Comparison of 2 Serum Free Light Chain Assays with Creatinine Normal and Abnormal Populations Demonstrates the Need for Standardization.

BACKGROUND: The objective of this study was to compare The Binding Site's Freelite on Optilite and Diazyme's Kappa/Lambda free light chains (K/L FLC) on Abbott Architect c8000 with healthy and renal insufficient populations and to evaluate their respective reference intervals for serum free light chains (sFLCs). METHODS: Two hundred sixty serum samples were measured for creatinine and sFLCs by both assays and a subset by immunofixation electrophoresis. Verification of manufacturer-defined reference intervals was assessed. RESULTS: Kappa free light chains (KFLC) showed excellent correlation of 0.998 R2 with a slope of 0.73. For Lambda free light chains (LFLC), an acceptable correlation of 0.953 R2 was found with a slope of 1.50 as well as a skewness-based difference with a -12.70 intercept. Healthy estimated glomerular filtration rate (eGFR) ≥60 reference interval verification of central 95% could not be confirmed for either Freelite or Diazyme although LFLC was much closer than KFLC for both assays with Freelite KFLC recovering only 37% of values within reference interval claims. The K/L FLC ratio did not meet 100% claim for both Freelite (91%) and Diazyme (95%) among those with eGFR ≥60. Samples with eGFR ≤59 had increasingly higher levels of KFLC and LFLC for both assays. When comparing worsening eGFR status, Freelite recovered increasingly higher ratios while Diazyme recovered increasingly lower ratios. CONCLUSIONS: Healthy reference intervals could not be verified for either Freelite or Diazyme. Renal reference intervals for Freelite are currently warranted while they are not recommended for Diazyme. The differences between these 2 assays can be minimized by standardization efforts such as recalibration.

Understand the FDA-cleared fentanyl testing: A clinical evaluation of the SEFRIA fentanyl immunoassay.

BACKGROUND: Screening for fentanyl has been adopted by many clinical laboratories to detect illicit drug use and monitor medication adherence. However, compared to other urine drug testing, fentanyl screening assays are relatively new and therefore their clinical performances are largely unknown. This study extensively evaluated the clinical performance, positive cutoff, and interference profile of SEFRIA fentanyl immunoassay in real patient settings. METHODS: The FDA-cleared cutoff of 1.0 was verified with 21 urine samples with low or undetectable levels of fentanyl. After assay implementation, all screened-positive samples were confirmed by liquid chromatography-tandem mass spectrometry. A new cutoff was derived from the numeric values of the false positive (FP) screening results. The FP rates before and after implementing the new cutoff were compared. Interferences were identified by an untargeted drug analysis and confirmed by spiking experiments. RESULTS: A total of 3951 screening results were reviewed in the first two months of the assay utilization, 410 were screened-positive, and 157 (38 %) were FP. After a new cutoff of 1.3 was implemented, the FP rate was reduced to 17 % based on 11119 screening results. Trazodone, labetalol, and haloperidol were identified as major interferents, accounting for 56 % of the FP results using the cutoff of 1.3. CONCLUSION: By applying the new cutoff and including an interference comment to positive screening results, the FP rate was reduced from initial 38 % to 7.5 % (17 % times 56 %).

Pediatric reference interval verification for 16 biochemical markers on the Alinity ci system in the CALIPER cohort of healthy children and adolescents.

OBJECTIVES: Special chemistry parameters are useful in the diagnosis and management of inherited disorders, liver disease, and immunopathology. Evidence-based pediatric reference intervals (RIs) are required for appropriate clinical decision-making and need to be verified as new assays are developed. This study aimed to evaluate the applicability of pediatric RIs established for biochemical markers on the ARCHITECT for use on newer Alinity assays. METHODS: An initial method validation was completed for 16 assays, including precision, linearity, and method comparison. Sera collected from approximately 100 healthy children and adolescents as part of the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) were also analyzed on the Alinity c system. Percentage of results within established ARCHITECT RIs were calculated and considered verified if ≥90 % fell within established limits. New RIs were established for three electrolytes, glucose, and lactate wherein no data were previously reported. RESULTS: Of the 11 assays for which CALIPER pediatric RIs were previously established on ARCHITECT assays, 10 met the verification criteria. Alpha-1-antitrypsin did not meet verification criterion and a new RI was established. For the other 5 assays, de novo RIs were derived following analysis of 139-168 samples from healthy children and adolescents. None required age- and sex-partitioning. CONCLUSIONS: Herein, pediatric RIs were verified or established for 16 chemistry markers in the CALIPER cohort on Alinity assays. Findings support excellent concordance between ARCHITECT and Alinity assays with one exception (alpha-1-antitrypsin) as well as robustness of age- and sex-specific patterns originally reported by CALIPER in healthy Canadian children and adolescents.

Pediatric reference interval verification for 17 specialized immunoassays and cancer markers on the Abbott Alinity i system in the CALIPER cohort of healthy children and adolescents.

OBJECTIVES: Clinical laboratory investigation of autoimmune, metabolic, and oncologic disorders in children and adolescents relies on appropriateness of reference intervals (RIs). The Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) previously established comprehensive pediatric RIs for specialized immunoassays on the Abbott ARCHITECT system. Herein, we aim to verify performance on new Alinity i assays by evaluating sera collected from healthy children as per Clinical and Laboratory Standards Institute (CLSI) EP-28A3C guidelines. METHODS: Precision, linearity, and method comparison experiments were completed for 17 specialized Alinity immunoassays, including cancer antigens, autoimmune peptides, and hormones. Sera collected from healthy children and adolescents (birth-18 years, n=100) were evaluated. CLSI-based verification was completed using previously established CALIPER RIs for ARCHITECT assays as the reference. RESULTS: Of 17 specialized immunoassays assays, only anti-cyclic citrullinated peptides (anti-CCP) did not meet acceptable verification criterion (i.e., ≥90% of results within ARCHITECT reference CI). Anti-thyroglobulin, anti-thyroid peroxidase, and carcinoembryonic antigen did not require age-specific consideration beyond one year of age, with 63, 91, and 80% of samples equalling the limit of detection, respectively. Estimates were separated by sex for relevant assays (e.g., sex hormone binding globulin, total and free prostate specific antigen). CONCLUSIONS: Findings support transferability of pediatric RIs on ARCHITECT system to the Alinity system for 16 specialized immunoassays in the CALIPER cohort and will be a useful resource for pediatric clinical laboratories using Alinity assays. Further work is needed to establish evidence-based interpretative recommendations for anti-CCP and continue to evaluate pediatric RI acceptability for newly available assay technologies.

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.

Tissue distribution and pharmacokinetics of stable polyacrylamide nanoparticles following intravenous injection in the rat.

A variety of polymer nanoparticles (NP) are under development for imaging and therapeutic use. However, little is known about their behavior. This study examined pharmacokinetics, distribution and elimination of stable polyacrylamide (PAA) nanoparticles (~31 nm average diameter). PAA NPs and polyethylene glycol-coated PAA NPs were injected into the tail veins of healthy male rats. Blood, tissues and excreta were collected at times ranging from 5 min to 120 h and their radioactive content was quantified. A mathematical model was then applied to analyze the distribution dynamics of both NPs. Elimination from the blood could be accounted for by a quick but finite relocation to the major organs (about 20%, 0.6 to 1.3h half-lives), and a slower distribution to the carcass (about 70%, 35 to 43 h half-lives). Excreted urinary levels correlated with blood concentrations. Combined cumulative urinary and fecal output accounted for less than 6% of the dose at 120 h. Compared to five other polymeric nanoparticles, the studied particles are at the highest half-lives and Area Under the Curve (4000 to 5000%-h). These two parameters decrease by three orders of magnitude when nanoparticle size increases from the 30 nm range up to 250 nm. For similar sizes, pegylated nanoparticles are more persistent in the blood than non-pegylated ones, but this difference is much smaller in the 30 nm and relatively high dose range than above 100 nm. Persistence of PAA NPs is not associated with acute toxicity signs as measured by typical serum markers of inflammation and cellular damage.

Photoreceptor cell apoptosis induced by the 2-nitroimidazole radiosensitizer, CI-1010, is mediated by p53-linked activation of caspase-3.

The nitroimidazole radiosensitizer CI-1010 ((R)-alpha-[[(2-bromoethyl)-amino]methyl]-2-nitro-1H-imidazole-1-ethanol monohydrobromide) causes selective, irreversible, retinal photoreceptor apoptosis in vivo. The mouse 661 W photoreceptor cell line was used as a neuronotypic model of CI-1010-mediated retinal degeneration. Exposure to CI-1010 for 24 h induced apoptosis in 661 W cells, as determined by ultrastructural analysis, agarose electrophoresis and analysis of TUNEL-positive nuclei. CI-1010 caused a loss of viability in 661 W cells, as measured by the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). A clear link was established between the onset of apoptosis and activity of caspase-3 and caspase-8, prior to poly[ADP-ribose]polymerase (PARP) cleavage. Pretreatment with caspase inhibitors, ZVAD.fmk or DEVD-CHO, prevented morphological changes in most CI-1010-treated cells. Evaluation of mitochondrial inner membrane potential (Deltapsi(m)) in live 661 W cells using the fluorescent dye, tetramethylrhodamine methyl ester revealed retention of (Deltapsi(m)) until after caspase activation. Absence of cytochrome c in the cytoplasm in treated cells further supports the hypothesis of a mitochondrial-independent mechanism of cell death. Significant increase in DNA crosslinks observed in 661 W cells correlates with induction and phosphorylation of p53 at multiple serine sites. Cell cycle analysis of 661 W cells reveals a G(2) arrest in response to CI-1010-induced DNA damage and neuronal cell death. Increased protein expression of Bax, Fas, and FasL, concomitant to the loss of Bcl-xL in treated 661 W cells may be modulated by p53. This study evaluates in vitro mechanisms of CI-1010-induced cell death in photoreceptors and provides evidence in support of a p53-linked activation of caspase-3 in response to DNA damage caused by CI-1010.