Limit Of Blank Research Articles

Limit of Blank (LOB), Limit of Detection (LOD), and Limit of Quantification (LOQ)

Limit of Blank (LOB), Limit of Detection (LOD), and Limit of Quantification (LOQ)

Detection capability of quantitative faecal immunochemical tests for haemoglobin (FIT) and reporting of low faecal haemoglobin concentrations.

Faecal immunochemical tests for haemoglobin (FIT) are widely used in asymptomatic population screening for colorectal (bowel) cancer. FIT are also used to assist with the assessment of patients presenting with lower abdominal symptoms. Quantitative FIT allow the generation of numerical estimates of faecal haemoglobin (f-Hb) concentrations. There is now great interest in "low" f-Hb concentrations in these clinical settings: in consequence, knowledge of the detection capability is very important for f-Hb concentration examinations. There are a number of current problems associated with the reporting of low f-Hb concentrations and wide misunderstanding of the metrological aspects of examinations of f-Hb at low concentrations. These would be solved if the detectability characteristics of f-Hb concentration examinations, namely, the limit of blank (LoB), limit of detection (LoD) and limit of quantitation (LoQ), were generated, validated and used in reporting systems exactly as recommended in the EP17-A2 guideline of the Clinical Laboratory Standards Institute. LoB and LoD are statistical concepts, but the LoQ depends on definition of analytical performance specifications (APS). In this Opinion Paper proposals for interim APS are made, based on the current state of the art achieved with examinations of faecal samples. It is proposed that LoQ is determined at an examination imprecision of CV≤10% using faecal samples naturally positive for Hb rather than faeces spiked with haemolysate. Detailed proposals for reporting f-Hb data at low concentrations are also made.

Nonlinear Regression Improves Accuracy of Characterization of Multiplexed Mass Spectrometric Assays

The need for assay characterization is ubiquitous in quantitative mass spectrometry-based proteomics. Among many assay characteristics, the limit of blank (LOB) and limit of detection (LOD) are two particularly useful figures of merit. LOB and LOD are determined by repeatedly quantifying the observed intensities of peptides in samples with known peptide concentrations and deriving an intensity versus concentration response curve. Most commonly, a weighted linear or logistic curve is fit to the intensity-concentration response, and LOB and LOD are estimated from the fit. Here we argue that these methods inaccurately characterize assays where observed intensities level off at low concentrations, which is a common situation in multiplexed systems. This manuscript illustrates the deficiencies of these methods, and proposes an alternative approach based on nonlinear regression that overcomes these inaccuracies. We evaluated the performance of the proposed method using computer simulations and using eleven experimental data sets acquired in Data-Independent Acquisition (DIA), Parallel Reaction Monitoring (PRM), and Selected Reaction Monitoring (SRM) mode. When the intensity levels off at low concentrations, the nonlinear model changes the estimates of LOB/LOD upwards, in some data sets by 20-40%. In absence of a low concentration intensity leveling off, the estimates of LOB/LOD obtained with nonlinear statistical modeling were identical to those of weighted linear regression. We implemented the nonlinear regression approach in the open-source R-based software MSstats, and advocate its general use for characterization of mass spectrometry-based assays.

Assessing matrix, interferences and comparability between the Abbott Diagnostics and the Beckman Coulter high-sensitivity cardiac troponin I assays.

Analytical evaluation of high-sensitivity cardiac troponin (hs-cTn) assays, with particular attention to imprecision, interferences and matrix effects, at normal cTn concentrations, is of utmost importance as many different clinical algorithms use concentration cutoffs <10 ng/L for decision-making. The objective for the present analytical study was to compare the new Beckman Coulter hs-cTnI assay (Access hsTnI) to Abbott's hs-cTnI assay in different matrices and for different interferences, with a focus on concentrations <10 ng/L. The limit of blank (LoB) and the limit of detection (LoD) were determined in different matrices for the Beckman hs-cTnI assay. Passing-Bablok regression and difference plots were determined for 200 matched lithium heparin and EDTA plasma samples for the Beckman assay and 200 lithium heparin samples for the Abbott assay. Both EDTA and heparin plasma samples were also evaluated for stability under refrigerated conditions, for endogenous alkaline phosphatase interference and for hemolysis and icterus. The Beckman hs-cTnI assay LoB was 0.5 ng/L with the following range of LoDs=0.8-1.2 ng/L, with EDTA plasma yielding lower concentrations as compared to lithium heparin plasma (mean difference=-14.9%; 95% CI=-16.9 to 12.9). Below 10 ng/L, lithium heparin cTnI results from the Beckman assay were on average 1.1 ng/L (95% CI=0.7 to 1.5) higher than the Abbott results, with no difference between the methods when using EDTA plasma (mean difference =-0.1 ng/L; 95% CI=-0.3 to 0.2). Low cTnI concentrations were less effected by interferences in EDTA plasma. The Access hsTnI method can reliably detect normal cTnI concentrations with both lithium heparin and EDTA plasma being suitable matrices.

Performance evaluation of cobas HBV real-time PCR assay on Roche cobas 4800 System in comparison with COBAS AmpliPrep/COBAS TaqMan HBV Test.

Hepatitis B virus (HBV) nucleic acid amplification testing (NAAT) is important for the diagnosis and management of HBV infection. We evaluated the analytical performance of the cobas HBV NAAT (Roche Diagnostics GmbH, Mannheim, Germany) on the cobas 4800 System in comparison with COBAS AmpliPrep/COBAS TaqMan HBV Test (CAP/CTM HBV). Precision was evaluated using three levels of cobas HBV/HCV/HIV-1 Control Kit, and linearity was evaluated across the anticipated measuring range (10.0-1.0×109 IU/mL) at seven levels using clinical samples. Detection capability, including limit of blank (LOB), limit of detection (LOD) and limit of quantitation (LOQ), was verified using the 4th WHO International Standard for HBV DNA for NAT (NIBSC code: 10/266). Correlation between the two systems was compared using 205 clinical samples (102 sera and 103 EDTA plasma). Repeatability and total imprecision (coefficient of variation) ranged from 0.5% to 3.8% and from 0.5% to 3.5%, respectively. Linearity (coefficient of determination, R2) was 0.999. LOB, LOD and LOQ were all acceptable within the observed proportion rate (85%). Correlation was very high between the two systems in both serum and plasma samples (correlation coefficient [r]=0.995). The new cobas HBV real-time PCR assay on the cobas 4800 System showed reliable analytical performances.

Performance evaluation of the chemiluminescence immunoassay for mycoplasma pneumonia antibody detecting

Objective Assessing the detection performance of testing mycoplasma pneumonia(MP)type-specific antibodies by Chemiluminescence immunoassay(CLIA), in order to evaluate the feasibility of screening MP infection by CLIA. Methods Total of 280 cases of respiratory disease patients, 20 examples infected mycoplasma pneumonia and 20 cases health volunteers as the control group were enrolled in this study from August 2016 to October 2017 in the Nanfang Hospital, Southern Medical University, testing MP antibodies by CLIA, Enzyme linked immunosorbent assay(ELISA)and Passive agglutination method(PA) respectively. According to the performance evaluation scheme, we evaluate the performance indexs of detecting MP antibodies by CLIA, including lower limit of detection, intra-batch precision, inter-batch precision, linearity range, clinical coincidence rate and consistency compared with ELISA and PA, and the results were analyzed by EXCEL and SPSS version 22.0. Results MP-IgG CLIA reagent: Limit of blank, Limit of detection and Limit of quantitation were 1.9 AU/ml, 4.5 AU/ml and 5.1 AU/ml respectively; Coefficient Variation (CV) of intra-batch precision in high and low concentration levels were 2.98% and 2.45% respectively; CV of inter-batch precision in high and low concentration levels were 6.44% and 6.83% respectively; both the Linear range and Clinical report range are from 2.0 AU/ml to 253.0 AU/ml; the linear regression equation R2≥0.990 0, 0.85≤b≤1.15. MP-IgM CLIA reagent: CV of intra-batch precision in high and low concentration levels were 2.55% and 2.86% respectively; CV of inter-batch precision in high and low concentration levels were 4.82% and 5.46% respectively. The total clinical coincidence rate of MP-IgG and MP-IgM detected by CLIA were 90.0% and 97.5% respectively. The kappa values of MP-IgG and MP-IgM detected by CLIA and ELISA were 0.763 (P=0.000) and 0.804 (P=0.023) respectively, with Consistent percentage of 88.9% and 91.4% respectively. The kappa value of CLIA and PA was 0.541 (P=0.063) with a consistent percentage of 79.6%. Conclusions The results of study show that detecting MP type-specific antibodies by CLIA meet the prescribed performance indexes. Detecting MP type-specific antibodies by CLIA, which is precise, speedy and automated, could be applied to clinical and replace ELISA and PA, becoming the prior choice in clinical for MP infection screening.(Chin J Lab Med, 2017, 40: 965-969) Key words: Chemiluminescence immunoassay; Mycoplasma Pneumonia; Type-specific antibodies; Performance evaluation; Clinical coincidence rate

1335P - EGFR T790M detection in TKI-naïve NSCLCs carrying sensitive EGFR mutations

Background: To date, the frequency of EGFR T790M in TKI-naïve patients remains unclear, ranging from 2% to 80% depending on the sensitivity and specificity of the methods. In this study we aimed to identify the frequency of EGFR T790M in NSCLCs before TKI treatment, comparing the detection rate of three highly sensitive molecular methods. Methods: Among 1100 NSCLCs (adenocarcinomas at stage IIIB or IV), we identified 130 NSCLCs with EGFR TKI-sensitive mutations, by MALDI-TOF mass spectrometry (MALDI-TOF MS) and Myriapod® Lung Status kit. The diagnostic performance in detecting de-novo EGFR T790M in these 130 tumors was evaluated comparing three methods, namely MALDI-TOF MS, Real Time AS-PCR (Easy®EGFR kit) and ddPCR™ (QX200 Droplet Digital PCR, PrimePCR™ Assays). Sensitivity and specificity of each method were defined by using a DNA reference standard set (Horizon). Limit of blank (LOB) of AS-PCR and ddPCR were determined by measuring replicates of 16 wild-type EGFR DNA samples obtained from peripheral blood lymphocytes and from formalin-fixed paraffin embedded (FFPE) normal lung tissues. Results: Comparison of the three methods was possible for 91 of the 130 NSCLCs. Overall, we identified a total of 16 de-novo T790M in the analyzable tumors (18%). In detail, 4 cases were identified by MALDI-TOF MS and confirmed by AS-PCR and ddPCR. Two T790M-mutated cases were additionally detected by AS-PCR. ddPCR confirmed EGFR T790M in all these tumors and additionally identified 10 mutated cases. Most of mutated cases showed a mutant-allele frequency between 5% and 0.1%. Titration experiments using a DNA reference standard set demonstrated higher sensitivity of ddPCR (0.1%) than AS-PCR (1%) and MALDI-TOF MS (5%). Analysis of wild-type EGFR DNA from FFPE samples was crucial for the determination of LOB of ddPCR in order to maximize sensitivity, avoiding loss of specificity. Conclusions: In this study, 18% of TKI-naïve NSCLCs show EGFR T790M mutation together with an EGFR activating mutation. Most of mutated cases showed a mutant-allele frequency between 5% and 0.1%. ddPCR is a robust method enabling the detection of mutant-allele frequencies as low as 0.1%. However, a careful preliminary evaluation of the specificity of this test is mandatory, especially when FFPE tissues are investigated. Legal entity responsible for the study: Asst-Sette Laghi Ospedale di Circolo Varese Funding: AstraZeneca Disclosure: All authors have declared no conflicts of interest.

Analytical evaluation of the new Beckman Coulter Access high sensitivity cardiac troponin I immunoassay.

This study was aimed to evaluate the analytical performance of the novel chemiluminescent and fully-automated Beckman Coulter Access hsTnI high-sensitivity immunoassay for measurement of cardiac troponin I (cTnI). The study, using lithium heparin samples, included assessment of limit of blank (LOB), limit of detection (LOD), functional sensitivity, linearity, imprecision (within run, between-run and total), calculation of 99th percentile upper reference limit (URL) in 175 healthy blood donors (mean age, 36±12 years; 47% women) and comparison with two other commercial cTnI immunoassays. The LOB, LOD and functional sensitivity of Access hsTnI were 0.14, 0.34 and 1.35 ng/L, respectively. The within-run, between-run and total imprecision was 2.2%-2.9%, 4.6%-5.4%, and 5.4%-6.1%, respectively. The linearity was excellent in the range of cTnI values between 0.95 and 4195 ng/L (r=1.00). The 99th percentile URL was 15.8 ng/L. Measurable cTnI values were found in 173/175 healthy subjects (98.9%). Good agreement of cTnI values was found with AccuTnI+3 (r=0.97; mean bias, -9.3%), whereas less satisfactory agreement was found with Siemens Dimension Vista cTnI (r=0.95; mean bias, -55%). The results of our evaluation of the Beckman Coulter Access hsTnI indicate that the analytical performance of this fully-automated immunoassay is excellent.

Abstract 565: Analytical performance of TruSight® Tumor 170 in the detection of gene fusions and splice variants using RNA from formalin-fixed, paraffin-embedded (FFPE) solid tumor samples

Abstract Recent studies have highlighted the importance of gene fusions and splice variants in solid tumor profiling1. Next-generation sequencing can be an effective means of detecting these alterations in FFPE samples using RNA rather than DNA, as a single chimeric RNA transcript could result from numerous alterations in DNA2. To that end, Illumina developed TruSight® Tumor 1703, a comprehensive, hybrid capture-based NGS assay targeting 170 key cancer genes. Along with a DNA workflow, the assay includes a RNA workflow for the identification of splice variants and gene fusions. Following sequencing on the NextSeq® or HiSeq® instruments, TruSight® Tumor 170 offers an analytical pipeline which initiates variant calling. These algorithms were first optimized against the simulated read data from &amp;gt;350 fusions and splice variants reported in the RNA content of the gene panel. A hybrid approach of read alignment and assembly was used to enhance the fusion calling sensitivity. Deliberate filters were designed to reduce false positive calling from sequence homologs, polymerase read-through, or FFPE artifacts. For splice variant calling, a panel of FFPE non-cancerous samples were used to capture false positive mutation calls. With endogenous RNA splicing in cellular physiology, exon-boundary probes were added in the hybrid capture to enhance enrichment efficiency. To the best of our knowledge, there is not yet a standard definition for the limit of detection (LoD) in detecting gene fusions and splice variants from NGS data. We propose to define the LoD of a fusion calling and splice variant NGS panel as the lowest molecule count of a chimeric transcript that could be reliably detected with a sufficient number of supporting sequencing reads. To determine the LoD of TruSight® Tumor 170 using this definition, we mixed cell lines expressing a panel of known fusions and splice variants to measure the copy number of each chimeric transcript. Using these samples we examined the ability of the assay to confidently detect the alterations using 40 ng of RNA input. To demonstrate the analytical sensitivity and specificity of this NGS based assay, we compiled a panel of 49 mixed samples and validated the molecule count to be near the LoD of 5 copies per ng RNA input by PCR. The sensitivity was &amp;gt;98% for fusions and 100% for splice variants. For understanding the limit of blank (LoB) of the assay, another panel of 40 samples not harboring fusions and splice variants was also assessed by TruSight® Tumor 170. These samples demonstrated a ~97% specificity for fusion calling and &amp;gt;95% specificity for splice variant calling. These results indicate that the TruSight® Tumor 170 panel analysis can identify lowly expressed fusions and splice variants from a small amount of compromised RNA from solid tumor samples at high analytical sensitivity and specificity. 1 Klijn et al. (2015) 2 Maher et al. (2009) 3 For Research Use Only. Citation Format: Tingting Du, June Snedecor, Jennifer S. LoCoco, Xiao Chen, Laurel Ball, Allan Castaneda, Danny Chou, Katie Clark, Brian Crain, Anthony Daulo, Manh Do, Sarah Dumm, Yonmee Han, Mike Havern, Chia-Ling Hsieh, Tingting Jiang, Suzanne Johansen, Scott Lang, Rachel Liang, Jaime McLean, Yousef Nassiri, Austin Purdy, Jason Rostron, Jennifer Silhavy, Natasha Talago, Li Teng, Kevin Wu, Clare Zlatkov, Chen Zhao, Ali Kuraishy, Karen Gutekunst, Sohela De Rozieres, Matthew Friedenberg, Anne C. Jager, Han-Yu Chuang. Analytical performance of TruSight® Tumor 170 in the detection of gene fusions and splice variants using RNA from formalin-fixed, paraffin-embedded (FFPE) solid tumor samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 565. doi:10.1158/1538-7445.AM2017-565

Heart Fatty Acid Binding Protein for the Diagnosis of Myocardial Ischemia and Infarction

Abstract Objective To establish the analytical performance of a heart fatty acid binding protein (HFABP) method suitable for routine clinical use and examine its role for the diagnosis of myocardial ischemia and myocardial infarction. Methods Analyses of HFABP were performed on an Advia 2400 (Siemens Healthcare Diagnostics). Imprecision, limit of detection (LOD), limit of blank (LOB), and linearity were assessed using standard methods. Stability was assessed at 4 °C, −20 °C, and with 3 repeated freeze-thaw cycles. Clinical diagnostic performance was assessed using chest pain in patients, with a final diagnosis according to the universal definition of myocardial infarction with cardiac troponin I (cTnI) measured on the Siemens Advia Centaur (cTnI Ultra method, 99th percentile 50 ng/L, 10% CV 30 ng/L). Ischemia was detected using sampling pre- and postangioplasty. Results LOD and analytical imprecision exceeded the manufacturer's specification (LOD 1.128 μg/L, 20% CV 1.3 μg/L, 10% CV 2.75 μg/L). Clinical diagnostic efficiency was less than cTnI. Addition of HFABP to cTnI produced a modest increase in diagnostic sensitivity at a cost of significant loss of specificity. Conclusions Although the test had excellent analytical performance, it did not contribute to the clinical diagnosis of patients with chest pain. HFABP appears to be a marker of myocardial infarction not myocardial ischemia.

Analytical validation of Gentian NGAL particle-enhanced enhanced turbidimetric immunoassay (PETIA)

ObjectivesThis study was designed to validate the analytical performance of the new Gentian particle-enhanced enhanced turbidimetric immunoassay (PETIA) for measuring neutrophil gelatinase-associated lipocalin (NGAL) in serum samples. Design and methodsAnalytical validation of the Gentian NGAL assay was carried out on a Roche Cobas c501 and was based on assessment of limit of blank (LOB), limit of detection (LOD), functional sensitivity, imprecision, linearity and concordance with the BioPorto NGAL test. ResultsThe LOB and LOD of Gentian NGAL were found to be 3.8ng/mL and 6.3ng/mL, respectively. An analytical coefficient of variation (CV) of 20% corresponded to a NGAL value of 10ng/mL. The intra-assay and inter-assay imprecision (CV) was between 0.4 and 5.2% and 0.6 and 7.1% and the total imprecision (CV) was 3.7%. The linearity was optimal at NGAL concentrations between 37 and 1420ng/mL (r=1.00; p<0.001). An excellent correlation was observed between values measured with Gentian NGAL and BioPorto NGAL in 74 routine serum samples (r=0.993). The mean percentage bias of the Gentian assay versus the Bioporto assay was +3.1% (95% CI, +1.6% to +4.5%). ConclusionsThese results show that Gentian NGAL may be a viable option to other commercial immunoassays for both routine and urgent assessment of serum NGAL.

Continuous ambulatory peritoneal dialysis, ascitic and pleural body fluids evaluation with the Mindray BC-6800 hematology analyzer.

Accurate evaluation of hematology analyzers is recommended before these devices can be broadly introduced for the routine testing of continuous ambulatory peritoneal dialysis (CAPD), ascitic, and pleural fluids. We evaluated the performance of Mindray BC-6800 for white blood cell (WBC) and differential cell count in 50 CAPD, 60 ascitic and 40 pleural compared with manual microscopy. Within-run precision, limit of blank (LoB), limit of detection (LoD), limit of quantitation (LoQ), and carryover were assessed. The Passing-Bablok regression in all fluids showed the following equations: yWBC =1.05x+3.31 (95%CI slope 0.95 to 1.12; intercept -0.25 to 5.52); yMN =0.85x+15.63 (95%CI slope 0.72 to 1.05; intercept -24.18 to 84.47); and yPMN =1.21x+13.37 (95%CI slope 1.03 to 1.35; intercept 4.00 to 32.47) with bias 78 cells/μL. The AUC for clinical PMN cut-off was 0.88 (95%CI: 0.77 to 0.98). In ascitic, pleural, and CAPD fluids the AUC for clinical PMN cut-off were 0.88 (95%CI: 0.63 to 1.00), 0.83 (95%CI: 0.68 to 0.99), and 1.00 (95%CI: 1.00 to 1.00) respectively. CV ranged from 3%-34%. LoB of 3cell/μL was verified. LoD and LoQ reported the same result (8cells/μL). Carry over never exceeded 0.05%. The effectiveness of BC-6800 to categorize cells from different body fluids was not compromised by the slight positive bias observed. This conclusion is supported by the high AUC and agreement between the automated method and the reference method. The results show that BC-6800 offers rapid, accurate, and reproducible results for clinical management of CAPD, ascitic, and pleural fluids.

Characterization of a Blood Spot Creatine Kinase Skeletal Muscle Isoform Immunoassay for High-Throughput Newborn Screening of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive, lethal X-linked neuromuscular disorder with an average worldwide incidence of 1:5000. Blood spot creatine kinase (CK) enzyme assays previously used in newborn screening programs for DMD are nonspecific because measured CK enzyme activity is attributable to 3 isoenzyme forms of CK (CK-MM, CK-MB, and CK-BB) and it is the CK-MM isoform that is found predominantly in skeletal muscle. CK-MM is increased in boys with DMD owing to muscle damage. We describe a sensitive and specific automated immunoassay for CK-MM to screen for DMD in blood spots. The prototype assay was developed on the PerkinElmer GSP® analyzer to enable high-throughput screening. CK-MM was assayed using a solid phase, 2-site immunofluorometric system. Purified human CK-MM was used to create calibrators and controls. The limit of blank (LOB), detection (LOD), and quantification (LOQ) values were <1, 3, and 8 ng/mL, respectively. The analytical measurement range was 4-8840 ng/mL. Interassay (n = 40) imprecision was <7% across the analytical range. Cross-reactivity was <5% for CK-MB and 0% for CK-BB. The mean recovery of CK-MM was 101% (range 87%-111%). Blood spots from newborn infants (n = 277) had a mean CK-MM concentration of 155 ng/mL and a 99th centile of 563 ng/mL. The mean blood spot CK-MM concentration from 10 cases of DMD was 5458 ng/mL (range 1217-9917 ng/mL). CK-MM can be reliably quantified in blood spots. The development of this CK-MM assay on a commercial immunoassay analyzer would enable standardized and high-throughput newborn blood spot screening of DMD.

Measuring zinc on the Roche cobas c502 analyzer-Validation, comparison, and pre-analytic aspects.

Characterization of an in vitro diagnostic zinc assay (LT-SYS) on a Roche cobas c502 analyzer and evaluation of the influence of pre-analytic factors on zinc concentration measurements. Imprecision, bias, linearity, limit of blank (LoB), and limit of detection (LoD) were established and method comparisons were performed based on the respective CLSI guidelines. The influence of time elapsed until analysis, the usage of a pneumatic tube delivery system (PTDS) and of special trace element sample tubes was evaluated as well. Estimates of imprecision ranged from 0.9% to 5.0% and bias was low with 1.3% and 1.5% deviation from target value. Linearity was met for the measuring range of 1.15-34.7μmol/L (7.51-226.9μg/dL), LoB and LoD were 0.17μmol/L (1.11μg/dL) and 0.73μmol/L (4.77μg/dL) respectively. The method comparison revealed an average deviation of 8.44% (y=0.542+1.036x). Plasma samples had 7.3% higher zinc values than serum samples on the average. Zinc values of uncentrifuged serum and plasma samples increased 20% in 8hours, while after centrifugation no significant increase could be detected. Usage of PTDS increased zinc values by 17% and usage of trace element sample tubes showed no advantage over normal ones. The LT-SYS zinc assay showed a fully acceptable performance with good degrees of imprecision and bias, no deviation from linearity and both a very low LoB and LoD. Samples for zinc analysis should be centrifuged timely and transport over PTDS should be avoided.

Quantification of human diamine oxidase

ObjectivesDiamine oxidase (DAO) is essential for extracellular degradation of histamine. For decades activity assays with inherent limitations were used to quantify the relative amounts of DAO. No reference DAO standard is available. Absolute DAO amounts cannot be determined. Controversy exists, whether DAO is circulating or not in non-pregnant individuals. The role of DAO as biomarker in various diseases is ambiguous. It is not clear, whether precise quantification of human DAO antigen using commercially available enzyme-linked immunosorbent assays (ELISAs) is possible. The objective was to develop a precise and robust ELISA to quantify DAO in various biological fluids. Design and methodsA research prototype ELISA was established using a mouse monoclonal antibody for capturing and a polyclonal rabbit serum IgG fraction for the detection of human DAO. The limit of blank (LoB), limit of detection (LoD) and estimated limit of quantification (eLoQ) and normal DAO concentrations in serum and plasma were determined. ResultsThe LoB, LoD and eLoQ derived from 42 standard curves are 0.27, 0.48 and 0.7ng/mL respectively. The detection range using the LoD as the lower and the highest DAO standard as the upper boundary is 0.5 to 450ng/mL. Serum and plasma mean/median concentrations are between 0.5 and 1.5ng/mL in healthy volunteers (n=58) and mastocytosis patients (n=19) and plateau at approximately 145ng/mL (n=16) during pregnancy. Accurate quantification was not influenced by heparin (DAO is a heparin-binding protein), lipaemic or hemolytic serum. The measured DAO antigen concentrations are in close agreement with published enzymatic activity data using radioactive putrescine as substrate. ConclusionsThis research prototype ELISA is able to reliably and accurately quantify human DAO in different biological fluids. The potential of DAO as biomarker in various diseases can be evaluated.

1162P - Analytical performance of a new liquid biopsy mutation panel for detection of clinically actionable variants

Assessing genomic alterations in circulating tumor DNA (ctDNA) from liquid biopsies may better reflect tumor heterogeneity, facilitate monitoring of tumor evolution and overcome the challenges of obtaining tissue biopsies. Analytic performance of such assays should be established on a per sample basis, using clinically relevant variants at levels representative of ctDNA. Here we report the analytic performance of a 17-gene panel (Oncotype SEQ™, Liquid Select) and illustrate its ability to detect ctDNA in cancer patients. Analytical specificity and sensitivity were characterized through determination of Limit of Blank (LoB) and Limit of Detection (LoD) respectively. 73 cell-free DNA (cfDNA) samples from 60 healthy donors were used to determine the LoB and set detection thresholds. A model system using cell line DNA harboring clinically actionable variants was then used to determine the allele fraction (AF)/copy number (CN) required for a 95% rate of detection (LoD), using 30-50ng DNA input. Repeatability and reproducibility was assessed using pools of cfDNA from cancer patients. Finally, liquid biopsies from 15 stage II-IV cancer patients (on or after therapy) were assayed for genomic alterations. Detection thresholds were set above the LoB corresponding to >99% per sample specificity. LoD was calculated using 105 samples for each variant tested. Mean LoDs were as follows; single nucleotide variants (SNVs), 0.56% AF; insertions/deletions (indels), 0.19% AF; fusions, 0.37% AF, and CN gain, 2.7 copies. Accuracy was verified using additional variant positive and variant negative standards. In the repeatability and reproducibility study using cfDNA pools, on average >95% of expected variants were detected in each run. 10 SNVs and 2 indels were found in the 15 patient plasma samples, ranging from <0.1-32% AF. The 17-gene panel (Oncotype SEQ™, Liquid Select) provides high sensitivity, detecting ctDNA at <0.1% in stage III or later disease. In addition, its high specificity and reproducibility ensures reliable patient reporting.

Characterization and Validation of the LT-SYS Copper Assay on a Roche Cobas 8000 c502 Analyzer.

Validation of the LT-SYS quantitative in vitro copper assay on a Roche Cobas 8000 c502 analyzer and comparison with a BIOMED assay on a Roche Cobas Mira analyzer. Imprecision and bias were quantified at different concentration levels (serum and plasma) over a 20-day period. Linearity was assessed covering a range from 4.08 µmol/L to 33.8 µmol/L. Limit of blank (LoB) and limit of detection (LoD) were established based on a total of 120 blank and low-level samples. The method comparison was based on 58 plasma samples. Within-run imprecision ranged from 0.7% to 1.2% and within-laboratory imprecision from 1.4% to 3.3%. Relative bias for the 2 serum pools with known target values was less than 2.5%. The assay did not deviate from linearity over the tested measuring range. LoB and LoD were 0.12 µmol/L and 0.23 µmol/L, respectively. The method comparison revealed an average deviation of 11.5% (2.016 µmol/L), and the linear regression fit was y = 1.464 + 0.795x. The LT-SYS copper assay characterized in this study showed a fully acceptable performance with good degrees of imprecision and bias, no deviation from linearity in the relevant measuring rangem, and very low LoB and LoD.

Optimization of Cellular analysis of Synovial Fluids by optical microscopy and automated count using the Sysmex XN Body Fluid Mode

BackgroundThis study was planned to assess the impact of pre-treating synovial fluid (SF) samples with hyaluronidase (HY), defining the best procedure for optical microscopy (OM) analysis and evaluating the performance of Sysmex XN-9000 Body Fluid module (XN-BF). MethodsThe cell count by OM was carried out both with and without HY pre-treatment, and using 3 different types of staining reagents. The evaluation of XN-BF included data comparison with OM (100 SFs), carryover, Limit of Blank (LoB), Limit of Detection (LoD), Limit of Quantitation (LoQ) and linearity. ResultsUnlike cell count in Burker's chamber and staining with Stromatol, pre-treatment with HY and staining with Methylene Blue and Turk's promoted cell clustering. The SF samples pre-treated with HY displayed excellent morphological quality, contrary to samples without HY pre-treatment. Excellent correlation was found between total cells counting with both OM and XN-BF. Satisfactory agreement was also observed between polymorphonuclear neutrophils compared to XN-BF parameter, whereas mononuclear cell count on XN-BF had suboptimal agreement with OM. The carryover was negligible. The LoB, LoD, LoQ and linearity were excellent. ConclusionXN-BF displays excellent performance, which makes it a reliable and practical alternative to OM for SF samples analysis in clinical laboratories.

Analytical evaluation of the novel Lumipulse G BRAHMS procalcitonin immunoassay

ObjectivesThis study was designed to evaluate the analytical performance of the novel Lumipulse G1200 BRAHMS procalcitonin (PCT) immunoassay. Design and methodsThis analytical evaluation encompassed the calculation of the limit of blank (LOB), limit of detection (LOD), functional sensitivity, intra- and inter-assay imprecision, confirmation of linearity and a comparison with the Vidas BRAHMS PCT assay. ResultsThe LOB, LOD and functional sensitivity were 0.0010ng/mL, 0.0016ng/mL and 0.008ng/mL, respectively. The total analytical imprecision was found to be 2.1% and the linearity was excellent (r=1.00) in the range of concentrations between 0.006–75.5ng/mL. The correlation coefficient with Vidas BRAHMS PCT was 0.995 and the equation of the Passing and Bablok regression analysis was [Lumipulse G BRAHMS PCT]=0.76×[Vidas BRAHMS PCT]+0.04. The mean overall bias of Lumipulse G BRAHMS PCT versus Vidas BRAHMS PCT was −3.03ng/mL (95% confidence interval [CI]: −4.32 to −1.74ng/mL), whereas the mean bias in samples with PCT concentration between 0–10ng/mL was −0.49ng/mL (95% CI: −0.77 to −0.24ng/mL). The diagnostic agreement was 100% at 0.5ng/mL, 97% at 2.0ng/mL and 95% at 10ng/mL, respectively. ConclusionsThese results attest that Lumipulse G BRAHMS PCT exhibits excellent analytical performance, among the best of the methods currently available on the diagnostic market. However, the significant bias compared to the Vidas BRAHMS PCT suggests that the methods cannot be used interchangeably.

Abstract 505: Optimized 7-Plex cell-free DNA assay for clinically relevant KRAS mutations in colorectal cancer

Abstract Background: KRAS gene mutations are present in approximately 40% of colorectal adenocarcinomas and predict for nonresponse to the anti-epidermal growth factor receptor antibodies used in routine clinical practice. The most common activating mutations in this malignancy occur in codons 12 and 13 of exon 2. Determination of KRAS mutational status from tumor samples is an essential tool for managing patients with colorectal cancer (CRC) but requires tissue obtained by invasive percutaneous or surgical biopsies. Recent advances allow for the isolation and analysis of cell-free DNA (cfDNA) from the peripheral blood. A highly sensitive multiplex technology that uses low amounts of input DNA is needed to establish cfDNA as a liquid biopsy to personalize care. Methods: Digital droplet PCR (ddPCR) is a particularly sensitive method for detecting rare and low copy targets. Related work led to 7 individual assays, and to companion 4-plex and 3-plex assays, to accommodate the 7 common KRAS mutations and internal controls. A single 7-plex assay is favored to maximize efficiency and minimize cost. We optimized the RainDrop Digital PCR System to collectively detect the G12/G13 KRAS mutations, given that documentation of a mutation (not the specific mutation) is the factor of clinical relevance. Commercially available cell lines and genomic DNA aliquots were used as reference standards and for determinations of sensitivity and limit of blank (LOB). Initial clinical validation samples included tumor DNA derived from FFPE tissue (n = 10), cfDNA from healthy donors (n = 10), and cfDNA from metastatic CRC patients with tumors of known KRAS mutation status (n = 4). All assays were run in triplicate to confirm reproducibility. Results: Custom Taqman MGB probes were selected for KRAS wild type (WT), G12A, G12C, G12D, G12R, G12S, G12V, and G13D to allow for testing in a single reaction. LOB was 9 per 1×10(6) droplets on the basis of WT controls, including cfDNA samples from healthy donors. Evaluation of cell line DNA, genomic DNA aliquots, and FFPE samples demonstrates that the assay detects mutant KRAS with 100% accuracy and 0.1% mutant DNA in a total input of 10 ng WT/mutant DNA. With the clinical samples, concordant KRAS findings between cfDNA and tissue were obtained for two patients with WT tumors and one patient with a KRAS G12S mutant tumor. cfDNA analysis did not detect the G12V mutation identified in the colon primary of the other patient. Evaluation of additional clinical samples is in progress. Conclusions: Our 7-plex G12/G13 KRAS assay has excellent accuracy and is able to detect the KRAS mutations of interest at low (0.1%) mutation frequencies. Additional studies are being performed on prospectively collected plasma samples from patients with metastatic CRC of known KRAS mutation status. Large scale correlation between tumor mutation status and cfDNA findings will be performed to validate our assay for clinical implementation. Citation Format: Rajeswari Avula, Benjamin R. Kipp, Jesse S. Voss, Keegan E. Haselkorn, W. Edward Highsmith, Kevin C. Halling, Jeremy C. Jones, Steven R. Alberts, Michael B. Campion, Cassandra J. Nelson, Jin Jen, Eric D. Wieben, Julie M. Cunningham, Minetta C. Liu. Optimized 7-Plex cell-free DNA assay for clinically relevant KRAS mutations in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 505.