be used for diagnosing persons with diabetes mellitus. The aim of this study was to evaluate the analytical performance of the POC Afinion HbA1c system in the hands of the users, and to investigate which predictors that were associated with good participant performance. Methods External quality assurance (EQA) data from seven surveys in 2017-2018 with a total of 5809 Afinion participants from a POC total quality system in Norway were included in this study (response rate 90%). The control materials were freshly drawn pooled EDTA whole blood. Each participant was evaluated against the analytical performance specification of ±6% from the target value, while the Afinion system was evaluated against the pooled within-laboratory CV <2%, the between-laboratory CV <3.5%, and bias <0.3%HbA1c. Logistic regression analyses were used to investigate which factors were associated with good participant performance. Results The participant pass rates for each survey varied from 98.2% to 99.7%. The pooled within-laboratory CV varied from 1.3% to 1.5%, the between-laboratory CV varied from 1.5% to 2.1%, and bias varied between -0.17 and -0.01 %HbA1c in all surveys. Reagent lot was the only independent factor to predict good participant performance. Conclusions Afinion HbA1c fulfilled the analytical performance specifications and is robust in the hands of the users. It can therefore be used both in diagnosing and monitoring persons with diabetes mellitus, given that the instrument is monitored by an EQA system.

1299. Cluster of False-Positive “Fifth-Generation” HIV Test Results During Implementation of a Routine HIV Screening Program in an Emergency Department

BackgroundIn November 2014, the University of Illinois Hospital (UI Health) introduced an electronic medical record (EMR)-driven HIV screening program in the emergency department (ED). In October 2016, our hospital laboratory introduced “Fifth-generation” HIV testing using the Bio-Rad BioPlex 2200 HIV Antigen/Antibody diagnostic assay. Fifth-generation HIV testing has the advantage of separately detecting and reporting HIV antibody and HIV-1 p24 antigen. Although the literature and manufacturer report high sensitivity and specificity of this test, we encountered higher than expected rates of false-positive tests during the introduction of this test.MethodsWe retrospectively reviewed the results of our ED HIV screening program from October 2016 to March 2019 to describe the outcomes of HIV testing, determine the rates of false-positive HIV tests and determine if false-positive rates were temporally clustered. We also investigated various potential causes of higher than expected false positives including pre-analytical and analytical error. We defined a false-positive test as a repeatedly reactive initial HIV antigen and/or HIV-1 antibody result with a subsequent negative or indeterminate HIV-1/2 antibody differentiation immunoassay and negative HIV-1 nucleic amplification test.ResultsDuring the review period, out of a total of 17,385 HIV tests which were performed, 85 tests were confirmed positive and 27 were false positives. This represents an HIV prevalence of 0.5%. Eighteen of the 27 false positives occurred during an 8 month period between October 2016 and April 2017 (see Figure 1). During our investigation of potential causes of the false-positive tests, we discovered that a reagent lot for the test was changed in June 2017 which resulted in a significant decrease in the false-positive rate (0.33% to 0.07%).ConclusionWe provide data which suggests that a reagent lot may have been the cause of higher than expected false-positive tests for HIV testing. Monitoring of testing outcomes during implementation of a routine HIV testing program can help identify potential root causes of false-positive tests. Disclosures All authors: No reported disclosures.

Application of Differential Pulse Polarography for Detection of Riboflavin from Pharmaceutical Samples and Vegetables

AbstractRiboflavin (vitamin B2) plays an important role in living processes as a precursor of coenzymes. The core structure of riboflavin with isoalloxazine ring participates in enzyme‐catalyzed electron transfer processes of many important metabolites. Several methodologies have been developed for the determination of riboflavin, but those methods have disadvantages like long analysis time, consumption of a lot of reagents, and expensiveness. Hence, to overcome these problems, the DPP technique is proposed in this study. The DPP method is used for the quantitative analysis of riboflavin. The experimental conditions are optimized to obtain the best characterized peak in terms of peak height with analytical validation of the method. The proposed methods are applied for the analysis of riboflavin in pharmaceutical samples and vegetables. The riboflavin is found to adsorb and undergo irreversible reduction reaction at the working mercury electrode.

Performance Evaluation of a New Mobile POC Lipids Analyzer

Hyperlipidemia, excessive amounts of fat and fatty substances in the blood, is a major risk factor for cardiovascular disease. The measuring of fat or fatty substance in blood is usually obtained through a blood lipid test which includes total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL) and high-density lipoprotein (HDL). In the Medical Device industry, Point-of-care (POC) methods provide fast, portable and easy to use devices suitable for rapid determination of Lipid levels. Here, we evaluated the analytical performance of PixoTest POCT Lipid System (iXensor Co., Ltd.), a newly developed POC lipid panel analyzer. PixoTest POCT Analyzer accuracy evaluation with the Cobas c111 Analyzer (Roche Diagnostics Ltd.) as a reference method was carried out according to CLSI EP 09-A3 guidelines. This study enrolled one hundred and twenty (120) subjects and conducted over three (3) clinical sites (40 subjects each), administered through nine (9) trained operators (total) performed under manufacturer’s protocol using three (3) reagent lots per blood sample (capillary & venous). Linear regression, bias plot and other statistical analyses were extrapolated from the test data in this study. Passing-Bablok regression analysis of the data between PixoTest POCT System and Roche Cobas c111 Analyzer with the venous blood showed a slope of 1.03, an intercept of − 0.1 and correlation coefficient of 0.99. The results for capillary blood showed a slope of 1.01, an intercept of − 0.04 and correlation coefficient of 0.99. Passing-Bablok regression analysis of the data (n = 120) from the comparison between the PixoTest POCT System and Roche Cobas c111 Analyzer with the TC venous blood of total 3 lots at 3 sites showed a slope of 0.99, an intercept of − 0.337 and correlation coefficient of 0.995 and the TC capillary blood showed a slope of 0.99, an intercept of − 0.105 and correlation coefficient of 0.984. TG venous blood of total 3 lots at 3 sites showed a slope of 0.99, an intercept of 0.0246 and correlation coefficient of 0.998 and TG capillary blood showed a slope of 0.99, an intercept of − 0.329 and correlation coefficient of 0.998. HDL venous blood showed a slope of 0.99, an intercept of − 0.0297 and correlation coefficient of 0.987 and the HDL capillary blood showed a slope of 0.99, an intercept of − 0.0403 and correlation coefficient of 0.989. The evaluation results of PixoTest POCT system’s accuracy, linearity, and correlation with Roche Cobas c111 Analyzer, the PixoTest POCT System displayed great performance results for all evaluated data points. Thus, the PixoTest POCT System is capable of providing accurate and reliable lipid measurements in the POC setting.

Analytical and Clinical Validation of Expressed Variants and Fusions From the Whole Transcriptome of Thyroid FNA Samples.

Introduction: The Afirma® Xpression Atlas (XA) detects gene variants and fusions in thyroid nodule FNA samples from a curated panel of 511 genes using whole-transcriptome RNA-sequencing. Its intended use is among cytologically indeterminate nodules that are Afirma GSC suspicious, Bethesda V/VI nodules, or known thyroid metastases. Here we report its analytical and clinical validation.Methods: DNA and RNA were purified from the same sample across 943 blinded FNAs and compared by multiple methodologies, including whole-transcriptome RNA-seq, targeted RNA-seq, and targeted DNA-seq. An additional 695 blinded FNAs were used to define performance for fusions between whole-transcriptome RNA-seq and targeted RNA-seq. We quantified the reproducibility of the whole-transcriptome RNA-seq assay across laboratories and reagent lots. Finally, variants and fusions were compared to histopathology results.Results: Of variants detected in DNA at 5 or 20% variant allele frequency, 74 and 88% were also detected by XA, respectively. XA variant detection was 89% when compared to an alternative RNA-based detection method. Low levels of expression of the DNA allele carrying the variant, compared with the wild-type allele, was found in some variants not detected by XA. 82% of gene fusions detected in a targeted RNA fusion assay were detected by XA. Conversely, nearly all variants or fusions detected by XA were confirmed by an alternative method. Analytical validation studies demonstrated high intra-plate reproducibility (89%-94%), inter-plate reproducibility (86–91%), and inter-lab accuracy (90%). Multiple variants and fusions previously described across the spectrum of thyroid cancers were identified by XA, including some with approved or investigational targeted therapies. Among 190 Bethesda III/IV nodules, the sensitivity of XA as a standalone test was 49%.Conclusion: When the Afirma Genomic Sequencing Classifier (GSC) is used first among Bethesda III/IV nodules as a rule-out test, XA supplements genomic insight among those that are GSC suspicious. Our data clinically and analytically validate XA for use among GSC suspicious, or Bethesda V/VI nodules. Genomic information provided by XA may inform clinical decision-making with precision medicine insights across a broad range of FNA sample types encountered in the care of patients with thyroid nodules and thyroid cancer.

Mobile Health for Diagnosis-PixoTest HbA1c POCT System

Glycated hemoglobin (HbA1c) has been recommended as a standard indicator of long-term glycemic control for diabetic and non-diabetic patients, within the in vitro diagnostic (IVD) industry that is focused on the development of point-of-care (POC) methods with improved operational convenience and shorter measurement time. In this study, we evaluated the measurement accuracy performance of a recently commercialized POC HbA1c analyzer, PixoTest POCT System (iXensor Co., Ltd.) that is portable, intuitive and wireless data transmission enabled. For PixoTest POCT accuracy evaluation, TOSOH G7 Analyzer (TOSOH Bioscience GmbH) was used as a reference method and performed in accordance with CLSI EP 09-A3 guidelines. This study enrolled one hundred and twenty (120) subjects from three clinical sites (40 subjects each), administered through nine (9) trained operators (total) performed under manufacturer’s protocol using three reagent lots per blood sample (capillary & venous). Linear regression, bias plot and other statistical analyses were extrapolated from the test data. Passing-Bablok regression analysis of the data between PixoTest POCT System and TOSOH G7 Analyzer with the venous blood showed a slope of 1.03 and an intercept of − 0.1 and a correlation coefficient of 0.99. The results for capillary blood showed a slope of 1.01 and an intercept of − 0.04 and a correlation coefficient of 0.99. The PixoTest POCT System’s performance is similar to TOSOH G7 Analyzer in accuracy and linearity. Therefore, the system’s HbA1c test capability is ideal for the management of chronic disease, as a user convenient and efficient POC solution.

Failure of internal quality control in detecting significant reagent lot shift in serum creatinine measurement.

BackgroundInternal quality control (IQC) in clinical laboratories is carried out to monitor analytical stability. Usually, the satisfactory results of the IQC ensure the acceptability of the examination results. Here, we reported that patients' creatinine results are unreliable, although the internal quality control is satisfactory.MethodsCreatinine levels were analyzed from two quality control materials and twenty patients' specimens using two different lots of reagents. Lot‐to‐lot comparison was performed. The daily median values of serum creatinine levels of patients were calculated from the test results recorded in our laboratory information system.ResultsAlthough IQC was consistent, serum creatinine concentrations were higher using lot B (median: 153 μmol/L; interquartile range: 122‐522 μmol/L) than using lot A (median: 133 μmol/L; interquartile range: 76‐508 μmol/L) for 20 patients (P = .001). The Deming linear regression showed a best fit of y = 0.9394 × x + 45.66. R 2 = .8919, and mean percentage difference between two lots was 34%. The new lot was considered unacceptable. Likewise, the median serum creatinine level from the 360 patients using lot B was 102 μmol/L, which was significantly higher than the daily medians of patients using lot A (median: 66 μmol/L; range: 61‐70 μmol/L) in the previous month.ConclusionThe variations in creatinine concentrations proved to be due to different lots of reagents. However, IQC materials tested using both lots of reagent exhibited minimal variation. Therefore, IQC alone is insufficient for assessing laboratory analytical results. This finding prompts us to be vigilant in potential pitfall of interpreting test results based on satisfactory IQC alone.

Analytical Verification Performance of Afirma Genomic Sequencing Classifier in the Diagnosis of Cytologically Indeterminate Thyroid Nodules

Background: Fine needle aspiration (FNA) cytology, a diagnostic test central to thyroid nodule management, may yield indeterminate results in up to 30% of cases. The Afirma® Genomic Sequencing Classifier (GSC) was developed and clinically validated to utilize genomic material obtained during the FNA to accurately identify benign nodules among those deemed cytologically indeterminate so that diagnostic surgery can be avoided. A key question for diagnostic tests is their robustness under different perturbations that may occur in the lab. Herein, we describe the analytical performance of the Afirma GSC.Results: We examined the analytical sensitivity of the Afirma GSC to varied input RNA amounts and the limit of detection of malignant signals with heterogenous samples mixed with adjacent normal or benign tissues. We also evaluated the analytical specificity from potential interfering substances such as blood and genomic DNA. Further, the inter-laboratory, intra-run, and inter-run reproducibility of the assay were examined. Analytical sensitivity analysis showed that Afirma GSC calls are tolerant to variation in RNA input amount (5–30 ng), and up to 75% dilution of malignant FNA material. Analytical specificity studies demonstrated Afirma GSC remains accurate in presence of up to 75% blood or 30% genomic DNA. The Afirma GSC results are highly reproducible across different operators, runs, reagent lots, and laboratories.Conclusion: The analytical robustness and reproducibility of the Afirma GSC test support its routine clinical use among thyroid nodules with indeterminant FNA cytology.

Analytical evaluation of the microarray-based FluChip-8G Influenza A+B Assay

BackgroundInfluenza causes a significant annual disease burden, with characterization of the infecting virus important in clinical and public health settings. Rapid immunoassays are fast but insensitive, whereas real-time RT-PCR is sensitive but susceptible to genetic mutations and often requires multiple serial assays. The FluChip-8G Influenza A+B Assay provides type and subtype/lineage identification of influenza A and B, including non-seasonal A viruses, in a single microarray-based assay with same day turnaround time. ObjectiveTo evaluate key analytical performance characteristics of the FluChip-8G Influenza A+B Assay. Study designAnalytical sensitivity, cross-reactivity, and multi-site reproducibility were evaluated. ResultsThe limit of detection (LOD) for the FluChip-8G influenza A+B Assay ranged from 5.8 × 102–1.5 × 105 genome copies/mL, with most samples ∼2 × 103 genome copies/mL (∼160 genome copies/reaction). Fifty two (52) additional strains were correctly identified near the LOD, demonstrating robust reactivity. Two variant viruses (H1N1v and H3N2v) resulted in dual identification as both “non-seasonal influenza A” and A/H1N1pdm09. No reproducible cross-reactivity was observed for the 34 organisms tested, however, challenges with internal control inhibition due to crude growth matrix were observed. Lastly, samples tested near the LOD showed high reproducibility (97.0% (95% CI 94.7–98.7)) regardless of operator, site, reagent lot, or testing day. ConclusionThe FluChip-8G Influenza A+B Assay is an effective new method for detecting and identifying both seasonal and non-seasonal influenza viruses, as revealed by good sensitivity and robust reactivity to 52 unique strains of influenza virus. In addition, the lack of cross-reactivity to non-influenza pathogens and high lab-to-lab reproducibility highlight the analytical performance of the assay as an alternative to real-time RT-PCR and sequencing-based assays. Clinical validation of the technology in a multi-site clinical study is the subject of a separate investigation.

Analytical validation of a tumor-agnostic integrated multianalyte circulating tumor DNA (ctDNA) assay in early-stage cancer.

3057 Background: ctDNA sequencing has been rapidly adopted for the identification of targetable somatic alterations (alts) in patients with advanced cancers. However, early stage disease detection has been hindered by low levels of ctDNA in circulation and the presence of confounding non-tumor-related somatic alts. We developed and validated a ctDNA assay that combines somatic and epigenomic signals to detect early stage tumors without tumor tissue or white blood cells (WBC). Methods: Using a single input sample, our assay integrates the sensitive detection of genomic alts with quantification of epigenomic signals associated with cancer. Non-tumor alts (e.g., clonal hematopoiesis of indeterminate potential; CHIP) are excluded using a newly developed bioinformatic classifier. To assess analytical sensitivity, specificity, and positive and negative reproducibility, we tested 337 clinical and contrived samples. Results: Clinical specificity was determined using 80 plasma samples from 50-75 year old presumptive cancer-free donors, and resulted in a single false positive (99% specificity). Analytical sensitivity (limit of detection) was established using a dilution series of 4 different late stage CRC pts tested in triplicate at a clinically relevant DNA input (30 ng) across multiple batches. 100% sensitivity was maintained even at the lowest tested level (estimated 0.1% tumor level). Positive/negative reproducibility was assessed by testing triplicates of diluted late stage samples and age-matched healthy donors, respectively, across different cfDNA inputs, and multiple reagent lots. Both positive and negative calls were 100% concordant across all replicates. Independent estimation of tumor levels from epigenomic or genomic signals produced highly concordant results (correlation r-value: 0.82, p-value: 3e-16). Conclusions: We designed and validated a highly specific ctDNA assay that integrates both genomic and epigenomic signals to allow for accurate and quantitative tumor level detection in early stages of the disease without requiring tumor tissue or WBC.

Multicenter evaluation of the cobas® HIV-1 quantitative nucleic acid test for use on the cobas® 4800 system for the quantification of HIV-1 plasma viral load

Background and objectivesMeasurement of HIV-1 viral load (VL) is necessary to monitor treatment efficacy in patients receiving antiretroviral therapy. We evaluated the performance of the cobas® HIV-1 quantitative nucleic acid test for use on the cobas® 4800 system (“cobas 4800 HIV-1”). MethodsLimit of detection, linearity, accuracy, precision, and specificity of cobas 4800 HIV-1, COBAS® AmpliPrep/COBAS® Taqman® HIV-1 version 2.0 (CAP/CTM HIV-1 v2) and Abbott RealTime HIV-1 were determined in one or two out of three sites. ResultsThe limit of detection of the cobas 4800 HIV-1 for 400 μL and 200 μL input volumes was 14.2 copies/mL (95% CI: 12.5–16.6 copies/mL) and 43.9 copies/mL (37.7–52.7 copies/mL), respectively. Cobas 4800 HIV-1 demonstrated 100% specificity, and results were linear for all analyzed group M HIV-1 subtypes. Precision was high (SD < 0.19 log10) across all measured ranges, reagent lots and input volumes. Correlation between cobas 4800 HIV-1 and CAP/CTM HIV-1 v2 or RealTime HIV-1 was high (R2 ≥ 0.95). Agreement between cobas 4800 HIV-1 and CAP/CTM HIV-1 v2 was 96.5% (95.0%–97.7%) at a threshold of 50 copies/mL, and 97.2% (95.8%–98.3%) at 200 copies/mL. Agreement between cobas 4800 HIV-1 and RealTime HIV-1 was 96.6% (93.4%–98.5%) at 50 copies/mL, and 97.0% (94.0%–98.8%) at 200 copies/mL. The mean difference between cobas 4800 HIV-1 and CAP/CTM HIV-1 v2 or RealTime HIV-1 was -0.10 log10 or 0.01 log10, respectively. ConclusionsThe cobas 4800 HIV-1 test is highly sensitive, accurate and correlated well with other assays, including agreement around clinically relevant thresholds, indicating minimal overall VL quantification differences between tested platforms.

Fingerstick Precision and Total Error of a Point-of-Care HbA1c Test.

Point-of-care (POC) HbA1c tests hold the promise of reducing the rates of undiagnosed diabetes, provided they exhibit acceptable analytical performance. The precision and total error of the POC (Afinion™ HbA1c Dx) test were investigated using whole blood samples obtained by fingerstick and venipuncture. Fingerstick samples spanning the assay range were collected from 61 subjects at three representative POC sites. At each site, six fingerstick samples were obtained from each subject and tested on the POC test across two (Afinion AS100) instruments. Repeatability, between-operator, and between-instrument components of variance were calculated using analysis of variance (ANOVA). Four venous samples (low, threshold, medium, and high HbA1c) were measured in duplicate across three instruments using three reagent lots, twice per day over 20-days. Repeatability, between-run, between-day, between-lot, and between-instrument components of variance were calculated. These fingerstick and venous blood results, combined with estimates of imprecision and bias from a prior investigation, allowed for the calculation of the total coefficient of variation (CV) and total error of the POC test using fingerstick and venous whole blood samples. The total imprecision ranged from 1.30% to 2.03% CV using fingerstick samples and from 1.31% to 1.64% CV using venous samples. The total error ranged from 2.87% to 4.75% using fingerstick samples and from 2.93% to 3.80% using venous samples. The POC test evaluated here is precise across its measuring range using both fingerstick and venous whole blood. The calculated total error of the test is well under the accepted quality requirement of ≤6%.

Diagnostic Accuracy of Conventional Cell Blocks Along with p16INK4 and Ki67 Biomarkers as Triage Tests in Resource-poor Organized Cervical Cancer Screening Programs

Background:Liquid based cytology with dual biomarkers has improved sensitivity and specificity in detecting high grade cervical intraepithelial neoplasia (CIN). In low resource settings, especially in organized camps, LBC is costly and immunohistochemistry on conventional pap smears is difficult to standardize with consumption of lots of reagents. In present study, to improve the accuracy of conventional pap smears and reduce the cost of biomarker testing, we evaluated conventional cell blocks (CCBs) preparations with biomarkers to detect high-grade CIN in resource-poor organized screening programs. We also studied feasibility of using CCB as primary screening test.Material and Methods:A total of 350 participants were included in the cross-sectional evaluation of the screening tests. A conventional Papanicolaou (Pap) smear was obtained, and another sample was then collected and placed in 10% neutral buffered formalin for CB preparation. All abnormal Pap tests and CBs were stained for the biomarkers p16INK4a and Ki67. Histopathology with p16INK4a expression was considered the gold standard. Diagnostic tests were compared using MacNemar’s test and receiver operating curves were plotted.Results:The sensitivity, specificity, and diagnostic accuracy of CCB cytology, CB + p16 cytology and CB + p16Ki67 cytology for detecting CIN2+ lesions were 85.71%, 100%, 97.44%; 100%, 93.75%, 94.87%; and 85.71%, 100%, 97.44%, respectively. The Ki67 index could further categorize low grade lesions into lesions with low proliferative index and with high proliferative index (Pearson chi-square p value <0.001).Conclusion:If CB preparation is standardized, CCB cytology with biomarkers can have better diagnostic accuracy than conventional cytology, can classify low grade lesions likely to progress and can be used in field settings as primary screening test.

A Non-aqueous Potentiometric Titration Method for Validation of Imatinib mesylate from Pharmaceutical Dosages

A simple precise, rapid accurate and sensitive non-aqueous potentiometric titration method was developed for quantitative determination of imatinib mesylate from pharmaceutical dosage form. The titration was carried out using standardized 0.1 N perchloric acid. The proposed method was found to be precise with % RSD 0.999) between 20% to 100% of 500 mg of drug substance weight. The percentage recovery of imatinib mesylate in the optimized method was between 100.087 to 100.692%. The method is also found to be rugged when checked by different analysts and using different lots of reagents and different makes of titrators.