Contrast thresholds for detection of various iodine concentrations in subtraction CT and dual-energy CT systems.

OBJECTIVE: To estimate the minimum iodine concentrations detectable in simulated vessels of various diameters for both subtraction computed tomography (CT) and dual-energy CT systems. METHODS: Fillable tubes (diameters: 1, 3, and 5 mm) were filled with a variety of iodine concentrations (range: 0-20 mg/ml), placed in the center of 28-mm cylindrical rods and surrounded with water. Rods with and without fillable tubes were placed in a 20-cm cylindrical solid-water phantom to simulate administration of iodine in blood vessels. The phantom was scanned with clinical subtraction CT (SCT) and dual-energy CT (DECT) head protocols to assess the detection of minimum iodine concentrations in both systems. The SCT and DECT images were evaluated quantitatively with a MATLAB script to extract regions of interest (ROIs) of each simulated vessel. ROI measurements were used to calculate the limit of detectability (LOD) and signal-to-noise ratio of Rose criteria for the assessment of the contrast thresholds. RESULTS: Both SNRRose and LOD methods agreed and determined the minimum detectable iodine concentration to be 0.4 mg/ml in the 5-mm diameter vessel for SCT. However, the minimum detectable concentration in the 5-mm vessel with DECT was 1 mg/ml. The 3-mm vessel had a minimum detectable concentration of 0.8 mg/ml for SCT and 2 mg/ml for DECT. Lastly, the minimum detectable iodine concentration for the 1-mm vessel was 10 mg/ml for SCT and 10 mg/ml for DECT. CONCLUSION: In this phantom study, SCT showed the capability to detect lower iodine concentrations compared to DECT. Contrast thresholds varied for vessels of different diameters and the smaller vessels required a higher iodine concentration for detection. Based on this knowledge, radiologists can modify their protocols to increase contrast enhancement.

Quantitative differential analysis of norovirus outbreak samples using RT-ddPCR.

Noroviruses cause acute gastroenteritis with symptoms of diarrhoea and vomiting, and their high infectivity allows outbreaks to readily occur. Quickly identifying and isolating potential contaminants is an effective method to prevent the spread of outbreaks. A total of 376 samples collected from nine outbreaks were categorized as either patient, asymptomatic individual, cook or environmental samples, according to the source of contamination. Using real-time PCR and sequencing analysis, norovirus GII genotypes were detected in 34·9% of samples from patients, 19·2% from asymptomatic individuals, 2·4% from the environment and 1·4% from cooks. Our findings showed contrasting results in samples categories quantified based on the limit of blank and detection limit by reverse transcription droplet digital PCR, which is a more sensitive testing method than real-time-PCR.

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.

Calculating detection limits and uncertainty of reference-based deconvolution of whole-blood DNA methylation data.

DNA methylation (DNAm)-based cell mixture deconvolution (CMD) has become a quintessential part of epigenome-wide association studies where DNAm is profiled in heterogeneous tissue types. Despite being introduced over a decade ago, detection limits, which represent the smallest fraction of a cell type in a mixed biospecimen that can be reliably detected, have yet to be determined in the context of DNAm-based CMD. Moreover, there has been little attention given to approaches for quantifying the uncertainty associated with DNAm-based CMD. Here, analytical frameworks for determining both cell-specific limits of detection and quantification of uncertainty associated with DNAm-based CMD are described. This work may contribute to improved rigor, reproducibility and replicability of epigenome-wide association studies involving CMD.

How to Design and Validate a Clinical Flow Cytometry Assay.

Multiparametric flow cytometry assays are long recognized as an essential diagnostic test for leukemias and lymphomas. Lacking Food and Drug Administration-approved standardized tests, these assays remain laboratory developed tests. The recently published guidelines, CLSI H62, are the most detailed and up-to-date instructions for designing and validating clinical flow cytometry assays. This review provides a historical background for the current situation, summarizes key points from the CLSI guidelines, and lists practical points for assay development gained from personal experience.

Development of an automated chemiluminescent immunoassay for cancer antigen 72-4 and the evaluation of its analytical performance.

Objectives: Cancer antigen (CA) 72-4 assay is widely used for monitoring gastric and ovarian cancers. The antigen is a mucin-like, tumor-associated glycoprotein known as TAG-72. It has been identified and characterized using two different monoclonal antibodies, CC49 and B72.3, which recognize its glycochain epitopes, Galß(1-3) sialyl-Tn and sialyl-Tn antigens, respectively. This study describes the quantitative analytical performance of a newly developed CA 72-4 assay, ARCHITECT CA 72-4. Design: and Methods: The ARCHITECT CA 72-4 assay was developed using the ARCHITECT i2000SRs and three ARCHITECT i1000SRs. The assay performance was evaluated based on guidance from CLSI (Clinical and Laboratory Standards Institute) and correlation against Elecsys CA 72-4. Results: In the total precision study, the minimum coefficient of variation (CV) for Control/Panel samples over 4 U/mL was 1.1%. The measuring interval was from 0.95 to 200 U/mL with good linearity; and limits of blank (LoB), detection (LoD), and quantitation (LoQ) were 0.09, 0.18, and 0.95 U/mL, respectively. High dose hook effect; differences among specimen tube types; and interference of common drugs, potential cross-reactants, and endogenous substances were not observed. Significantly, this assay has high biotin tolerance at 4875 mg/mL and correlates well with the Elecys CA 72-4 assay (correlation coefficient: 0.95). Conclusions: ARCHITECT CA 72-4 is a highly sensitive and precise assay for CA 72-4 measurement in human sera and plasma.

Comparison of ADVIA Centaur ultra-sensitive and high-sensitive assays for troponin I in serum.

Cardiac troponin I (cTnI) is a standard biomarker for the diagnosis of acute myocardial infarction (AMI). While older, ultra-sensitive cTnI (us-cTnI) assays use the 99th percentile as the reference threshold, newer high-sensitive cTnI (hs-cTnI) assays use the limit of detection or functional sensitivity instead. However, little has been done to systematically compare these two methods. The present study also served as a validation of hs-cTnI in our laboratory. Here, we compared the results obtained from the blood serum obtained from 8810 patients using the us-cTnI and the hs-cTnI assays run in tandem on the ADVIA Centaur XP analyser. We found that in 2279 samples the concentration of cTnI measured with the ultra-sensitive method was below the detection limit, while with the high-sensitive method, only 540 were below the detection limit. We also compared results from these assays with the ultimate diagnosis of a subset of individuals. The analysis of the results below cut-off with the ultra-sensitive method showed that this method would not detect 96 cases related to heart disorder. Overall, the main finding of our research is that hs-cTnI is the preferable option and is able to be deployed effectively in the laboratory setting.

High-throughput nuclear magnetic resonance measurement of citrate in serum and plasma in the clinical laboratory.

OBJECTIVES: Despite reports highlighting citrate association with different diseases, serum citrate is scarcely used for diagnosis. Existing methods to quantify citrate are limited by their complexity and practicality of implementation. A simple and rapid NMR-based method to measure circulating citrate is described here, and its analytical performance evaluated. DESIGN: and Methods: Citrate was quantified from NMR spectra using a non-negative linear least squares deconvolution algorithm. The analytical characteristics of the assay were evaluated using CLSI guidelines. To determine if the assay has adequate sensitivity to measure clinically relevant concentrations of citrate, the assay was used to quantify citrate in apparently healthy adults (n â€‹= â€‹553), and in the general population (n â€‹= â€‹133,576). RESULTS: The LOQ for the assay was determined to be 1.48 â€‹mg/dL. Linearity was demonstrated over a wide range of concentrations (1.40-4.46 â€‹mg/dL). Coefficients of variation (%CV) for intra- and inter-assay precision ranged from 5.8-9.3 and 5.2-9.6%, respectively. Substances tested did not elicit interference with assay results. Specimen type comparison revealed <1% bias between serum and plasma samples, except for heparin plasma (3% bias). Stability was demonstrated up to 8 days at room temperature and longer at lower temperatures. In a cohort of apparently healthy adults, the reference interval was <1.48-2.97 â€‹mg/dL. Slightly higher values were observed in the general population. CONCLUSIONS: The newly developed NMR-based assay exhibits analytical characteristics that allow the accurate quantification of clinically relevant citrate concentrations. The assay provides a simple and fast means to analyze samples for research and clinical studies.

Digital PCR can provide improved BCR-ABL1 detection in chronic myeloid leukemia patients in deep molecular response and sensitivity of standard quantitative methods using EAC assays.

BCR-ABL1 molecular detection using quantitative PCR (qPCR) methods is the golden standard of chronic myeloid leukemia (CML) monitoring. However, due to variable sensitivity of qPCR assays across laboratories, alternative methods are tested. Digital PCR (dPCR) has been suggested as a robust and reproducible option. Here we present a comparison of droplet dPCR with routinely used reverse-transcription qPCR (RT-qPCR) and automated GeneXpert systems. Detection limit of dPCR was above 3 BCR-ABL1 copies, although due to background amplification the resulting sensitivity was 0.01% BCR-ABL1 (MR4.0). Nevertheless, in comparison with GeneXpert, dPCR categorized more than 50% of the patients into different MR groups, showing a potential for improved BCR-ABL1 detection.

Assessment of Access hsTnI 99th percentiles upper reference limits following IFCC recommendations.

BACKGROUND: The detection of an increase and/or decrease of cardiac troponin (cTnI) values, with at least one value above the 99th percentile of the upper reference limit (URL) have a central role in acute myocardial infarction (AMI) diagnosis. The employment of sex specific 99th percentile URLs and High-sensitivity (Hs) assays are recommended. We assessed sex specific 99th percentile URL for Access Hs-cTnI and AccuTnI3+ (Beckman Coulter) using European donor reference population following recent International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) recommendations. METHODS: 300 males and 300 females plasma samples were collected. Both chemiluminescent immunoenzymatic assays were performed on UniCel DxI 800 platform (Beckman Coulter). RESULTS: For Access hsTnI, the observed sex-specific 99th percentile URLs were 5.5 (90% CI: 4.4-7.6) for females and 13.9 ng/L (90% CI: 7.4-17.4) for males. For AccuTnI+3 we could not establish them because the assay couldn't report detectable values of troponin for most of the analyzed samples. CONCLUSION: The sex-specific 99th percentile URLs established for Access hsTnI assay were significantly lower than those declared by the manufacturer caused by the different choice of population selection, age groups and sample types: for those reasons, we maintain the 99th URLs provided by manufacturer.

A high-throughput test for diabetes care: An evaluation of the next generation Roche Cobas c 513 hemoglobin A1C assay.

OBJECTIVES: The level of glycated hemoglobin A (HbA1C) in blood is the preferred marker for diabetes monitoring and treatment. Here, we evaluate the analytical performance of the Roche Diagnostics Cobas c 513, a stand-alone HbA1C immunoassay analyzer. DESIGN AND METHODS: Performance was assessed with regards to imprecision, accuracy, linearity, method comparison against the Roche Cobas Integra 800 CTS, specimen stability, interference from common hemoglobin variants and hemoglobin F, and throughput. RESULTS: Within-run and between-run precisions were 0.5-0.7 and 0.8-1.3%CV, respectively. An average bias of -1.6% to proficiency survey samples was observed. The c 513 correlated well with the Integra (slope = 0.94, y-intercept = 0.50, and correlation coefficient = 0.998). The effect of hemoglobin variants on this assay was negligible while specimens containing ≥10% HbF demonstrated a negative bias. The c 513 instrument can process up to 340 samples per hour. CONCLUSIONS: The c 513 is a precise, accurate, automated high throughput analyzer for measuring HbA1C in large laboratories.

Trial design for assessing analytical and clinical performance of high-sensitivity cardiac troponin I assays in the United States: The HIGH-US study.

BACKGROUND: High-sensitivity cardiac troponin I (hs-cTnI) assays have been developed that quantify lower cTnI concentrations with better precision versus earlier generation assays. hs-cTnI assays allow improved clinical utility for diagnosis and risk stratification in patients presenting to the emergency department with suspected acute myocardial infarction. We describe the High-Sensitivity Cardiac Troponin I Assays in the United States (HIGH-US) study design used to conduct studies for characterizing the analytical and clinical performance of hs-cTnI assays, as required by the US Food and Drug Administration for a 510(k) clearance application. This study was non-interventional and therefore it was not registered at clinicaltrials.gov. METHODS: We conducted analytic studies utilizing Clinical and Laboratory Standards Institute guidance that included limit of blank, limit of detection, limit of quantitation, linearity, within-run and between run imprecision and reproducibility as well as potential interferences and high dose hook effect. A sample set collected from healthy females and males was used to determine the overall and sex-specific cTnI 99th percentile upper reference limits (URL). The total coefficient of variation at the female 99th percentile URL and a universally available American Association for Clinical Chemistry sample set (AACC Universal Sample Bank) from healthy females and males was used to examine high-sensitivity (hs) performance of the cTnI assays. Clinical diagnosis of enrolled subjects was adjudicated by expert cardiologists and emergency medicine physicians. Assessment of temporal diagnostic accuracy including sensitivity, specificity, positive predictive value, and negative predictive value were determined at presentation and collection times thereafter. The prognostic performance at one-year after presentation to the emergency department was also performed. This design is appropriate to describe analytical characterization and clinical performance, and allows for acute myocardial infarction diagnosis and risk assessment.

Analytical performance of a single epitope B-type natriuretic peptide sandwich immunoassay on the Minicare platform for point-of-care diagnostics.

Point-of-care B-type natriuretic peptide (BNP) testing with adequate analytical performance has the potential to improve patient flow and provide primary care givers with easy-to-use advanced diagnostic tools in the management of heart failure. We present the analytical evaluation of the Minicare BNP immunoassay under development on the Minicare I-20 platform for point-of-care testing. Analytical performance was evaluated using EDTA venous whole blood, EDTA plasma and capillary whole blood. Method comparison with a lab-testing system was performed using samples from 187 patients. Normal values were determined based on 160 healthy adults, aging from 19 to 70 years. Limit of blank (LoB), limit of detection (LoD) were determined to be 3.3 ng/L, 5.8 ng/L. Limit of quantitation (LoQ) in whole blood at 20% and 10% coefficient of variation (CV) was found < 9 ng/L and <30 ng/L respectively without significant differences between EDTA whole blood and EDTA plasma. Total CV was found to be from 6.7% to 9.7% for BNP concentrations between 92.6 and 3984 ng/L. The sample type comparison study demonstrated correlation coefficients between 0.97 and 0.99 with slopes between 1.03 and 1.09 between the different samples. Method comparison between Minicare BNP and Siemens ADVIA Centaur BNP demonstrated a correlation coefficient of 0.92 with a slope of 1.06. The 97.5% URL of a healthy population was calculated to be 72.6 ng/L. The Minicare BNP assay is a robust, easy-to-use and sensitive test for rapid determination of BNP concentrations that can be used in a near-patient setting.

Fundamentals of Counting Statistics in Digital PCR: I Just Measured Two Target Copies-What Does It Mean?

Current commercially available digital PCR (dPCR) systems and assays are capable of detecting individual target molecules with considerable reliability. As tests are developed and validated for use on clinical samples, the need to understand and develop robust statistical analysis routines increases. This chapter covers the fundamental processes and limitations of detecting and reporting on single molecule detection. We cover the basics of quantification of targets and sources of imprecision. We describe the basic test concepts: sensitivity, specificity, limit of blank, limit of detection, and limit of quantification in the context of dPCR. We provide basic guidelines how to determine those, how to choose and interpret the operating point, and what factors may influence overall test performance in practice.

Variance functions, detectability and bias: a re-evaluation.

Background A recent study used simulated internal quality control data (4 specimens × 40 replicates) to investigate the use of variance functions in estimating limit of blank and limit of detection, as per ISO definitions. Small systematic negative biases were found (typically <1%), but subsequent investigation has shown that these estimates had unacceptably large uncertainties because of an inadequate simulation size. Methods The previous data generation and variance function estimations were repeated 25 times using a different random number generator seed on each occasion. The study was further extended by increasing data quantities 100-fold and by reducing the number of replicates per specimen (40 through 20, 10, 5 and 2). Results The previously reported negative biases were shown to be an artefact, and this was confirmed by simulations using 100-fold more data. Biases were <|0.1%| throughout with replication ≥20, but positive biases were found at lower replication; up to + 1.23% in the case of duplicates and large variances (e.g. some immunoassays) and up to + 0.2% in the case of duplicates and small variances. Conclusions The variance function provides essentially unbiased estimates of limit of blank and limit of detection at data replication ≥20 (bias: <1 part in 1000) and minimal biases at lower replication when measurement errors are small.

Performance evaluation of enzyme immunoassay for voriconazole therapeutic drug monitoring with automated clinical chemistry analyzers.

OBJECTIVE: Voriconazole is a triazole antifungal developed for the treatment of fungal infectious disease, and the clinical utility of its therapeutic drug monitoring has been evaluated. Recently, a new assay for analyzing the serum voriconazole concentration with an automated clinical chemistry analyzer was developed. We evaluated the performance of the new assay based on standardized protocols. METHODS: The analytical performance of the assay was evaluated according to its precision, trueness by recovery, limit of quantitation, linearity, and correlation with results from liquid chromatography-tandem mass spectrometry (LC-MS/MS). The evaluation was performed with the same protocol on two different routine chemistry analyzers. All evaluations were performed according to CLSI Guidelines EP15, EP17, EP6, and EP9 [1-4]. RESULTS: Coefficients of variation for within-run and between-day imprecision were 3.2-5.1% and 1.5-3.0%, respectively, on the two different analyzers for pooled serum samples. The recovery rates were in the range of 95.4-102.2%. The limit of blank was 0.0049 µg/mL, and the limit of detection of the samples was 0.0266-0.0376 µg/mL. The percent recovery at three LoQ levels were 67.9-74.6% for 0.50 µg/mL, 75.5-80.2% for 0.60 µg/mL, and 89.9-96.6% for 0.70 µg/mL. A linear relationship was demonstrated between 0.5 µg/mL and 16.0 µg/mL (R2 =0.9995-0.9998). The assay correlated well with LC-MS/MS results (R2 =0.9739-0.9828). CONCLUSIONS: The assay showed acceptable precision, trueness, linearity, and limit of quantification, and correlated well with LC-MS/MS. Therefore, its analytical performance is satisfactory for monitoring the drug concentration of voriconazole.

Development and analytical performance of a new ARCHITECT automated dipeptidyl peptidase-4 immunoassay.

BACKGROUND: Dipeptidyl peptidase-4 (DPP-4) may be a suitable biomarker to identify people with severe asthma who have greater activation of the interleukin-13 (IL-13) pathway and may therefore benefit from IL-13-targeted treatments. We report the analytical performance of an Investigational Use Only immunoassay and provide data on the biological range of DPP-4 concentrations. METHODS: We assessed assay performance, utilising analyses of precision, linearity and sensitivity; interference from common endogenous assay interferents, and from asthma and anti-diabetic medications, were also assessed. The assay was used to measure the range of serum DPP-4 concentrations in healthy volunteers and subjects with diabetes and severe, uncontrolled asthma. RESULTS: The total precision of DPP-4 concentration measurement (determined using percentage coefficient of variation) was ≤5% over 20 days. Dilution analysis yielded linear results from 30 to 1305 ng/mL; the limit of quantitation was 19.2 ng/mL. No notable endogenous or drug interferences were observed at the expected therapeutic concentration. Median DPP-4 concentrations in healthy volunteers and subjects with asthma or Type 1 diabetes were assessed, with concentrations remaining similar in subjects with diabetes and asthma across different demographics. CONCLUSION: These analyses indicate that the ARCHITECT DPP-4 Immunoassay is a reliable and robust method for measuring serum DPP-4 concentration.

Using the variance function to estimate limit of blank, limit of detection and their confidence intervals.

BACKGROUND: Implementing International Organization for Standardization definitions of limit of blank and limit of detection requires precision estimates from specimens devoid of analyte (blank specimens) and also from specimens located close to zero. Calculations are straightforward if errors are constant over the relevant concentration range but estimation of the relationship between variability and concentration (variance function) is necessary in the general case when errors are not constant. This study investigated the efficacy of incorporating the variance function into estimation of limit of blank, limit of detection and their confidence intervals. METHODS: Simulated data, designed to encompass the range of properties that would typically be observed in practice, consisted of four distinct relationships between variance and concentration, in combination with large and small variances and three concentration ranges. Four methods of estimating limit of blank were evaluated together with the accuracy of variance function derived estimates of limit of detection and the accuracy of symmetrical 95% confidence intervals constructed from limit of blank and limit of detection constituent variables. RESULTS: Most limit of blank estimates and all limit of detection estimates showed systematic negative bias but, provided the data concentration range is not too small, the biases were consistently <1% with confidence interval coverages ranging from 92% to 95%. Estimating limit of blank by extrapolating the variance function to zero lost little in comparison with methods based on blank specimen data. CONCLUSIONS: The variance function provides a convenient and reliable way of analyzing data from experiments evaluating detection capability and, provided certain assumptions are tenable, of estimating limit of blank and limit of detection as part of routine internal quality control.

Evaluation and analytical validation of a handheld digital refractometer for urine specific gravity measurement.

OBJECTIVES: Refractometers are commonly used to determine urine specific gravity (SG) in the assessment of hydration status and urine specimen validity testing. Few comprehensive performance evaluations are available demonstrating refractometer capability from a clinical laboratory perspective. The objective of this study was therefore to conduct an analytical validation of a handheld digital refractometer used for human urine SG testing. DESIGN AND METHODS: A MISCO Palm Abbe™ refractometer was used for all experiments, including device familiarization, carryover, precision, accuracy, linearity, analytical sensitivity, evaluation of potential substances which contribute to SG (i.e. "interference"), and reference interval evaluation. A manual refractometer, urine osmometer, and a solute score (sum of urine chloride, creatinine, glucose, potassium, sodium, total protein, and urea nitrogen; all in mg/dL) were used as comparative methods for accuracy assessment. RESULTS: Significant carryover was not observed. A wash step was still included as good laboratory practice. Low imprecision (%CV, <0.01) was demonstrated using low and high QC material. Accuracy studies showed strong correlation to manual refractometry. Linear correlation was also demonstrated between SG, osmolality, and solute score. Linearity of Palm Abbe performance was verified with observed error of ≤0.1%. Increases in SG were observed with increasing concentrations of albumin, creatinine, glucose, hemoglobin, sodium chloride, and urea. Transference of a previously published urine SG reference interval of 1.0020-1.0300 was validated. CONCLUSIONS: The Palm Abbe digital refractometer was a fast, simple, and accurate way to measure urine SG. Analytical validity was confirmed by the present experiments.

Determining lower limits of detection of digital PCR assays for cancer-related gene mutations.

Digital PCR offers very high sensitivity compared to many other technologies for processing molecular detection assays. Herein, a process is outlined for determining the lower limit of detection (LoD) of two droplet-based digital PCR assays for point mutations of the epidermal growth factor receptor (EGFR) gene. Hydrolysis probe mutation-detection assays for EGFR p.L858R and p.T790M mutations were characterized in detail. Furthermore, sixteen additional cancer-related mutation assays were explored by the same approach. For the EGFR L8585R assay, the assay sensitivity is extremely good, and thus, the LoD is limited by the amount of amplifiable DNA that is analyzed. With 95% confidence limits, the LoD is one mutant in 180,000 wild-type molecules for the evaluation of 3.3 µg of genomic DNA, and detection of one mutant molecule in over 4 million wild-type molecules was achieved when 70 million copies of DNA were processed. The measured false-positive rate for the EGFR L8585R assay is one in 14 million, which indicates the theoretical LoD if an unlimited amount of DNA is evaluated. For the EFGR T790M assay, the LoD is one mutant in 13,000 for analysis of a 3.3 µg sample of genomic DNA, and the dPCR assay limit sensitivity approaches one mutant in 22,000 wild-type molecules.