bluuebunny/crossref_metadata_2025_split huggingface dataset:
B-052 Establishment of New Antibodies and ELISA System to Detect the Potato Alkaloids α-Solanine and α-Chaconine
Authors: Okada K , Matsuo K 
Published: September 2023

Abstract

Background
Food poisoning by potato alkaloids, α-solanine (SO) and α-chaconine (CHA), currently occurs worldwide, however laboratory methods have not yet been established to detect these toxins in biological samples. In this study, we established new antibodies to detect SO and CHA and evaluated the practicality of enzyme-linked immunosorbent assays (ELISAs) using these antibodies in laboratory medicine.

Methods
Solanidine, a chemical compound found in both SO and CHA, was conjugated with bovine albumin, and this complex was repeatedly injected into two rabbits for two months. Two types of polyclonal solanidine antibodies were then purified from rabbit serum using affinity column chromatography. Using these antibodies, two forms of direct ELISAs (Sold ELISA-1, 2) were constructed. The basic detection performance of the ELISAs was confirmed by measuring the levels of SO and CHA diluted in phosphate-buffered saline (PBS). To simulate biological samples from patients with potato food poisoning, SO and CHA powders were mixed with commercially available human serum and urine, and SO and CHA levels were then measured using ELISA. Lastly, SO and CHA levels were detected in extracts from rotten potato (Irish Cobbler) sprouts, peels, and tubers.

Results
In samples of SO diluted in PBS, the limit of quantification (LoQ) was determined as 2.56 ng/mL and 9.55 ng/mL for Sold ELISA-1 and -2, respectively. In CHA diluted in PBS, the LoQs of Sold ELISA-1 and -2 were 1.36 ng/mL and 4.8 ng/mL, respectively. The LoQs of SO in the serum were calculated as 14.25 ng/mL and 19.41 ng/mL using Sold ELISA-1 and -2, respectively. In contrast, the LoQs of CHA in the serum were 12.48 ng/mL and 16.92 ng/mL using Sold ELISA-1 and -2, respectively. The LoQs of SO in the urine were determined as 30.28 ng/mL and 45.16 ng/mL using Sold ELISA-1 and -2, respectively. Conversely, the LoQs of CHA in the urine were 22.92 ng/mL and 38.15 ng/mL using Sold ELISA-1 and -2, respectively. Using Sold ELISA-1, both SO and CHA in the extracts from rotten potato tubers and peels were detected as approximately 1.7- and 2.3-fold higher, respectively, in each part than those in the extracts from fresh potatoes. When both SO and CHA were measured in the same samples using Sold ELISA-2, the levels were approximately 1.6- and 2.4-fold higher, respectively, in the tubers and peels than those in fresh potato samples. Furthermore, the potato sprout extract contained approximately 80-fold more SO and CHA than the tuber and 8-fold more than the peel.

Discussion and Conclusion
Based on our results, Sold ELISA-1 presented greater performance in detecting SO and CHA than Sold ELISA-2. The non-specific reactions in the serum samples and influence of urea in the urine samples may underlie the reduced detection performance of SO and CHA in ELISA, respectively. Although the sensitivity depends on the sample type, these ELISAs may be effective as future clinical and food testing methods.
B-055 Comparison of Sensitivity Between Tissue Anti-Transglutaminase and Anti-Endomysium IgA and IgG in Serological Screening for Celiac Disease in a Large Clinical Laboratory in Brazil
Authors: de Souza J F , do Sacramento Santos D , Pinto F A , Justino Feo A F , Vidotto Frade de Oliveira V , Zampieri G , Costa Gurgel Castelo M H , Correa Wengerkievicz Lopes A 
Published: September 2023

Abstract

Background
Celiac Disease (CD) is a genetic and autoimmune disorder characterized by permanent intolerance to gluten found in foods, resulting in small intestine injury in children and adults. Anti-tissue transglutaminase (anti-tTG) and anti-endomysium (anti-EM) antibodies are important serological markers of this disease. The aim was to analyze the analytical sensitivity of these markers in a large-scale Clinical Laboratory routine.

Methods
We employed a pure sample and a series of dilutions in the analysis of the functional sensitivity of each marker (anti-tTG IgA/IgG and anti-EM IgA/IgG) and a database survey of a large Clinical Laboratory in Sao Paulo/Brazil, from January to December 2021, analyzing 60 000 samples with concomitant results of anti-tTG IGA and anti-EM IgA and 7075 samples with results of anti-tTG IgG and anti-EM IgG, from patients with no defined clinic condition. The anti-tTG tests were performed by fluorescence enzyme immunoassay (FEIA) and the anti-EM tests by indirect immunofluorescence (IFI), with an initial dilution of 1:10, following the manufacturer's recommendations.

Results
The determination of functional sensitivity by checking the maximum dilution of antibody detection showed higher sensitivity of the anti-tTG IgA and IgG test (dilution factor 0.3 for both) compared to the anti-EM IgA and IgG (dilution factor 0.7 for both). Of the 60 000 samples analyzed for IgA markers, 59 083 (98.5%) were negative for both tests. Of the remaining 917 samples (1.5%), 609 (66.4%) were positive for both anti-tTG IgA and anti-EM IgA, 308 (33.6%) were positive only for anti-tTG IgA, and we did not observe any sample anti-tTG IgA negative with anti-EM IgA positive. Of the 7075 samples analyzed for IgG markers, 7032 (99.4%) were negative for both tests. Of the remaining 43 samples (0.6%), 18 (41.9%) were positive for anti-tTG IgG and anti-EM IgG, 14 (32.6%) were positive only for anti-tTG IgG, and 1 (2.3%) were positive only for anti-EM IgG.

Conclusion
The study showed greater sensitivity of anti-tTG compared to anti-EM for both IgA and IgG, in agreement with the literature data. The anti-tTG IgA test, in individuals without immunoglobulin A (IgA) deficiency, is recommended as the initial test in suspicion of celiac disease by current scientific guidelines on celiac disease. Based on our findings and literature data, in individuals with IgA deficiency, we suggest a screening using two automated IgG markers, tTG IgG and deamidated gliadin IgG (DG IgG). In addition to the lower sensitivity of anti-EM, indirect immunofluorescence (IFI), compared to the fully automated FEIA method, is a more subjective, observer-dependent, and labor-intensive methodology to be used as screening in large-scale Clinical Laboratory routines.
B-068 Reference Intervals for Serum Free Light Chains when Ruling out Monoclonal Gammopathies in Subjects with Normal or Minimally Variant Electrophoretic Findings
Authors: Stezar L , Elhady R , Zari H , Winston-McPherson G , Carey J , Cook B 
Published: September 2023

Abstract

Background
Serum free light chains (SFLC) analysis for detection and initial diagnosis of monoclonal gammopathies (MC) is a critical tool in the comprehensive assessment of risk and in management of established disease. Our reference intervals (RIs) for SFLC were based on the seminal report by Katzmann. Recent literature indicates with current analytic platforms and reagent formulations, significantly different RIs might be more appropriate.

Methods
The study sought to establish RIs for the population typically evaluated for possible MC and was expected to include acute/chronic inflammation, polyclonal elevations of gamma-globulins, hypogammaglobulinemia and chronic liver disease. The reference population inclusion criteria were: serum submitted for a serum monoclonal protein evaluation and SPE (± IFIX); lacked any direct or indirect evidence of a MC; normal or minimally variant SPE pattern; no prior history of leukemia, lymphoma, myeloma or MGUS; normal eGFR (CKD1–2 or &gt;60 mL/min). CKD-EPI calculation was based only on IDMS-standardized creatinine measurements without race modifiers. Of the 202 cases encountered over 15 days, 135 met the inclusion criteria. Specimens were tested using a Binding Site Optilite system.

Results
The mean subject age was 62 years. There was a mild female predominance (80 females vs 55 males) and females tended to be younger than males (58 vs 68 years). Two approaches used to establish the central 95% interval (mean ± 2SD and ranked-order analysis for 2.5 and 97.5 percentiles) yielded similar limits for SFLC and SFLC ratio (Table). These results agree well with recent reports from others.

Conclusion
As international guideline prognostic thresholds for SFLCR in plasma cell neoplasia are based upon the original Binding Site free light chain analysis, we will maintain those RIs, adding an appended comment to all SFLC reports to indicate for screening purposes, the HFH laboratory-established SFLC ratio interval is 0.8–1.9:1.
B-096 Are we truly Choosing Wisely? An Assessment of non-specific testing, time to diagnosis and costs related to the diagnosis of Rheumatoid Arthritis
Authors: Borunda Duque T , Joy V 
Published: September 2023

Abstract

Background
Treatment of Rheumatoid Arthritis (RA) early in the disease course can significantly impact patient quality of life by preventing the development of joint erosion and slowing down disease progression. Timely diagnosis is a critical aspect of early treatment. Most patients initially present within a primary care (PC) setting where a variety of non-specific tests/test algorithms are used. We reviewed 8 years’ worth of RA real-world laboratory data to assess time to diagnosis (TTD) between different tests/test combinations.

Methods
Data extraction spanning 2014–2021 was pulled from a US laboratory, using Rheumatoid Factor (RF) and Anti-Cyclic Citrullinated Peptide (CCP) positivity as the primary inclusion criteria. Additionally, sex, age, ordering physician specialty, ICD-10 codes, test order date and test codes (ESR, CRP, ANA screen, RF isotypes) were pulled longitudinally.

Results
This data set includes 3024 patients with RA (RA ICD code). Of these, 45% were first suspected and evaluated within PC. TTD between patients initially tested with only CRP or ESR had a median TTD of 920 days and those tested with ANA only had a median TTD of 685 days. Those tested with RF + CCP or RF + CCP + ANA had a median TTD of 367 days and 290 days, respectively.

Conclusion
Our data set highlights the impact of test methods on TTD of patients with RA, with tests combinations more specific for RA significantly decreasing TTD. Further analysis will be conducted to determine the downstream implications resulting between these diagnostic delays. Standardizing and implementing initial testing algorithms could potentially decrease RA TTD, and directly impact burden of care.
B-126 Comparison of Liftover Tools for Conversion to Genome Reference Consortium Human Build 38 from Human Genome 19
Authors: Park K , Park J 
Published: September 2023

Abstract

Background
Genome Reference Consortium Human Build 38 (GRCh38) was released with improvements such as accuracy and completeness over human genome 19 (hg19). However, GRCh38 has not been widely adopted in clinical laboratories because re-alignment is time-consuming and computationally-expensive. To address this issue, faster and more convenient liftover tools have been developed to convert genome coordinates between assemblies. The aim of this study was to compare the performance of liftover tools for conversion to GRCh38 from hg19.

Methods
Two variant call format (VCF) files aligned to hg19 (No. of clinical variants = 1 570 644) and GRCh38 were downloaded from ClinVar (clinvar_20221217.vcf.gz). The liftover tools such as CrossMap, NCBIRemap, and UCSCliftOver were used to convert genome coordinates from hg19 to GRCh38. We investigated the conversion failure of clinical variants and evaluated classification of variants provided by multiple submitters with assertion criteria and evidence according to ClinVar review status, to ensure the authenticity of the variants.

Results
The conversion rates of CrossMap, NCBIRemap, and UCSCliftOver were 99.64% (1 567 848/1 570 644), 99.52% (1 565 963/1 570 644) and 99.81% (1 570 560/1 570 644), respectively. The variants which failed conversion were identified in ABCC9 (n = 2), ACACA (n = 1), CNOT3 (n = 3), CYFIP1 (n = 1), DGAT1 (n = 9), EMG1 (n = 1), GAS2L2 (n = 5), GDF2 (n = 38), GPR179 (n = 96), HNF1B (n = 132), HYOU1 (n = 2), MBOAT7 (n = 5), MYO19 (n = 7), NSDHL (n = 15), ODAD3 (n = 1), ORAI1 (n = 2), PCGF2 (n = 3), PQBP1 (n = 19), PRPF31 (n = 56), PUF60 (n = 9), RBP3 (n = 52), RPS17 (n = 1), SARM1 (n = 1), SCARF2 (n = 1), SLC35A2 (n = 27), SLC39A4 (n = 25), SLC52A2 (n = 86), SURF1 (n = 65), TAF15 (n = 8), TFE3 (n = 2), TSEN34 (n = 15), WDR45 (n = 56), ZNHIT3 (n = 1) genes. The numbers of pathogenic or likely pathogenic variants among variants which failed conversion by CrossMap, NCBIRemap, and UCSCliftOver were 67, 115, and 0, respectively. Classification of converted variants from GRCh37 to GRCh38 remained unchanged.

Conclusions
This study demonstrated that UCSCliftOver generates most reliable results. This study suggests that liftover tools might be one of the practical alternatives for genome conversion in case that re-alignment approaches were not possible.
B-170 Identifying Metabolic Phenotyping Variability in a Cellular Model of Barth Syndrome
Authors: Kodger J 
Published: September 2023

Abstract

Introduction
Barth Syndrome is an X-linked multisystem disease caused by the mutation in the TAZ gene (TAZ, G 4.5, OMIM 300394) that encodes for acyltransferase taffazin. Taffazin is a mitochondrial protein highly expressed in heart and skeletal muscle and has a central role in cardiolipin remodeling process. TAZ is the only known single gene defect in cardiolipin remodeling, with more than a hundred TAZ mutations identified. However, no systematic correlation has been established between TAZ genotypes and clinical or biochemical phenotypes in Barth syndrome. There is a large phenotypic heterogeneity among affected individuals sometimes even within the same family. This raises a possibility of genetic and metabolic modifiers involved in TAZ clinical and metabolic phenotypes. The aim of this study is to apply LC-MS/MS untargeted and targeted metabolic analyses to explore possible correlations between two TAZ genotypes and metabolome in a cellular model of Barth Syndrome.

Methods
Mutant TAZ 3(c.647G&gt;T), TAZ 4(c.778-24_778-7delinsA) fibroblast and matching by donors’ age and gender control fibroblast lines (healthy#1, healthy #2) were cultured under identical conditions. Cells (n = 6 for each genotype) were harvested and extracted followed Bligh and Dyer protocol. Extracted metabolites were analyzed by Agilent 6545 Q-TOF coupled with 1290 Infinity II HPLC. Chromatographic separation was achieved by using a Water Premier BEH Z-HILIC Column (2.5 µM, 2.1 × 150 mm) and Waters X-select HSS T3 (2.5 µM, 2.1 × 100 mm) columns for polar and non-polar metabolites respectively. Raw data was normalized with internal standard and total cellular protein amounts. Statistical and pathway enrichment analyses were performed with a chemometric platform Mass Profiler Professional (Agilent) and Metaboanalyst webtool.

Results
Data was treated first as for two separate genotypes (healthy vs TAZ) followed and compared to the analysis with four separate genotypes (healthy#1, healthy #2 TAZ c.647G&gt;T, TAZ c.778-24_778-7delinsA). Metabolites contributing to discrimination of the genotypes based on metabolic profiles were identified by using a univariate analysis (P &lt; 0.05) and a supervised PLSDA (VIP&gt;1 for small polar metabolites and VIP &gt; 3 for non-polar molecules). The differentially expressed metabolites included amino acids, acylcarnitine, Krebs cycle intermediates, ATP, 3-methylglutaconic acid, fatty acids and a variety of phospholipids. Pathway enrichment analysis revealed that several metabolic pathways were involved.

Conclusions
Our analyses reveal that genetic variations in TAZ gene cause the differences in the abundance of certain metabolites and affected pathways. This leads us to the conclusion that from the point of view of metabolic pathophysiology, TAZ should be considered beyond a single-gene defect and a more personalized approach is needed in search for potential therapies.
B-182 Advantage of Core-Shell Particle Morphology Over Fully Porous HPLC Column for the Analysis of Vitamin K Compounds by LC-MS/MS
Authors: Huq S W , Aqeel Z 
Published: September 2023

Abstract

Background
Vitamin K’s are a group of structurally similar, fat-soluble compounds. There are two natural forms K1 (Phylloquinone) and K2 (Menaquinone), where the later has short and longer chain homologues called Vitamin K2-MK-4 and Vitamin K2-MK-7 respectively. Due to the extremely hydrophobic nature and poor ionization characteristics of these compounds, LC-MS/MS analysis at low concentrations becomes challenging. In this communication we make parallel comparisons of three HPLC columns packed with fully porous and core-shell particles to showcase the superiority of core-shell columns for sensitive detection of vitamin K analytes by LC-MS/MS.

Method
We compared fully porous (Gemini and Luna, Phenomenex, Torrance, CA) and core-shell (Kinetex EVO, Phenomenex) C18 columns for this study. All columns evaluated were 50 × 2.1 mm with a 5 μm particle size to maintain low back pressure for the viscous organic modifier utilized. Deionized water and a combination of isopropanol and acetonitrile (1:1; v/v) were used as mobile phase A and B respectively for chromatographic separation. An Agilent 1290 Infinity series LC system was employed with a SCIEX 6500 QTRAP mass spectrometer under APCI for MS detection. Analyst software 1.7.2 used for data analysis.

Results
The LC-MS/MS method had a 4-minute runtime for chromatographic separation of the analytes including 1.5 min of equilibration time. The comparison data revealed the Gemini C18 column shows some higher selectivity and resolution values, whereas both EVO and Luna C18 columns were somewhat comparable. However, the core-shell EVO C18 column displayed much higher efficiency with narrower and compressed peak widths, which dramatically increased the peak height for all 3 analytes exhibiting the highest signal/noise ratio.

Conclusion
The core-shell Kinetex 5 μm, EVO C18 column with its unique organo-silica grafting shows much higher efficiency and sensitive detection capability for vitamin K analysis by LC-MS/MS compared to that of conventional fully porous silica particles.
B-194 Development and Validation of Rapid LC-MS/MS Method for Simultaneous Determination of 8-iso-PGF2α and 11-DHTXB2 in Human Urine
Authors: Abdelkader N , Odat B 
Published: September 2023

Abstract

Background
Cardiovascular disease (CVD) is one of the leading causes of death globally, the prevalence ofCVD is expected to increase from 36.9% to 40.5% between 2010 and 2030 in the United States.Atherosclerosis (AS) is thought to be the primary cause of cardiovascular diseases (CVDs),however there are several others. About half of Americans between ages 45 and 84 haveatherosclerosis and don’t know it. Eicosanoids (8-iso-PGF2α and 11-Dehydrothromboxane B2) arethe most common biomarkers for predicting atherosclerosis. The objective of this study was todevelop a rapid LC-MS/MS method for simultaneous determination of 8-iso-PGF2α and 11-DehydrothromboxaneB2 (11-DHTXB2) in human urine.

Methods
One milliliter of human urine spiked with 0.5 µL of each internal standard (1 µg/mL) was mixedwith 1 mL of heptane, 1 mL of ethyl acetate, and 1 mL of Acetonitrile in a glass tube followed by theaddition of 1 mL of pure water. The mixture was vortexed for 1 min and then centrifuged (4000rpm) for f 5 min. The middle organic layer was transferred to another tube and dried undernitrogen and resuspended with 50 µL of the initial mobile phase composition (70:30 v/v H2O:ACN) for the analysis. One milliliter of human urine spiked with 0.5 µL of each internal standard(1 µg/mL) was loaded to a conditioned SPE cartridge, then washed with 1 mL of 10% ACN afterthat analytes eluted with 1 mL of pure ACN at the end eluent is collected, dried and reconstitutedin 50 µL of (70:30 v/v H2O: ACN). LC-MS/MS was performed using AB Sciex 5500 QTRAPtriple quadrupole tandem mass spectrometer coupled to Shimadzu HPLC with 10 minutesgradients for both samples.

Results
The method was optimized by using standard solutions of 8-iso-PGF2α and 11-dehydrothromboxane B2 and showed separation at 5.27 and 5.45 min respectively. The methodapplied on pooled urine sample from healthy individuals and showed No bias in retention time.Two purification methods were used to extract the two analytes from pooled urine samples, threephase liquid extraction (3PLE) and solid phase extraction (SPE). 3PLE showed a better resultsSPE method in eicosanoids extraction which results in higher intensity signals by 2-fold for 8-iso-PGF2α and 5-fold for 11-DHTXB2.The method was shown to be linear from 0.1 to 5 ng/mL forboth analytes (R2 ≥ 0.98).

Conclusion
The three-phase liquid extraction method extracted the two analytes better than the solid phaseextraction method, which is the most used purification method for eicosanoids. Furthermore, arapid and simple LC-MS/MS method for simultaneously measuring urinary 8-iso-PGF2α and 11-DHTXB2 was successfully developed.
B-214 Serial Changes of Donor-derived Cell-free DNA in Pancreas Transplant Recipients
Authors: Lee H , Shin K , Choi B , Lee M , Shin K 
Published: September 2023

Abstract

Background
Pancreatic transplant patients need invasive pancreatic biopsies to diagnose graft rejection. Organ damage following organ donation yields circulating tissue-isolated donor-derived cell-free DNA (ddcf-DNA). Based on the ddcf-DNA ratio or quantity in several transplantations, graft rejection is predicted. Thus, we examined a 2-week ddcf-DNA monitoring study after pancreatic transplantation at this medical institution.

Methods
The current prospective observational study examined five pancreas transplant recipients who were sampled on postoperative days (PODs) 4, 7, 14, 30, 60, 90, 120, and 180 following the study protocol. Serum levels were determined for the following: amylase, lipase, and tacrolimus. Cell-free DNA (cf-DNA) was isolated from the plasma using a NucleoSnap cf-DNA extraction kit. The QX200 Droplet Digital PCR System was used to measure ddcf-DNA. To analyze transplant rejection and differentiate between donor and recipient, all selected single nucleotide polymorphisms (SNPs) met the following criteria: known and validated minor allele frequency of 43% in Koreans and reported ethnicities from global populations.

Results
The median patient age was 35 years. All recipients achieved euglycemia without exogenous insulin immediately after the surgery. All 6-month postoperative hemoglobin A1c levels were within the normal range (4%–6%). One (20%) of the five patients with pancreas transplant had a T cell-mediated rejection event on POD 531. For PODs 4, 7, 14, 30, 60, 90, and 180, the percentage (median, range) of ddcf-DNA was 2.7 (0.4–5.5), 0.6 (0.2–4.4), 3.7 (0.4–12.7), 7.1 (0.4–7.9), 2.1 (0.1–5.4), 2.0 (0.3–8.5), and 0.3 (0.2–7.6), respectively; and amylase (median, range) was 68.0 (30.0–80.0), 82.0 (57.0–151.0), 74.0 (66.0–157.0), 81.0 (50.0–137.0), 74.0 (39.0–111.0), and 80.0 (27.0–98.0). (43.0–105.0), respectively. The ddcf-DNA value of POD 14 was greater than 10% in individuals who had TCMR on POD 530.

Conclusion
In our study, patients with high ddcf-DNA on POD 14 after transplantation respond poorly to immunosuppressive agents, and rejection was later confirmed.