Reagent Lots Research Articles

A validated non-aqueous potentiometric titration method for quantitative determination of clopidogrel bisulphate from pharmaceutical preparation

A simple precise, rapid accurate and sensitive non-aqueous potentiometric titration method was developed for quantitative determination of terbutaline sulphate 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 150% of 0.400 mg of drug substance weight. The percentage recovery of terbutaline sulphate in the optimized method was between 99.39 to 99.64%. The method is also found to be rugged when checked by different analysts and using different lots of reagents and different makes of titrators.

Analytical and Clinical Validation of a Molecular Diagnostic Signature in Kidney Transplant Recipients

Context: The TruGraf test is a blood-based assay that provides non-invasive, accurate assessment of adequacy of immunosuppression in kidney transplant recipients. TruGraf relies on gene-expression “signatures” that differentiate a state of Transplant eXcellence (TX, indicating adequately immunosuppressed) from not-TX. Objective: To evaluate the performance of the TruGraf test. Design: Analytical performance studies to characterize stability of RNA in blood during collection and shipment, analytical sensitivity (input RNA concentration), analytical specificity (interfering substances) and assay performance (clinical validity, and intra-assay, inter-assay, inter-laboratory reproducibility). Results: Total RNA extracted from whole blood specimens collected in PAXgene Blood RNA tubes was stable up to 3 days at room temperature (stable RNA yield). Under routine ambient shipping conditions, storage and shipping temperatures did not affect results. However, specimen shipments exposed to temperatures >400°C or to ambient temperatures for >3 days were unacceptable for processing. Analytical sensitivity studies demonstrated tolerance to variation in RNA input (50 to 400 ng per 3’ IVT (in vitro transcript] labeling reaction). Specificity studies using genomic DNA spiked into 3 ’IVT reactions at 10-20% demonstrated negligible assay interference. The test was reproducible across operators, runs, reagent lots, and laboratories. External validation demonstrated that the TruGraf blood test accurately classified patients in 72% of 295 samples. Conclusions: Analytical sensitivity, analytical specificity, robustness, quality control, and clinical validity of the TruGraf blood test were successfully verified, indicating its suitability for clinical use.

Analytical Validation of the nCounter®-based Lymphoma Subtyping Test (LST) for Cell-of-Origin (COO) Identification in Formalin-Fixed Paraffin-Embedded (FFPE) Diffuse Large B-Cell Lymphoma (DLBCL) Specimens

Introduction: Diffuse large B-cell lymphoma (DLBCL) is an aggressive form of non-Hodgkin’s lymphoma with two distinct molecular subtypes defined by Cell-of-Origin (COO): germinal center B-cell (GCB) and activated B-cell (ABC). COO is being evaluated as a predictive biomarker in multiple DLBCL clinical trials hence the need for an accurate and precise companion diagnostics (CDx). NanoString LST is an investigational multiplexed digital gene expression assay performed on the nCounter® Dx Analysis System that identifies COO from FFPE tissue with a turnaround time of 2-3 days (Wallden B et al, JCO 2015; Storhoff J et al, Blood 2015). The test is based on the Lymph2Cx gene expression assay which profiles 15 classifier genes and 5 housekeeping genes to compute a linear predictor score (LPS) and determine the COO subtype (Scott D et al, Blood 2014). To establish a CDx, both analytical and clinical validations are required using the locked algorithm and assay procedure. Clinical validity of the LST is currently being evaluated in a global Phase 3 study in which the assay is used to prospectively select patients with ABC-type DLBCL to receive in a randomized fashion R2-CHOP or R-CHOP (Nowakowski G et al, JCO 2016). The analytical validation studies described herein were designed to evaluate the analytical performance of the test across multiple laboratory sites, operators, instruments, reagent lots, and RNA input levels.Design: Reproducibility was measured across 3 sites and 6 operators by testing replicate tissue sections from 43 FFPE DLBCL tumor blocks following independent pathology review at each site. The testing included both core needle and surgical biopsy specimens. The standard deviation (SD) of the LPS output was analyzed as a continuous variable using a linear mixed model to estimate assay variance, and the site-to-site concordance was evaluated. Analytical precision was measured across 3 sites and 6 operators by testing 5 pooled DLBCL tumor RNA samples representing each COO subtype including samples near the subtype thresholds. The sensitivity of the assay was evaluated by testing 4 RNA levels within the recommended input range (62.5 ng, 125 ng, 500 ng, 1000 ng) and 2 input levels outside of this range (50 ng and 1250 ng) using 14 DLBCL tumor RNA samples and 2 reagent lots. The percentage of test samples passing all QC metrics was determined at each input level, and the subtype concordance was evaluated compared to the nominal test input level (500 ng). Interference of human genomic DNA was assessed by omitting DNase from the assay procedure. The impact of including non-tumor tissue was assessed by omitting the tissue macrodissection step from the assay.Results: In the multi-site reproducibility study, the average site-to-site LST subtype concordance with independent pathology review was >97% with no ABC-to-GCB discordances (or vice versa). The overall range of LPS spanned approximately 5000 units. The total SD was <100 LPS units including all sources of variation (site/pathologist, operator, and between-run/residual). No differences in variance were observed between excisional and core needle biopsies and <1% of the variance was attributed to sites and operators. In the multi-site precision study, the total SD of LPS was 46 including all sources of variation (site, operator, between-runs/day, input level, and within-run/residual). There were no significant differences between sites, operators or days of run. A separate precision study showed that reagent lot and instrument do not significantly contribute to assay variability. In the RNA input study, all input levels inside and outside the specified operating range had 100% assay pass rate. The estimated rate of samples switching from ABC to GCB was <0.00001% for all input levels. In the interference study, genomic DNA contamination resulted in a negative bias in LPS (mean bias = -186), indicating the necessity of DNase treatment in this assay.Conclusions: The investigational NanoString LST has been analytically validated for identifying COO subtypes across multiple testing laboratories. The test provides a highly precise, sensitive and rapid method for measuring COO on FFPE DLBCL tumor specimens, including both excisional and core needle biopsies. DisclosuresChen:NanoString Technologies, Inc.: Employment, Other: Stock option. Dennis:NanoString Technologies, Inc.: Employment, Other: Stock option. Danaher:NanoString Technologies, Inc.: Employment, Other: Stock option. Wallden:NanoString Technologies, Inc.: Employment. Hood:NanoString Technologies, Inc.: Employment, Other: Stock option. Ren:NanoString Technologies, Inc.: Employment, Other: Stock option. Liu:NanoString Technologies, Inc.: Employment, Other: Stock option. Dowidar:NanoString Technologies, Inc.: Employment, Other: Stock option. Sullivan:NanoString Technologies, Inc.: Employment, Other: Stock option. Haffner:NanoString Technologies, Inc.: Employment, Other: Stock option. Cesano:NanoString Technologies, Inc.: Employment, Other: Stock option. Ferree:NanoString Technologies, Inc.: Employment, Other: Stock option, Patents & Royalties: NanoString Technologies, Inc.. Storhoff:NanoString Technologies, Inc.: Employment, Other: Stock option.

Molecular polymorphism of the ABO system in Moroccan blood donors suggesting an A2 allele depression by B allele

BackgroundSeveral previous studies have determined the phenotypic and genetic frequencies of ABO blood groups as well as their distributions in different regions of Morocco. However, there are no detailed published data concerning ABO genetic polymorphism up to date. Regarding the ABO system importance in transfusion medicine and anthropology. Our aim is to determine the ABO phenotypic and genetic polymorphism within Moroccan blood donors.Subjects and MethodsThe ABO phenotype was determined using Olympus PK 7300 Automate and two lots of reagents. Genotyping of 120 subjects taken randomly and of 60 subjects with AB blood groups was performed by allele‐specific polymerase chain reaction (AS‐PCR) technique. The direct sequencing concerned five DNA of individuals whose genotypes were undetermined by AS‐PCR.ResultsThe ABO allelic frequencies found among 9271 blood donors are 0·681, 0·205 and 0·114 for O, A and B alleles respectively. A total of 10 alleles and 20 genotypes have been revealed in 120 blood donors. The ABO genetic frequencies of which are 0·346, 0·233, 0·121, 0·113, 0·058, 0·021, 0·0125, 0·0042, 0·0042 and 0·0042 for ABO*O02, ABO*O01, ABO*B101, ABO*A101, ABO*A201, ABO*A102, ABO*O03, ABO*O306, ABO*O107 and ABO*Onv alleles respectively. Furthermore, our results suggested the existence of A2 allele depression by B allele among 16·18% of AB subjects. This depression concerns 96·66% among A201/B101 and 3·34% among A206/B101 subjects.ConclusionThis study is the first in Morocco concerning the ABO genetic polymorphism. The direct sequencing of ABO gene taking into account a large population lets us precise the genetic diversity and consolidate the hypothesis of the existence of the A2 allele depression by B allele within the Moroccan population.

Multicenter evaluation of analytical characteristics of the Elecsys® Periostin immunoassay

ObjectiveThe multifunctional cytokine IL-13 is thought to play a central role in Type 2 inflammation in asthma. Serum periostin has been explored as a candidate biomarker for evaluating IL-13 activity in the airway. We describe the technical performance characteristics of a novel, fully automated immunoassay for the determination of periostin in serum. Design and methodsLimit of blank [LoB], limit of detection [LoD] and limit of quantitation [LoQ], linearity, precision and reproducibility across sites and lots were evaluated according to Clinical and Laboratory Standards Institute guidelines. Interferences and sample stability were also investigated. ResultsThe pre-specified values for LoB (2ng/mL), LoD (4ng/mL) and LoQ (10ng/mL) were met. The assay was linear throughout the measuring range (10–160ng/mL) with recoveries within ±10% of target at concentrations >30ng/mL and within ±3ng/mL at concentrations ≤30ng/mL. Recovered periostin concentrations were also within ±10% of target in presence of 43 potentially interfering substances and drugs. Samples were stable across various storage conditions and durations (24h at room temperature, 7days at 4°C, 12weeks at −20°C, and 3 freeze/thaw cycles). Repeatability experiments resulted in CVs across samples and controls ranging from 0.9–1.5%. Intermediate precision was 1.2–1.7% and reproducibility including 3 testing sites and 3 reagent lots was 1.7–3.1%. The final assay correlates to the assay version used in previous clinical trials (Pearson's r=0.998, bias at 50ng/mL=1.2%). ConclusionThe performance evaluation of the Elecsys® Periostin immunoassay including a multicenter precision analysis demonstrated that the assay is suitable for measuring serum periostin at clinically important concentrations around 50ng/mL.

The establishment and application of internal quality control system for real-time quantitative PCR detection of BCR-ABL (P210) transcript levels

目的制备BCR-ABL (P210）实时定量PCR（RQ-PCR）检测的室内质控物，建立RQ-PCR检测BCR-ABL (P210）转录本水平的室内质控方法。方法取K562细胞和HL-60细胞，制备RQ-PCR检测BCR-ABL（P210）转录本水平的高、低浓度质控物，2013年8月至2015年10月用RQ-PCR法与临床标本同步检测BCR-ABL（P210）184次，按试剂批号（批号20130303、20131212、20140411和20150327分别简称为R1、R2、R3、R4）统计分析标准曲线斜率、截距及相关系数，并按试剂批号和质控物批号（批号20130725、20140611分别简称为Q1、Q2）统计分析高、低浓度质控物检测结果，对斜率、截距及质控物检测结果采取Levey-Jennings质控图结合Westgard多规则质控方法进行质控判断。结果①标准曲线斜率、截距：R1检测53次，斜率、截距均未失控；R2检测37次，斜率失控6次，且第2~8次在x−s限值下侧，第12~37次在x值上侧，截距失控9次，且第1~8次在x+s限值上侧，第12~37次出现在x值下侧；R3检测80次和R4检测14次，斜率、截距均未失控。②质控物结果：Q1批号质控物，R1检测49次未失控，R2检测23次失控1次；Q2批号质控物，R2检测14次、R3检测72次及R4检测14次均未失控。结论利用K562细胞和HL-60细胞制备定量检测BCR-ABL（P210）转录本水平的高、低浓度室内质控物，制备方便，检测结果可靠、稳定，使用质控物检测结果结合标准曲线斜率、截距进行室内质控，可更有效地保证临床检测结果的准确性和稳定性。

Automatic Detection of Mitosis and Nuclei From Cytogenetic Images by CellProfiler Software for Mitotic Index Estimation.

Mitotic Index (MI) estimation expressed as percentage of mitosis plays an important role as quality control endpoint. To this end, MI is applied to check the lot of media and reagents to be used throughout the assay and also to check cellular viability after blood sample shipping, indicating satisfactory/unsatisfactory conditions for the progression of cell culture. The objective of this paper was to apply the CellProfiler open-source software for automatic detection of mitotic and nuclei figures from digitized images of cultured human lymphocytes for MI assessment, and to compare its performance to that performed through semi-automatic and visual detection. Lymphocytes were irradiated and cultured for mitosis detection. Sets of images from cultures were analyzed visually and findings were compared with those using CellProfiler software. The CellProfiler pipeline includes the detection of nuclei and mitosis with 80% sensitivity and more than 99% specificity. We conclude that CellProfiler is a reliable tool for counting mitosis and nuclei from cytogenetic images, saves considerable time compared to manual operation and reduces the variability derived from the scoring criteria of different scorers. The CellProfiler automated pipeline achieves good agreement with visual counting workflow, i.e. it allows fully automated mitotic and nuclei scoring in cytogenetic images yielding reliable information with minimal user intervention.

The Importance of Reagent Lot Registration in External Quality Assurance/Proficiency Testing Schemes.

Providers of external quality assurance (EQA)/proficiency testing schemes have traditionally focused on evaluation of measurement procedures and participant performance and little attention has been given to reagent lot variation. The aim of the present study was to show the importance of reagent lot registration and evaluation in EQA schemes. Results from the Noklus (Norwegian Quality Improvement of Primary Care Laboratories) urine albumin/creatinine ratio (ACR) and prothrombin time international normalized ratio (INR) point-of-care EQA schemes from 2009-2015 were used as examples in this study. The between-participant CV for Afinion ACR increased from 6%-7% to 11% in 3 consecutive surveys. This increase was caused by differences between albumin reagent lots that were also observed when fresh urine samples were used. For the INR scheme, the CoaguChek INR results increased with the production date of the reagent lots, with reagent lot medians increasing from 2.0 to 2.5 INR and from 2.7 to 3.3 INR (from the oldest to the newest reagent lot) for 2 control levels, respectively. These differences in lot medians were not observed when native patient samples were used. Presenting results from different reagent lots in EQA feedback reports can give helpful information to the participants that may explain their deviant EQA results. Information regarding whether the reagent lot differences found in the schemes can affect patient samples is important and should be communicated to the participants as well as to the manufacturers. EQA providers should consider registering and evaluating results from reagent lots.

Antibody levels against GLURP R2, MSP1 block 2 hybrid and AS202.11 and the risk of malaria in children living in hyperendemic (Burkina Faso) and hypo-endemic (Ghana) areas.

Background Differences in parasite transmission intensity influence the process of acquisition of host immunity to Plasmodium falciparum malaria and ultimately, the rate of malaria related morbidity and mortality. Potential vaccines being designed to complement current intervention efforts therefore need to be evaluated against different malaria endemicity backgrounds.MethodsThe associations between antibody responses to the chimeric merozoite surface protein 1 block 2 hybrid (MSP1 hybrid), glutamate-rich protein region 2 (GLURP R2) and the peptide AS202.11, and the risk of malaria were assessed in children living in malaria hyperendemic (Burkina Faso, n = 354) and hypo-endemic (Ghana, n = 209) areas. Using the same reagent lots and standardized protocols for both study sites, immunoglobulin (Ig) M, IgG and IgG sub-class levels to each antigen were measured by ELISA in plasma from the children (aged 6–72 months). Associations between antibody levels and risk of malaria were assessed using Cox regression models adjusting for covariates.ResultsThere was a significant association between GLURP R2 IgG3 and reduced risk of malaria after adjusting age of children in both the Burkinabe (hazard ratio 0.82; 95 % CI 0.74–0.91, p < 0.0001) and the Ghanaian (HR 0.48; 95 % CI 0.25–0.91, p = 0.02) cohorts. MSP1 hybrid IgM was associated (HR 0.85; 95 % CI 0.73–0.98, p = 0.02) with reduced risk of malaria in Burkina Faso cohort while IgG against AS202.11 in the Ghanaian children was associated with increased risk of malaria (HR 1.29; 95 % CI 1.01–1.65, p = 0.04).ConclusionThese findings support further development of GLURP R2 and MSP1 block 2 hybrid, perhaps as a fusion vaccine antigen targeting malaria blood stage that can be deployed in areas of varying transmission intensity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1146-4) contains supplementary material, which is available to authorized users.

Analytical performance of a bronchial genomic classifier.

BackgroundThe current standard practice of lung lesion diagnosis often leads to inconclusive results, requiring additional diagnostic follow up procedures that are invasive and often unnecessary due to the high benign rate in such lesions (Chest 143:e78S-e92, 2013). The Percepta bronchial genomic classifier was developed and clinically validated to provide more accurate classification of lung nodules and lesions that are inconclusive by bronchoscopy, using bronchial brushing specimens (N Engl J Med 373:243–51, 2015, BMC Med Genomics 8:18, 2015). The analytical performance of the Percepta test is reported here.MethodsAnalytical performance studies were designed to characterize the stability of RNA in bronchial brushing specimens during collection and shipment; analytical sensitivity defined as input RNA mass; analytical specificity (i.e. potentially interfering substances) as tested on blood and genomic DNA; and assay performance studies including intra-run, inter-run, and inter-laboratory reproducibility.ResultsRNA content within bronchial brushing specimens preserved in RNAprotect is stable for up to 20 days at 4 °C with no changes in RNA yield or integrity. Analytical sensitivity studies demonstrated tolerance to variation in RNA input (157 ng to 243 ng). Analytical specificity studies utilizing cancer positive and cancer negative samples mixed with either blood (up to 10 % input mass) or genomic DNA (up to 10 % input mass) demonstrated no assay interference. The test is reproducible from RNA extraction through to Percepta test result, including variation across operators, runs, reagent lots, and laboratories (standard deviation of 0.26 for scores on > 6 unit scale).ConclusionsAnalytical sensitivity, analytical specificity and robustness of the Percepta test were successfully verified, supporting its suitability for clinical use.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2153-0) contains supplementary material, which is available to authorized users.

Multiplexed color-coded probe-based gene expression assessment for clinical molecular diagnostics in formalin-fixed paraffin-embedded human renal allograft tissue.

Histopathologic diagnoses in transplantation can be improved with molecular testing. Preferably, molecular diagnostics should fit into standard-of-care workflows for transplant biopsies, that is, formalin-fixed paraffin-embedded (FFPE) processing. The NanoString(®) gene expression platform has recently been shown to work with FFPE samples. We aimed to evaluate its methodological robustness and feasibility for gene expression studies in human FFPE renal allograft samples. A literature-derived antibody-mediated rejection (ABMR) 34-gene set, comprised of endothelial, NK cell, and inflammation transcripts, was analyzed in different retrospective biopsy cohorts and showed potential to molecularly discriminate ABMR cases, including FFPE samples. NanoString(®) results were reproducible across a range of RNA input quantities (r = 0.998), with different operators (r = 0.998), and between different reagent lots (r = 0.983). There was moderate correlation between NanoString(®) with FFPE tissue and quantitative reverse transcription polymerase chain reaction (qRT-PCR) with corresponding dedicated fresh-stabilized tissue (r = 0.487). Better overall correlation with histology was observed with NanoString(®) (r = 0.354) than with qRT-PCR (r = 0.146). Our results demonstrate the feasibility of multiplexed gene expression quantification from FFPE renal allograft tissue. This represents a method for prospective and retrospective validation of molecular diagnostics and its adoption in clinical transplantation pathology.

Harmonizing the International Normalized Ratio (INR) : Standardization of Methods and Use of Novel Strategies to Reduce Interlaboratory Variation and Bias.

To reduce interlaboratory variation and bias in international normalized ratio (INR) results, as used to monitor patients receiving vitamin K antagonist therapy, including warfarin, in a large pathology network (n = 27 laboratories) by procedural standardization and harmonization. Network consensus to standardize to common instrument and reagent platforms was established, following development of hemostasis test specifications. Subsequent installations and implementation occurred after conclusion of a government tender process. Network-wide application of simple novel process of verification harmonization of local international sensitive index and mean normal prothrombin time initiated for each new lot of INR reagent that does not require ongoing use of reference thromboplastin or calibration/certified plasma sets. We achieved reduction of different instrument manufacturers (from four to one), instrument types (10 to three), reagent types (four to one), and instrument/reagent combinations (12 to three), plus substantial reduction in INR variability and bias. Results infer significant improvement in local patient management, with positive implications for other laboratories. For the United States in particular, lack of US Food and Drug Administration-cleared certified plasmas may compromise INR accuracy, and our novel approach may provide a workable alternative for laboratories and networks.

Using Quartz Crystal Microbalance for Field Measurement of Liquid Viscosities

The field measurement of liquid viscosities, especially the high viscous liquids, is challenging and often requires expensive equipment, long processing time, and lots of reagent. We use quartz crystal microbalances (QCMs) operating in solution which are also sensitive to the viscosity and density of the contacting solution. QCMs are typically investigated for sensor applications in which one surface of QCM completely immersed in Newtonian liquid, but the viscous damping in liquids would cause not only large frequency shifts but also large losses in the quality factorQleading to instability and even cessation of oscillation. A novel mass-sensitivity-based method for field measurement of liquid viscosities using a QCM is demonstrated in this paper and a model describing the influence of the liquid properties on the oscillation frequency is established as well. Two groups of verified experiments were performed and the experimental results show that the presented method is effective and possesses potential applications.

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

A simple precise, rapid accurate and sensitive non-aqueous potentiometric titration method was developed for quantitative determination of lafutadine 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 0.99) between 20% to 100% of 100 mg of drug substance weight. The percentage recovery of lafutadine in the optimized method was between 99.878 to 101.315%. The method is also found to be rugged when checked by different analysts and using different lots of reagents and different makes of titrators.

Validation of the ANSR(®) Listeria monocytogenes Method for Detection of Listeria monocytogenes in Selected Food and Environmental Samples.

Work was conducted to validate performance of the ANSR(®) for Listeria monocytogenes method in selected food and environmental matrixes. This DNA-based assay involves amplification of nucleic acid via an isothermal reaction based on nicking enzyme amplification technology. Following single-step sample enrichment for 16-24 h for most matrixes, the assay is completed in 40 min using only simple instrumentation. When 50 distinct strains of L. monocytogenes were tested for inclusivity, 48 produced positive results, the exceptions being two strains confirmed by PCR to lack the assay target gene. Forty-seven nontarget strains (30 species), including multiple non-monocytogenes Listeria species as well as non-Listeria, Gram-positive bacteria, were tested, and all generated negative ANSR assay results. Performance of the ANSR method was compared with that of the U.S. Department of Agriculture, Food Safety and Inspection Service Microbiology Laboratory Guidebook reference culture procedure for detection of L. monocytogenes in hot dogs, pasteurized liquid egg, and sponge samples taken from an inoculated stainless steel surface. In addition, ANSR performance was measured against the U.S. Food and Drug Administration Bacteriological Analytical Manual reference method for detection of L. monocytogenes in Mexican-style cheese, cantaloupe, sprout irrigation water, and guacamole. With the single exception of pasteurized liquid egg at 16 h, ANSR method performance as quantified by the number of positives obtained was not statistically different from that of the reference methods. Robustness trials demonstrated that deliberate introduction of small deviations to the normal assay parameters did not affect ANSR method performance. Results of accelerated stability testing conducted using two manufactured lots of reagents predicts stability at the specified storage temperature of 4°C of more than 1 year.

Analytical Performance of Nanostring's Companion Diagnostic (CDx) Lymphoma Subtyping Test (LST) with Diffuse Large B-Cell Lymphoma Core Needle Biopsy Samples

Background: Diffuse large B-cell lymphoma (DLBCL) is an aggressive non-Hodgkin's lymphoma with two distinct molecular cell-of-origin (COO) subtypes known as germinal center B-cell (GCB) or activated B-cell (ABC). DLBCL subtypes have been reported to be prognostic and potentially predictive of treatment benefit, underscoring the need for a precise and accurate CDx test (Roschewski, Nat Rev Clin Oncol, 2014). NanoString's LST was developed to enable identification of the COO subtypes on the nCounter® Dx Analysis System using formalin fixed paraffin embedded (FFPE) tissue specimens based on the previously developed Lymph2Cx gene expression profiling assay (Scott, Blood 2014). The test assigns the COO subtypes based on a Linear Predictor Score (LPS) that is calculated using a weighted sum of the gene expression. The LST is currently being used as a CDx in a phase III global clinical validation trial (clinicaltrials.gov NCT02285062) to identify newly diagnosed DLBCL patients of the ABC type who may preferentially respond to lenalidomide.The LST was previously reported to be a highly precise and accurate method of determining DLBCL subtypes (ABC and GCB) when using FFPE excisional/surgical biopsy tissue specimens as a test input (Wallden, JCO 2015). Although excisional biopsies are recommended for DLBCL diagnosis by current ESMO and NCCN guidelines, recent studies have reported excellent sensitivity and specificity in diagnosing malignant lymphomas by core needle biopsy (CNB) leading to increased usage of this method (Hu Q, Am J Clin Pathol, 2013, de Kerviler, Best Pract Res Cl Ha, 2012). The aim of the current study was to assess the analytical performance of the LST when using CNB samples as a test input.Methods: Recently archived CNB and small biopsy DLBCL samples were procured from Oregon Health & Science University (Portland, OR) (n=23), Hôpital Saint-Louis (Paris, France) (n=19), and Asterand Bioscience (n=12). Both nodal and extra-nodal tissue sites were included. Pathology review was performed on an H&E stained slide from each tissue sample to identify the area of DLBCL. Unstained slide mounted FFPE tissue sections (5 µm) were prepared from each sample for assay processing. The RNA extraction and LST procedures were the same as reported previously for surgical biopsy samples (Wallden, JCO 2015). The number of sections used for each RNA extraction was based on the tumor surface area (TSA) measured on the slide (typically 4-6 slides for TSA<10 mm2; 2-4 slides for TSA≥10 mm2). Multiple extractions were performed and subsequently tested independently for each sample (n=148 total). Additional variables tested in this study were user (n=2), reagent lot (n=2), and instrument (n=2). The reproducibility of the test outputs were evaluated using a linear mixed model (LMM) for LPS variance and concordance for subtype call. Principal component analysis (PCA) was performed on the gene expression profile to determine the major source(s) of variability.Results: Of the 54 total samples tested, 51 provided test results (failures were due to RNA degradation). With the 51 passing samples, an assay pass rate of 97% (144/148) was achieved. The total tissue area input into the test was correlated to RNA yield (R2 =0.54). The LPS was highly reproducible across sample replicates (Figure 1) with a total assay standard deviation of 59 LPS units (<2% of the LPS range). This estimate was similar to surgical biopsy samples (76 LPS units). The subtype concordance was estimated as 96-98% (between users) where 2 of 51 samples with LPS scores close to the thresholds moved from ABC or GCB to unclassified, or vice versa. A PCA of the gene expression data demonstrated that the first principle component is highly correlated to the LPS providing further evidence that the biology underlying the LST is the major driver of algorithm gene expression in CNB.Conclusions: In this study we demonstrated robust performance of the LST using FFPE DLBCL CNB/small biopsy samples. Our data support that sufficient RNA yield and gene expression quality can be achieved from biopsy samples with measured TSA of 2 mm2 or greater. We also show that the analytical performance of the LST is comparable between CNB/small biopsy samples and surgical biopsies and that the principal component of gene expression variation is LPS. The LST sample input requirements for the ongoing phase III trial will be updated to include CNB/small biopsy samples to accommodate clinical practice. [Display omitted] DisclosuresStorhoff:NanoString Technologies, Inc.: Employment, Other: Stock . Wallden:NanoString Technologies, Inc.: Employment, Other: Stock. Braziel:Oregon Health & Sciences University: Employment. Thieblemont:St. Louis Hospital, Paris, France: Employment. Hood:NanoString Technologies, Inc.: Employment, Other: Stock. Ravi:NanoString Technologies, Inc.: Employment, Other: Stock. Dennis:NanoString Technologies, Inc.: Employment, Other: Stock. Dowidar:NanoString Technologies, Inc.: Employment, Other: Stock. Danaher:NanoString Technologies, Inc.: Employment, Other: Stock. Dunlap:Oregon Health & Sciences University: Employment. Briere:St. Louis Hospital, Paris, France: Employment. de Kerviler:St. Louis Hospital, Paris, France: Employment. Ferree:NanoString Technologies, Inc.: Employment, Other: Stock, Patents & Royalties: NanoString Technologies, Inc..

Detecting long-term drift in reagent lots.

Between-reagent lot verification is a routine laboratory exercise in which a set of samples is tested in parallel with an existing reagent lot and a candidate reagent lot (before the candidate lot is committed to test patient samples). The exercise aims to verify and maintain consistency in the analytical performance of a test. We examined the limitations of a routine between-reagent lot verification procedure in detecting long-term analytical drift and looked for a more sensitive alternative. Via numerical simulations, we examined the statistical power of the current regression-based (weighted Deming regression) procedure for between-reagent lot verification in detecting proportional bias and constant bias. An alternative procedure applying the Student t-test to separately examine cumulative regression slopes and intercepts across multiple reagent lots was proposed and evaluated by numerical simulations. The regression-based procedure had poor statistical power in detecting proportional bias and constant bias when small numbers of samples were used in each between-reagent lot verification exercise. Furthermore, the method failed to detect long-term drifts in analytical performance. The proposed approach based on the Student t-test can detect long-term (cumulative) drifts in regression slopes and intercepts. This method detected a mild downward drift in the serum sodium assay in our hospital that was missed by routine between-reagent lot verification. The proposed method objectively and systematically detects long-term proportional and constant bias separately. However, the statistical power of this procedure remains unsatisfactory when used with small sample sizes. Sharing of information between laboratories may provide sufficient statistical power to detect clinically important analytical shifts.

Advantages of the lipoprotein-associated phospholipase A2 activity assay

Objectives: Lipoprotein-associated phospholipase A2 (Lp-PLA2) is increased in circulation in patients at higher risk of coronary heart disease (CHD) events and stroke. Therefore, measurement of Lp-PLA2 can be used as an adjunct to traditional cardiovascular risk factors for identifying individuals at higher risk of cardiovascular events. Recently, a reagent for measuring Lp-PLA2 activity (diaDexus, San Francisco, CA) received FDA approval. Here we evaluate the assay performance of the Lp-PLA2 activity assay.Methods: Lp-PLA2 activity assay reagent performance was evaluated on an open user-defined channel on a Cobas 6000/c501 (Roche Diagnostics, Indianapolis, IN) using a 5-point calibration curve (0–400nmol/min/mL). Analytical performance was established for the following parameters: precision, linearity, accuracy, analytical sensitivity, analytical specificity, reference interval, reagent lot-to-lot comparison, specimen type, on-board reagent stability, and sample stability.Results: Assay limit of detection was determined to be 7.8nmol/min/mL with an average %CV of 2.8%. Precision studies revealed a coefficient of variation ≤1.6% between 79 and 307nmol/min/mL and accuracy was demonstrated between 4.8–368.7nmol/min/mL. Comparable results were generated in paired SST serum and EDTA plasma. No age association was found with Lp-PLA2 activity at the 95th percentile however a gender association was identified resulting in gender-specific 95th percentile limits in a healthy reference population. No bias was found when comparing results from several different lots of assay reagent. Lp-PLA2 activity results are extremely stable in both serum and EDTA plasma under refrigerate and frozen storage conditions up to 31days.Conclusions: Lp-PLA2 activity assay displays accurate and precise performance characteristics on the Cobas c501 platform. The assay performance is significantly improved over the predecessor immunoassay allowing for adoption of Lp-PLA2 activity in clinical practice.

Prognostic Significance of Diffuse Large B-Cell Lymphoma Cell of Origin Determined by Digital Gene Expression in Formalin-Fixed Paraffin-Embedded Tissue Biopsies.

To evaluate the prognostic impact of cell-of-origin (COO) subgroups, assigned using the recently described gene expression-based Lymph2Cx assay in comparison with International Prognostic Index (IPI) score and MYC/BCL2 coexpression status (dual expressers). Reproducibility of COO assignment using the Lymph2Cx assay was tested employing repeated sampling within tumor biopsies and changes in reagent lots. The assay was then applied to pretreatment formalin-fixed paraffin-embedded tissue (FFPET) biopsies from 344 patients with de novo diffuse large B-cell lymphoma (DLBCL) uniformly treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) at the British Columbia Cancer Agency. MYC and BCL2 protein expression was assessed using immunohistochemistry on tissue microarrays. The Lymph2Cx assay provided concordant COO calls in 96% of 49 repeatedly sampled tumor biopsies and in 100% of 83 FFPET biopsies tested across reagent lots. Critically, no frank misclassification (activated B-cell-like DLBCL to germinal center B-cell-like DLBCL or vice versa) was observed. Patients with activated B-cell-like DLBCL had significantly inferior outcomes compared with patients with germinal center B-cell-like DLBCL (log-rank P < .001 for time to progression, progression-free survival, disease-specific survival, and overall survival). In pairwise multivariable analyses, COO was associated with outcomes independent of IPI score and MYC/BCL2 immunohistochemistry. The prognostic significance of COO was particularly evident in patients with intermediate IPI scores and the non-MYC-positive/BCL2-positive subgroup (log-rank P < .001 for time to progression). Assignment of DLBCL COO by the Lymph2Cx assay using FFPET biopsies identifies patient groups with significantly different outcomes after R-CHOP, independent of IPI score and MYC/BCL2 dual expression.

Clinically relevant lot-to-lot reagent difference in a commercial immunoturbidimetric assay for glycated hemoglobin A1c

ObjectivesHemoglobin A1c (HbA1c) is employed for diagnosis and therapy monitoring of diabetes mellitus. The effect of a change of reagent lot on the measured values of a commercial immunoturbidimetric HbA1c assay (A1C3) was investigated. Design and methodsComparison measurements of A1C3 and an automated affinity chromatography method (VIIT) were performed in 15 samples for the initial and in 20 samples for the subsequent A1C3 lot. The results of 27 and 19 measurements of a normal and of 28 and 20 of a pathological control (before and after the switch of the A1C3 reagent lot, respectively) were evaluated. Finally, the results of 6463 patient samples that had been measured with the initial and 434 that had been measured with the subsequent A1C3 lot were investigated. ResultsVIIT yielded significantly higher results than the initial A1C3 lot (bias: 0.41% HbA1c, 4.5mmol/mol) but agreed well with the subsequent lot (bias: −0.01% HbA1c, −0.1mmol/mol). Changing to the subsequent reagent lot resulted in significant increases of the mean of the normal control of 0.316% HbA1c (3.5mmol/mol) and of the pathological control of 0.749% HbA1c (8.2mmol/mol). The median of patient samples measured with the subsequent lot was significantly higher by 0.40% HbA1c (4.4mmol/mol). ConclusionsThe subsequent A1C3 reagent lot yields significantly higher measurement results than the initial by approximately 0.5% HbA1c (5.5mmol/mol). This difference is considered as clinically relevant. A combined effort of manufacturers and notified bodies is necessary to minimize lot-to-lot variation.