bluuebunny/crossref_metadata_2025 huggingface dataset:
B-033 Impact of Red Blood Cell Sediment on HbA1c Measurement
Authors: Nanthakhan T , Jaru-arayanun M
Published: September 2023

Background

HbA1c testing using EDTA whole blood as specimens is essential for monitoring glycemic control in diabetic patients. In the laboratory that using total laboratory automation system, the transportation process might be impact the quality of HbA1c result according to red blood cell sedimentation. This study, we aimed to evaluate the impact of HbA1c result from red blood cell sediment which happen in transportation process of total laboratory automation system.

Methods

HbA1c samples (n = 50) were mixed once and HbA1c levels were determined by using an Alinity c analyzer at an initial time point (T0 = 0 min.), followed by three additional time points (T1 = 7 min., T2 = 15 min., and T3 = 30 min.). The HbA1c levels and samples analyzed at each time point were then used to calculate correlation coefficient, slope, intercept values. The percent difference in these parameter values from all time points was calculated using the analytical criteria of 5% bias according to the National Glycohemoglobin Standardization Program (NGSP) criteria.

Results

We discovered that the correlation coefficient, slope, and intercept values obtained from the samples at T1 are 0.999, 0.994, 0.008, respectively, T2 are 0.999, 0.982, 0.042, respectively and T3 are 0.999, 0.979, 0.028, respectively. The percent difference of all criteria was 0.00% which were not significantly different from those at the initial time point (T0).

Conclusion

This study shows that, the time that using in transportation process in total laboratory automation system at 7, 15 and 30 minutes will not impact quality of HbA1c result even though red blood cell might be sediment before aspirate in the analyzer.
B-050 A Versatile Immunoassay Platform for Fast Multiplex Detection of Protein Biomarkers Based on A Novel Magnetic Particle System
Authors: Cho Y , Moon D , Han K , Choi H , Kim J , Ro Y , Oh B , Song S , Jung Y
Published: September 2023

Background

A major challenge of clinical diagnostic applications in the field of emergency department is the not only fast but also accurate measurement of multiple protein biomarkers with broad dynamic range spectrum. Here, we describe the development of a versatile immunoassay platform, the VEUS (Versatile, Easy, and User-friendly System), with compact size which occupies minimal space.

Methods

The VEUS is designed to enable fast multiplex detection (i.e., 15∼60 minutes) of target proteins at a concentration with a low limit of detection (i.e., <1 pg/mL) by automation of overall immunoassay processes.The system possesses four core technologies enabling entirely automated rapid diagnostics: (1) novel magnetic particles for enabling multiplexed assay using various length of rod-shaped magnetic particles (RSMPs) with conjugated individual antibodies, (2) RSMP movement controlling (up & down/well to well) for increasing antibody-antigen reaction and washing efficiency, (3) a unique optical module for highly sensitive imaging under large field of view for a whole imaging well, and (4) an artificial intelligence (A.I.) based intrinsic program for fast and accurate fluorescent signal analysis which covers detection and classification of RSMP based on their length, and then measurement of fluorescent signal and conversion to biomarker concentration. We evaluated clinical performance of the VEUS platform to diagnose traumatic brain injury (TBI), an emergency disease, as a proof of concept of the versatile immunoassay platform. Among TBI, mild TBI (mTBI) is prevalent in younger population with high level of physical activities that have been linked to increased rate of incidences. Although mTBI is rarely resulted in death, unidentified by the Computed Tomography (CT) and prolonged mTBI as a stealth pathology, may cause life-long disability.

Results

We determined the assay cut-off value from critical concentration of GFAP (Glial fibrillary acidic protein) and UCH-L1 (Ubiquitin carboxy-terminal hydrolase L1) as of 24 pg/mL and 138 pg/mL, respectively. The VEUS performance for a TBI clinical test showed 94.38 % sensitivity and 98.99 % specificity with the clinical serum samples of 89 TBI patients and 97 healthy controls within 50 minutes from the multiplexed assay of GFAP and UCH-L1.

Conclusions

As a versatile immunoassay platform, the VEUS system showed high specificity and sensitivity from the clinical test for the TBI diagnosis. Thus, the VEUS system can contribute to proper treatment for the TBI patients with rapid and accurate clinical results and be also applied for an independent and a precise diagnosis of multiple emergency diseases, such as sepsis, myocardial infraction, heart failure, and stroke which are common in the field of emergency department.
B-066 Evaluation on Effectiveness of Colloidal Gold Immunochromatography Assay for Rapid Detection of Carbapenemase-producing Enterobacteriaceae
Authors: Yang G , Yan J , Zhang Z , Yao W , Wang Y , Wang H , Su Y , Zhou Z
Published: September 2023

Background

The identification, treatment, and control of carbapenem-resistant Enterobacteriaceae (CRE) infections are a major challenge for health care institutions and diagnostic laboratories worldwide. A novel Carbapenemase-producing Bacteria Test Kit (Lateral Flow Assay) from Dynamiker Biotechnology (Tianjin) Co., Ltd. is a qualitative test that was easy to operate and can be widely accepted by clinical and primary medical. Results can be read in just 15 minutes using colloidal gold immunochromatography, which can detect double and multiple carbapenemase types (Fig. 1). We analyzed the effectiveness of Dynamiker carbapenemase-producing bacteria test kit for rapid detection of CRE carbapenemase.

Methods

A total of 120 strains of Enterobacterales in blood culture were collected, including 80 strains of CRE and 40 strains of CSE (carbapenem-susceptibility Enterobacteriaceae). PCR was used to detect KPC, NDM, IMP, VIM and OXA-48 genes as gold standard. Dynamiker carbapenemase-producing bacteria test kit was used to detect the carbapenemase produced by CRE, which was conducted by the performance evaluation combined with PCR results.

Results

The 80 strains of CRE expressing carbapenemase genes were all positive for the detection of carbapenemasstre by Dynamiker carbapenemase-producing bacteria test kit, which includes 39 strains producing KPC, 26 strains producing NDM, 8 strains producing IMP, 5 strains producing VIM and 2 strains producing OXA-48 enzymes; the 40 strains of CSE were negative and no carbapenemase were detected. Compared with the PCR results, the sensitivity and specificity of the four enzymes by Dynamiker carbapenemase-producing bacteria test kit were both 100% (Fig. 2).

Conclusion

Dynamiker carbapenemase-producing bacteria test kit can be applied as a simple, rapid, sensitive and specific diagnostic method for the detection of different CRE carbapenemase types, which is of great value for the accurate use of clinical anti-infective therapy.Fig. 1.Operating procedures of colloidal gold immunochromatography.Fig. 2.Results of Dynamiker carbapenemase-producing bacteria test kit C: Control, K: KPC, N: NMD, I: IMP, V: VIM, O: OXA-48.
B-072 Analysis of Anti-SARS-Cov-2 S1-RBD and N IgG in Saliva and Serum
Authors: Shin K , Lee H , Yu S , Lee S , Lee S , Kim W
Published: September 2023

Background

Mucosal antibody testing may be a significant and more practical tool for measuring humoral immunity since it is non-invasive. The aim of this study was to evaluate the SARS-CoV-2-specific antibody prevalence in saliva compared to serum in 2022.

Methods

From February to December 2022, the paired serum and saliva specimens were collected twice over a period. The saliva specimens were collected using a Salivette Cortisol (Sarstedt AG & Co. KG, Germany) device according to manufacturers’ instructions. Antibody tests were performed using nucleocapsid protein-based Elecsys anti-SARS-CoV-2 (anti-N IgG) and spike protein targeted SARS-CoV-2 IgG II Quant (anti-S1-RBD IgG).

Results

162 (97.6%) of 166 had anti-S1-RBD IgG in their serum. At the 1st visit and 2nd visit, 79 (47.5%) and 97 (58.4%) patients, respectively, had anti-N IgG in their serum. At 1st visit and 2nd visit, respectively, 14 (8.4%) and 13 (7.8%) patients had anti-S1-RBD IgG in their saliva. Four (2.4%) and five (3.0%) patients had anti-N IgG in their saliva during the 1st visit and 2nd visit, respectively. The correlation coefficient of the results of anti-S1-RBD IgG and anti-N IgG quantitative results between at 1st visit and 2nd visit were low (r = 0.1721 to 0.6448). All patients in the anti-S1-RBD IgG seronegative group (N = 4) had anti-N IgG in their serum, but neither anti-S1-RBD IgG nor anti-N IgG were detected in their saliva. All anti-N IgG seronegative patients (N = 68) had anti-S1-RBD IgG in serum, while 3 patients had anti-S1-RBD IgG and 7 patients had anti-N IgG in saliva.

Conclusion

Mucosal antibodies were detected in certain seronegative patients. The assessment of SARS-CoV-2-specific saliva antibodies should be regarded as a supplemental non-invasive diagnostic to serum to evaluate the prevalence and transmission of COVID-19.
B-079 Identifying Heterogeneity of Systemic Connective Tissue Diseases by Applying Joint Dimension Reduction and Cluster Analysis to Immunomarkers
Authors: Yu J , Tseng Y , Lin W , Wang Y , Lin T , Chang C , Wang H
Published: September 2023

Background

Accurate identification of systemic connective tissue diseases (SCTDs) can be challenging due to the heterogeneity of each disease. This study aims to explore the heterogeneity of different SCTDs by applying non-supervised learning to immunomarker data and proposes an alternative classification. Additionally, the study investigates the clinical implications of these different groups.

Methods

We utilized multiple correspondence analysis and k-means clustering to analyze the immunomarker data of patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and Sjögren’s syndrome (SS) in Taiwan between 2001 and 2016. To investigate the clinical correlations of the classification results, we conducted a comprehensive review of the clinical findings and compared the results across different groups.

Results

A total of 11 923 patients with the three SCTDs were classified into six distinct clusters based on their immunomarker patterns. In this clustering system, RA patients were predominantly grouped in cluster 1, whereas SLE and SS patients had a more dispersed distribution. Among patients with SLE, renal involvement was more frequent in clusters 3 and 6 (52% and 51%, respectively) than in other clusters, and discoid lupus was more frequent in cluster 3. Among SS patients, typical SS findings such as inflammation or infection of eyes and allergic reactions were more common in cluster 2 (54% and 51%, respectively). In contrast, patients in cluster 3 had more unspecified benign neoplasms (58%) and were more likely to experience immune disorders (63%).

Conclusion

The immunomarker-driven clustering yields an alternative classification result with new clinical insights. The data-driven approach would provide a more evidence-based classification and aid in a more accurate diagnosis and management for SCTDs.
B-091 Assessing Postvaccination SARS-CoV-2 Neutralizing Antibodies at Different Timepoints: a Laboratory-Based Study
Authors: Souza G M , Carvalho G C , Nunes R A , Cristaldi F M , Ramadan D R , Tufik S
Published: September 2023

Background

Serum-neutralizing antibodies (NAbs) against SARS-CoV-2 may be produced as a result of either viral infection or vaccination. NAbs can disrupt the connection between the RBD (Receptor-Binding Domain) of the Spike protein and the ACE2 receptor, hindering the virus's entry into cells. However, only a portion of these antibodies are able to neutralize the virus and prevent it from infecting new cells. While the presence of NAbs should not be considered a guarantee of neither immunity nor vaccine effectiveness, it can serve as an indicator for seroprevalence studies in healthcare settings. The purpose of this study was to compare results for NAbs from a group of healthcare individuals that received either CoronaVac/Sinovac (CVC/S) or Oxford-AstraZeneca (O/AZ) in São Paulo, Brazil.

Methods

Study Population and Characteristics: The current study evaluated a total of 33 and 34 healthcare professionals that received, respectively, O/AZ and CVC/S in the year 2021. Blood samples were collected 15 days (15D1) after the first dose of vaccination and at 30 (30D2), 60 (60D2), or 90 (90D2) days after the second dose. The presence of neutralizing antibodies was assessed with the cPassTM SARS-CoV-2 Neutralization Antibody Detection Kit GenScript. Results were classified into three categories based on reference values: not reactive (0–20%), undetermined (20–30%), and reactive (greater than 30%).

Results

Among the 67 health professionals, mostly were women in both vaccination groups: CVC/S (85.3%) and O/AZ (90%). At 15 days after receiving the first dose, the presence of neutralizing antibodies was detected in individuals who received both vaccines. However, a total of 32.3% (11/34) and 15.1% (5/33) individuals for CVC/S or O/AZ respectively do not show reactive antibodies at this first period evaluated. NAbs values obtained at 15D1, 30D2, 60D2, and 90D2 for CVC/S and O/AZ were 53.9%, 79.3%, 74.7%, 66.7% (for CVC/S) and 68.2%, 87.7%, 82.9%, 69.6% (for O/AZ). For both vaccination groups, higher levels for neutralizing antibodies were detected at 30 days after the second dose, and decreased at 60 and 90 days.

Conclusion

Oxford-AstraZeneca induced higher levels of neutralizing antibodies in this group of healthcare individuals. Serum follow-up samples taken at 30D2 elicited increased NAbs compared to other timepoints.
B-107 Serodiagnosis of Anti-glomerular Basement Membrane Disease Using a Newly Developed Chemiluminescence Immunoassay
Authors: Sabalza M , Bestmann Y , Hartwig L , Kühnl A , Dähnrich C , Schlumberger W
Published: September 2023

Background

Autoantibodies against the glomerular basement membrane (GBM) are important markers in the diagnosis and monitoring of autoimmune glomerulonephritides. Fast and reliable detection of these autoantibodies is crucial as anti-GBM disease can progress rapidly with fatal outcome. Here, we investigated the diagnostic performance of a newly developed, standardized anti-GBM chemiluminescence immunoassay (ChLIA).

Methods

The diagnostic performance of the EUROIMMUN Anti-GBM ChLIA (IgG), processed on the EUROIMMUN RA Analyzer 10, was assessed using sera from 67 clinically characterized anti-GBM disease patients and 221 disease controls. Results were compared with those obtained by the EUROIMMUN Anti-GBM ELISA (IgG). Inter-assay concordance, measurement range and interference were determined in a subset of samples.

Results

The ChLIA reached 100% sensitivity at a specificity of 98.6%, while the ELISA was less sensitive (89.6%) and more specific (100%). High qualitative concordance between both assays was evidenced by positive and negative agreement rates of 100% and 95.6%, respectively, and a kappa score of 0.901. The ChLIA showed linearity within a measurement range of 3.8–517.3 CU/mL. Coefficients of variation were calculated as 1.2%–3.3% (intra-lot) and 1.6%–4.2% (inter-lot). No interference was observed for hemolyzed, lipemic or icteric samples.

Conclusion

These validation results demonstrate a high quality of the novel Anti-GBM ChLIA. Given its excellent performance compared to the corresponding ELISA, it represents a promising alternative tool for accurate anti-GBM assessment in routine diagnostic settings with the advantage of rapid turnaround time and fully automated random-access processing. Future studies will address the assay’s suitability for monitoring anti-GBM levels during follow-up.
B-112 Routine Application of PR3- and MPO-ANCA Chemiluminescence Immunoassays Demonstrates Enhanced Detection Rates
Authors: Venkataraman I , van Beers J , Damoiseaux J , Dähnrich C , Kühnl A , Schlumberger W
Published: September 2023

Background

Sensitive and accurate PR3- and MPO-ANCA detection are crucial in the diagnosis and monitoring of patients with ANCA-associated vasculitides (AAV) and the specific diseases categorised thereof, including granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (EGPA). Chemiluminescence immunoassays (ChLIA) and bead technology have emerged as promising tools for fast, sensitive and accurate autoantibody detection. Applied to PR3- and MPO-ANCA, they could improve limitations in sensitivity, narrow measuring range and level of quantification seen in other assay formats. Here, we examined the routine performance of ChLIAs using human native MPO as well as human recombinant PR3 antigens.

Methods

The Anti-PR3 ChLIA (IgG) and prototype Anti-Myeloperoxidase ChLIA (IgG) were processed on the RA Analyzer 10 (EUROIMMUN) and the EliA PR3S and EliA MPOS on the Phadia250 (Thermo-Fisher Scientific), both in accordance to the recommendations by the manufacturers. For a comparative analysis of the routine performance, 675 sera from 615 individuals requested for PR3-ANCA, MPO-ANCA or both at the Central Diagnostic Laboratory at Maastricht University Medical Center were measured with the respective assays from both automation systems. The results were compared in respect to the qualitative outcome and numerical correlation.

Results

Of the total 675 samples, 585 and 556 were subjected to PR3-ANCA and MPO-ANCA analysis, respectively. For both ANCA types, all samples found positive with EliAs were also positive in the corresponding ChLIAs. 24 and 26 samples resulted positive for PR3- and MPO-ANCA by ChLIA, respectively, but were below cut-off in the relevant EliA test. Of those, 50.0% (n = 12 for PR3) and 96.2% (n = 25 for MPO) were from individuals in follow-up after AAV diagnosis, in the remaining cases an ANCA involvement was suspected based on symptoms of the patients. The overall concordance between the two assay systems was 96.0% for PR3-ANCA and 95.4% for MPO-ANCA detection.

Conclusion

In a routine scenario, the ChLIAs fully covered the PR3- and MPO-ANCA detection by the compared reference test system. Moreover, further positive results in samples found negative by EliA demonstrated enhanced detection rates by both ChLIAs, predominantly in samples from individuals with a known history of AAV. The clinical relevance will be subject to further studies.
B-136 Indirect Estimation and Verification Tool for Reference Intervals Using Unsupervised Machine Learning
Authors: Vieira L M , Dias A C , Oliveira D d , Dias C M , Almeida T , Campos J R
Published: September 2023

Background

Determining the Reference Intervals (RI) of quantitative laboratory tests is crucial. This aspect is also part of the accreditation process, which can be challenging. The goal of our study was to create and validate a model using the R programming language that can estimate and verify Reference Intervals using an indirect method by mining data from the Laboratory Information System.

Methods

The new model integrates algorithms that carry out preliminary processing, grouping, and statistical reduction of results. The algorithm identifies unusual outcomes by analyzing asymmetry and kurtosis and applying the Expectation-Maximization (EM) algorithm. During the grouping and reduction phases, the EM algorithm computes the mean, standard deviation, and weight of the distribution for each group. Additionally, the model integrates algorithms that determine the global mean/median, modes, antimodes, kurtosis, and skewness, to find values that best represent the reference population’s distribution. The distribution is then truncated to the defined IR partition through the combination of algorithms (Fig. 1). The Bias Ratio was used to evaluate the difference between the RI determined by our model and those obtained from the Kosmic and Refine R models. The model’s validity was tested using simulated and real data from anonymous reference individuals.Fig. 1.

Results

The effectiveness of the model was evaluated using was assessed for each test under varying levels of contamination. The overall performance showed excellent results with an accuracy of 0.996, kappa of 0.882, sensitivity of 0.945, specificity of 0.979, positive predictive value of 0.864, negative predictive value of 0.992, and F1-score of 0.896.

Conclusions

The indirect estimation and verification tool for Reference Intervals is a cost-free and valuable resource for clinical laboratories. It supports the accurate interpretation of patient´s results.
B-165 Establishment of a Labile Bound Copper and Labile Bound Copper Ratio Reference Interval in a Healthy Adult Population
Authors: Bitzer A , Day P , Fox J , Wermers M , Jannetto P , Bornhorst J
Published: September 2023

Background

While clinical tests are available to aid in the diagnosis of Wilson’s Disease (WD), some such as total copper determination lack sensitivity and specificity, while others such as liver tissue copper assessment are costly and invasive. WD affects copper metabolism and is known to cause low serum copper concentrations. In addition, an elevated percentage of total serum copper exists as non-protein bound or labile bound copper (LBC). Previous studies have shown that measuring the ratio of labile bound copper to total serum copper may aid in the diagnosis of WD. To support the utility of a laboratory developed test using serum LBC ratios to aid in the diagnosis of WD, serum samples from healthy donors were used to establish a reference range for LBC and the ratio of LBC to total copper in adults.

Methods

Serum samples were collected in trace metal free tubes from 214 donors (n = 110 male, n = 104 female) between the ages of 19–80 years old, selected against pre-determined exclusion criteria including acute hepatitis, chronic hepatitis, renal failure, biliary cirrhosis, celiac disease, and leukemia. Prior to analysis, samples went through a centrifugation-based preparation process to isolate the LBC fraction using molecular weight size exclusion filters and EDTA chelation. Samples were then analyzed via ICP-MS in kinetic energy discrimination (KED) mode to directly measure the LBC and total copper concentrations. LBC ratio values were determined from the LBC/total copper concentrations.

Results

Preliminary reference ranges were developed for both LBC and the LBC ratio based on the 2.5th and 97.5th percentiles with 95% confidence intervals. Established sex specific reference intervals for total copper were deemed acceptable per CLSI guidelines in document EP28-A3c, with ≤10% of results from this sample set falling outside of the established intervals. No statistically significant relationship was found in the mid-95th percentile between LBC concentration or ratio with age or sex. The reference intervals for LBC concentration were calculated as 13.41–104.66 and 11.69–106.50 ng/mL for females and males respectively. The reference intervals for LBC ratio were calculated as 1.04–8.12 and 1.17–10.48 for females and males respectively.

Conclusions

A reference range of LBC and LBC ratio has now been determined for the healthy adult population. Further testing can now be done on disease state populations to determine the sensitivity and specificity of LBC ratio in the detection, monitoring, or diagnosis of WD.
B-205 A Simple Method for Opioids, Synthetic Opioids and PCP Detection in Hair Samples by LC-MS/MS
Authors: Paulo B F , Junior L F , Zauli D A
Published: September 2023

Background

Hair analysis is useful for monitoring exposure to drugs and is a unique body material for the retrospective detection of drug consumption due to its large detection window, and it is easy to collect, store, and transport. Establishing a LC-MS/MS protocol for opioids determination may be challenging due to similarities among the molecular structures and the MS/MS fragmentation profile. This study aimed to develop and validate a simple, rapid, and sensitive LC-MS/MS method for quantification of 6-acetylcodeine, 6-acetylmorphine, codeine, dihydrocodeine, EDDP, PCP, fentanyl, heroin, hydrocodone, hydromorphone, meperidine, methadone, morphine, oxycodone, oxymorphone and tramadol in hair samples.

Methods

In this method, 15 mg of hair in a 2.0 mL microtube was decontaminated sequentially with dichloromethane and methanol. The sample was dried, and 0.75 mL of extraction solvent and a labeled internal standards solution were added. The hair sample was pulverized in a FastPrep-24™ 5G from MP-Biomedicals and then sonicated at 60°C for 60 min. The sample was centrifuged and filtered with Captiva filtration plates. Then, 100 µL of supernatant was added to 100 uL of ultrapure water and 5.0 uL was injected directly into the LC-MS/MS system. Chromatographic separation was performed on an Agilent 1290 Infinity II equipped with HALO Biphenyl column, and employing a mobile phase constituted by acetonitrile with 0.1% of formic acid and 5 mM of ammonium formate. The detection was performed with a Sciex 6500+ QTrap mass spectrometer.

Results

The current method was successfully validated for carryover, linearity, precision, recovery, limits of detection and quantification, and the measurement of uncertainty was calculated. The results were summarized in the Table 1.

Conclusion

The developed LC-MS/MS method was simple, rapid, and sensitive to detect opioids in hair samples.
B-216 Development of a Point-of-care Multiplexed Room Temperature Stable Environmental Toxin Test
Authors: Aithal S , Gupta S , Duong K , Kumar A , Ho N , Guo S , Ando Y , Liu R , Warden J , Ho D H
Published: September 2023

Background

Hememics™ Biosensor System (HBS) is a platform technology enabling antigen and molecular testing in the field. The system consists of a reader, HemBox™, and a biosensing chip, HemChip™. The HemBox™ is a battery-operated, cell phone size with Bluetooth and WIFI module that can be integrated into the Health Management System. The biosensing chips can be programed with bio-receptors to detect pathogens in less than 5 min. This study presents performance characteristics of HBS as a rapid POC to detect toxins from environmental samples.

Methods

The testing protocol consists of insert a HemChip™ into the HemBox™ and mixing the sample with the buffer and apply a drop of this mixture to the chip. In less than 5 min, results are displayed on the screen of HemBox™ or transmitted to user’s cellphone via Bluetooth. Samples were prepared by spiking solutions of mud, serum, vegetable wash, and milk with Staphylococcal enterotoxin B (SEB) and ricin.

Results

HemChip™ was functionalized to recognize Ricin and SEB targets. For testing, these targets were mixed in various matrix. To achieve statistical significance, each sample tested represented a minimum of 10 data points.

Conclusion

Hememics™ Biosensor System (HBS) is designed to be the ultimate Portable Lab Diagnostics for field use. The ability to test for environmental toxins in the field is significant because it allows for quick and on-site detection of potentially harmful substances. Our biosensor system also eliminates the need for sample processing, collection and transportation, which can take significant time and resources. The results of field testing can be available almost immediately, allowing for prompt action to be taken in the case of high toxin levels.
B-225 Assessment of the Genotype Frequency of Thiopurine Methyltransferase (TPMT) Deficiency in a Large Cohort of Patients With Immune Mediated Inflammatory Disease and Cancer
Authors: Stachelski J , Pham D , Hughes P , McLachlan O , Dervieux T
Published: September 2023

Background

Thiopurine methyltransferase (TPMT) alleles (*1/*2/*3A/*3B/*3C) are routinely genotyped to determine the metabolizer status of patients that plan to undergo thiopurine therapies (6-Mercaptopurine and Azathioprine). Screening of these alleles allows for personalized patient dosing and reduces the risk of severe or even fatal myelosuppression. The objective of this study was to determine the genotype frequency of TPMT deficiency in a large clinical cohort of patients with immune mediated inflammatory diseases and cancer.

Methods

All genotyping was performed in a reference clinical laboratory. Patient specimens were tested using genomic DNA isolated from whole blood. Clinical patient diagnoses were physician provided (international classification of disease, ICD-10). The genomic DNA was tested for the presence of three single nucleotide polymorphisms (SNPs) in the TPMT gene coding region and known to associate with reduced TPMT activity (rs1800462-Exon 5, rs1800460-Exon 7 and rs1142345-Exon 10). These polymorphisms determine the presence of *2/*3A/*3B/*3C alleles. Testing occurred from 10/26/2000 to 9/9/2022. Three different testing technologies were used during the 22 years of testing (PCR-RFLP from 2000 to 2003, Taqman™ chemistries from 2004 to 2015, and Luminex™ xMAP™ bead-based since 2016).

Results

A total of 219 129 patients were tested (mean age: 38 yrs, 56% female). As presented in the Table the genotype frequency for the wild type *1/*1 was 90.8% (95%CI: 90.7–90.9%), it was 9.0% (95%CI: 8.8–9.1%) for the heterozygous and 0.22% (95%CI: 0.020–0.024%) for homozygous variant, with similar frequency across disease states. The TPMT *1/*3B genotype frequency was very rare (<0.1%, n = 5) and the *3B/*3C homozygous variant (which cannot be distinguished from the *1/*3A) was calculated to have a frequency of 1:7 706 230 (95%CI: 1:7 043 228–1:8 369 232) and lower than initial estimates.

Conclusion

We estimated the frequency of TPMT genotypes in the largest cohort reported to date. The occurrence of the *3B/*3C genotype may be rarer than initially estimated.
B-246 The Evaluation of Actionable Mutations By Multiplex Analysis Using IntelliPlex® Lung Cancer Panel for Non-Small Cell Cancer Management
Authors: Cheng W
Published: September 2023

Background

Understanding the genetic alterations which occur in genes associated with various cancers can improve therapy outcomes by promoting tailored treatments for each patient. Molecular profiling is therefore a crucial tool for precision medicine and enables for a personalized approach to cancer management. Current guidelines for molecular testing of Non-Small Cell Lung Cancer (NSCLC) patients involves treatment selection of a targeted Tyrosine Kinase Inhibitor based on the minimal testing of EGFR, ALK and ROS1 genes and strongly recommending the additional testing of KRAS, BRAF, MET, RET, NTRK and ERBB2 (Her2) genes to fully evaluate the patient’s mutational status.

Method

The IntelliPlex Lung Cancer Panel, based on PlexBio’s multiplexing πCode® technology, is an in vitro molecular amplification assay intended for the qualitative identification of 74 DNA mutations in the KRAS, NRAS, PIK3CA, BRAF, EGFR, HER2, MEK1 and AKT1 genes and 28 gene variants of the ALK, ROS1, RET, NTRK1 and MET genes. The assay is suitable for the use with formalin-fixed paraffin-embedded (FFPE) tumor tissues from NSCLC patients. The performance of IntelliPlex Lung Cancer Panel was evaluated on FFPE specimens by comparing the results with two commercially available molecular amplification assays. Discrepant samples were resolved by a corresponding digital PCR method kit.

Results

The IntelliPlex Lung Cancer Panel can detect DNA mutations at very low allele frequencies (as low as 0.1%) while only requiring DNA quantities of 10ng. Most RNA rearrangements are detected with 10 or more copies present in the sample while only low RNA input quantities (50 ng) are required. The IntelliPlex Lung Cancer Panel can analyze the mutational status of 102 targets in less than 5.5 h from sample to results and allows screening of up to 48 samples in parallel. Among 50 FFPE DNA specimens tested, the overall agreement for EGFR and KRAS mutations between IntelliPlex system and the comparison method were both found to be 98%. And the overall agreement after resolved the discrepancies by digital PCR was 100%.

Conclusion

The multiplex amplification and detection capabilities of the IntelliPlex Lung Cancer Panel provides a comprehensive and efficient way to evaluate the individual mutational status from lung cancer patients with high sensitivity and specificity. Accurate genetic analysis provides valuable information which can support precision medicine decisions for better cancer management. This can also be used further with non-invasive whole blood sampling (i.e., “liquid biopsy”) for subsequent treatment monitoring.
B-247 Rapid and Field-deployable Microchip-based Real-time PCR for Hepatitis B Virus DNA Detection
Authors: Liu Y , Gao Y , Song Q , Hong Y , Wen W
Published: September 2023

Background

Hepatitis B is one of the most common infectious diseases with global significance, seriously causing chronic liver complications and a heavy health burden in developing countries. There is no doubt that real-time polymerase chain reaction (RT-PCR) has been a golden standard for monitoring the Hepatitis B virus (HBV) level in clinical health management. However, the conventional PCR protocol cannot fulfill the field test requirement in some resource-poor areas due to its drawbacks, such as huge size, long turnaround time, and demand for professional laboratory facilities. For solving these problems, a portable microfluidic chip-based PCR detection system was developed to enable the nucleic acid test in a flexible, rapid, and economical way. With the efficient temperature control module and advanced optical path design, this novel panel can achieve fast thermal circulation with a 30°C/s heating rate and multiple fluorescent detection with a throughput of 6 samples at a time.

Methods

In this study, we assessed the detection performance of the system using a commercial HBV nucleic acid detection kit. The reproducibility, limit-of-detection (LoD), and linearity were validated by reference samples with gradient concentrations. We evaluated the clinical sensitivity and specificity of the microdevice by a total of 200 extracted serum samples (100 positive and 100 negative cases). The analytical results were compared with a traditional PCR device with FDA certification.

Results

The variation coefficients of the microdevice for reference samples with different concentrations are 1.78%, 1.98%, and 1.50% (n = 10), respectively. The field-deployable PCR system can provide the HBV DNA assay with a detection limit of 300 IU/ml, whose reaction time was approximately 67 mins rather than the 108 mins performed by the comparator. During the establishment of the standard curve, this fast PCR test shows a strong linear correlation (R2 = 0.9919) between targeted gene concentration and dilution factor. Additionally, among the 200 clinical samples, we used the microchip-based system to rapidly complete quantitative detection for each sample with high sensitivity of 100% (100/100) and specificity of 100% (100/100), which is critically consistent with the traditional PCR method.

Conclusion

Hence, the point-of-care microfluidic PCR system exhibits great feasibility in offering timely, accurate, and reliable molecular diagnosis for early epidemic disease screening and therapeutic guidance of chronic infections.
B-293 Non-renal Sources of Erythropoietin: Complications for Interpreting Erythropoietin Assays
Authors: Alshamali R , Marrow J P , Platt M J , Brunt K R , Simpson J A
Published: September 2023

Background

Erythropoietin (EPO) is a glycoprotein that stimulates the production of red blood cells, in response to systemic hypoxia. Generally, the kidney is thought to be the sole producer of EPO. Emerging data shows Epo mRNA expression can occur in non-renal tissues, yet the significance of this remains uninvestigated. Clinically, EPO assays are primarily used to identify the underlying cause of anemia and polycythemia. Interestingly, EPO serum levels are elevated following a myocardial infarct (MI); the source is always assumed to be kidney with no further investigations into non-renal sources of EPO. Here we investigate the heart as a novel source of serum EPO following a MI. We hypothesize that the heart, not the kidney, produces EPO in response to organ-specific hypoxia (i.e., MI).

Methods

To assess which organs are producing EPO following a MI, we examined EPO mRNA expression. mRNA levels correlate strongly with EPO protein synthesis, as EPO is exclusively regulated at the transcription level. To induce a MI, CD1 mice were subjected to ligation of the lateral anterior descending artery. Left ventricle and kidney tissues were collected at 24 h, 4 weeks and 18 weeks following MI or sham surgery; tissue was snap frozen and stored at −80°C for qPCR analysis. Hematocrit was measured at 2-, 4-, and 18-weeks following MI or sham surgery.

Results

24 h following a MI, the left ventricle showed a ∼100-fold increase in EPO mRNA expression in comparison to sham group (P = 0.0012); the kidney showed no significant differences in EPO mRNA between MI and sham group. Hematocrit was measured from the carotid artery. At 2- and 4- weeks following surgery, hematocrit in MI group was significantly elevated in comparison to baseline hematocrit levels (Baseline: 40.8% ± 4, 2-week: 47.8% ± 3.1, and 4-week: 46% ± 2.6 (P = 0.0003, P = 0.0103, respectively). Whereas there were no significant differences in hematocrit between baseline and the sham group.

Conclusion

Here we show that following a MI, the heart, not the kidney, produces EPO. Subsequently, there is an increase in hematocrit as a result of cardiac-specific EPO production. Further, perturbations in EPO serum levels are not disease specific. In cases where EPO assays are conducted on MI patients, interpretation of EPO levels should be cautioned as it may not be exclusively diagnostic of the underlying cause of anemia or other bone marrow disorders. Therefore, an alternative diagnosis may be warranted for elevated EPO levels in this specific patient population.
B-294 Delineating the Diversity of Recombinant Monoclonal Antibodies Isolated From Alpaca Polyclonal Mixture
Authors: Narang D , Le Bihan T , De Villavicencio Diaz T N , Ma B
Published: September 2023

Introduction

Polyclonal antibodies are a mixture of antibodies produced by plasma B-cells and are often obtained by injecting a specific antigen into a host system. They are commonly used for research and diagnostic purposes and are inexpensive and relatively quick to produce. But they lack batch-to-batch reproducibility making them less reliable than recombinant monoclonal antibodies (mAbs). Our team designed a polyclonal sequencing method to sequence the mAbs directly from the immunoserum and then used recombinant technology to reliably express the mAbs. The diversity of the epitopes of these recombinant mAbs are then examined with Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS).

Methods

In this study, we used a novel mass-spectrometry-based approach to do de novo sequencing of the Alpaca IgGs directly from the immunoserum without analyzing the RNA-seq data. Using our established REpAb sequencing platform and a machine learning-based bioinformatic analysis, we successfully compiled complete amino acid sequences of individual alpaca IgG clones found in the original polyclonal mixture. Using this approach, we isolated ten unique sequences from the polyclonal mixture and expressed them recombinantly. In addition, we performed Hydrogen-deuterium exchange Mass spectrometry experiments on 4 of the ten mAbs to identify their binding sites. For HDX analysis, the antigen digestion was performed using Pepsin/protease XIII column, and the peptides were identified using Waters SELECT SERIES Cyclic IMS Mass spectrometer.

Preliminary data

The four mAbs which showed strong binding to the antigen were chosen to characterize further using HDX-MS experiments. The preliminary MS/MS experiments provided us with approximately 90% of the amino acid sequence coverage of the antigen. Interestingly, the HDX-MS results showed that different mAbs bind to different antigen regions, which is evident from the deuterium exchange. Overall, our results showed that the monoclonal antibodies isolated from the polyclonal mixture bind to different antigen regions and can represent the diversity in the polyclonal mixture.

Novel aspect

De novo sequencing functional mAbs from immunoserum and studying their epitope diversities with HDX-MS may help discover novel therapeutics antibodies.
B-306 Performance Evaluation of an Automated Assay for Measurement of Tacrolimus* on the ADVIA Centaur XP System
Authors: Chuang T , Swan R , Sankiewicz O , Casson N , Baxter S
Published: September 2023

Background

Tacrolimus (FK506) (PROGRAF) is a macrolide antibiotic of fungal origin (Streptomyces tsukubaensis). Tacrolimus is an inhibitor of calcineurin, a phosphatase that activates T-cell proliferation. At the cellular level, tacrolimus binds a family of binding proteins termed FK506-binding proteins (FKBPs). The correlation between steady-state trough concentration and area under the curve (AUC) provides reliable therapeutic monitoring for tacrolimus exposure at the trough level. The objective of this study was to determine the performance characteristics of the Tacrolimus assay on the ADVIA Centaur® XP System.

Methods

The ADVIA Centaur Tacrolimus assay is a competitive immunoassay using direct chemiluminescent technology. Free tacrolimus in the patient sample competes with the bound tacrolimus in the solid phase for binding with the acridinium ester-labeled anti-tacrolimus antibody. The amount of tacrolimus present in the patient sample has an inverse relationship to the amount of relative light units detected by the system.

Results

The ADVIA Centaur Tacrolimus assay demonstrated a mean %CV of 3.1% for intra-assay and 6.0% for total precision (across a sample range of 3.10 to 26.15 ng/mL). Close correlation to LC-MS/MS was demonstrated, with r, slope, and intercept of 0.957, 0.973, and 0.475, respectively. LoD and LoQ were 0.7 ng/mL and 1.9 ng/mL, respectively. Likewise, comparison to the Atellica IM assay resulted in r, slope, and intercept of 0.966, 1.09, and -0.508, respectively. Linearity was demonstrated across a range from LoQ to 30.0 ng/mL. Tacrolimus metabolite percent cross-reactivity ranged from 1.7 to 34.2%. A panel of potential interferants including the following were tested and found not to interfere (≤10%): cephalosporin to 100 µg/mL, erythromycin to 13.8 mg/dL, phenobarbital to 10 mg/dL, rapamycin to 5 µg/mL, sulfamethoxazole to 150 µg/mL, tobramycin to 3.3 mg/dL, trimethoprim to 4.2 mg/dL, bilirubin (conjugated) to 60 mg/dL, bilirubin (direct) to 60 mg/dL, and rheumatoid factor to 500 IU/mL.

Conclusion

The resulting data demonstrates that the ADVIA Centaur Tacrolimus assay is an accurate and precise method of quantifying tacrolimus concentration in EDTA whole-blood samples. *Assay under development by Randox Laboratories Ltd. for Siemens Healthineers. Not available for sale. Future availability cannot be guaranteed.
B-323 Urinary Thebaine Improves Opioid Interpretation in Pain Management
Authors: Pourfarrokh N , Nguyen N
Published: September 2023

Background

Poppy seed from Papaver somniferum plant is used in bakery results in positive urine test for Morphine and Codeine up to 48 h. This leads to prescription compliance failure in the setting of clinical pain management. To mitigate misinterpretation, Thebaine testing was introduced to confirmatory opioid panel. Thebaine is naturally found in poppy seeds but not in prescription opiate formularies. A positive Thebaine result helps identify poppy seed consumption rather than from opioid prescription abuse.

Method

Each of the seven healthy adult volunteers (age 20–50) consumed one poppy seed filled kolache. Urine samples were then collected from each person at pre and post-consumed time points at 2, 4, and 24 h. All samples were screened on Abbott Architect c16000 immunoassay (Abbott Park, IL) for opioids at 300 ng/mL cutoff. The samples were quantitated using ThermoFisher Vanquish Quantis LC-MS/MS (Waltham, MA) for Codeine, Morphine, and Thebaine at AMR 10–500 ng/mL, 10–500 ng/mL, and 5–250 ng/mL, respectively. Data acquisition/analyses were performed on TraceFinder software. The deuterated internal standards were purchased from Cerilliant Corporation (Round Rock, TX). All samples met calibrators and quality controls quality criteria.

Results

All post-consumption immunoassay results were positive for opioid. All LC-MS/MS positive results showed various levels of Codeine, Morphine, and Thebaine at 230–4300 ng/mL, 9–290 ng/mL, and 5–210 ng/mL, respectively (Fig. 1).Fig. 1.

Conclusion

Poppy seed consumption results in positive opioid class by immunoassay and positives Codeine, Morphine by LC-MS/MS. Detection of Thebaine in these samples would enable accurate clinical interpretation for poppy seed dieters and abusers of Codeine and Morphine prescription.
B-325 Retrospective Study Comparing Immunoassay Screen and Mass Spectrometry Confirmation Results in an Opioid Dependant Population
Authors: Bennett M J , Luong J , Kinniburgh D W
Published: September 2023

Background

Retrospective analysis of clinical laboratory data is employed to monitor the performance of instruments, evaluate test utilization and support operational changes. Urine drug testing in the context of opioid use disorder is used to assist in the management of patients. This testing is typically performed by an immunoassay screen followed by mass spectrometry confirmation. Immunoassay screens identify drug class and can cross-react with off-target substances to produce false-positive or false-negative results. Mass spectrometry confirmations provide more sensitive and specific results. The objective of this study was to utilize historical data to determine the concordance between immunoassay and mass spectrometry results from patients in an opioid dependency treatment program.

Methods

Urine drug screening results spanning a 3-year period (2020–2023) were mined to identify samples that screened positive for amphetamines, benzodiazepines, opiates, fentanyl/carfentanil, oxycodone/oxymorphone, and cocaine. These results were compared to the mass spectrometry confirmation results. The immunoassay results were collected on an Olympus AU480 instrument and the mass spectrometry results were collected using an in-house developed dynamic multiple reaction monitoring method on both Agilent 6470 and 6460 triple quadrupole instruments.

Results

Of the 15 850 urine drug records reviewed, 28.8% screened positive for amphetamines, 23.5% screened positive for benzodiazepines, 14.2% screened positive for opiates, 23.8% screened positive for fentanyl/carfentanil, 4.2% screened positive for oxycodone/oxymorphone, and 11.2% screened positive for cocaine. Of the amphetamine class samples that screened positive, 95.3% confirmed positive and 4.7% confirmed negative. Of the benzodiazepine class samples that screened positive, 50.8% confirmed positive and 49.2% confirmed negative. Of the opiate class samples that screened positive, 79.8% confirmed positive and 20.2% confirmed negative. Of the fentanyl/carfentanil samples that screened positive, 97.2% confirmed positive and 2.8% confirmed negative. Of the oxycodone/oxymorphone samples that screened positive, 58.5% were confirmed positive and 41.5% confirmed negative. Of the cocaine samples that screened positive, 99.8% confirmed positive and 0.2% confirmed negative.

Conclusion

Our findings determined there were discrepancies among all classes when comparing immunoassay vs mass spectrometry results. There was good agreement between immunoassay and mass spectrometry results in the detection of fentanyl/carfentanil, cocaine and amphetamines. The differences between methods were most significant in the benzodiazepine, opiate and semi-synthetic opioid classes. This may be due to different cut-off levels between methods, specificity limitations of immunoassays and/or changing drug use patterns resulting in mass spectrometry multiple reaction monitoring false negative results.