Assay Cutoff Research Articles

Abundant Drug-Resistant NS3 Mutants Detected by Deep Sequencing in Hepatitis C Virus-Infected Patients Undergoing NS3 Protease Inhibitor Monotherapy

The high genetic variation of hepatitis C virus (HCV) results in rapid selection of drug resistance mutations (DRMs) during monotherapy with direct-acting antivirals (DAAs). It has been proposed that each possible single mutant preexists in infected individuals; however, the levels of preexisting DRMs are too low to be directly quantified in most patients using current techniques. In this study, we evaluated the presence of DRMs in HCV-infected patients treated with the HCV protease inhibitors GS-9256 or GS-9451 as monotherapy using deep sequencing in 137 longitudinal samples from 45 patients. Software was developed to analyze deep-sequencing results with an assay cutoff of 0.25%. No NS3 DRMs that confer resistance to GS-9256 and GS-9451 (R155K, A156T, and D168V/E) were observed in 33 baseline samples at >0.25%. In contrast, these and other substitutions at NS3 positions 155, 156, and 168 were detected in 19/27 patients at day 2 (24 h) and 21/21 at day 4 (84 h) of monotherapy but not in placebo-treated patients. Based on the DRM growth kinetics during drug treatment, pretreated NS3 mutations at amino acids 155, 156, and 168 were estimated on average at 0.025% and 0.015% per genotype 1a and 1b HCV-infected patients, respectively. Relative fitness of the DRM viruses was shown to be significantly lower than the wild type. Deep-sequencing analyses of NS3 protease inhibitor-treated HCV-infected patients suggest a limit of HCV viral load suppression of 3.6 to 3.8 log(10) with NS3 protease inhibitor monotherapy that does not suppress the identified preexisting NS3 DRMs and thus a need for a combination therapy.

Safety, Reactogenicity and Immunogenicity of the Human Rotavirus Vaccine in Preterm European Infants

Rotavirus disease is more severe in preterm infants than in full-term infants. This study assessed the safety, reactogenicity and immunogenicity of a human rotavirus vaccine, RIX4414, in European preterm infants. A total of 1009 preterm infants were randomized (2:1, vaccine:placebo) and stratified into 2 groups: 20% of early (27-30 weeks, group 1) and 80% of late (31-36 weeks, group 2) gestational age preterm infants in each group. Two doses of RIX4414/placebo were administered to these preterm infants according to the recommended chronologic age for full-term infants with an interval of 30-83 days between doses. Serious adverse events were recorded throughout the study period. Solicited and unsolicited adverse events were recorded for 15 and 31 days post-each dose. Antirotavirus IgA concentrations (enzyme-linked immunosorbent assay cutoff = 20 U/mL) and geometric mean concentration were determined pre-dose 1 and 30-83 days post-dose 2 in a subset of 300 infants. This study is registered with ClinicalTrials.gov, number NCT00420745 (eTrack106481). Serious adverse events were reported at a similar frequency in both groups (P = 0.266). Fifty-seven infants reported at least 1 serious adverse event (5.1% [3.5-7.0] in the RIX4414 group and 6.8% [4.3-10.0] in the placebo group). During the 15-day postvaccination follow-up period, diarrhea, vomiting and fever occurred at a similar frequency in both groups; fever could have been due to concomitant vaccines. Five cases (RIX4414 = 3, Placebo = 2) of rotavirus gastroenteritis were reported. The onset of rotavirus gastroenteritis in the RIX4414 group was 1-5 days after vaccination (vaccine strain identified in all cases) and in the placebo group it was 3-4 days after receiving placebo (wild-type rotavirus identified from both cases). Antirotavirus IgA seroconversion rates at 30-83 days post-dose 2 were 85.7% (79.0-90.9) in the RIX4414 group and 16.0% (8.8-25.9) in the placebo group. Geometric mean concentrations were 202.2 U/mL (153.1-267.1) in the RIX4414 group and <20 U/mL in the placebo group. Seroconversion rate in groups 1 and 2 in RIX4414 recipients were 75.9% (95% confidence interval [CI]: 56.5-89.7%) and 88.1% (95% CI: 80.9-93.4%), respectively; the geometric mean concentrations in the respective groups were 110.2 U/mL (95% CI: 56.1-216.5) and 234.8 U/mL (95% CI: 173.4-318.0; exploratory analysis). Two doses of RIX4414 were immunogenic and well-tolerated in European preterm infants.

Correlation of improved hepatitis B surface antigen detection limits with hepatitis B virus DNA nucleic acid test yield in blood donations

This study provides data on the quantitative relationship between hepatitis B surface antigen (HBsAg; ng/mL) and hepatitis B virus (HBV) nucleic acid test (NAT; copies/mL) and addresses whether HBsAg assays with improved sensitivity would impact the detection of HBV-positive samples from occult or early seroconversion window period infections. Plasma samples were tested with an HBsAg assay (PRISM, Abbott Laboratories; sensitivity, 0.08-0.1 ng/mL) and with two HBsAg research prototype assays: HBsAg Prototype 1 (sensitivity, 0.032-0.045 ng/mL) and HBsAg Prototype 2 (sensitivity, 0.009-0.017 ng/mL); NAT assays were used to determine HBV DNA copy levels. Samples from 10 hepatitis B seroconversion panels covering the ramp-up phase were utilized to examine the relationship between detection of HBsAg using improved assays and viral load using quantitative HBV DNA polymerase chain reaction. For these samples, detection at the HBsAg assay cutoff (sample-to-cutoff ratio, 1.0) corresponded to 206 copies/mL HBV DNA for the HBsAg Prototype 1 assay and 329 copies/mL for the PRISM HBsAg assay. Compared to the PRISM HBsAg and HBsAg Prototype 1 assays, the HBsAg Prototype 2 assay detected two additional samples of 32 HBV DNA-positive samples obtained from blood donors with occult HBV and one of seven from blood donors with early window period infections. Increased sensitivity HBsAg assays result in the detection of samples containing lower viral loads. Improvements in the analytic sensitivity of HBsAg prototype assays allow the detection of additional HBV DNA-positive samples from donors with window period or occult infections compared to PRISM HBsAg. Improved HBsAg assays should allow for incremental detection of HBV infection.

Seroprevalence of Toxoplasma gondii in Dogs in Shandong, Henan, and Heilongjiang Provinces, and in the Xinjiang Uygur Autonomous Region, People's Republic of China

Seroprevalence of Toxoplasma gondii infection was determined in sera of 632 dogs (551 pets, 81 strays) from Shandong, Henan, and Heilongjiang Provinces, and in the Xinjiang Uygur Autonomous Region, People's Republic of China (PRC), using the indirect hemagglutination assay (cutoff titer 1:64 or higher); 11.1% were seropositive. The seroprevalence in stray dogs and in ≥3-yr-old dogs was significantly higher (P < 0.05) than that in household dogs and in <3-yr-old dogs. There were no significant differences in terms of gender, breed, or locality (P ≥ 0.05). The results indicate that T. gondii infections are common in dogs in PRC.

Measuring Enzymatic HIV-1 Susceptibility to Two Reverse Transcriptase Inhibitors as a Rapid and Simple Approach to HIV-1 Drug-Resistance Testing

Simple and cost-effective approaches for HIV drug-resistance testing are highly desirable for managing increasingly expanding HIV-1 infected populations who initiate antiretroviral therapy (ART), particularly in resource-limited settings. Non-nucleoside reverse trancriptase inhibitor (NNRTI)-based regimens with an NRTI backbone containing lamivudine (3TC) or emtricitabine (FTC) are preferred first ART regimens. Failure with these drug combinations typically involves the selection of NNRTI- and/or 3TC/FTC- resistant viruses. Therefore, the availability of simple assays to measure both types of drug resistance is critical. We have developed a high throughput screening test for assessing enzymatic resistance of the HIV-1 RT in plasma to 3TC/FTC and NNRTIs. The test uses the sensitive “Amp-RT” assay with a newly-developed real-time PCR format to screen biochemically for drug resistance in single reactions containing either 3TC-triphosphate (3TC-TP) or nevirapine (NVP). Assay cut-offs were defined based on testing a large panel of subtype B and non-subtype B clinical samples with known genotypic profiles. Enzymatic 3TC resistance correlated well with the presence of M184I/V, and reduced NVP susceptibility was strongly associated with the presence of K103N, Y181C/I, Y188L, and G190A/Q. The sensitivity and specificity for detecting resistance were 97.0% and 96.0% in samples with M184V, and 97.4% and 96.2% for samples with NNRTI mutations, respectively. We further demonstrate the utility of an HIV capture method in plasma by using magnetic beads coated with CD44 antibody that eliminates the need for ultracentifugation. Thus our results support the use of this simple approach for distinguishing WT from NNRTI- or 3TC/FTC-resistant viruses in clinical samples. This enzymatic testing is subtype-independent and can assist in the clinical management of diverse populations particularly in resource-limited settings.

Allele-Specific Real-Time PCR System for Detection of Subpopulations of Genotype 1a and 1b Hepatitis C NS5B Y448H Mutant Viruses in Clinical Samples

The Y448H mutation in NS5B has been selected by GS-9190 as well as several benzothiadiazine hepatitis C virus (HCV) polymerase inhibitors in vitro and in vivo. However, the level and the evolution kinetics of this resistance mutation prior to and during treatment are poorly understood. In this study, we developed an allele-specific real-time PCR (AS-PCR) assay capable of detecting Y448H when it was present at a level down to 0.5% within an HCV population of genotype 1a or 1b. No Y448H mutation was detected above the assay cutoff of 0.5% in genotype 1b-infected Con-1 replicons prior to in vitro treatment. However, the proportion of replicons with the Y448H mutation rapidly increased in a dose-dependent manner upon treatment with GS-9190. After 3 days of treatment, 1.2%, 6.8%, and >50% of the replicon population expressed Y448H with the use of GS-9190 at 1, 10, and 20 times its 50% effective concentration, respectively. In addition, plasma from 65 treatment-naïve HCV-infected patients (42 and 23 with genotype 1a and 1b, respectively) was tested for the presence of Y448H by AS-PCR and population sequencing. As expected, all patient samples were wild type at NS5B Y448 by population sequencing. AS-PCR results were obtained for 62/65 samples tested, with low levels of Y448H ranging from 0.5% to 3.0% detected in 5/62 (8%) treatment-naïve patient samples. These findings suggest the need for combination therapy with HCV-specific inhibitors to avoid viral rebound of preexisting mutant HCV.

Short Communication Prevalence of Susceptibility to Etravirine by Genotype and Phenotype in Samples Received for Routine HIV Type 1 Resistance Testing in the United States

Abstract The prevalence of susceptibility to etravirine was investigated among clinical samples submitted for routine clinical testing in the United States using two separate weighted genotypic scoring systems. The presence of etravirine mutations and susceptibility to etravirine by phenotype of clinical samples from HIV-1-infected patients, submitted to Monogram Biosciences for routine resistance testing between June 2008 and June 2009, were analyzed. Susceptibility by genotype was determined using the Monogram and Tibotec etravirine-weighted genotypic scoring systems, with scores of ≤3 and ≤2, respectively, indicating full susceptibility. Susceptibility by phenotype was determined using the PhenoSense HIV assay, with lower and higher clinical cut-offs of 2.9 and 10, respectively. The frequency of individual etravirine mutations and the impact of the K103N mutation on susceptibility to etravirine by genotype were also determined. Among the 5482 samples with ≥1 defined nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations associated with resistance, 67% were classed as susceptible to etravirine by genotype by both scoring systems. Susceptibility to etravirine by phenotype was higher (76%). The proportion of first-generation NNRTI-resistant samples with (n=3598) and without (n=1884) K103N with susceptibility to etravirine by genotype was 77% and 49%, respectively. Among samples susceptible to first-generation NNRTIs (n=9458), >99% of samples were susceptible to etravirine by phenotype (FC <2.9); the remaining samples had FC ≥2.9-10. In summary, among samples submitted for routine clinical testing in the United States, a high proportion of samples with first-generation NNRTI resistance was susceptible to etravirine by genotype and phenotype. A higher proportion of NNRTI-resistant samples with K103N than without was susceptible to etravirine.

Immunobiomarkers in Small Cell Lung Cancer: Potential Early Cancer Signals

We investigated the presence of autoantibodies as immunobiomarkers to a panel of tumor-associated antigens in a group of individuals with small cell lung cancer (SCLC), a disease group that has a poor overall cancer prognosis and therefore may benefit most from early diagnosis. Sera from 243 patients with confirmed SCLC and normal controls matched for age, sex, and smoking history were analyzed for the presence of these early immunobiomarkers (i.e., autoantibodies to p53, CAGE, NY-ESO-1, GBU4-5, Annexin I, SOX2, and Hu-D) by ELISA. Autoantibodies were seen to at least 1 of 6 antigens in 55% of all the SCLC patients' sera tested, with a specificity of 90% compared with controls. Using a higher assay cutoff to achieve a specificity of 99%, autoantibodies were still detectable in 42% of SCLC patients (receiver operator characteristic area under the curve = 0.76). There was no significant difference in sensitivity when analyzed by stage of the cancer or by patient age or gender. The frequency of autoantibodies to individual antigens varied, ranging from 4% for GBU4-5 to 35% for SOX2. Levels of Annexin I autoantibodies were not elevated in patients with SCLC. Antibodies were also detected in 4 separate patients whose sera were taken up to 3 months before tumor diagnosis. The presence of an autoantibody to one or more cancer-associated antigens may provide an important addition to the armamentarium available to the clinician to aid early detection of SCLC in high-risk individuals.

Establishing assay cutoffs for HLA antibody screening of apheresis donors.

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related deaths. Donor HLA antibodies have been implicated in TRALI cases. Blood centers are implementing TRALI risk reduction strategies based on HLA antibody screening of some subpopulations of ever-pregnant apheresis platelet (PLT) donors. However, if screening assay cutoffs are too sensitive, donation loss may adversely impact blood availability. Pregnancy history and HLA antibody screening and single-antigen bead data from blood donors in the Retrovirus Epidemiology Donor Study-II Leukocyte Antibody Prevalence Study were evaluated for correlations between assay screening values, HLA antibody titer, and number of HLA antigen specificities. The probabilities of matching a cognate antigen in a recipient were calculated and examined in association with total number of specificities observed and screening values. The relative impact of imposing various screening assay cutoffs or pregnancy stratification was examined in relation to detection of HLA antibody-reactive donations and loss of donors and donations. We provide evidence that higher HLA antibody screening assay values are associated with maintaining higher screening signals upon dilution and an increased breadth of specificities compared with lower screening values; the latter correlated with an increased risk of a cognate antigen match in potential recipients. Depending on the TRALI risk reduction strategy used, the potential loss of donations ranged between 0.9 and 6.0%. This analysis should enable blood centers to decide upon a TRALI risk reduction strategy for apheresis PLTs that is consistent with how much donation loss the blood center can tolerate.

Effect of Low-Level HCV Viraemia at Week 24 on HCV Treatment Response in Genotype 1 Patients

We examined the detection of low-level viraemia at week 24 as a predictor of sustained virological response (SVR) and viral relapse/breakthrough, and the agreement between the Roche Cobas TaqMan™ HCV RNA assay (TaqMan) and Roche Cobas(®) Amplicor HCV qualitative assay (Amplicor; both Roche Molecular Diagnostics, Pleasanton, CA, USA) for detection of low-level viraemia. A total of 871 treatment-naive HCV genotype 1 patients participating in an induction-dose pegylated interferon therapy study had virological responses assessed using TaqMan. A total of 151 patients with HCV RNA levels ≤500 IU/ml had samples tested in parallel using the Amplicor and TaqMan assays. SVR was significantly lower and relapse/breakthrough significantly higher in patients with low-level residual viraemia at week 24 compared with those who had undetectable viraemia: SVR was 72%, 29% and 14% (P<0.0001) and relapse/breakthrough 28%, 71% and 86% (P<0.0001) in patients with viraemia that was undetectable, detectable <15 IU/ml and detectable 15-<50 IU/ml, respectively, at week 24. The negative predictive value (NPV) for a week-24 virological response for SVR was 86%, 90% and 90% using TaqMan cutoffs of undetectable, <15 IU/ml and <50 IU/ml, respectively. The percentage agreement between Amplicor and TaqMan was similarly high for TaqMan cutoffs of 50 IU/ml and 15 IU/ml, but lower for undetectable viraemia (83%, 83% and 70%, respectively). These data emphasize the importance of achieving undetectable HCV RNA during pegylated interferon therapy to maximize SVR; however, the current 24-week stopping rule of undetectable HCV RNA appears too stringent when using sensitive PCR assays given the observed lower NPV for SVR using the TaqMan undetectable cutoff. Our data also suggest that a TaqMan <15 IU/ml result is comparable to an Amplicor-negative result (that is, below the assay cutoff value) when monitoring viral response.

GADD45α induction in the greenscreen HC indicator assay does not occur independently of cytotoxicity

Mammalian chromosomal integrity assays are influenced by cytotoxicity, a phenomenon which impacts data interpretation, assay specificity, and regulatory testing guidelines. Concordance of the GADD45α GreenScreen HC indicator assay to established in vitro and in vivo genetic toxicological assays has previously been described, yet a detailed description in the manner by which cytotoxicity influences its performance has not. Here we present a post-hoc analysis of a previously tested set of 91 proprietary and nonproprietary compounds investigating the influence of cytotoxicity on GADD45α induction and how varying assay cutoff criteria impacts assay performance. Significant cytotoxicity was identified to accompany the majority (72%) of compounds classified as genotoxic by GADD45α induction. Decreasing the GADD45α genotoxic induction criteria (from a 50 to a 30% increase over solvent controls) resulted in an increased assay sensitivity (from 30 to 68%) and concordance (from 55 to 68%), though a concomitant decrease in specificity was also observed (from 97 to 68%). We conclude that GADD45α induction in the GreenScreen HC indicator assay is influenced by cytotoxicity and that assay performance can be improved if different cutoff criteria are implemented.

Determination of Mean Recency Period for Estimation of HIV Type 1 Incidence with the BED-Capture EIA in Persons Infected with Diverse Subtypes

The IgG capture BED enzyme immunoassay (BED-CEIA) was developed to detect recent HIV-1 infection for the estimation of HIV-1 incidence from cross-sectional specimens. The mean time interval between seroconversion and reaching a specified assay cutoff value [referred to here as the mean recency period (ω)], an important parameter for incidence estimation, is determined for some HIV-1 subtypes, but testing in more cohorts and new statistical methods suggest the need for a revised estimation of ω in different subtypes. A total of 2927 longitudinal specimens from 756 persons with incident HIV infections who had been enrolled in 17 cohort studies was tested by the BED-CEIA. The ω was determined using two statistical approaches: (1) linear mixed effects regression (ω(1)) and (2) a nonparametric survival method (ω(2)). Recency periods varied among individuals and by population. At an OD-n cutoff of 0.8, ω(1) was 176 days (95% CL 164-188 days) whereas ω(2) was 162 days (95% CL 152-172 days) when using a comparable subset of specimens (13 cohorts). When method 2 was applied to all available data (17 cohorts), ω(2) ranged from 127 days (Thai AE) to 236 days (subtypes AG, AD) with an overall ω(2) of 197 days (95% CL 173-220). About 70% of individuals reached a threshold OD-n of 0.8 by 197 days (mean ω) and 95% of people reached 0.8 OD-n by 480 days. The determination of ω with more data and new methodology suggests that ω of the BED-CEIA varies between different subtypes and/or populations. These estimates for ω may affect incidence estimates in various studies.

Identification of specificities of antibodies against human leukocyte antigens in blood donors

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related mortality. Blood centers are implementing TRALI risk reduction strategies based on screening apheresis donors for antibodies to human leukocyte antigens (HLA). HLA antibody screening was performed on 7920 blood donors from the Leukocyte Antibody Prevalence Study (LAPS) using Luminex-based normalized background (NBG) cutoff ratios of 10.8 (Class I) and 6.9 (Class II). Single antigen bead (SAB) assay cutoffs of 2500 median fluorescence intensity units (Class I) and 1500 (Class II) were established based on results of two subpopulations of LAPS donors. Antibody frequencies against HLA A, B, C, DR, DQ, and DP antigens were determined for screen-reactive donors with prior pregnancies. SAB reactivity for samples above our multiantigen bead NBG cutoffs was 78% for Class I and 79% for Class II. The SAB-positive rate increased among women with zero to four or more pregnancies (0.3%-15.6% Class I and 0.4%-18% Class II; p < 0.00001). The highest frequency antibodies were DR11 and B15 (4.4% of women with prior pregnancies). The majority of Class I positives contained more than five specificities. For Class II, antibody-positive women segregated into two groups: a single specificity or more than five specificities. Identification of HLA antigen specificities supports pregnancy associations previously found with screening assays. The significance of particular HLA specificities for inducing TRALI is currently being evaluated in a large lookback study of recipients of high-plasma-volume components from this donor cohort.

Immunogenicity of a Reduced Schedule of Pneumococcal Conjugate Vaccine in Healthy Infants and Correlates of Protection for Serotype 6B in the United Kingdom

Pneumococcal conjugate vaccine (PCV) was introduced in the United Kingdom immunization schedule in September 2006. This study was conducted to establish the immunogenicity of licensed PCV (Prevenar) at a reduced, 2 priming dose schedule (2+1) and to evaluate functional responses in the context of vaccine effectiveness. Infants were randomized to receive PCV at 2 and 3 months or 2 and 4 months of age. Boosters were administered at the same time as Haemophilus influenzae type B/meningococcal C conjugate and Measles, Mumps and Rubella or with Measles, Mumps and Rubella alone (www.ClinicalTrials.gov NCT00197808). PCV at 2/3 months of age was poorly immunogenic and recruitment to this arm was terminated. PCV at 2/4 months of age resulted in lower than expected responses to serotypes 6B and 23F. Functional analysis of serotype 6B by OPA revealed that an enzyme-linked immunosorbent assay cutoff of 0.2 microg/mL was a better predictor of OPA positivity than a cut off of 0.35 microg/mL. PCV booster responses were excellent and no interference from concomitant vaccines was noted. An interval of at least 8 weeks is required when starting PCV vaccination at 2 months of age although not all serotypes are equally immunogenic. Correlates of protection derived from enzyme-linked immunosorbent assay values may not be equally appropriate for all serotypes as illustrated by results for 6B in this study.

Complement-Dependent Cytotoxicity Assay for Differentiating West Nile Virus from Japanese Encephalitis Virus Infections in Horses

A complement-dependent cytotoxicity (CDC) assay was established to measure antibodies to the West Nile virus (WNV) nonstructural protein 1 (NS1) in horses. Sera collected from a WNV-infected horse mediated lysis of WNV NS1-expressing cells in a dose-dependent manner at higher percentages than sera from a Japanese encephalitis virus (JEV)-infected horse. The percentages of specific lysis for sera diluted 1:10 to 1:80 were <19.8% (assay cutoff) for almost all of the 100 JEV-infected or uninfected horses tested, in contrast to 55 to 76% in WNV-infected horses. Experimental infection revealed that horses became anti-WNV NS1 antibody positive 10 days after WNV infection. This study demonstrated the utility of this assay for differentiating WNV from JEV infections in horses.

Cross-reactivity between cockroach and ladybug using the radioallergosorbent test

Home infestations from Harmonia axyridis (ladybug) occur throughout the United States. IgE-mediated sensitization with allergic disease has been reported. The prevalence of ladybug sensitization has been studied by questionnaire and allergy testing in patients diagnosed as having allergic disease. Cross-reactivity with cockroach exists. To determine the prevalence of ladybug specific IgE in the general population by specific IgE immunoassay and to examine cross-reactivity to cockroach. An experimental solid phase for use in immunoassay was prepared using a ladybug extract, and performance characteristics were determined. Serum samples from 100 adult blood bank donors were tested using the ladybug specific IgE immunoassay. Known ladybug IgE-positive serum samples obtained from symptomatic patients were used to study crossreactivity with German cockroach by specific IgE immunoassay inhibition. The mean background response of the assay solid phase was 51 fluorescent units with an analytical cutoff of 59 fluorescent units. It was estimated that a response of 88 fluorescent units corresponds to a specific IgE concentration of 0.1 kUa/L. The extinction dilution curve was linear to 0.1 kUa/L. The assay cutoff was set at 0.1 kUa/L. Of the 100 blood donor serum samples, 10 were positive for ladybug specific IgE. Inhibition assays revealed partial cross-reactivity with German cockroach. Although an immunoassay solid phase prepared with ladybug whole body extract will identify persons in a general population sensitized to epitopes found in the extract, clinicians performing allergy testing using whole body ladybug extract should be aware that a positive result may or may not indicate that exposure to actual ladybug allergens is causing sensitization.

59-P: Flow crossmatch (FCXM) interpretation: The disconnect between assay cutoff and the presence of donor specific antibody (DSA)

59-P: Flow crossmatch (FCXM) interpretation: The disconnect between assay cutoff and the presence of donor specific antibody (DSA)

PX-28 A comparison study of the biomerieux nucelisens easyMAG and the QIAGEN QIAsymphony extractors

Measurement of thyroid peroxidase autoantibodies (TPOAb) is useful in diagnosing patients with autoimmune thyroid disease. Measurement of thyroglobulin autoantibodies (TgAb) is used to detect potential interferences with thyroglobulin immunoassays and in limited situations for the diagnosis of autoimmune thyroid disease.The limit of detection, imprecision, reference interval, method comparison and diagnostic concordance for the ADVIA Centaur, ARCHITECT i2000, AxSYM, Immulite 2000, Modular E170 (TPOAb only), and UniCel DxI 800 (TgAb only) methods were evaluated. The Advantage was used as the comparison method.Total imprecision ranged from 2.6% to 14.9% for TgAb and 2.1% to 15.8% for TPOAb. Passing–Bablok slopes ranged from 0.51 to 10.4 (TgAb) and 1.05 to 7.12 (TPOAb) with correlation coefficients of 0.48 to 0.82 (TgAb) and 0.66 to 0.78 (TPOAb). Assay cutoffs were adjusted using a common set of reference interval samples. Concordance with the Advantage assay using the new cutoffs was found to be improved and ranged from 68.5% to 84.7% (TgAb) and 77.5% to 84.7% (TPOAb).Although all assays generally performed well, assay concordance for a negative or positive result ranged from 54.2 to 84.7%. Quantitative agreement between methods was generally poor and methods could not be used interchangeably. Additional standardization efforts are required to improve inter-method agreement.

Sensitive Troponin I Assay in Early Diagnosis of Acute Myocardial Infarction

Cardiac troponin testing is central to the diagnosis of acute myocardial infarction. We evaluated a sensitive troponin I assay for the early diagnosis and risk stratification of myocardial infarction. In a multicenter study, we determined levels of troponin I as assessed by a sensitive assay, troponin T, and traditional myocardial necrosis markers in 1818 consecutive patients with suspected acute myocardial infarction, on admission and 3 hours and 6 hours after admission. For samples obtained on admission, the diagnostic accuracy was highest with the sensitive troponin I assay (area under the receiver-operating-characteristic curve [AUC], 0.96), as compared with the troponin T assay (AUC, 0.85) and traditional myocardial necrosis markers. With the use of the sensitive troponin I assay (cutoff value, 0.04 ng per milliliter) on admission, the clinical sensitivity was 90.7%, and the specificity was 90.2%. The diagnostic accuracy was virtually identical in baseline and serial samples, regardless of the time of chest-pain onset. In patients presenting within 3 hours after chest-pain onset, a single sensitive troponin I assay had a negative predictive value of 84.1% and a positive predictive value of 86.7%; these findings predicted a 30% rise in the troponin I level within 6 hours. A troponin I level of more than 0.04 ng per milliliter was independently associated with an increased risk of an adverse outcome at 30 days (hazard ratio, 1.96; 95% confidence interval, 1.27 to 3.05; P=0.003). The use of a sensitive assay for troponin I improves early diagnosis of acute myocardial infarction and risk stratification, regardless of the time of chest-pain onset.

‘False-Positive’ and ‘False-Negative’ Test Results in Clinical Urine Drug Testing

The terms 'false-positive' and 'false-negative' are widely used in discussions of urine drug test (UDT) results. These terms are inadequate because they are used in different ways by physicians and laboratory professionals and they are too narrow to encompass the larger universe of potentially misleading, inappropriate and unexpected drug test results. This larger universe, while not solely comprised of technically 'true' or 'false' positive or negative test results, presents comparable interpretive challenges with corresponding clinical implications. In this review, we propose the terms 'potentially inappropriate' positive or negative test results in reference to UDT results that are ambiguous or unexpected and subject to misinterpretation. Causes of potentially inappropriate positive UDT results include in vivo metabolic conversions of a drug, exposure to nonillicit sources of a drug and laboratory error. Causes of potentially inappropriate negative UDT results include limited assay specificity, absence of drug in the urine, presence of drug in the urine, but below established assay cutoff, specimen manipulation and laboratory error. Clinical UDT interpretation is a complicated task requiring knowledge of recent prescription, over-the-counter and herbal drug administration, drug metabolism and analytical sensitivities and specificities.