bluuebunny/crossref_metadata_2025_split huggingface dataset:
B-167 Quantification of Thyroglobulin in Serum for Clinical Research using SISCAPA Workflow Combined with LC-MS/MS
Authors: Davey L , Foley D , Razavi M , Calton L J , Trudeau M , Wardle R , Mira N 
Published: October 2024

Abstract

Background
Accurate measurement of Thyroglobulin (Tg) using existing immunoassay-based techniques can be challenging in the presence of anti-Tg antibodies (TgAb), which can prevent the binding of Tg to assay antibodies thus leading to non-quantifiable Tg concentrations. The Stable Isotope Standards and Capture with Anti-Peptide Antibodies (SISCAPA™) Workflow combined with LC-MS/MS has been successfully employed to circumvent this problem by digesting the serum sample thus eliminating interfering TgAb and measuring a Tg-specific surrogate peptide instead.

Methods
Using a Andrew+™ Pipetting Robot, denaturation and reduction buffer was added to 250µL serum samples and incubated at 37°C. Internal standard and trypsin were added and samples incubated at 37°C. The samples were quenched with protease inhibitor and Anti-Tg FSP mAb SISCAPA was added to the serum samples and mixed for 60 minutes. Using a magnetic array plate, the beads were left to pull down, samples were discarded, and the beads were twice washed with PBS/CHAPS prior to elution with acetonitrile with formic acid. Using an ACQUITY™ UPLC™ I-Class PLUS FL System, samples were injected onto a Waters™ XSelect™ HSS T3 Column using a water/acetonitrile/formic acid gradient elution profile and the Tg FSP peptide was quantified using a Waters Xevo™ TQ Absolute Mass Spectrometer.

Results
The method demonstrated no significant carryover or matrix effects and was shown to be linear from 0.1 - 50 ng/mL. Analytical sensitivity investigations indicate the analytical sensitivity of this method would allow precise quantification (&lt;20%) at 0.1 ng/mL with S/N (PtP) &gt;10:1. Coefficients of variation (CV) for total precision and repeatability on 5 analytical runs for low, mid and high QCs were all &lt; 9.5% (n = 25) for both automated and manual precision assessments. Comparison with samples from the UK NEQAS scheme demonstrated significant method bias of -40% for the developed LC-MS/MS method. Re-assignment of the calibrator concentrations using the EQA samples reduced the bias to -5.5%, indicating differences in the calibration materials used for these measurements.

Conclusions
A LC-MS/MS clinical research method for serum thyroglobulin was developed using SISCAPA, followed by analysis using ACQUITY UPLC I-Class PLUS FL System and the Xevo TQ Absolute Mass Spectrometer. The method provides analytical sensitivity down to 0.1 ng/mL from 250 µL serum, while providing sufficient sample for re-analysis. The method demonstrates excellent linearity across the calibration range, with no significant carryover, interferences, and matrix effects. Total reproducibility and repeatability of the method was ≤9.5% RSD for manual and automated sample preparation, using the Andrew+ Pipetting Robot. In addition, the Andrew+ Pipetting Robot can minimize user touch-time, allowing a full plate to be prepared within four hours without user intervention.
B-218 Inconclusive results in the Fetal Sex Determination from maternal plasma assay may indicate miscarriage
Authors: Levi J , Xavier T D , Caciatore M , Lourenço G P , Almeida S S , Andrade C E , Mello L A , Carpinetti M L , Batistutti V P , Osório P S 
Published: October 2024

Abstract

Background
The purpose of this study was to evaluate the miscarriage rates from patients presenting inconclusive results in a cell-free DNA fetal sex determination test at Dasa, a Brazilian laboratory, between 2021 and 2023.

Methods
In validating the fetal sex determination test, a Ct value range was established to assign the presence of the Y chromosome on maternal plasma and a gray zone for determining an inconclusive result. In this case, our operational procedure is to repeat the analysis from the beginning, due to plasma volume limitation. When the result remains inconclusive upon repetition a new sample is requested.After the 2nd sample collection with inconclusive results, cases are forwarded to specialized medical contact, when patient is informed and potential explanations for the rare situation of a repeat-indeterminate sex determination result are discussed.At the end of 2022, the need to widen the sex-determining range and consequently the inconclusive zone was routinely perceived. The main reason for this change was to reduce unnecessary repetitions.

Results
The adjustments of range reduced unnecessary repetition, requests for new samples and medical contacts (Table). Between 2021 and 2023 the median gestational age at 1st collect of medical contact was 9 weeks, and in cases of miscarriages was 10 (2022) and 8 weeks (2023).

Conclusions
The widening of the Y chromosome Ct range proved to be efficient on a routine basis without changing the result error rates, which since 2021 have remained at 0.05%. In addition to providing more precision to the assay, the newly established gray zone increased the positive predictive value for miscarriage. Our results, show that what was thought to be a limitation of the technique in determining the fetal sex may in fact, in many cases, be associated to fetal loss
B-221 Combined RT-PCR and Whole Exome Sequencing Pharmacogenetic Testing in Polypharmacy Patient: A Case Report
Authors: Rubio Prieto J L , Segura E , Pérez López E , Cordero Matia E , Rodríguez Rodríguez A , García de Veas Silva J , Guerrero J , Macher H 
Published: October 2024

Abstract

Background
Pharmacogenetics has become one of the main branches of Precision Medicine owing to its usefulness for patients’s optimal therapeutic management. Notwithstanding, pharmacogenetic testing results must be taken carefully, specially in multimorbidity patients with a variety of drugs prescribed. The aim of the present work is to introduce a combined RT-PCR and Whole Exome Sequencing Approach for pharmacogenetic testing in a patient with multiple drug-related adverse events.

Case Background
A 67 years old female was derived to our Clinical Chemistry Unit for pharmacogenetic testing due to multiple drug-related adverse events: nifedipine-voriconazole interaction, voriconazole-related neurological toxicity and moxifloxacin-related dizziness. Aditionally, she had been receiving morphine, corticoid for her reumatoid arthritis and antituberculous therapy (r isoniazid / pyrazinamide / rifampin and ethambutol).

Methods
Patient’s DNA was extracted from a blood sample and undergone by pharmacogenetic testing in two parallel ways: -A first genotyping assay was carried out using Taqman® OpenArray™ PGx Express 120 panel (ThermoFisher™), according to manufacturer’s instructions.
-Simultaneusly, in order to cover possible copy number variants and SNPs not covered by our RT-PCR array, a whole exome sequencing run was performed on IlluminaTM NextSeq 1000. Sample library was prepared applying a custom GRCh38 pipeline.

Results
Relevant variants related to patient’s therapy found by RT-PCR are shown in table 1.
SNPs genotyped by RT-PCR were confirmed by NGS. Moreover, whole exome sequencing revealed a deletion in exon 5 of SLCO1B1 gene, which corresponds to SLCO1B1*49 (loss of function); and some variants in NAT2 that may cause toxicity in antituberculous treatment (diplotype NAT2*6C/*7C).

Conclusions
In those challenging cases that involve multimorbidity and polypharmacy patients, pharmacogenetic testing must be approached taking into account variants that are not covered by the analytical method routinely used and the possibility of complement it with alternative proccedures.
B-222 Receptor-donor CYP3A5 and CYP3A4 genotype testing in liver transplant patients and its influence in tacrolimus blood levels: a retrospective study
Authors: Rubio Prieto J L , Macher H , Gómez Bravo M Á , García de Veas Silva J L , Suárez Artacho G , Rodríguez Rodríguez A , Sánchez Pozo C , Guerrero J , Rubio A 
Published: October 2024

Abstract

Background
Optimal immunosupression is crucial for transplant success. In orthohepatic transplants, blood target levels of tacrolimus, when coadministred with corticoids and mycophenolic acid, are between 6 and 10 ng/mL during the first three months post-transplant. Notwithstanding, achieving tacrolimus concentrations into this range may be tough because of factors such as liver function status, age, sex, drug-drug interactions, etc. In this way, pharmacogenetic testing of CYP3A5 and CYP3A4 genes have demonstrated to be useful in tacrolimus dosing optimization. Nonetheless, there is no evidence enough for pharmacogenetic based adjusting in liver transplant, specially if receptor and graft’s genotypes differ in these loci.
The aim of the present study is to find out how the receptor and donor genotypes in CYP3A5 and CYP3A4 genes affect tacrolimus pharmacokinetics.

Methods
Genotypic profiling of 108 receptor-donor's pairs biopsies gathered from patients who were underwent hepatic transplant from 2012 to 2019 were carried out using Taqman® OpenArray™ PGx Express 120 panel (ThermoFisher™). We focused on CYP3A5*3, *6 and *7; and CYP3A4*1B, *3 and *22 alleles. Next to pharmacogenetic testing, each patient was classified as very poor, poor, intermediate, rapid or ultrarapid metabolizer according to their own genotype, graft’s genotype or both of them. Patient’s initial dose, at seven days after treatment start and after discharge of tacrolimus and their trough concentrations (C0) were collected from their electronic health records.
Tacrolimus levels were measured by affinity chrome-mediated immunoassay. C0 was normalized by weight-adjusted dosage (C/D ratio); then, median C/D ratios between different metabolic profiles were compared through Kruskal-Wallis test.

Results
When patients were classified based on donor’s genotype isolated or combined with receptor’s genotype, lower C/D ratios were found as metabolizer ability increases (p=0.045 for donors and p=0.027 for combined genotypes, respectively). No statistically differences were found for C/D ratios at seven days and discharge, though that differences were almost significant for donor’s genotypes (p=0.06 in both cases).

Conclusions
These results show that previous information about both patient’s and specially donor’s CYP genes genotype may improve initial immunosupression and avoid tacrolimus induced toxicity. Pharmacogenetics has been currently proposed as a guide for tacrolimus treatment of some solid organ transplants as kidney transplanted patients. Better understanding of how receptor and donor’s CYP genotype affects its pharmacokinetics, along with liver’s damage markers and other genetic polymorphisms will allow clinicians, in the era of personalized medicine, to improve immunosupressive management.
B-228 Performance Comparison of three viral RNA pathogens in MK Buffered Solution for Diagnostic Based Detection using quantitative PCR vs droplet digital PCR
Authors: Casto M , Hicks R , Brazeau D , Karamchi M 
Published: October 2024

Abstract

Background
Droplet digital PCR (ddPCR) by partitioning a single reaction into many thousands of nano-droplets allows for the absolute quantitation of a given DNA/RNA target and represents the gold standard for the identification and quantification of pathogens in clinical and environmental samples. While nucleic acid based molecular diagnostics have become fundamental for the accurate and rapid quantification of specific pathogens, these instruments are still based upon quantitative PCR (QPCR) assays. The goal of this study was to assess the compatibility of a MK Buffered solution with three different RNA viruses combined into two standard clinical negative matrices and compare performance of ddPCR and QPCR for all three pathogens.

Methods
Serial dilutions of heat-inactivated Sars-CoV-2 (NR-52286), Influenza A (ATCC VR-1469) and RSV-A (ATCC strain VR-26) were combined into a single tube and added to two confirmed clinically negative matrices, Nasal and Sputum (Lee Biosolutions, Maryland Heights, MO). Serial dilutions of the combined pathogens were added to sample tubes containing 500 µL of MK buffered solution yielding final concentrations of each pathogen over 4 orders of magnitude in each clinical matrix. For each sample 300 µl was processed for RNA isolation using standard RNA isolation kits (Zymo Viral RNA Kit). For ddPCR samples were quantified for all three pathogens using a BioRad® Droplet Digital PCR System (QXD200). For QPCR, samples were quantified using Quantitative real-time PCR using Promega GoTaq® Probe 1- Step RT-qPCR System. The same TAQMan® probes specific for each pathogen were used for both systems.

Results
Yields of all three viral RNAs were significantly lower from sputum samples as compared to nasal samples. For all three pathogens the ddPCR assay was an order of magnitude more sensitive than observed with QPCR. QPCR and ddPCR values showed a significant linear relationship for all three pathogens (Sars-CoV-2, r2 = 0.984; RSV A, r2 = 0.814; Influenza A, r2 = 0.985).

Conclusions
QPCR and Droplet digital PCR accurately quantified concentrations of all three RNA pathogens in both clinically negative nasal and sputum matrices across four orders of magnitude, though detected yields were significantly lower in samples spiked into the sputum matrix. For all three pathogens there was a significant linear relationship with decreasing concentration. ddPCR was an order of magnitude more sensitive than QPCR for all three pathogens.
B-254 Analytical and Clinical Performance Evaluation of Plasma pTau217 in a Clinical Diagnostic Laboratory
Authors: Encarnacion M , Kumar P , Mousavi A , Hallett K , Biehl D , Pflueger S , Mammel A , Frykman H 
Published: October 2024

Abstract

Background
Blood-based biomarkers provide an accessible and cost-effective tool for diagnosing Alzheimer’s disease (AD). Among these biomarkers, phosphorylated tau 217 (pTau217) shows promise for both AD diagnosis and differential diagnosis, as well as for facilitating access to emerging disease-modifying therapies. The ALZpath SIMOA pTau-217 v2 assay is a digital immunoassay designed for quantifying pTau-217 in human EDTA plasma samples. This study assesses the analytical performance of plasma pTau217 assays as a Laboratory Developed Test (LDT) appropriate for diagnostic application.

Methods
In this analytical study, we evaluate accuracy, precision, detection limits, linearity, and analyte stability. Plasma pTau217 levels were measured using the ALZpath pTau217 v2 assay on a single Quanterix HD-X SIMOA Analyzer platform at BC Neuroimmunology Lab. Inc. Vancouver, British Columbia, Canada. As a clinical testing laboratory, our validation procedures are in accordance with CAP and CLIA requirements, with a primary focus on adhering to CLSI guidelines for both analytical and clinical performance testing. To determine a reference range and cut-off, samples of Amyloid PET-confirmed cases (n=263) and healthy subjects between the ages of 55 and 95, with a roughly equal ratio of males to females (n=219), were tested.

Results
The ALZpath pTau217 assay demonstrates an analytical measurement range of 0.0046 - 3.4056 ng/L, with a limit of blank (LoB) of 0.0019 ng/L, a limit of detection (LoD) of 0.0046 ng/L, and a limit of quantification (LoQ) of 0.03 ng/L. Linearity spans from 0.06 to 1.75 ng/L, with repeatability showing intra-laboratory precision above LoQ ≤ 20% CV. Stability is maintained at 4⁰C for ≤ 1 week, at room temperature for ≤ 72 hrs, at -20⁰C for ≤ 2 weeks, and at -70⁰C for ≤ 3 weeks, with freeze/thaw cycles tolerated up to 6 times. Moreover, interference is not detected at the maximum concentrations tested for albumin, unconjugated bilirubin, biotin, hemoglobin, creatinine, and heterophilic antibodies. The reference range was determined using the 95% reference interval, resulting in 0.398 ng/L. ROC analysis yielded an AUC of 0.932 with a Youden index of 0.338 ng/L. At the Youden index, sensitivity was 90.0% with a specificity of 85.6%. Specificity reached 95.5% at the upper cut point of 0.471 ng/L, while sensitivity was 94.8% at the lower cut point of 0.242 ng/L.

Conclusions
The outstanding analytical performance of pTau217 supports its adoption as a reliable screening and diagnostic tool in clinical practice, aiding clinicians in the accurate diagnosis of AD.
B-266 Therapeutic Drug Monitoring of Natalizumab and Anti-Natalizumab Antibodies
Authors: Chun K , Bauer B , Bastidas M , Zikry M , Yang J 
Published: October 2024

Abstract

Background
Natalizumab (NTZ) is a monoclonal antibody directed against the α4-integrin subunit of α4β1 and α4β7 integrins. NTZ is used to treat relapsing and remitting forms of multiple sclerosis. Although a majority of patients on standard dosing (intravenous 300 mg, every 4 weeks) reach therapeutic drug concentrations, about 10% of patients will eventually fail NTZ therapy due to the development of anti-NTZ antibodies. NTZ and anti-NTZ antibody concentrations in serum are used together to provide patient-specific pharmacokinetic and immunogenic assessment to aid in patient management.

Methods
Serum measurements of NTZ and anti-NTZ antibody levels are performed by lab developed electro-chemiluminescent immunoassays where assay design and principle are equivalent to biologic therapeutic drug monitoring described in our previous publication. The natalizumab drug assay is an immunoassay validated to measure free drug (pharmacodynamically active, ADA-unbound) in serum. The anti-natalizumab antibody assay utilizes a solution phase bridging method designed to measure total binding antibodies (including IgM & IgG subtypes). All anti-natalizumab antibody positive samples are confirmed for drug-specificity by an additional signal suppression step. The anti-natalizumab antibody assay is quantitative and drug-tolerant, meaning that circulating drug in patient serum does not interfere with anti-NTZ detection and quantitation.

Results
Here, we report concomitant measurement of NTZ and anti-NTZ antibody in 160 patient serum samples. Positive anti-NTZ levels from 24 to 235 ng/mL were found in 7.5% (12/160) patients. Anti-NTZ-free samples (148/160) had NTZ drug levels ranging from undetectable (&lt;1.0) to 85 ug/mL. The mean and median drug level in 90 samples (results &gt;1.0) was 10.8 and 2.7, respectively. 61/90 samples had therapeutic drug concentrations (at least 2.0 ug/mL). In the presence of anti-NTZ, 67% (8/12) had undetectable drug level and (4/12) had mean and median drug levels of 1.2 and 1.3, respectively, which is 9- or 2-fold lower than the mean and median drug levels of the anti-NTZ Ab -free group.

Conclusions
Low serum NTZ and high anti-NTZ are associated with loss of clinical efficacy, while elevated NTZ may increase progressive multifocal leukoencephalopathy risk. Assays to measure biologic drugs and their anti-drug antibodies in patient serum may be useful tools in dose optimization and patient management.
B-267 Validation of the Newly Developed Fully Automated Chemiluminescent Enzyme Immunoassay (CLEIA) Reagent for Measurement of Cyclosporine in the Blood
Authors: Tanaka M , Yui H , Baba K , Oshimo A , Hara A , Tanaka M , Ishii Y , Kaneko A , Aoyagi K 
Published: October 2024

Abstract

Background
Cyclosporine (CsA) is an immunosuppressive drug requiring therapeutic drug monitoring (TDM) for its narrow-ranged medication level. Approximately 60% of CsA in the blood exist as molecular complexes with proteins like cyclophilin in lymphocytes and erythrocytes. Measurement of CsA in the blood by immunoassay or liquid chromatography-tandem mass spectrometry (LC-MS/MS) requires an extraction process for CsA from the whole blood specimen, which is often manual complicated and time-consuming process causing a risk of variation in the measurement values among different operators. In addition, the small (cyclic polypeptide composed of 11 amino acids) and hydrophobic property of CsA, make it difficult to establish antibodies for a sandwich immunoassay. In this study, we have validated a novel fully automated sandwich immunoassay reagent for measurement of blood CsA that used a combination of newly-established CsA antibodies and iTACT® (immunoassay for total antigen including complex via pretreatment) technology.

Methods
The reagent was developed and validated on a fully automated immunoassay system, LUMIPULSE® L2400 (FUJIREBIO INC). Twenty uL whole blood specimens are sampled after mixed with suction/discharge stirring to prevent that blood cells settle to the bottom of a container on the analyzer, and then treated with a pretreatment solution to obtain free CsA. The treated samples are assayed by a two-step sandwich immunoassay. Limit of Detection (LoD), Limit of Quantitation (LoQ), within-laboratory precision, linearity, no interference, and the correlation of this assay and LC-MS/MS were validated, following the current CLSI guidelines protocols. Data were analyzed using Microsoft Excel statistical tool Analyse-it.

Results
The LoD and the LoQ were 2.7 ng/mL and 10.8 ng/mL respectively. The within-laboratory precision showed CV 1.6-2.9 %. Linearity was demonstrated over the range 13.1-2381.5 ng/mL. No interference was observed with unconjugated (≤19.7 mg/dL) or conjugated bilirubin (≤20.1 mg/dL), chyle (≤1480 FTU), RF (rheumatoid factor, ≤1000 IU/mL), and HAMA (human anti-mouse antibody, ≤1010 ng/mL). The correlation coefficient and the regression slope of this assay and LC-MS/MS were 1.0 and 1.0, respectively (N=120).

Conclusions
The performance of our novel CsA assay, which use LUMIPULSE L2400, was satisfactory for routine analysis. A unique fully-automated process, including the pretreatment process of whole blood specimens, for measurement would contribute the low LoQ and the low Within-laboratory CV. In addition, the results are obtained in 35-minute. We expect this novel immunoassay could reduce the burden on operators and turnaround time.
B-269 Development and CYP3A4 Characterization of a Population-based Urinary Buprenorphine and Norbuprenorphine Nomogram
Authors: Twum K , Deharvengt S J , Khan W A , Tsongalis G J , Korpi-Steiner N 
Published: October 2024

Abstract

Background
Buprenorphine (Bup), routinely prescribed for opioid use disorder, metabolizes to norbuprenorphine (NBup) primarily via cytochrome P450 (CYP) 3A4. Confirmation of urinary Bup and NBup helps monitor patient adherence to therapy and potential misuse. This study aims to develop and characterize a population-based urinary NBup/Bup nomogram using pharmacogenetics (PGx) to facilitate opioid result interpretations.

Methods
Adult urinary Bup and NBup results between 09/01/2022 and 09/30/2023 at UNC Health were retrieved from the EMR. Results outside the clinically reportable range (CRR; 5-25,000 ng/mL) were assigned the lower or upper CRR value, respectively. A scatterplot of Bup versus NBup/Bup on logarithmic scale was generated using Excel. Buffy coats from 14 EDTA whole blood samples were extracted and isolated using EasySep buffer (StemCell) with centrifugation at 8000rpm for 10 minutes without braking and stored at -80°C until shipment to DHMC. Genomic DNA was extracted using an EZ1 DNA procedure (Qiagen) and libraries (Agilent SureSelect) were prepared by hybridization capture of a custom PGx panel. Paired-end sequencing was performed on a MiSeq prior to diplotype-metabolizer function assignments in AUGMET. 22 CYP3A4 gene hotspots were detected by NGS and analyzed for gene-drug-variant relationships.

Results
3593 results from 896 patients were retrieved. The NBup/Bup nomogram revealed 3 subgroups from this population (Figure). CYP3A4 genotyping showed 13 wildtype calls and 1 CYP3A4 *1/*22 star allele genotype, with reduced enzyme activity. Wildtype results corresponded with Bup/NBup in subgroups 1(n=1), 2 (n=11) and 3 (n=1); CYP3A4 *1/*22 result corresponded with subgroup 3.

Conclusions
We developed a novel NBup/Bup nomogram revealing 3 subgroups in our patient population. Overlap of wildtype CYP3A4 in subgroups 1, 2 and 3 as well as CYP3A4 *1/*22 genotype in subgroup 3 suggest the NBup/Bup subgroups are independent of CYP3A4 phenotype. The characterization of population-based urinary NBup/Bup nomogram shows promise for aiding in opioid result interpretations.