bluuebunny/crossref_metadata_2025 huggingface dataset:
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

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

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 >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-086 High Precision T Cell Testing Using the T-SPOT Technology: Improved Cell Yields From Lower Volumes of Blood When the T-SPOT Technology is Used With the T-Cell Select Reagent Kit for Cell Isolation
Authors: Jackson-Soutter T , Brignall R , Quinn M H , Ahmed R S , Dudek M M
Published: September 2023

Background

T cells play a critical role in protecting humans from disease, and are a powerful diagnostic tool when used to measure immune responses to infection. However, the use of T cells in a clinical setting has been limited due to challenges associated with T cell testing methodologies. One of the key challenges has been the ability to scale up T cell testing to meet the needs of clinical laboratories. The T-SPOT technology is most widely known for the T-SPOT.TB test, an Interferon-Gamma Release Assay (IGRA) diagnostic test based on the detection of Mycobacterium tuberculosis-specific effector T cells using a single cell resolution platform (ELISPOT). The conventional method of running the T-SPOT.TB test was to use density gradient isolation on 6 mL of whole blood to isolate peripheral blood mononuclear cells (PBMCs) for high precision T cell testing. However, this testing was not accessible to some clinical laboratories due to the hands-on time associated with isolating PBMCs from whole-blood. The T-Cell Select reagent kit was developed to positively select mononuclear cells and enhance cell separation efficiency for the T-SPOT technology. The T-Cell Select reagent kit offers the opportunity to automate the T-SPOT technology which improves scalability and reliability, but it also means that an improved cell yield can be achieved from a lower volume of blood. This makes high precision T cell testing more accessible, whilst maintaining the high sensitivity and specificity of the T-SPOT technology.

Methods

PBMCs from 242 samples were isolated from 6 mL of whole blood, and were processed according to the T-SPOT.TB test package insert for density gradient isolation. Matched samples were used to isolate PBMCs from 3.5 mL of whole blood using magnetic bead-based positive selection according to the T-Cell Select reagent kit package insert.

Results

Cell yields were compared using matched samples for density gradient and the T-Cell Select reagent kit isolated cells. Cell count means were 6.6 vs 6.7 × 10^6 cell/mL for the T-Cell Select reagent kit and density gradient isolation methods, respectively. Of the 242 samples tested, two samples had insufficient yields to run the T-SPOT.TB test with the density gradient method, whilst the T-Cell Select reagent kit isolated sufficient numbers from all samples. Results of the T-SPOT.TB test had an overall agreement between the two methods of 97%, negative agreement 99% and positive agreement 94%.

Conclusion

When used with the T-SPOT technology, the T-Cell Select reagent kit provides an improved cell isolation methodology, requiring up to 50% lower blood volumes, and reducing the hands-on time associated with T cell testing. This makes T cell testing more accessible to labs, meaning that fewer blood samples need to be redrawn due to low cell numbers, providing an advantage in the immunosuppressed.
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

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

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

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>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>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 < 0.05) and a supervised PLSDA (VIP>1 for small polar metabolites and VIP > 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

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

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

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.
B-221 Quantitation of DNA in Antibody-based and RNA-based Drugs by Quantitative Polymerase Chain Reaction (qPCR)
Authors: Su J , Uriu J , Aguilar E N , Tobias R , Marr G
Published: September 2023

Background

Current FDA regulation defined acceptance level of 10 ng per dose of residual DNA in drug products poses great challenges for accurate quantification of residual DNA, particularly for the products used in high doses or formulated in complex matrixes. The purpose of this study is to develop and qualify a qPCR method for the quantification of Chinese Hamster Ovary (CHO) DNA in antibody-based drugs and for quantitation of human DNA in RNA-based drugs.

Methods

An 8-point standard curve consisting of 0.005, 0.05, 0.5, 5, 50, 500, 1000 and 5000 pg CHO DNA and a 5-point standard curve consisting of 0.3, 3, 30, 300 and 3000 pg human DNA was generated by serial dilutions. The antibody sample (300 µg) was spiked with 2.5, 10, and 50 pg CHO DNA. The RNA sample (1.6 µg) was spiked with 0.3, 3 and 30 pg human DNA. The samples were analyzed by qPCR using a Real-Time PCR instrument. Commercial PCR test kits (TaqMan™ Probe and TaqMan™ Universal Master Mix), and sequence specific forward and reverse primers were used.

Results

For CHO DNA in antibody product, the linearity slope and R2 are −3.5 and 1.00, respectively, which met system suitability criteria. The accuracy by spiked recovery of 2.5 pg, 10 pg and 50 pg CHO DNA in 300 µg antibody, 150 pg CHO DNA in formulation buffer, and 150 pg CHO DNA in 1000 pg human DNA are 70%, 76%, 86%, 93% and 95%, respectively. The precision of the assay for 2.5 pg, 10 pg, and 50 pg of CHO DNA spiked in 300 µg antibody are 6%, 7% and 2%, respectively. The QL of the assay is 2.5 pg CHO DNA per 300 µg of antibody product.For human DNA in RNA product, the linearity slope and R2 are −3.3 and 1.00, respectively, which met system suitability criteria. The accuracy by spiked recovery of 0.3 pg, 3 pg, and 30 pg human DNA in 1.6 µg RNA, 3 pg human DNA in formulation buffer, 1.5 pg human DNA in 150 pg CHO DNA are 113%, 104%, 102%, 97% and 88%, respectively. The precision of the assay for 0.3 pg, 3 pg and 30 pg human DNA spiked in 1.6 µg RNA are 6%, 5% and 2%, respectively. The QL of the assay is 0.3 pg human DNA per 1.6 µg of RNA product.

Conclusion

Two qPCR methods were developed to quantify residual DNA in drug products. One method is specific to quantitation of low levels of CHO DNA in antibody product. Another method is specific to quantitation of low levels of human DNA in RNA product.
B-264 Identification of Potential Biomarkers Related to Major Depressive Disorder Using a Meta-Analysis Approach
Authors: de Carvalho A H , de Lima A B , Dias H D , Zauli D A
Published: September 2023

Background

Major depressive disorder (MDD), also known as depression, has become the most prevalent mood disorder today. Currently, the diagnosis remains a major challenge given the disease heterogeneity and have been made by associating signs and symptoms with other diagnostic criteria. Several treatment options are available for MDD. However, less than 35% of patients achieve remission while 60% may show some degree of resistance to treatment even when receiving antidepressant treatment according to consensus guidelines and using measurement-based care. Between difficulties associated with the management of MDD, the lack of consensus on classification, diagnosis, and treatment stands out. Because of the incomplete understanding of MDD pathogenesis, it is urgent to develop studies that can help in the elucidation of the mechanisms related to the disease and the identification of biomarkers that allow its correct classification. Thus, this work aimed to perform a meta-analysis study to identify potential molecular biomarkers from different NGS databases.

Methods

Datasets from seven association studies stored in the IEU Open GWAS Project Database were selected. The meta-analysis was performed using the METAL tool (https://genome.sph.umich.edu/wiki/METAL_Documentation), which combines the relevance of the p-values, sample size, and the direction of the effect size. The criteria adopted for selecting the datasets were: (1) Specification of “depression”, “major depressive disorder” or “major depression” as the only phenotype; and (2) description of allele frequency data. The threshold for results significance was defined as P-value ≤5 × 10−8. After performing the analyses, only the SNPs whose direction of effect was the same in all original studies were evaluated, considering only the set of SNPs that were not significant in the original studies.

Results

After performing the meta-analysis, 348 new SNPs with P-value ≤5 × 10−8 were identified and, of those, 133 SNPs were mapped next to 17 genes. None of these genes was previously associated with depression. However, the GABBR1 gene, which encodes a type B GABA receptor subunit, has been associated with alcoholism, while the SORCS3 gene has been associated with attention deficit/hyperactivity disorder and Alzheimer’s disease.

Conclusions

The meta-analysis findings could help elucidate genes and polymorphisms involved in MDD pathogenesis. Due to MDD heterogeneity is nearly impossible to restrict and select a single disease biomarker. It is worth noting that only the identification of genetic polymorphisms involved in MDD development cannot be the only source of potential biomarkers for MDD. Indeed, it is necessary to extend the analysis to other components, such as regulatory regions, epigenetics, metabolites, etc. However, this work provide new insights into the identification of MDD potential biomarkers that can be better investigated in association to others factors related to the disease.
B-271 Molecular EQA Programs Verify the Use of a Quick Lysis Reagent in a Wide Range of Matrices and Pathogens
Authors: Martínez-Santolaria M , Sota-Diez C , Gaston-Galvez B , García-Manrique B , Machetti-Mareca E , Gil-Rodríguez C
Published: September 2023

Background

External Quality Assessment (EQA) programmes, like Quality Control for Molecular Diagnostics (QCMD), allow laboratories to verify the correct functionality of the molecular workflows used in the diagnosis of different pathogens. The aim of this study was to evaluate whether a quick molecular workflow had a good functionality for the diagnosis of different pathogens.

Methods

A total of 90 frozen samples from 2022 QCMD programmes were analysed. 20 transport medium (TM), 9 TM or saline and 1 saline samples from respiratory panels (QAV164188, QAV164189, QAM174193), 20 synthetic faecal matrix samples from gastrointestinal panels (QAP124154, QAB124153), 13 TM, 4 simulated swabs and 3 urines from sexual transmitted infections (STI) panels (QAB154177, QAM174201), and 18 TM and 2 synthetic cerebrospinal fluid from central nervous system (CNS) panels (QAV174195, QAM174196). Samples were processed using an in-house processing method called quick lysis reagent (QLR) (Certest Biotec S. L.). In parallel, samples were also extracted using the automatic robot KingFisher (Thermo Fisher Scientific). Nucleic acids were analysed at least in duplicate with qPCRs from VIASURE using the CFX96™ Real-Time PCR Detection System (Bio-Rad). Results were compared to the final diagnosis provided by QCMD and with the automatic extractor results.

Results

The in-house processing method QLR, in combination with VIASURE qPCRs, could process and identify bacteria, parasites and viruses from respiratory, gastrointestinal, STI and CNS panels. Negative samples were correctly detected. This workflow obtained a 100% of sensitive and specificity, same as the automatic extractor.

Conclusion

These EQA programmes tested the correct functionality of a quick molecular workflow, based on the VIASURE diagnostic products, that could detect different pathogens (bacteria, viruses, and parasites) from different origins (respiratory, gastrointestinal, STI and CNS).
B-299 Liquid Chromatography-mass Spectrometry Measurement of Drugs of Abuse and Alcohol Biomarker Phosphatidylethanol (peth 16:0/18:1) Using Volumetric Dried Blood Spot Device
Authors: Göksu Gürsu G , Maviş M E , Yılmaz H
Published: September 2023

Background

Reliable biomarkers have great significance in the investigation of alcohol consumption patterns and in the treatment of alcohol-induced diseases. Direct biomarkers to detect alcohol intake are non-oxidative products of ethanol metabolism (1, 2). Phosphatidylethanol (PEth), blood-based direct alcohol biomarker, represents a group of unnatural phospholipids formed on leukocytes and predominantly erythrocyte membranes. The presence of PEth in blood samples is an indisputable indicator of ethanol consumption (3, 4). Moreover, detecting drugs of abuse (DOA) including opiates, amphetamines, cocaine metabolites, benzodiazepines, THC-metabolites, MDMA, methadone, fentanyl etc. is important to establish patterns of illicit drug usage. In recent years, DBS has been gaining interest in plenty of research areas owing to ease of specimen collection and storage (5, 6).The main objective of this study was to determine the concentration of PEth-16:0 18:1 in DBS samples collected using a volumetric dried blood spot (DBS) device (HemaxisTM DB) and to identify/quantify drugs of abuse (including 119 parameters-Jasem Clinical Toxicology mixture) using another blood spot punch of the same DBS sample.

Methods

According to Jasem method, DBS samples were prepared by sampling from spiked 20 µL of blood drop located onto paraffin film. PEth-16:0 18:1 and DOA were extracted from entire blood spot of DBS specimens implementing two different extraction reagents at room temperature for 10 min then subjected to HPLC system equipped with Agilent 6470 TQ. The total run times from injection to injection for PEth and DOA were 4.0 min and 12.0 min respectively.

Results

The linearity and accuracy of the methods were evaluated using 5 DBS calibrators levels for PEth and 6 DBS calibrators levels for DOA. Linearity was confirmed in the range 10 to 1000 ng/mL (r² > 0.995) for PEth 16:0/18:1 and in the range 2 to 100 ng/mL for DOA (r² > 0.99). For two methods linearity, RSD% (inter-intraday) and accuracy were within the analytical acceptable ranges.

Conclusion

The measurement of blood PEth levels provides a precious tool to determine the chronological profile based on retrospective alcohol intake in a reliable way. Besides, simultaneous analysis of DOA containing 119 parameters ensures comprehensive analytical solution and clinically relevant information. Both of Jasem LC-MS/MS approaches are centred on simple sample preparation following non-invasive sample collection method. Prior to rapid extraction, applying volumetric DBS device assures to collect fixed volume of blood regardless of blood haematocrit level making the measurements more consistent.
B-307 Direct Quantitation of 77 Therapeutic and Clinical Toxicology Drugs in Dried Blood Spots Using the Fully Automated Transcend DSX-1 System
Authors: Guo J , Patterson C , Gibson R , van Natta K , Samra S
Published: September 2023

Background

The dried blood spot (DBS) sampling technique is advantageous over the traditional liquid blood collection due to its minimal invasiveness, smaller sample volume, improved analyte stability, and ease of storage and transportation, resulting in its increasing usage in therapeutic drug monitoring and clinical toxicology. Here, we describe a fully automated workflow to rapidly extract and quantify a wide range of therapeutic drugs and drugs of abuse in DBS using the Thermo Scientific™ Transcend™ DSX-1 system, fulfilling the routine drug monitoring needs in the clinical laboratories.

Methods

Calibrators in blood are spotted on the DBS cards, analytes are extracted with the innovative flow-through desorption technology, and their corresponding internal standards (IS) are introduced via an automated IS addition module in the dried spot autosampler. The 2-dimensional TurboFlow technology allows the removal of interferences from the extracted samples before analytical separation. An integrated software controls every step of the sample desorption and separation. Analyte quantitation is performed on the Thermo Scientific™ TSQ Altis™ Plus triple quadrupole mass spectrometer, and the data is analyzed in TraceFinder™ software.

Results

A total of 77 therapeutic and clinical toxicology drugs from eleven classes, including anticonvulsants, antidepressants, antihistamines, antipsychotics, benzodiazepines, cocaine, dissociatives, opioids, and stimulants, are quantified in a single injection from DBS cards using a rapid automated method on a Transcend DSX-1 system. DSX-1 combines a dried spot module for direct analyte extraction with Transcend™ UHPLC for online sample separation using the TurboFlow technology. The method only takes 4.3 min from analyte extraction to MS detection. Good calibration curves with R2 > 0.98 are achieved using a weighting factor of 1/x, and the limit of quantification (LOQ) values are established with % RSD and % CV < 15, │% Diff│ < 20, and relative ion ratio < %20. The LOQ values are all in the low ng/mL levels, which largely meet the screening and confirmation sensitivity needs of analytical methodologies in routine clinical laboratories.

Conclusion

A fully automated LC-MS-based workflow is developed to rapidly and reliably quantitate 77 compounds therapeutic and clinical toxicology drugs in dried blood spots.
B-316 Retrospective Data Analysis Reveals Unusual Metabolism Pattern of Ethanol in Pediatrics as Compared to Adult and Geriatric Populations
Authors: Baryeh K , Zhou R , Zuromski L M , McMillin G A , Krasowski M D , Johnson-Davis K L
Published: September 2023

Background

Ethanol is utilized in many pharmaceutical formulations and in the treatment of methanol poisoning. It is also consumed recreationally and is the most abused drug globally. About 95% of consumed ethanol is metabolized by alcohol dehydrogenase into acetaldehyde, followed by conversion of the acetaldehyde into acetic acid by acetaldehyde dehydrogenase. Less than one percent is metabolized via nonoxidative pathways, which may include: glucuronidation, sulfation, and the formation of fatty acid esters of ethanol. In neonates the glucuronidation pathway has been reported to be underdeveloped and matures with age. In this work, the patterns and concentrations of phase II nonoxidative metabolites of ethanol, ethyl glucuronide (EtG) and ethyl sulfate (EtS), were assessed in random urine collections of pediatric, adult, and geriatric patients.

Methods

Test results (n = 63498) from urine samples tested for EtG and EtS by quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) at our facility were utilized for this study. For both analytes, the lower limit of positivity was 100 ng/mL. The age range under consideration was 0–100 years. Data was partitioned into age groups: 0–17 years, 18–80 years and 81–100 years. The EtG and EtS concentrations across the partitions were compared.

Results

Across all ages, 60%–65% of patients had both EtG and EtS present in urine. Between 5%–10% had EtG present without EtS, and 25%–35% of patients had neither of the ethanol metabolites present. In all age groups there was a low positivity rate of EtS without the presence of EtG (0%–1%). All age partitions showed a similar pattern with the highest percentages of patients showing the presence of both metabolites, and the lowest percentage of patients showing the presence of EtS only. Markedly, there were no pediatric patient samples that had the sulfated metabolite present in the absence of the glucuronidated metabolite; this was statistically significantly different than this proportion in adults (Fisher’s Exact test, P = 0.025). This finding suggests greater glucuronidation activity in the pediatric population.

Conclusion

From the data presented in this work, EtG is more prevalent relative to EtS in urine samples of patients assessed for ethanol exposure. This work also shows that glucuronidation may be the predominant pathway for ethanol metabolism in pediatric populations. Although the level and frequency of ethanol exposure, as well as the time between exposure and sampling were unknown for the patients studied, the data offers insights into the relative distribution of EtG and EtS in random urine samples for pediatric and adult populations.
B-321 Development of Chemiluminescent Enzyme Immunoassay (CLEIA) for Detecting Methotrexate in Blood
Authors: Inagaki E , Baba K , Imai Y , Kaneko A , Aoyagi K
Published: September 2023

Background

Methotrexate (MTX) is a folate metabolism antagonist used in the treatment of rheumatoid arthritis, psoriasis, and antineoplastic agents. Therapeutic drug monitoring (TDM) is required to avoid serious adverse reactions of high-dose MTX/leucovorin rescue therapy or in patients with possible delayed excretion due to renal dysfunction or other factors. Although there are many reagents for MTX measurement based on the immunoassay method, they have a narrow measurement range to 2 µmol/L. Therefore, high concentration samples often make users conduct dilution and retest. In addition, most reagents have a cross-reactivity rate of over 40% with 2,4-diamino-N10-methylpteroic acid (DAMPA), one of the metabolites of MTX, so users must take note in the interpretation of measurement results. We have established an antibody with high specificity against MTX, and have developed a fully-automated, chemiluminescence immunoassay for LUMIPULSE® L2400 system for detecting MTX in blood, Lumipulse Presto® Methotrexate, which can significantly improve the issues described above.

Methods

MTX assay is a one-step competitive immunoassay on the LUMIPULSE® L2400 System (FUJIREBIO INC.), which requires 10 µL of sample. The resulting reaction signals are derived within 30 min/sample, and are proportional to the amount of MTX in the sample allowing quantitative determination of MTX in plasma or serum.

Results

The detection limit of the assay was 0.019 µmol/L, and the limit of quantitation was 0.030 µmol/L. A 20-day precision study was performed during a 31-day period using two controls and three panel specimens, and within-laboratory precision showed CV ≤ 3.1 %. Linearity was demonstrated over the range 0.017–28.821 µmol/L. No interference was observed with unconjugated (≤20.1 mg/dL) or conjugated bilirubin (≤20.2 mg/dL), chyle (≤1770 FTU), hemoglobin (≤450 mg/dL), and RF (rheumatoid factor, ≤5522 IU/mL). The correlation coefficient and the regression slope of this assay and LC-MS/MS were 1.00 and 1.0, respectively (N = 171). Compared to existing immunoassay reagents, the assay showed much lower cross-reactivity to DAMPA.

Conclusion

The performance of our novel MTX assay using LUMIPULSE® L2400 system was satisfactory, suggesting it is useful for routine analysis. In particular, the measurement range is wide, 0.030 to 25.000 µmol/L, which greatly reduces the frequency of dilution and retest of high concentration samples. Used with the auto dilution system in LUMIPULSE® L2400, it is possible to measure almost all clinical specimen automatically. Because of the low cross-reactivity with DAMPA, it is expected to be utilized in patient receiving glucarpidase.

Acknowledgment: Some of the samples were provided in accordance with “Guidelines for clinical measurement and diagnosis technology improvement project”. The guidelines are promoted by Tsukuba Medical Laboratory of Education and Research Center, and University of Tsukuba Hospital, Japan.
B-322 Assessment of MassTrak™ Immunosuppressant Calibrator and Control Sets: Confidence in Your Calibrators
Authors: Hammond G , Balloch S , Calton L
Published: September 2023

Background

The analytical performance of the Waters MassTrak Immunosuppressant Calibrator and Quality Control Sets (IVD) were evaluated using a laboratory developed LC-MS/MS method, to analyze all four immunosuppressants in a single run.

Methods

The MassTrak Immunosuppressant Calibrator and Quality Control Sets were used to quantify cyclosporine, everolimus, sirolimus and tacrolimus in human whole blood. Samples (50 µL) were treated with zinc sulfate and acetonitrile containing internal standards. A water/methanol/ammonium acetate gradient was used with a Waters C18 HSS SB Column on a Waters ACQUITYTM UPLTM I-Class FL and Xevo TQ-S micro Mass Spectrometer with an injection-to-injection time of less than 2 min.

Results

No system carryover was observed from high concentration samples. Precise quantification (≤20% CV, ≤15% bias) at concentrations equal to or lower than the lowest concentration calibrator was demonstrated. Total precision and repeatability (3 pools, 5 replicates, 5 days; n = 25) were determined to be ≤7.1% CV. The method was linear over the measuring interval of 19.3–1500 ng/mL (cyclosporine) and 0.77–39 ng/mL (everolimus, sirolimus and tacrolimus). Addition of high concentrations of several endogenous materials did not affect quantification. External quality assurance samples for all drugs met the scheme acceptance criteria (cyclosporine: n = 39, range 0–2658 ng/mL, mean % bias +1.0%; everolimus: n = 35, range 0–21.9 ng/mL, mean % bias +0.9%; sirolimus: n = 34, range 0–74.2 ng/mL, mean % bias −7.5%; tacrolimus: n = 40, range 0–23.0 ng/mL, mean % bias −0.5%).

Conclusion

Performance of the Waters MassTrak Immunosuppressant Calibrator and Quality Control Sets, with the developed method described provide good method performance. The accuracy data demonstrates confidence in the MassTrak Immunosuppressant Calibrator Set, providing harmonization across laboratories. This method is an example of an application using the instrumentation, software and consumables described in this document. This method has not been cleared by any regulatory entity for diagnostic purposes. The end user is responsible for completion of the method development and validation. MassTrak Immunosuppressant Calibrator and Quality Control Sets are not available for sale in all countries.
B-342 Fentanyl Urine Drug Screen Comparison of the ARK II and Lin Zhi Immunoassays
Authors: Lam K , Menlyadiev M , Suhandynata R , Fitzgerald R , Song L , Metushi I
Published: September 2023

Background

Given the opioid epidemic, screening for fentanyl in urine has become increasingly important. Routine opiate immunoassays cannot identify fentanyl, given its structural variance from the more common opiates such as morphine and codeine. Therefore, the detection of fentanyl by immunoassay urine drug screen depends on antibody specificity for fentanyl. The ARK II is a widely used immunoassay that will detect fentanyl and norfentanyl. Recently, a novel fentanyl assay by Lin-Zhi has become available on the Roche platform; here, we evaluate its performance.

Methods

One hundred twenty nine urine samples were analyzed for fentanyl across the Lin-Zhi and ARK II assay on the Cobas c502 platform. Samples were either analyzed immediately upon request for drug of abuse screening or frozen for subsequent analysis. Liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) and/or prescription was used as confirmation for the presence of fentanyl. Our of the total 129 tested samples, so far 59 have been confirmed and additional studies are ongoing. The mass spectrometry assay has a limit of detection of 1 ng/mL for fentanyl/norfentanyl. The ARK II immunoassay has a 1 ng/mL cut-off for fentanyl and 7% cross-reactivity for norfentanyl; the Lin Zhi has a 5 ng/mL cut-off for norfentanyl and 131.58% cross reactivity for fentanyl.

Results

There were 9/129 discrepant samples between ARK II and Lin Zhi immunoassays as shown in Fig. 1. From the 59 confirmed samples Ark II resulted in one false positive sample and seven false negative samples (Fig. 1B) while the Lin Zhi assay resulted in 9 false negatives.

Conclusion

Given the current data set, there appear to be some discrepant results between the ARK II and Lin Zhi immunoassays. Most of the discrepant results observed have concentrations of fentanyl and norfentanyl that are close to the lower limit of immunoassay detection.