bluuebunny/crossref_metadata_2025_split huggingface dataset:
B-053 Four years summary of flow cytometry live cell based assay for detecting serum anti-aquaporin 4 IgG and anti-myelin oligodendrocyte glycoprotein IgG in a Korean reference laboratory
Authors: Moon S , Min J , Hahm C , Choi J , Oh G 
Published: October 2024

Abstract

Background
In the diagnosis of neuromyelitis optica spectrum disorder (NMOSD), it is important to sensitively detect anti-aquaporin 4 (AQP4) IgG and anti-myelin oligodenrocyte glycoprotein (MOG) IgG in patients' serum. NMOSD is a disease that shows 3-7 times more common prevalence in Far East Asia than in the West. According to the IPND diagnostic criteria, it is recommended to diagnose as a cell based assay using full length recombinant protein. In this study, we would like to review 4 years results of AQP4, MOG requested to Eone laboratories in Korea performed with flow cytometry live cell based assay (FC-CBA).

Methods
FC-CBA was performed using piRES2 cell transfected with AQP4, MOG and stained with DsRed2. Subcultured cells were counted with 10^6, and reacted with patients' serum for 1 hour in a refrigerated state. Thereafter, reaction with FITC anti-human IgG for 2nd antibody for 30 minutes, and 10,000 cells were read from Navius (Beckman Coulter). The mean fluorescence intensity (MFI) ratio , which is the ratio obtained by dividing the MFI value of both FITC and DsRed2 positive cells and DsRed2 positive cells to negative cells, was used to determine positive, borderline, and negative for the serum sample.
AQP4 reviewed cases during the period from November 2018 to January 2024, and MOG reviewed cases during the period from December 2019 to January 2024. Indirect immunofluorescence assay using formalin fixed cells is commercially available and compared to the FC-CBA of this institution.

Results
During the study period, 4,733 cases of AQP4 were performed, and 23,540 cases of MOG were performed by FC-CBA. Among them, AQP4 was positive in 405 cases (8.5%) and borderline in 71 cases (1.5%), and all others were negative. MOG was 2,597 positive (11%), borderline in 1,144 cases (4.8%), and all others were negative. The positive and borderline cutoffs for MFI ratio were changed during the study period, and the positive rate was significantly changed.

Conclusions
Eone laboratories are the only institution in Korea that performs live cell based assay using flow cytometry for AQP4 and MOG IgG, and receives samples from patients suspected of NMOSD at university hospitals. When reading with MFI ratio, it is important to properly set the cutoff to determine the positivity rate.
B-055 Comparison of two different methods for quantitation of anti-dsDNA antibody
Authors: Choi J , Oh G 
Published: October 2024

Abstract

Background
Anti-dsDNA antibody is a specific marker of rheumatic disorder, primarily systemic lupus erythematosus (SLE). Early detection of these diseases is most important in treatment and prognosis. We evaluated two different quantitation systems of anti-dsDNA antibody: the BioPlex 2200 multiplex simultaneous detect system and the single detection method, which is the Phadia 250 system.

Methods
53 serum samples were analyzed by two different methods, which are BioPlex 2200, Bio-Rad Laboratories, Hercules, CA, and Padia 250, Thermo Fisher Scientific, Uppsala, Sweden, respectively. The method of the BioPlex 2200 system is multiplexed bead assay, and that of Phadia 250 is Fluorescence Enzyme Immunoassay (FEIA).

Results
The quantitation results of BioPlexx 2200 were 1 to 126 IU/mL, and those from Phadia 250 were 0.08 to 109.53 IU/mL, respectively. These two groups of the anti-dsDNA antibody values showed excellent correlations (r=0.9036).

Conclusions
The quantitation of anti-dsDNA antibody were performed mainly by a single analyzing method, Phadia 250, in Korea. Bioplex 2200 system is multiplex bead assay; it can analyze eight different Extractable Nuclear Antigen Antibodies (anti-ENA) simultaneously: Anti Ribosomal P Ab, Anti SSa/Ro Ab, Anti SSb/La Ab, Anti Centromere Ab, Anti Sm Ab, Anti RNP Ab, Anti SCL-70 Ab, Anti Jo-1 Ab as well. Nevertheless, these anti-ENA results showed semiquantitative results.
B-116 Cost Estimation of Laboratory Test Overuse in 14 Ordered Parameters: Experience From 6 hospitals and 65 Primary Care Centers
Authors: Puigví L , Cebollero A , Ruiz M , Regidor D , Carbonell M , Miró S , Esteve C , Massaro M , Romero C , Mariné J , Guillén J , Miró J , París X , Benítez M 
Published: October 2024

Abstract

Background
Inappropriate retesting and test request are considered causes of laboratory overutilization. The former can be managed through minimum retesting intervals (MRI) and the latter with expert rules in the laboratory information system (LIS). The aim of this study was to assess the effectiveness of both tools.

Methods
We performed a retrospective analysis of 14 laboratory routine tests subject to MRI or expert rules among 6 hospitals and 65 primary care centers from March 2018 through December 2023. Both tools were gradually implemented in the LIS (Servolab4®, Siemens, Berlin, Germany). Data from an export software was used for analysis (Health Analytics, Siemens Healthineers, Madrid, Spain).

Results
During the observation period, a total of 493.640 tests requests were analyzed and 60.151 violations were counted (Table 1).
All violations represented an economic saving of 494.603€ (8.760€ per month). The most popular rejected test was 1,25-DihydroxyvitaminD (34,5%), followed by thyroid peroxidase antibodies (20,3%) and 25-HydroxyVitaminD (19%). These three tests were responsible for 73,8% of the total cost savings.

Conclusions
The real cost estimation of inappropriateness is difficult to assess, but the generated costs for the health system are certainly high and not negligible. Moreover, addressing overutilization would ensure standardization and high-quality care.
B-121 Optimizing Test Ordering: Leveraging Heuristics in Naming Conventions - A PSA Case Study
Authors: Orahoske C , Wu A , Lynch K 
Published: October 2024

Abstract

Background
A common screening test for prostate cancer is the Prostate-Specific Antigen (PSA) test, which may also be referred to as total PSA or total PSA plus free PSA, among other variations. Looking at the test menus of two major reference laboratories, it's apparent that PSA can be ordered under thirteen different test names. From an end-user perspective, the question arises: which of these thirteen tests should be ordered, and why are there so many options? This plethora of choices can lead to confusion and uncertainty for clinicians and patients alike. According to various guidelines, ordering Free PSA is only warranted when PSA values exceed 4 ng/ml. In such cases, the ratio of free PSA to total PSA can aid in further clinical decision-making and patient workup. At Zuckerberg San Francisco General Hospital (ZSFGH), we offer two tests for PSA. The first is named “Prostate Specific Antigen” is total PSA done in house, while the second is labeled “Prostate Spec. Ag. Total and Free.” which is sent out to a reference lab. Upon reviewing our send-out orders, we observed a significant increase (376%) from 2022 to 2023 in the use of the send out order.

Methods
The ordering data for our providers was collected from February 2022 to January 2024 for both in-house and send-out orders. The data was analyzed using descriptive statistics in Microsoft Excel.

Results
In our population, we observed an abnormal concentration of send-outs originating from a select group of physicians. Additional we also identified a group of providers who only selected the send out test order. Analysis of in-house assay results for PSA revealed that only 15% (1292 out of 8136) exceeded the cutoff of 4 ng/ml, necessitating further testing to inform clinical decision-making. Yet our send out volume of 4157 test is in discordance with how many PSA would need further evaluation. Based on a cost analysis reducing the send out volume by 80% could result in $53,210 savings. Therefore, to reduce the send out amount and to keep the naming convention similar we decided to implement a new test name that would reflex the PSA to Free PSA if the PSA is above 4 ng/ml. We decided on the name “Prostate Specific Antigen total with reflex to Free Prostate Specific Antigen”.

Conclusions
Our investigation into the variability in PSA test ordering revealed a significant disparity in the number of send-out orders, particularly originating from a select group of physicians. However, the disproportionately high volume of send-out tests suggested an inefficiency in the current testing approach. To address this issue and align with clinical guidelines, we decided to process a request to our medical executive committee for approval for this reflex test in order to meet reimbursement compliance with the Office of the Inspector General. This change aims to streamline the process, reduce unnecessary send-out tests, and ultimately improve the efficiency and accuracy of prostate cancer screening at our institution.
B-151 Development and Validation of Plasma Catecholamines by Mass Spectrometry
Authors: Kemp J , Taylor R , Maus A , Grebe S K 
Published: October 2024

Abstract

Background
Our established method for measuring plasma catecholamines utilized a Shimadzu HPLC coupled to an ESA Coulochem III electrochemical detector. Chromatographic run times were long, resulting in slow turnaround times. Instrument maintenance was labor intensive, and the assay was prone to interferences. To overcome these limitations, we changed the method to liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurements.

Methods
After the addition of a labeled internal standard for each analyte, samples are loaded onto 50 mg of activated alumina at pH 8.6, utilizing a Tris-HCl, sodium carbonate and EDTA buffer. After incubation the alumina is then washed with 50% acetonitrile followed by water. Elution off the alumina is performed with 2% acetic acid. Derivatization is then performed on the eluate utilizing acetaldehyde and a cyanoborohydride buffer. Following derivatization, the samples are analyzed for epinephrine, norepinephrine, and dopamine by LC-MS/MS using a Sciex 7500 coupled to a ThermoFisher Scientific TLX-4 equipped with Vanquish pumps. Chromatographic separation was obtained using a Phenomenex Kinetex 2.6 um BiPhenyl 50x3 mm analytical column.

Results
Runtimes were significantly shorter than with the previous method, and no interferences were observed. Assay validation consisted of: intra/inter assay precision, accuracy, recovery, linearity, carryover, limit of detection (LOD), and interferences. Three levels were assessed for intra assay precision and each of the analytes monitored yielded %CVs of less than 6%. Four levels were assessed for inter assay precision and each of the analytes monitored yielded %CVs of less than 10%. When using linear regression analysis to compare results with the previous clinical assay, the slope, y-intercept and R2 were the following; dopamine: 1.06, 2.74, 0.978; epinephrine: 0.905, 2.20, 0.989; norepinephrine: 0.919, 9.75, 0.953. Spike and recovery from five samples yielded a mean average recovery of 93.4% for dopamine, 100% for epinephrine and 98.1% for norepinephrine. For linearity 4 samples were diluted and the mean % difference from expected was -1.66% for dopamine, -0.25% for epinephrine and 1.78% for norepinephrine. Blanks were injected following the high standard and carryover was negligible. LOD was determined by running a low sample pool along with a blank over 20 assays. The LOD was determined to be less than the LLOQ. Sixty-two common drugs/vitamins along with varying levels of hemolysis, lipemia, and bilirubin were also tested to ensure no interferences were seen.

Conclusions
The LC-MS/MS method exceeded the performance of the historical Shimdazu HPLC system with ESA Coluochem III electrochemical detector method. We anticipate that the better analytical performance will improve the testing quality of our patients.
B-153 Improving Accuracy of IgG subclasses Quantitation in Serum Using Liquid Chromatography - Tandem Mass Spectrometry (LC-MS/MS) on SCIEX 7500
Authors: Yi R , Holmes D T , Mattman A 
Published: October 2024

Abstract

Background
Serum IgG subclasses (IgGSC) are most often measured for identification of selective immunodeficiency disease and IgG4-related disease (IgG4RD). Traditional nephelometric (IN) assays suffer from errors related to antigen excess and cross-reactivity that can impact the accuracy of results. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been the routine testing methodology for serum IgG subclasses at St. Paul’s Hospital since 2015 due to the simplicity and freedom from inter-subclass cross-reactivity problems. In brief, 20 μL of serum sample is denatured, reduced, alkylated and digested, processed semi-automatically on Hamilton Starlet liquid handler. This is followed by analysis on a SCIEX 5500 QTRAP LC-MS/MS. However, when the method was migrated to the SCIEX 7500, it suffered from calibration curve splitting and poor between-device comparability. It was hypothesized that this could be caused by detuning of some high concentration analytes, suboptimal internal standard (IS) concentrations and, suboptimal IS multiple reaction monitoring (MRM) transition choices, and matrix effects from artificial calibrators.

Methods
Several modifications were made to improve accuracy and robustness: 1) new in-house calibrators were prepared using patient sample pool instead of using Binding Site IgG Subclass Kit calibrators. 2) More IS MRM transitions were added to the method with each analyte MRM quantitated using its own IS MRM using the same fragment ion. The IS concentration was also increased 5-fold for IgG1 and Total IgG. 3) To avoid problems arising from selectively ion detuning, the injection volume was reduced from 10 μL to 1 μL which permitted collision energies (CEs) to be normalized for each analyte/IS MRM pair.

Results
The new SCIEX 7500 method compares well with the original method (N=168) with Passing Bablok regression slopes of 0.94, 1.1, 1.0, 0.98, and 0.98 for IgG1-4 and IgG total respectively with corresponding intercepts of 0.3, -0.1, 0.0, 0.0, and 0.41 g/L and R^2 of 0.96, 0.95, 0.99, 0.98, and 0.95. Median differences were -1.76%, 1.07%, 1.06%, 6.13% and 1.91%. In a typical batch of 90 patient samples and four sets of calibrators, the calibration curves overlap well. Use of patient-based calibrators allowed calibration values to be centered in the range of typical patient sample values, particularly for IgG4, where the calibration curve was extended to 3.46 g/L compared with the previous high calibrator value of 0.48 g/L.

Conclusions
The new IgG subclass quantitation method on SCEIX 7500 LC-MS/MS system is robust with excellent reproducibility and between-device comparability.
B-159 Fast Cleanup for 19 Steroid Analytes in Serum Using Supported Liquid Extraction (SLE)
Authors: Chakravarti R , Marin S , Tackett B 
Published: October 2024

Abstract

Background
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful tool that holds promise in designing the future of steroid analysis in the clinical laboratory. It is already widely used in steroid analysis in endocrinology and is being utilized increasingly in newer applications for individual steroids and panels relevant in clinical research. LC-MS/MS is gaining user base owing to greater sensitivity and specificity and gradually even replacing some of the legacy test methods. Improved sample preparation, modern chromatography methods, and sensitive, faster scanning mass spectrometers have all played a role in improving LC-MS/MS. Steroid analysis for clinical research can require very low limits of detection which demand high recoveries and very clean extracted samples. Supported Liquid Extraction (SLE) can provide very clean extracted samples with less method development and a streamlined workflow compared to solid phase extraction (SPE). Laboratories desire high-throughput methods which consolidate analytes into one panel with fast chromatographic run times. Chromatographic separation of steroid isomers with the same m/z is necessary for accurate quantitation by LCMS/MS. Meeting these criteria can be challenging in a single LC-MS/MS method.

Methods
Each 200 µL serum sample was pretreated with 200 µL of pH 6 Ammonium Acetate buffer (aq) before being loaded onto a Novum™ PRO SLE, MAX 96-well plate, following the standard SLE protocol. Samples were eluted using 2 × 900 µL of Dichloromethane or Ethyl Acetate. After evaporation of the elution solvent, samples were reconstituted in 100 µL of 0.5 mM Ammonium Fluoride / Methanol (60:40, v/v). A Kinetex™ 2.6 μm C18, 50 x 3.0 mm column using a gradient of 0.5 mM Ammonium Fluoride (aq) and Methanol on an Agilent® 1290 Infinity UHPLC and SCIEX® 7500 Triple Quad™ were used for quantitation.

Results
Here, we present an effective sample extraction method and LC-MS/MS analysis method for the quantitation of a 19-analyte steroid panel from serum. The Kinetex 2.6 μm C18 column provided a fast 8-minute chromatographic separation and good selectivity for steroid analytes in both positive ion mode and negative ion mode. Calibration curves with a linear fit and 1/x weighting, showed good linearity with R2 of &gt;0.99 for all analytes except 17-OH-Pregnenolone and DHEAS when using Dichloromethane as the elution solvent, and R2 of &gt;0.980 for all analytes when using Ethyl Acetate as the elution the solvent. Accuracy of samples using Dichloromethane as the elution solvent were 80-112 %, except for 17-OH-Pregnenolone and DHEAS. Accuracy of all samples using the Ethyl Acetate elution solvent were 91-118 %.

Conclusions
The-96 well plate extraction method was robust, reproducible, and can be automated. SLE using Novum Pro MAX combined with a fast LC method provides a simple, rapid, high-throughput workflow for identification and quantitation of 19 steroids from serum.
B-166 Differentiation of Aziridine Functionality from Related Functional Groups in Protonated Analytes by Using Selective Ion-Molecule Reactions
Authors: Anyaeche R , Alzarieni K , Kumar R , Milton J , Kaur J , Sheng H , Kenttämaa H 
Published: October 2024

Abstract

Background
The development of better methods for the rapid and reliable identification of unknown compounds in mixtures is urgently needed in drug development. Aziridines, in particular, are used for drug synthesis but are potentially mutagenic and carcinogenic compounds. However, the detection and identification of these compounds pose challenges for many analytical methods, such as Nuclear Magnetic Resonance (NMR) spectroscopy, Fourier-transform infrared spectroscopy (FT-IR), and X-ray crystallography. These techniques are time-consuming and often require large quantities of highly pure compounds.

Methods
Therefore, this study demonstrates that selective gas-phase ion-molecule reactions of protonated analytes with neutral tris(dimethylamino)borane (TDMAB), followed by diagnostic collision-activated dissociation (CAD), can be used to differentiate aziridines from structurally related compounds in a modified linear quadrupole ion trap (LQIT) mass spectrometer. The experiments were performed using an atmospheric pressure chemical ionization (APCI) source in positive ion detection mode. TDMAB was purchased from Sigma-Aldrich and used without purification. It was introduced into the ion trap (2 mtorr of helium buffer gas) via an external reagent mixing manifold through a syringe pump at a flow rate of 10 μL h-1 and diluted with helium before entering the ion trap through a variable leak valve. All analytes were prepared at a concentration of 1 mM in methanol. The protonated analytes were transferred into the ion trap, isolated, and allowed to react with TDMAB (MS2 experiments).

Results
Product ions formed upon reactions of the analyte ions with TDMAB were subjected to a collision-activated dissociation (CAD) experiment (MS3 experiments). CAD on product ions produced diagnostic fragment ions with 25- and 43 m/z-units lower m/z values than the adduct - DMA product ions. Quantum chemical calculations were employed using the Gaussian 16 program calculated at the M06-2X/6-311++G(d,p) level of theory to provide theoretical foundation and to explore the underlying mechanisms of the observed gas-phase reactions. None of the tested structurally related analytes produced a similar fragmentation pattern

Conclusions
The innovative approach presented in this study offers a promising avenue for enhancing efficiency and accuracy in drug development. The successful differentiation of aziridine is an alternative method in overcoming the limitation of traditional techniques. The findings from the research contributes to the advancement of analytical techniques, addressing critical issues in identifying impurities in drug products. The versatility of the methods extends beyond drug developments, making it applicable in various other fields such as in molecular diagnostics, metabolism and laboratory medicines.
B-189 Alinity m You-Create Lab-Developed Test for hepatitis delta virus
Authors: Sasaki M , Wang X , Toolsie D , Cloherty G , Lucic D 
Published: October 2024

Abstract

Background
Lab developed tests (LDTs) play an important role in the realm of infectious diseases. Current molecular diagnostic platforms offer fully automated high-throughput performance capable of running LDTs. The Alinity m platform enables users to run LDTs concurrently with other FDA cleared or approved IVD assays with its Alinity m You-Create feature. Here, we investigated the performance of an LDT assay targeting hepatitis delta virus (HDV) using commercially available PCR reagents.

Methods
Alinity m You-Create LDT for HDV was evaluated using Applied Biosystems TaqMan Fast Virus 1-Step Multiplex Master Mix, No ROX, (ThermoFisher, Cat: 5555532). Sample extraction utilized Alinity m Sample Prep Kit 2. Alinity m You-Create HDV LDT analytical performance for sensitivity, linearity and precision was assessed using panels prepared by diluting the HDV 1st WHO international standard in negative plasma. Alinity m You-Create HDV LDT clinical performance was assessed by comparing 45 HDV positive samples previously tested on the m2000 HDV LDT. Assay specificity was assessed testing 97 HDV negative, HBV and/or HCV positive (by serology or nucleic acid amplification test).

Results
Alinity m You-Create HDV LDT had 100% detection as low as 2.5 IU/mL with SD ≤0.25 Log IU/mL for panels that were ≥2.5 IU/mL. Coefficient of correlation between Alinity m You-Create HDV LDT and m2000 HDV LDT was 0.969 and the mean bias was -0.16 Log IU/mL. All HDV negative specimens were undetected by Alinity m You-Create HDV LDT.

Conclusions
This study demonstrated successful implementation of an LDT for HDV on the Alinity m platform using the Alinity m You-Create feature using commercially available PCR reagents. The Alinity m You-Create feature for HDV has not been approved by FDA for use in the detection or diagnosis for HDV and its safety and effectiveness has not been established.