Pre-analytical considerations in the development of a prototype SARS-CoV-2 antigen ARCHITECT automated immunoassay.

OBJECTIVES: To evaluate pre-analytical challenges related to high-volume central laboratory SARS-CoV-2 antigen testing with a prototype qualitative SARS-CoV-2 antigen immunoassay run on the automated Abbott ARCHITECT instrument. METHODS: Contrived positive and negative specimens and de-identified nasal and nasopharyngeal specimens in transport media were used to evaluate specimen and reagent on-board stability, assay analytical performance and interference, and clinical performance. RESULTS: TCID50/mL values were similar for specimens in various transport media. Inactivated positive clinical specimens and viral lysate (USA-WA1/2020) were positive on the prototype immunoassay. Within-laboratory imprecision was ≤0.10 SD (<1.00 S/C) with a ≤10% CV (≥1.00 S/C). Assay reagents were stable on board the instrument for 14 days. No high-dose hook effect was observed with a SARS-CoV-2 stock of Ct 13.0 (RLU>1.0 × 106). No interference was observed from mucin, whole blood, 12 drugs, and more than 20 cross-reactants. While specimen stability was limited at room temperature for specimens with or without viral inactivation, a single freeze/thaw cycle or long-term storage (>30 days) at -20 °C did not adversely impact specimen stability or assay performance. Specificity of the prototype SARS-CoV-2 antigen immunoassay was ≥98.5% and sensitivity was ≥89.5% across two ARCHITECT instruments. Assay sensitivity was inversely correlated with Ct and was similar to that reported for the Roche Elecsys® SARS-CoV-2 Ag immunoassay. CONCLUSIONS: The prototype SARS-CoV-2 antigen ARCHITECT immunoassay is sensitive and specific for detection of SARS-CoV-2 in nasal and nasopharyngeal specimens. Endogenous proteases in mucus may degrade the target antigen, which limits specimen storage and transport times and complicates assay workflow.

Specificity and Confirmation of SARS-CoV-2 Serological Test Methods in Emergency Department Populations across the United States.

BACKGROUND: Serological testing for SARS-CoV-2 is integral for understanding prevalence of disease, tracking of infections, confirming humoral response to vaccines, and determining timing and efficacy of boosters. The study objective was to compare the specificity of serology assays in emergency department populations across the United States in 2019 (pre-pandemic) and early 2020, incorporating an automated confirmatory assay. METHODS: Patient specimens (n = 1954) were from 4 regions in the United States: New York, NY; Milwaukee, WI; Miami, FL; and Los Angeles, CA. Specimens were tested with SARS-CoV-2 anti-spike receptor-binding domain assays: SARS-CoV-2 IgG on the Abbott Alinity i (AdviseDx SARS-Cov-2 IgG II) and Beckman Coulter Access 2 (SARS-CoV-2 IgG II), and SARS-CoV-2 IgM on the Abbott Alinity i (AdviseDx SARS-CoV-2 IgM). Reactive samples were tested with a research use only angiotensin-converting enzyme 2 binding inhibition assay (Abbott ARCHITECT) for confirmation of SARS-CoV-2 neutralizing antibodies. Assay specificity was determined and comparisons performed with Fisher's exact test. RESULTS: Overall SARS-CoV-2 IgG specificity was 99.28% (95% confidence interval, 98.80%-99.61%), 99.39% (98.93%-99.68%), and 99.44% (98.99%-99.72%) for SARS-CoV-2 IgG by Abbott and Beckman, and SARS-CoV-2 IgM, respectively. Overall agreement for the two IgG assays was 99.28% (range for the 4 sites: 98.21% to 100%). There were no specificity differences between assays or sites. CONCLUSIONS: The specificity of the serological assays evaluated in a large, diverse emergency department population was >99% and did not vary by geographical site. A confirmatory algorithm with an automated pseudo-neutralization assay allowed testing on the same specimen while reducing the false positivity rate and increasing the value of serology screening methods.

Specificity and Confirmation of SARS-CoV-2 Serological Test Methods in Emergency Department Populations Across the United States in 2019 and Early 2020.

BACKGROUND: Serological testing for SARS-CoV-2 is integral for understanding prevalence of disease, tracking of infections, confirming humoral response to vaccines, and determining timing and efficacy of boosters. The study objective was to compare the specificity of serology assays in emergency department populations across the United States in 2019 (pre-pandemic) early 2020 incorporating an automated confirmatory assay. METHODS: Patient specimens (n = 1954) were from four regions in the United States: New York, NY; Milwaukee, WI; Miami, FL; and Los Angeles, CA. Specimens were tested with SARS-CoV-2 anti-spike receptor binding domain assays: SARS-CoV-2 IgG on the Abbott Alinity i (AdviseDx SARS-Cov-2 IgG II) and Beckman Coulter Access 2 (SARS-CoV-2 IgG II), and SARS-CoV-2 IgM on the Abbott Alinity i (AdviseDx SARS-CoV-2 IgM). Reactive samples were tested with a research use only ACE2 binding inhibition assay (Abbott ARCHITECT) for confirmation of SARS-CoV-2 neutralizing antibodies. Assay specificity was determined and comparisons performed with Fisher's Exact Test. RESULTS: Overall SARS-CoV-2 IgG specificity was 99.28% (95% confidence interval: 98.80%-99.61%), 99.39% (98.93%-99.68%), and 99.44% (98.99%-99.72%) for SARS-CoV-2 IgG by Abbott and Beckman, and SARS-CoV-2 IgM, respectively. Overall agreement for the two IgG assays was 99.28% (range for the four sites: 98.21%-100%). There were no specificity differences between assays or sites. CONCLUSIONS: The specificity of the serological assays evaluated in a large diverse emergency department population was >99% and did not vary by geographical site. A confirmatory algorithm with an automated pseudo-neutralization assay allowed testing on the same specimen while reducing the false positivity rate and increasing the value of serology screening methods.

Prevalence of Detectable Biotin in Five US Emergency Department Patient Cohorts.

BACKGROUND: The objective of this study was to estimate the prevalence of biotin supplementation in United States emergency department patients using a multi-site, geographically distributed sampling model. METHODS: Biotin was measured using an Abbott ARCHITECT Biotin research use only assay in 7118 emergency department patient serum or plasma samples from five US medical centers. Samples with biotin ≥10 ng/mL underwent additional LC-MS/MS confirmatory testing for biotin and its primary metabolites. The overall and site-specific prevalence of detectable biotin was determined using the screening assay while biotin speciation (i.e., prevalence of detectable metabolites) was determined using LC-MS/MS. RESULTS: Of 7118 samples screened, 291 (4.1%) had biotin ≥10 ng/mL and were considered positive. Across five medical centers, the fraction of positive samples ranged from 2.0% to 5.4%. The maximum biotin concentration observed was 355 ng/mL. Of the 285 positive screens that underwent additional LC-MS/MS testing, 89 (31%) showed detectable biotin, bisnorbiotin, and/or biotin sulfoxide. Biotin, bisnorbiotin, and biotinsulfoxide were detected in 82/89 (92.1%), 61/89 (68.5%), and 18/89 (20.2%) samples, respectively; biotin was detected in the absence of either metabolite in 18/89 (20.2%) samples. CONCLUSIONS: Using a screening assay, 4.1% of emergency department patient samples were found to be potentially susceptible to interference from biotin. Confirmatory testing showed detectable biotin and/or biotin metabolites in 31% of positive screens (1.3% overall). The prevalence of biotin ≥10 ng/mL varied 2-3-fold across US emergency department patient cohorts. Biotin metabolites were observed in 80% of samples confirmed to have detectable biotin species by LC-MS/MS, suggesting that rigorous assessments of assay susceptibility to biotin interference, often performed using in vitro studies, should consider the potential role of biotin metabolites present in vivo.

An improved HIV antigen/antibody prototype assay for earlier detection of acute HIV infection.

BACKGROUND: Early detection of acute HIV infection by HIV antigen/antibody assays depends on antigen sensitivity. Maintaining consistently high sensitivity across diverse HIV strains is critical to ensure equal detection. OBJECTIVES: The performance of an improved HIV antigen/antibody prototype, HIV Combo Next, was evaluated for detection of genetically-diverse HIV strains and seroconversion samples. STUDY DESIGN: Antigen sensitivity of the prototype was evaluated and compared to five FDA-approved HIV antigen/antibody assays using World Health Organization (WHO) HIV p24 antigen standard and reference panels, 17 virus isolates and 9 seroconversion panels. Antibody sensitivity and assay specificity of the prototype were also assessed with 1062 disease-staged and genotyped samples, and samples from 3000 blood donors and 955 individuals with low-risk for HIV infection. RESULTS: Compared with other assays evaluated, the prototype demonstrated the best analytical sensitivity for WHO antigen standard, reference panels including 12 HIV-1 variants (0.04 - 0.25 IU/ml) and one HIV-2 variant, and 17 HIV virus isolates including HIV-1 group M, N, P and O and HIV-2 (0.3 -16 pg/ml). The enhanced sensitivity was also observed for seroconversion samples, detecting more PCR-positive samples with detection up to 7 days earlier than the other assays. Improvement in antigen sensitivity did not compromise antibody sensitivity or assay specificity, detecting all HIV disease-staged and genotyped samples, with assay specificity of 99.97% for blood donors and 99.68% for the low-risk population. CONCLUSIONS: These data indicate that the new prototype HIV Combo Next assay will be of diagnostic value, providing improved early detection for acute HIV infection from divergent HIV strains.

A panel of selected serum protein biomarkers for the detection of aggressive prostate cancer.

Background: Current PSA-based tests used to detect prostate cancer (PCa) lack sufficient specificity, leading to significant overdetection and overtreatment. Our previous studies showed that serum fucosylated PSA (Fuc-PSA) and soluble TEK receptor tyrosine kinase (Tie-2) had the ability to predict aggressive (AG) PCa. Additional biomarkers are needed to address this significant clinical problem. Methods: A comprehensive Pubmed search followed by multiplex immunoassays identified candidate biomarkers associated with AG PCa. Subsequently, multiplex and lectin-based immunoassays were applied to a case-control set of sera from subjects with AG PCa, low risk PCa, and non-PCa (biopsy negative). These candidate biomarkers were further evaluated for their ability as panels to complement the prostate health index (phi) in detecting AG PCa. Results: When combined through logistic regression, two panel of biomarkers achieved the best performance: 1) phi, Fuc-PSA, SDC1, and GDF-15 for the detection of AG from low risk PCa and 2) phi, Fuc-PSA, SDC1, and Tie-2 for the detection of AG from low risk PCa and non-PCa, with noticeable improvements in ROC analysis over phi alone (AUCs: 0.942 vs 0.872, and 0.934 vs 0.898, respectively). At a fixed sensitivity of 95%, the panels improved specificity with statistical significance in detecting AG from low risk PCa (76.0% vs 56%, p=0.029), and from low risk PCa and non-PCa (78.2% vs 65.5%, p=0.010). Conclusions: Multivariate panels of serum biomarkers identified in this study demonstrated clinically meaningful improvement over the performance of phi, and warrant further clinical validation, which may contribute to the management of PCa.

Comparative evaluation of three FDA-approved HIV Ag/Ab combination tests using a genetically diverse HIV panel and diagnostic specimens.

BACKGROUND: HIV Ag/Ab combination assays are recommended by CDC for routine screening and several HIV Ag/Ab combination tests are now FDA-approved. Maintaining high specificity and consistent sensitivity across diverse HIV strains is critical for these assays to accurately detect HIV infection and expedite delivery of patient results. OBJECTIVES: To evaluate performance of three FDA-approved HIV tests: ARCHITECT HIV Combo (Abbott), ADVIA Centaur HIV Combo (Siemens) and BioPlex HIV Ag-Ab (Bio-Rad). STUDY DESIGN: Sensitivity and specificity were evaluated using an extensive panel of 28 HIV infected human specimens and 17 cultured virus isolates representing multiple genotypes, 6 seroconversion panels, 4 human samples with acute infection, WHO p24 standard and 4020 clinical specimens. RESULTS: The p24 limit of detection (LOD) for the WHO standard was 0.19IU/ml, 0.70IU/ml, and 1.77IU/ml in BioPlex, ARCHITECT, and Centaur respectively. The distribution of LODs across 15 HIV-1 isolates was substantially narrower in ARCHITECT (5-33pg/ml) than in BioPlex (11-198pg/ml) and Centaur (6-384pg/ml). All assays detected antibodies to the majority of HIV-1 and HIV-2 variants. However, reduced sensitivity was observed for Centaur in detection of antibodies to HIV-1 group M (CRF02_AG), O and N variants. BioPlex and ARCHITECT showed better seroconversion sensitivity than Centaur, detecting one bleed (3-7 days) earlier in 4 (BioPlex) and 3 (ARCHITECT) of 6 seroconversion panels. ARCHITECT demonstrated the highest specificity (99.90-100%) compared to BioPlex (99.80%) and Centaur (99.42%). CONCLUSIONS: The overall performance of ARCHITECT and BioPlex was superior to Centaur, especially for detection of acute HIV infection.

Serum leptin and pathological findings at the time of radical prostatectomy.

PURPOSE: Obesity has been associated with a higher risk of progression following radical prostatectomy (RP). Obese men have higher serum leptin, a hormone produced by adipocytes, which has also been shown to be an in vitro prostate cancer growth factor. We examined whether serum leptin correlates with advanced pathological findings at RP. MATERIALS AND METHODS: Preoperative serum from 225 men treated with RP between 1998 and 1999 was examined for serum leptin. Multivariate logistic regression analysis was used to determine whether serum leptin was predictive of extraprostatic extension (pT3a). RESULTS: Serum leptin highly correlated with body mass index (Spearman r = 0.602, p <0.001). Serum leptin was not associated with total or percent free prostate specific antigen (PSA), biopsy or prostatectomy Gleason score, age or height. On multivariate analysis with total and percent free PSA, clinical stage, age, biopsy Gleason score, body mass index, serum leptin, and height as variables considered for entry into the model, serum PSA (p = 0.009), clinical stage (p = 0.019) and serum percent free PSA (p = 0.041) were the only variables predictive of extraprostatic extension. Serum leptin was not significantly associated with pathological stage (pT3a). CONCLUSIONS: In the current study of predominantly white men with mainly low risk disease there was no statistically significant association between serum leptin and pathological stage (pT3a) at RP. In this cohort serum leptin was not a good biomarker for predicting advanced stage at RP.

Association between serum adiponectin, and pathological stage and grade in men undergoing radical prostatectomy.

PURPOSE: Adiponectin is a polypeptide hormone produced by adipocytes that has anti-angiogenic properties. Circulating adiponectin is lower in obese men. Obesity has been associated with advanced stage and a higher risk of biochemical progression following radical prostatectomy (RP) in several series. We examined whether serum adiponectin is associated with advanced disease stage at RP. MATERIALS AND METHODS: Adiponectin was measured by enzyme-linked immunosorbent assay in the preoperative serum of 236 men treated with RP between 1998 and 1999. The odds ratio (OR) of advanced stage (pT3a or greater) and high grade disease (pathological Gleason sum 7 or greater) associated with quartiles of adiponectin were estimated using multivariate logistic regression models. RESULTS: Serum adiponectin weakly correlated inversely with body mass index (Spearman r = -0.22, p = 0.01). Serum adiponectin was not associated with cancer stage or grade. However, in normal weight men adiponectin was positively associated with high stage disease (OR 1.14, 95% CI 1.02 to 1.29, p = 0.03), although there was no statistically significant association with high grade disease (OR 1.05, 95% CI 0.94 to 1.18, p = 0.38). In overweight and obese men adiponectin was inversely associated with high grade disease (OR 0.94, 95% CI 0.87 to 1.01, p = 0.09), although there was no statistically significant association with high stage disease (OR 0.97, 95% CI 0.91 to 1.04, p = 0.43). Further adjustments for body mass index had little impact on any ORs. CONCLUSIONS: These data provide evidence to suggest that adiponectin may be related to prostate cancer aggressiveness, although the direction of the associations may depend on the extent of adiposity and on cancer grade.

Complexed prostate-specific antigen as a staging tool for prostate cancer: a prospective study in 420 men.

Over time, the parameters commonly used to predict pathological stage in men with localized prostate cancer have changed, and there is now little stratification in pretreatment prostate-specific antigen (PSA) concentrations, clinical stages, and biopsy Gleason scores. This prospective study evaluated the utility of complexed PSA (cPSA ) for predicting organ-confined disease in a contemporary series of subjects. The age range of the 420 men was 39 to 72 years (58.2 +/- 6 years). Specimens were collected before radical prostatectomy, and total and free PSA (Hybritech Tandem assays, Beckman Access; Beckman Coulter, Inc., Brea, CA) and total and cPSA (Bayer Immuno 1; Bayer Corporation, Tarrytown, NY) were measured. Pathologic stage was determined from the prostatectomy specimen. Of the 420 men, 316 (75%) had organ-confined disease, and 104 (25%) had non-organ-confined disease (20.7% had extraprostatic extension, 2.6% had seminal vesicle involvement, and 1.4% had positive lymph nodes). Prebiopsy Gleason score distribution was as follows: organ-confined organ-confined, 6 (87%) and 7 (10%); non-organ-confined, 6 (66%) and 7 (30%). Of patients with organ-confined disease, 75% had clinical stage T1c disease compared with 56% for non-organ-confined disease. Using univariate logistic regression, the following variables predicted organ-confined disease: biopsy Gleason score, clinical stage, total PSA, percent free PSA, cPSA, percent cPSA (P <0.05). A multivariate model with biopsy Gleason score, clinical stage, and cPSA had a receiver operator characteristic area under the curve of 0.69. Replacing cPSA with total PSA in this model provided similar information. cPSA and total PSA were highly correlated (r = 0.985). In summary, cPSA was equivalent to total PSA in predicting organ-confined disease. Present and future models and nomograms using PSA as an indicator of pathological stage could consider use of cPSA.

Short-term stability of the molecular forms of prostate-specific antigen and effect on percent complexed prostate-specific antigen and percent free prostate-specific antigen.

Differences in stability of the free and complexed molecular forms of prostate-specific antigen (PSA) may influence the clinical utility of assays for these forms, as well as the calculated ratios to total PSA (tPSA), such as percent free PSA (fPSA) and percent complexed PSA (cPSA). The objective of this study was to directly compare the short-term stability of fPSA and cPSA under different storage conditions. Specimens (3 with prostate cancer, 3 biopsy-negative without cancer, 2 normal) from 8 men were analyzed at baseline within 2 hours of collection, and at 4 hours, 8 hours, 24 hours, 48 hours, and 1 week after storage at room temperature, 4 degrees C, or -20 degrees C. Serum specimens were analyzed in duplicate on the Bayer Immuno 1 analyzer (tPSA, cPSA) and on the Beckman Coulter Access analyzer (tPSA, fPSA Tandem assays). Baseline tPSA values ranged from 0.7 to 62.0 ng/mL, with a median of 7.9 ng/mL (Immuno 1). Overall, all forms of PSA were stable up to 24 hours at the 3 temperatures, with the exception of fPSA and percent fPSA, which decreased when stored at 4 degrees C. After 1 week, tPSA levels decreased when stored at room temperature and at 4 degrees C, as did cPSA stored at room temperature. Over the 7 days, percent cPSA was stable at room temperature, but increased at 4 degrees C. There were no significant changes in any PSA form or calculated ratio with storage at -20 degrees C for up to 1 week. In summary, in the short term (<1 week), fPSA is less stable with storage than tPSA or cPSA in a time- and temperature-dependent fashion. Thus, specimen handling should be considered when interpreting PSA results. It is recommended that specimens not analyzed the same day (within 8 hours of collection) be stored frozen at -20 degrees C.

Clinical utility of proPSA and "benign" PSA when percent free PSA is less than 15%.

OBJECTIVES: To investigate the clinical utility of the subforms of free prostate-specific antigen (PSA), namely proPSA and "benign" PSA (BPSA), to improve cancer detection when the percent free PSA level is less than 15%. Percent free PSA, while maintaining sensitivity, has greatly improved the specificity of PSA for the early detection of prostate cancer. A low percent free PSA value indicates a greater risk of cancer, but only 30% to 50% of men with percent free PSA levels of less than 15% actually have cancer at biopsy. METHODS: Archived sera from 161 consecutive men who were prospectively enrolled in our Early Detection Research Network prostate cancer early detection biomarker program with a percent free PSA value of less than 15% were included in the study. Total PSA, free PSA, proPSA, and BPSA were measured for each sample. RESULTS: The mean total PSA was 6.1 ng/mL (range 1.8 to 24.0). The mean age of the study group was 62 +/- 7 years. Prostate cancer was detected in 66 (41%) of 161 men. The area under the curve-receiver operating characteristic for total and percent free PSA was 0.51 and 0.54, respectively. BPSA and proPSA/BPSA both improved cancer detection compared with percent free PSA alone; the improvement was statistically significant (P <0.001) . The area under the curve-receiver operating characteristic for proPSA/BPSA was 0.72, giving a sensitivity and specificity of 90% and 46%, respectively. CONCLUSIONS: The results of our preliminary studies have suggested that the ratio of proPSA and BPSA can distinguish cancer with greater accuracy when the percent free PSA value is very low (less than 15%), and may, therefore, provide better clinical utility in this lower range of percent free PSA.

Proenzyme psa for the early detection of prostate cancer in the 2.5-4.0 ng/ml total psa range: preliminary analysis.

OBJECTIVES: To determine the clinical utility of using proenzyme prostate-specific antigen (pPSA) for early detection of prostate cancer in the 2.5 to 4.0 ng/mL total PSA range. pPSA, the precursor form of PSA that contains a 7 amino acid leader peptide, and truncated forms such as [-2]pPSA and [-4]pPSA can be measured in serum by research immunoassay. METHODS: Archival serum from 119 men (noncancer, 88; cancer, 31), obtained before biopsy and in the total PSA range of 2.5 to 4.0 ng/mL, were assayed for total PSA, free PSA (fPSA), and pPSA. pPSA was defined as the sum of the [-2], [-4], and [-7] forms, and the percent pPSA (%pPSA) was defined as pPSA/fPSA. RESULTS: pPSA averaged 4.6% +/- 0.4% (SEM) of total PSA and 39.3% +/- 3.5% of fPSA. PSA and %fPSA values were similar between the noncancer and cancer groups, and %pPSA tended to be higher in the cancer group (50.1% +/- 4.4%) compared with the noncancer group (35.5% +/- 6.7%; P = 0.07). Using receiver operating characteristic analysis to assess clinical utility, the area under the curve for %pPSA was 0.688 compared with 0.567 for %fPSA. At a fixed sensitivity of 75%, the specificity was significantly greater for %pPSA at 59% compared with %fPSA at 33% (P <0.0001). CONCLUSIONS: In the 2.5 to 4.0 ng/mL total PSA range, 75% of cancers can potentially be detected with 59% of unnecessary biopsies being spared using %pPSA; use of %fPSA would result in sparing only 33% of unnecessary biopsies. A large prospective clinical trial is needed to confirm these preliminary findings.