bluuebunny/crossref_metadata_2025 huggingface dataset:
A-377 The Selection and Implementation of POC Illicit Drug Toxicology Testing in Hamad Medical Corporation - Doha, Qatar
Authors: Al Kuwari E , Al-Wali D , Habib J S , Kabir Z , Abdelfadel A , Torato E , Dellil A
Published: September 2023

Background

In 2013, Qatar launched the National Mental Health Strategy which is further supported by National Health Strategy 2018–2022 and Qatar National Vision 2030, where the mental health has been identified as a priority area of development. In doing so, the Ministry of Public Health worked closely with the Ministry of Interior in providing the appropriate treatment rehabilitation. With all considerable advantages of point-of-care testing (POCT) in rapid diagnosis, POC toxicological test has been implemented and used in screening of patients for drugs of abuse. In this study, we highlighted the challenges in selection and implementation process of POC toxicological test in Hamad Medical Corporation (HMC).

Methods

Due to unavailability of all required drug panels, it had to be customized and tested for the verification purpose before starting the evaluation process. Maintaining HMC procurements regulations and Clinical Laboratory Improvement Amendments (CLIA) and College of American Pathologist (CAP) standards for POC toxicology tests, two POC illicit drug toxicology screening tests has been evaluated, comparison study has been performed against the HMC central laboratory. POCT team conducted training session ensuring the appropriate practice in place. The Extendable Laboratory Information System (LIS) functionality has been utilized for seamless data management and reporting for the automated analyzer.

Results

QUIDEL triage meter pro is an automated drug testing analyzer which detects Amphetamines, Barbiturates, Benzodiazepines, Cannabinoids, Methadone Metabolite, Methamphetamine, Opiates, Tricyclic and Cocaine Metabolites, while the customized manual testing kit FASTEP detects Tramadol (TRA), Methylenedioxymeth-amphetamine (MDMA), Phencyclidine (PCP), Buprenorphine (BUP), Oxycodone (OXY), Fentanyl (FYL), Ethyl Glucuronide (EtG), Cotinine (COT), Pregabalin (PGB) and Gamma Hydroxybutyrate (GHB). Both analytical performances were verified. Rapid bedside testing performed for more than 100 patients in last 8 months during admission, observation, and pre- discharge process. There is An Obvious improvement observed in testing turnaround time compared to the laboratory, easy results interpretation and reporting as well as cost-effective.

Conclusion

The implementation of POC illicit drug toxicology testing in HMC aligned with core pillars of the Qatar vision 2030 to deliver the right treatment at the right time and place. Its enhancing patient outcomes and overall Satellite health care sector in Qatar.
B-011 Analytical Validation of a New Vasoactive Intestinal Peptide Radioimmunoassay
Authors: Ashrafzadeh-Kian S , Bornhorst J , Algeciras-Schimnich A
Published: September 2023

Background

Vasoactive intestinal peptide (VIP), a potent vasodilator, is produced by neuronal cells located in the central nervous system, the digestive, respiratory, and urogenital tracts, and the exocrine, thyroid, and adrenal glands. Increased circulating VIP is clinically useful to detect VIP-producing tumors in patients with chronic diarrheal diseases.

Objective

Develop a sensitive replacement radioimmunoassay (RIA) for VIP quantitation in human plasma due to the diminished supply of the antibody used in the previous in-house method.

Method

Patient VIP in EDTA plasma competes with labeled (125I) VIP for a limited number of primary rabbit polyclonal antibody (BMA Biomedicals, Augst, Switzerland) binding sites. Antibody-bound VIP is then complexed with a goat anti-rabbit secondary antibody and precipitated out of the solution with the use of polyethylene glycol and centrifugation. After the supernatant is discarded, the 125I signal from the pellet is counted on the Wizard2 gamma counter (Perkin Elmer, Waltham, MA). The measured signal is inversely proportional to the amount of VIP present in the patient’s sample. Method validation of the new assay included determination of imprecision (pooled human EDTA plasma), limits of detection and quantification, analytical measurement range (AMR), accuracy by spike recovery, interferences, cross-reactivity, stability, reference interval determination, and agreement with the current Mayo laboratory-developed assay.

Results

Stability studies on freshly collected EDTA plasma showed VIP is stable for 3 days ambient, 7 days refrigerated, and 14 days frozen. Intra-assay imprecision studies showed 8.3, 2.3, and 1.3%CV for mean VIP concentrations of 41, 144, and 304 pg/mL. Inter-assay imprecision was 12.9, 7.6, 5.2, and 7.5%CV for mean VIP concentrations of 30, 81, 180, and 308 pg/mL. The assay limit of detection was 29 pg/mL. The limit of quantitation was 30 pg/mL (%CV = 13%) and was established via precision profile. The AMR was 30–500 pg/mL (slope of 0.98, intercept of 3.0 pg/mL, and R2 of 0.97). The mean % spike recovery using a different preparation lot of calibrator material was 89% (range 83%–93%). The assay was not affected by up to 1000 mg/dL hemoglobin, 1000 mg/dL triglycerides, or 10 mg/dL bilirubin. Peptide histidine methionine 27, neurotensin, gastric inhibitory peptide, motilin, secretin, and glucagon do not cross-react in the assay. The 97.5th reference limit was <86 pg/mL (95% CI 81–90, n = 127). Positive percent agreement (PPA), negative percent agreement (NPA), and overall percent agreement (OPA) with the current Mayo VIP assay were 97, 82, and 90% respectively.

Conclusion

We have developed a replacement VIP RIA. This assay has a lower limit of quantitation and a broader AMR than the current assay. Concordance with the current assay was acceptable.
B-020 Fully Automated and Scalable cfDNA Extraction Solution from up to 10 mL Samples
Authors: Lysen J , Baggs J , Butts T , Durvasula K
Published: September 2023

Background

The use of circulating, cell-free DNA (cfDNA) is rapidly evolving in the clinical landscape, becoming one of the fastest growing areas in recent years. It offers tremendous clinical potential as a screening method for tumor, cancer, disease progression, treatment response, and more. Circulating DNA is found in low quantities and often requires large sample input volumes to isolate the less abundant cfDNA to sufficient amounts needed for downstream analysis. The existing methods for extraction are often silica spin column-based, which involve an extensive amount of hands-on work, and they require large volumes of binding buffers, making it unsuitable for extractions involving sample volumes as large as 10 mL. These existing workflows impose certain limitations in terms of the sample volume and number of samples that they can process.

Methods

To tackle this necessity, Omega Bio-tek has developed a fully automated solution using Mag-Bind® cfDNA Kit (M3298) to extract cfDNA from up to 10 mL volumes from 48 samples in 2.75 h when integrated on Hamilton’s Microlab® STAR™ platform.

Results

The system is scalable and can extract from sample volumes ranging from 1–10 mL without any hardware modification or additional, expensive accessories on the Hamilton workstation. Here, we elucidate the workings of this automated protocol using Omega Bio-tek’s Mag-Bind® cfDNA Kit and compare its performance to a manual extraction protocol from 4 mL of plasma processed using the same kit. We also present additional solutions using the Mag-Bind® cfDNA kit to showcase extraction of fragments as small as 50 bp with minimal genomic DNA contamination.

Conclusion

Omega Bio-tek’s custom workflows provides the customers with different protocols to fit their different application needs. Thus, this kit offers researchers and scientists the flexibility to use the same kit for not only large volume sample processing but also for recovering short cfDNA fragments.
B-034 Evaluation of the Analytical Performance of Sebia CAPI 3 HbA1c Assay
Authors: Laryea E T , Bates M , Fan S
Published: September 2023

Background

HbA1c has been the most important marker for glycemic control and for the diagnosis of diabetes mellitus. The NGSP has invested significant efforts in the standardization of the HbA1c assays, and several analytical methods have been developed. We evaluated the analytical performance of the Sebia CAPI 3 HbA1c assay, which is determined by capillary electrophoresis. Additionally, samples with and without hemoglobin variants are analyzed, and results are compared to two HPLC-based methods.

Methods

Control materials are used in the precision study, which are included in the Sebia CAPI 3 HbA1c kit (Lisses, France). Linearity study is assessed with the linearity materials from LGC Maine Standards (Cumberland Foreside, ME). Carryover is determined by the EP Evaluator (Colchester, VT) with total 21 aliquots of a high and a low HbA1c sample assayed in a specific order. Accuracy study is determined by whole blood patient samples without hemoglobin variants (n = 40) and with hemoglobin variants (n = 8, mostly with Hb S of 30–40%). Results from Sebia CAPI 3 are compared to the current laboratory method (BioRad Variant II Turbo) (Hercules, CA) and the TOSOH G8 (Tokyo, Japan). Both are HPLC-based methods.

Results

The within-run precision at a mean of 5.5% HbA1c has a CV at 1% (n = 24), and the between-run precision at the level of 5.5% and 8.7% HbA1c has a CV at 1% (n = 20) and 2% (n = 20), respectively. Linearity study demonstrates the analytical measuring range is between 3.1% and 20.8%, although each laboratory might establish its own clinical reportable range. Carryover is passed based on the automatically calculated error limit in EP Evaluator (3 times the SD of the Low-Low results). The accuracy study shows the Sebia results correlate well with percent bias at 2% for BioRad Variant II Turbo and 3% for TOSOH G8 in samples with (n = 8) and without (n = 40) hemoglobin variants.

Conclusion

The Sebia CAPI 3 HbA1c assay is based on capillary electrophoresis principle, which is a recognized method by NGSP. The assay has demonstrated good precision, a wide analytical measuring range and minimum carryover. The results are comparable to two HPLC-based methods and are well within the total allowable error of 6% by NGSP in samples with and without hemoglobin variant.
B-035 Maintaining the Analytical Performance of Calcium and Phosphorus in 24-hour Urine Without Addition of 6n hcl in the Pre-Analytical Process
Authors: Canali D G , Zampieri G , Motta C M , Oliveira D C , Rosin C , Argolo S V , Satsuma W
Published: September 2023

Background

The measurements of Calcium (Ca) and Phosphorus (P) in 24-hour urine samples are associated with the evaluation of hyperparathyroidism, renal tubular acidosis, and monitoring of Falconi Syndrome and Nephrolithiasis. The aim is to demonstrate the maintenance of analytical performance without the addition of 6N hydrochloric acid (HCl) in the pre-analytical process.

Methods

The study was divided into stages and carried out for both analytes. In the first stage, correlation tests were performed on 40 samples with and without HCl within the range of linearity. Imprecision was processed at 3 clinically relevant concentrations for 5 days, repeated 5 times. In the second stage, stability testing of Ca and P was performed on 7 samples over 5 days, without HCl (pH 5–6.7), with HCl (pH < 3), alkalized (pH > 8), and neutral (pH 7–7.5). In the last stage, interferent testing was performed on 286 samples, where pH was measured after collection and after acidification with 200uL of HCl. A pH meter and pH strips were used to measure pH, and a Siemens® - Advia 2400 equipment was used for analytical measurement of Ca and P.

Results

In the correlation results, the variance test for both analytes resulted in F-test: 1.01 < 1.69, the difference analysis was less than 2.02, correlation coefficient and Bias% of 1.00 and 0.55 for Ca and 0.99 and 0.27 for P, respectively. Total error (TE) obtained for Ca was 9.69 and 9.41, lower than the allowed TE for both analytes. The repeatability and intralaboratory imprecision of the three levels of each analyte were less than 13.8% for Ca and 9.0% for P. There was 100% correlation of the results when the analyses were performed with samples at pH up to 7; at pH values > 7, the concentrations decreased by 60%. All 268 samples with different pH values, subjected to 200uL of HCl, were suitable for Ca and P measurement with pH below 5.7.

Conclusions

Samples with a pH greater >or = to 7 necessarily need to be acidified with HCl 6N. There is no analytical alteration if all samples in the routine of urinary Ca and P are submitted to 200uL of HCl 6N, even the most alkaline samples. The non-provision of preservative containers to patients reduces the risk of accidents, costs, and the need for legal licenses for handling in healthcare facilities.
B-047 Performance Evaluation of Enzymatic HbA1c Assay on Newly Launched Mindray BS-600M Chemistry Analyzer
Authors: Li M , Xie W , Zeng Y , Xu A , Ji L , Dai J
Published: September 2023

Background

HbA1c has important reference value in risk stratification, diagnosis, therapeutic monitoring and drug selection for diabetic patients. Therefore, the ability to detect HbA1c has attracted much attention. This study was designed to evaluate the performance of the enzymatic HbA1c assay on a newly launched Mindray BS-600M chemistry analyzer, which can be used to measure whole blood samples directly after mixing without centrifugation.

Methods

Total 309 clinical samples (EDTA anticoagulation) were analyzed using Mindray BS-600M (enzymatic method), Tosoh G8 (HPLC method) and Roche c501 (immunoassay method) analyzers for the performance comparison. And also, the analytical performance of BS-600M, such as precision, accuracy, linearity, interference of conventional interferents, hemoglobin variants, hemoglobin derivatives and common drugs, were evaluated using other fresh whole blood samples, eight frozen calibrators (IFCC) and forty frozen venous whole blood samples (NGSP).

Results

The within-run and between-run precision of the enzymatic method for HbA1c determination were less than 1% and linearity of HbA1c (%, NGSP unit) was excellent from 2.96% to 20.23%. The deviations between the results of 48 accuracy samples (IFCC and NGSP) and target values were within ±5%, and the sigma metrics of HbA1c at the high and low concentration levels were between 9.9∼19.0, which belongs to the "world-class" level. The results were excellently correlated with those obtained by Tosoh HLC -723 G8 and Roche Cobas c501 (r = 0.997, 0.992). The regression equations of HbA1c was y = 0.979x + 0.005 (n = 198) between BS-600M and G8 and y = 0.934x + 0.317 (n = 112) between BS-600M and c501. The deviation of interference from conventional interferents (bilirubin, Intralipid, vitamin C), hemoglobin variants (HbS, HbC, HbD, HbE), hemoglobin derivatives (LAlc, CarHb, AcetylatedHb), and commonly used antipyretic analgesics and hypoglycemic drugs were all within ±6%, except for the high level HbF (>10%); and also, BS-600M cannot be applied to severe anemia patients (HGB < 45 g/L), which may lead to falsely high HbA1c results.

Conclusion

The Mindray BS-600M proved to be a robust and reliable method for HbA1c measurement, and was suitable for routine clinical chemistry or hematology laboratory use. In addition, attention or comment need to be added for samples having high HbF concentrations and severe anemia patients in reporting HbA1c% result.
B-056 Performance Characteristics of a New, Fully Automated Planar Microarray Immunoassay for the Detection of IgG Auto-Antibodies Against Centromere Protein B (CENP-B)
Authors: Daubrosse C , Ghillani-Dalbin P , Mercier V , Pasion Galvan R , Bijlsma D , Lukasik E , Botti V , Gomez G , Moreau E , Hausmann M , Ginocchio C C , Miyara M
Published: September 2023

Background

Detection of auto-antibodies against Extractable Nuclear Antigens (ENA), including CENP-B, is key in identification of autoimmune (AI) diseases. Many current test methods are manual, slow, labor intensive or do not facilitate the simultaneous detection of several auto-antibodies, that could reduce time to provide comprehensive results to clinicians. We report the performance characteristics of MosaiQTM CENP-B (Quotient Suisse, Eysins, CH), a new planar microarray immunoassay designed for use with the fully automated, continuous random access, high throughput MosaiQ system for qualitative serological detection of CENP-B IgG auto-antibodies. CENP-B assay is one parameter of a multiplexed MosaiQ connective tissue disease panel in development.

Methods

MosaiQ CENP-B microarrays were prepared by printing CENP-B antigens onto epoxy-silane glass chips and assembled into magazines for processing on the MosaiQ 125 instrument (Fig. 1). Primary patient serum tubes and reagents are loaded on the instrument and automatically processed to generate immunoassay spot signals, interpreted by the instrument using a proprietary image analysis algorithm. Anonymized human serum samples (Hôpital Pitié Salpetrière, Paris, France), characterized as CENP-B auto-antibodies non-reactive or reactive with ≥1 CE-marked devices, were included in the study.

Results

After discordant analysis, MosaiQ system correctly identified 96/99 samples characterized as reactive by comparators and all 199 non-reactive samples: positive percent agreement 97.0% (95% CI, 91.4%, 99.4%), negative percent agreement 100% (95% CI, 98.2%, 100%), overall percent agreement 99% (95% CI, 97.1%, 99.8%). A reproducibility and repeatability evaluation showed, respectively, overall agreement of 99.7% and 100%.

Conclusion

MosaiQ CENP-B microarray provides a high level of clinical concordance with other CE-marked assays for detecting CENP-B auto-antibodies. The MosaiQ System has the potential to advance AI disease testing by increasing laboratory efficiency and productivity, with the ability to multiplex the detection of various auto-antibodies on a single microarray, and a capacity to automatically process thousands of samples per day.
B-059 Allergen-specific IgE Measurement for Peanut Ara h 2*
Authors: Chuang T , Ready N , Rowlands M , Chamberlain J , Hammer D , Sommer M , Hovanec-burns D
Published: September 2023

Background

Legumes such as peanuts are a major source of food allergies among children and adults. Ingestion is the primary route of exposure, although inhalation may also trigger an allergic response in patients with known hypersensitivity. Allergic reactions to peanut can include atopic dermatitis, angioedema, vomiting, diarrhea, and anaphylaxis. Several allergenic peanut proteins have been identified and linked with variable severity of patient symptoms. Ara h 2 is one of the proteins uniquely associated with severe peanut allergy. In this study, we evaluate the analytical performance of the Ara h 2 allergen containing reagent for use in the IMMULITE® 2000 3gAllergy® Specific IgE assay.

Methods

Serum samples were collected from 13 patient samples with documented histories of tree nut or legume-related allergies. The samples were analyzed for specific IgE reactivity to a commercially available recombinant Ara h 2 (rAra h 2) using the IMMULITE 2000 3gAllergy Specific IgE assay (Siemens Healthcare Diagnostics Inc., Tarrytown, NY). Precision, linearity, limit of detection, and endogenous interference were assessed.

Results

Repeatability and within-lab precision at all levels tested were less than 10% over a 20-day study. Regression analysis was performed following linearity assessment and resulted in a slope 95% CI of 0.9251–1.0492 (range 0.3–24 kU/L). Limit of blank and limit of detection were 0.02 kU/L and 0.08 kU/L, respectively. Endogenous interference testing showed <10% difference when spiked with hemoglobin (1000 mg/dL), intralipid (3000 mg/dL), and unconjugated bilirubin (40 mg/dL).

Conclusion

rAra h 2 allergen containing reagent for use in the IMMULITE 2000 3gAllergy Specific IgE assay is sensitive and capable of quantitatively measuring IgE in patients with clinical history of nut allergy.

*Assay under development. Not available for sale. Future availability cannot be guaranteed.
B-097 Storage Stability of HbA1c in Whole Blood Tubes at Room Temperature on the Abbott Alinity c Clinical Chemistry System
Authors: Berman M , Wang X , Cheng P , Kulasingam V
Published: September 2023

Background

Hospital laboratories frequently receive requests to order additional tests on an existing specimen. For this reason, laboratories routinely save specimens after initial testing has been completed for a defined period of time. The current literature lacks specific information regarding HbA1c stability in whole blood and the effects of red blood cell settling. In addition, literature references are not inclusive of various conditions which can cause gaps in data collection such as samples in queue for prolonged periods. To obtain information on prolonged storage of whole blood tubes, HbA1c was assessed for storage at room temperature (RT) (20–25°C) for up to 4 hours on the Alinity c system. In addition, potential carryover from a whole blood to a serum sample was assessed.

Methods

A minimum of 40 whole blood sample pools (approx. 8 mLs) were prepared from remnant samples. Sample pools were mixed, aliquoted, and stored at RT on the Alinity c system. The samples were tested as a single replicate continuously for up to 4 hours without mixing (minimum of 20 cycles) and compared to the baseline sample values. Carryover from a whole blood to a glucose serum sample was assessed after 4 hours continuous testing of whole blood to mimic a high volume testing laboratory. The stability of each HbA1c sample and carryover into a glucose sample were evaluated by comparing the mean absolute or percent difference from the respective sample baseline measurement. The allowed deviation from baseline was 5%.

Results

After 4 hours at RT, the whole blood cells had settled to approximately 50% from the top of the tube. The red blood cell settling had no significant impact on the results. Within sample precision for the 20 cycles of repetitive testing was </ = 1%. The 48 individual samples showed insignificant difference from the baseline value, with a mean change of 1.0% (Range 0–3.2%). Sample carryover from the whole blood samples to a serum glucose sample was not significant (2.71 Baseline vs 2.64 first replicate).

Conclusion

This study demonstrated that the storage stability of HbA1c is stable for up to 4 hours at RT on the Alinity c system.
B-137 Development of a Novel Software System for Providing Real-Time Laboratory Test Information to Clinicians at Point of Order Entry
Authors: Douglas C S , Zaydman M
Published: September 2023

Background

Clinicians depend on an increasingly complex set of laboratory tests to aid in the diagnosis and monitoring of patients. Staying informed about the proper usage of these tests requires active effort, especially as test offerings continually evolve. To aid in test decision making, institutions often implement some form of decision support, including: grouping of common “order sets”, “best practice alert” popups, and web-based “lab catalogs”. For many institutions, lab catalogs provide the most comprehensive information on available tests, but typically exist separate from the interface used to place test orders, and thus suffer from underutilization in actual clinical practice.

Methods

We developed a novel, open-source software to closely integrate our institution’s lab catalog at the point of order entry within our electronic medical record (EMR) system. The software provides an immediate pop-up window anytime a clinician needs additional information while placing orders. The software also provides an independent, customizable search function that helps steer clinicians away from commonly mis-ordered tests. Both functions are immediately available at the point of order entry, without requiring the user to leave the EMR or otherwise interrupt their normal workflow. We conducted a small usability pilot with a focus group of clinicians, using the System Usability Scale (SUS) (1).

Results

Our software reduced the number of clicks and keystrokes needed to access detailed lab information from 27 to 2. The focus group (n = 6) rated the product favorably on the SUS, with average rating 89 ± 9 out of 100.

Conclusion

This study demonstrates proof-of-concept for a novel software decision support tool that provides instant access to lab information at the point of order entry. Future work will focus on evaluating utility in a real-world clinical pilot.
B-186 Investigating LC Column Selectivity for 19 Steroids in a Clinical Research Setting
Authors: Freige M , Marin S J
Published: September 2023

Background

Steroids are a class of structurally related endogenous compounds. Several have the same chemical formula (and hence the same m/z) and must be chromatographically separated for accurate identification and quantitation by LC-MS/MS. Here, six bonded silica solid core or “core-shell” LC columns (see Table 1) were evaluated to determine selectivity and optimum separation conditions for a 19 analyte steroid panel for clinical research. The optimum column for the separation of estrone and estradiol was also established.

Methods

Analytical reference standards (Cerilliant®, Round Rock, TX) were used to prepare diluted samples for injection. Six different core-shell 2.6 µm, 50 × 2.1 mm LC columns (Kinetex™, Phenomenex, Torrance, CA) were evaluated (see Table 1). Detection employed a 7500 triple quadrupole LC-MS/MS equipped with an ESI source used in positive and negative mode (SCIEX®, Framingham, MA) coupled to an Agilent® 1290 liquid chromatograph (Agilent Technologies, Santa Clara, CA).

Results

The traditional C18 column worked best for the complete steroid clinical research panel. Estrogens had the best separation on the biphenyl phase, which demonstrated the highest level of selectivity for estrone and estradiol. The phenyl-hexyl column did not sufficiently separate all steroid isomers in the clinical research panel, however it was sufficient to separate estrone and estradiol.

Conclusion

The proposed HPLC conditions and the core-shell 2.6 μm 50 × 2.1 mm C18 column provided the best separation for 19 steroids with a fast 8 minute run time. For research focused on estrogens, the core-shell biphenyl column yielded the best separation.
B-196 LC/MS-MS Quantification of Myo-inositol: A Novel Biomarker for Kidney Disease
Authors: Omosule C , Blair C , Herries E , Farnsworth C , Dietzen D , Gaut J
Published: September 2023

Currently accepted clinical standards for assessing kidney function include the measurement of creatinine for calculating estimated glomerular filtration rates (eGFR) and albumin to creatinine ratios. However, creatinine, a product of muscle creatine metabolism, is an imperfect proxy for renal function because it is subject to non-renal interferences. There is an unmet need for extremely sensitive and precise biomarkers for evaluating renal function in patients given that acute kidney injury (AKI) and chronic kidney disease (CKD) are linked to high mortality, morbidity, and contribute considerably to health care expenditures.Myo-inositol oxygenase (MIOX) is a novel biomarker limitedly expressed in the proximal renal tubules. MIOX concentrations are elevated in mice with renal injury and in critically ill patients with acute kidney injury (AKI), and increases are observed 54 h prior to changes in serum creatinine are observed. Importantly, MIOX facilitates the conversion of myo-inositol to glucuronic acid. We developed a robust LC-MS/MS method to quantify myo-inositol in human plasma and serum, hypothesizing that following renal injury (acute and chronic), plasma concentrations of myo-inositol will increase, and will correlate with increases in serum creatinine concentrations.In patients with stable renal function, the central 95% distribution of plasma myo-inositol concentrations was 16.61–44.2 µM (Median 27.7 µM, 95% CI: 26.7–28–7). No sex-based differences in plasma myo-inositol were observed. We also observed excellent correlation between plasma myo-inositol and serum creatinine concentrations (r = 0.84, 95% CI: 80–88), as well as excellent sensitivity (99%) and specificity (100%) at a myo-inositol concentration of 50.22 µM for differentiating patients with stable kidney function from those with CKD. Patients with CKD exhibited higher plasma myo-inositol concentrations (P value < 0.0001) with a central 95% percentile of 51.2–309.1 µM (Median 104.1 µM, 95% CI: 92.3–113.6).These results imply that myo-inositol concentrations increase in accord with renal tubular injury and MIOX buildup in patients with kidney dysfunction, making it a potential biomarker for renal impairment.
B-212 Application of Annexin A5Coated Beads for Collection of Extracellular Vesicles and Their Content RNA as Liquid Biopsy for Lung Cancer
Authors: Mei-Chia W , Guan-Yu G , Chih-Liang W , Pi-Yueh C , Chiuan-Chian C
Published: September 2023

Background

Extracellular vesicles (EVs) carry molecules derived from cells, such as RNA. Measurement of tumor-derived extracellular RNA (exRNA) in EVs from plasma can be a promising approach for the development of blood-based cancer detection. However, the lack of an easy method for EVs separation limits its application. The aim of this study is to establish a procedure for EV collection and exRNA detection for non-small-cell lung cancer (NSCLC) diagnosis and monitoring.

Methods

Annexin A5-coated magnetic beads (ANX beads) were developed for EVs isolation. Next, we established a procedure for EV separation and exRNA detection from fluidic specimens using the ANX beads. Then blood samples from 22 NSCLC patients and 20 healthy controls were collected to evaluate the procedure and we detected different RNAs in the samples.

Results

We evaluated the ability of ANX beads in EVs separation and found that the beads could capture large and small EVs and could increase RNA concentration. Then we detected different RNAs in the samples and found that CK19 mRNA, MALAT1 lncRNA, and miR155 microRNA had good performance to differentiate NSCLC from healthy controls.

Conclusion

In conclusion, this assay protocol could separate EVs to enrich RNA concentration. The EVs separation employed was captured by magnetic beads which could be adopted into an automatic nucleic acid extract system to conveniently concentrate and extract nucleic acid from EVs.
B-228 Partition-free Digital Polymerase Chain Reaction: A Novel Approach to Enable Rapid Turnaround Time With Digital Accuracy
Authors: Erb P , Myrick J , Hensley M , Schreiber J , Sundberg S
Published: September 2023

Background

Partition-free digital PCR (dPCR) is a new approach developed to provide absolute quantification in less than 10 min. Partition-free eliminates the need for droplets or physical wells that need to be filled and sealed. The technique relies on rapid thermal cycling and microfluidics to limit diffusion and enable the identification of individual amplicon clusters originating from a single target nucleotide template. Rapid heating and cooling were achieved with a thermal cycling engine comprised of a broadband LED with a sapphire block that is both a light guide and heatsink. Sample is distributed through a custom microfluidic cartridge containing 64 microchannels for optical quantification.

Methods

The feasibility of partition-free dPCR was tested with primers targeting MTHFR c.665. A titration was performed on three 4X serial dilutions of cell line genomic DNA in triplicate. Fluorescent profiles were generated for all 64 microchannels from an image captured after 35 PCR cycles, where amplicon clusters were identified. Fluorescent peaks of the amplicon clusters were counted and quantified via a custom analysis algorithm, which uses Poisson statistics to calculate concentration.

Results

The quantification results obtained from the partition-free dPCR system were compared to concentration measurements taken via a Bio-Rad QX200 platform. Concentrations of 69.4 ± 0.8, 15.4 ± 1.5, and 3.3 ± 0.6 copies/µL (R2 = 0.9997) were measured on the partition-free system, and concentrations of 67.8 ± 3.6, 17.2 ± 1.1, and 2.8 ± 0.2 copies/µL (R2 = 0.9995) were measured on the Bio-Rad QX200.

Conclusion

It has been demonstrated that absolute quantification can be achieved in less than 10 min with a partition-free dPCR platform, and shows results comparable to a QX200 droplet dPCR system. The partition-free rapid dPCR technology described herein allows for the exploration of new avenues in molecular diagnostics, with the potential to bring absolute quantification of dPCR to a Point-of-Care setting.
B-259 Exocas-2 Rapid and Pure Isolation of Exosomes by Anionic Exchange using Magnetic Beads
Authors: Bae M , Shin S
Published: September 2023

Background

Extracellular vesicles (EVs) are considered essential biomarkers in liquid biopsies. Despite intensive efforts aimed at employing EVs in a clinical setting, workable approaches are currently limited owing to the fact that EV-isolation technologies are still in a nascent stage. This study introduces a magnetic bead-based ion exchange platform for isolating EVs called ExoCAS-2 (exosome clustering and scattering). Owing to their negative charge, exosomes can easily adhere to magnetic beads coated with a polycationic polymer. Owing to the features of magnetic beads, exosomes can be easily processed via washing and elution steps and isolated with high purity and yield within 40 min.

Methods

Cationic salt is coated on the surface of magnetic beads with carboxyl group surface residues. Inject the cation coated magnetic bead (ExoCAS) into the plasma solution and incubate in 4°C for 30 minutes. By charge interaction, negative charged EVs are attached to the cationic beads. And then, pH 6 washing buffer and 1M NaCl elution buffer were used for EV isolation.

Results

The present results confirmed the isolation of exosomes through analyses of size distribution, morphology, surface and internal protein markers, and exosomal RNA. Compared with the commercially available methods, the proposed method showed superior performance in terms of key aspects, including operation time, purity, and recovery rate.

Conclusion

This highlights the potential of this magnetic bead-based ion exchange platform for isolating exosomes present in blood plasma.
B-260 Innovative One-Step Extraction of Exosomal micro RNA Without Isolating Exosomes
Authors: Bae M , Shin S
Published: September 2023

Background

Extracellular vesicles (EVs) are receiving great attention as biomarkers in liquid biopsies since they are involved in physiological and pathological processes. It is important to obtain a high-yield of nucleic acids because it is possible to prediagnose through the exosome.

Methods

Here, we report an efficient one-step method for extraction of miRNAs by clustering EVs. When cationic salt is added to samples, anionic EVs tend to form clusters and precipitate within a short time. After centrifugation and adding lysis buffer, miRNA can be extracted with a spin column. Commercial methods were compared to determine the yield of the precipitation method using cationic salt. In addition, we compared with two reference methods to determine the miRNA extraction efficiency. The first one is consisted of two-step method of extracting miRNA from isolated exosome (2 step), which is a general method, where as the second reference method is a commercial product consisting of 1-step extracting miRNA from biofluids.

Results

Cluster precipitation efficiency using cationic salt was identified through cluster image, SEM, and Western blot. The present method for extract exosomal miRNA has an EV isolation efficiency of more than 3 times compared to the commercial EV isolation methods. In addition, it has excellent exosomal miRNA extraction efficiency from various biofluids such as Saliva, Urine, and Plasma, and can be used as a sample of breast cancer patients.

Conclusion

These results might be contributed by the sequence of precipitation of exosomes, exosome lysis, and nuleic acid extraction. The rapid and efficient method for extracting exosomal nucleic acids will make an innovative contribution to clinical applications.
B-262 Evaluation of the Genotypic Profile of the c.202 G>A Mutation in the G6PD Gene in Brazil
Authors: Silva J D , Rios J S , Zauli D A
Published: September 2023

Introduction

Glucose 6 phosphate dehydrogenase (G6PD) is an enzyme found in the cytoplasm of all human cells and plays an important role in preventing cellular damage to reactive oxygen species (ROS). G6PD deficiency is inherited in an X-linked recessive genetic disorder that can destroy red blood cells (hemolysis) leading to acute hemolytic anemia during periods of increased ROS production. The disease prevalence is more frequent in male individuals. To date, more than 400 G6PD variants have been identified, and molecular studies have shown that G6PD deficiency can be associated with different mutations. The c.202 G > A mutation (rs1050828; NM_000402.4) was described as the most frequent. The rapid and accurate diagnosis of this condition is extremely important in neonatal screening and cases of diagnostic suspicion. Therefore, this study aims to describe the genotypic profile of the c.202 G > A mutation in the G6PD gene in Brazilian samples by two molecular assays.

Methods

Genomic DNA samples from 130 individuals previously genotyped by Restriction Fragment Length Polymorphism PCR were used for validation. DNA isolation was performed in MagNA Pure 24 Total NA Isolation Kit at the automated platform MagNA Pure 24 System (Roche Diagnostics, Basel, Switzerland), according to manufacturer instructions. Genotyping for rs1050828 was performed using TaqMan SNP Genotyping Assays (Thermo Fisher, Foster City, CA) at 7500 Fast Real-Time PCR System (Thermo Fisher, Foster City, CA), according to manufacturer instructions. The results were analyzed using TaqMan Genotyper Software version 1.0.1.

Results

This study evaluated 130 blood samples, of which 81 were men (62.3%) and 49 were women (37.7%). Male carriers of rs1050828 A genotype (hemizygotes) predominated (56/81, 69.1%) and the remaining carriers for allele G (wild type) were 25/81 (30.9%). While female carriers of rs1050828 GG (wild type) predominated (39/49, 79.6%), heterozygotes were 5/49 (10.2%), and homozygotes also were 5/49 (10.2%). The number of male individuals with the presence of the mutation in hemi/homozygotes was considerably higher when compared to female individuals. This is explained by the fact that the mutation is inherited in an X-linked recessive manner. The results showed agreement and reproducibility of 100% for both methodologies. However, the qPCR assay sensibility and efficiency (>99%) were higher than the RFLP PCR assay.

Conclusions

Here, we described an efficient molecular method to detect the mutation c.202 G>A in the G6PD gene. The frequency of c.202 G>A described in this study was similar to that observed in others population studies in Brazil. Detection of the mutation target requests an additional evaluation of the G6PD activity level. Obtained results should be evaluated in association with the clinical aspects. The absence of the c.202 G>A in the G6PD gene does not exclude the existence of other mutations in the same region.
B-276 Preservation of Proteomic Inflammatory Biomarkers signal in a Saliva Multiomic Buffer at Room Temperature
Authors: Jensen M M , Maecker H , Nguyen T , Vu D , Vigerust D , Gupta R
Published: September 2023

Background

Saliva represents an attractive biospecimen as it is pain-free, simple for at home collections, and patients can provide multiple samples over time to provide time resolved data. However, few diagnostic tests have been developed using saliva due to poor stability and comparatively low concentration of analytes. Stabilization of biomarkers at ambient temperature in saliva would enable labs to take advantage of the benefits of saliva sample collections, while minimizing the risks posed by the degradation of already scarce analytes.

Methods

Matched samples of neat serum, saliva, and saliva collected with Saliva Multi-omic Device (SMD) preservative were collected from 30 volunteers (15 male & 15 female) ages 18–64 with no known morbidities. The Olink Inflammation panel was used to examine 92 inflammation-related proteins. The method utilized proximity extension assay technology, wherein antibody probe pairs reacted with their respective target proteins to generate a polymerase chain reaction target sequence through proximity-dependent DNA polymerization. This sequence was measured using real-time polymerase chain reaction and normalized in two steps, producing a semi-quantitative Normalized Protein eXpression (NPX) unit. To evaluate analyte stability specimens were measured initially and then again after 24 h at room temperature (23 ± 2°C).

Results

Protein expression (NPX) units for 36 of the 92 inflammatory markers had higher concentrations in saliva compared to serum. Serum (78/92) and saliva 78% (72/92) showed similar coverage for the number of markers in the panel where at least 75% of subjects had values exceeding the limit of detection (LoD). SMD preservation buffer improved the average stability of inflammatory protein biomarkers by 19.6% compared to neat saliva and 10.2% compared to serum over 24-hours at room temperature. However, the relative stability of individual between protein markers [DV1] [HM2] varied. For proteins in SMD buffer, initial concentrations of analytes were highly predictive [HM3] of values 24 h later, having Pearson correlation coefficients > 0.7, for 23 of the analytes and greater than 0.5 for 47. The change in protein marker in serum over time was only moderately predictive of the protein’s stability in saliva with or without preservative, having correlations of 0.33 ± 0.20 and 0.47 ± 0.19 (mean ± standard deviation), respectively.

Conclusion

This study demonstrates potential utility of saliva in proteomic applications utilizing the Olink platform. The SMD preservation buffer stabilized inflammatory biomarkers in saliva samples likely through protease inhibition and antimicrobial action. Preservation of self-collection salivary samples by subjects without cold storage enables noninvasive sampling and reliable measurements even when samples are collected by users at remote locations or under challenging conditions.
B-278 Pilot Clinical Study of the CarePoint Assay
Authors: Briggs1 J S , Brooks-Starks A H , Elenberger T J , Brenneman R , Scarpato K R , Chang S S , Bassett J C , Linehan J
Published: September 2023

Background

An estimated >710 000 individuals live with bladder cancer in the United States, with approximately 82 290 new cases expected this year (2023). Recurrence rates for bladder cancer are high: approximately 65% of patients with non-invasive or in situ tumors and 73% of patients with more advanced disease experience recurrence within five years. Consequently, patients treated for bladder cancer require prolonged surveillance to safeguard against tumor recurrence. The gold standard of surveillance, cystoscopy—an uncomfortable, expensive, and invasive procedure—is recommended every three to 12 months. Consequently, there is a need for an inexpensive, effective, noninvasive diagnostic tool as an alternative to cystoscopy.

The CarePoint Assay (CPA) is a 30-minute electrochemical immunoassay for the detection of disease-state biomarkers in human urine. The analytical performance of CPA has been evaluated for method comparison with a three-hour sandwich immunosorbent assay (ELISA) for bladder tumor antigen (BTA), a diagnostic biomarker for bladder cancer.

Method

Receiver operating characteristic (ROC) curves were calculated by simple logistic regression with Prism9 (GraphPad) using patient urine samples across multiple medical centers (n = 51), comparing BTA concentration (continuous variable) with high-grade tumor status (binary variable). Patient urine samples were evaluated by CPA using commercial potentiostats for comparison with a sandwich ELISA. The data was analyzed using simple linear regression.

Results

Patient urine evaluated by CPA was interpolated by simple linear regression using a three-point calibration with standard spiked into urine pooled from healthy donors. Data produced by sandwich ELISA was interpolated by five-parameter nonlinear regression using a 12-point standard curve. Both methods showed good correlation: (r) = 0.93. Simple logistic regression comparing analyte concentration to high-grade tumor status produced a sensitivity and specificity of 52% and 88% with an AUROC of 0.71 for the CPA, and a sensitivity and specificity of 72% and 77% with an AUROC of 0.77 for the sandwich ELISA. Sensitivities and specificities were calculated using Youden’s J statistic.

Conclusions

We established proof of concept for a rapid, electrochemical immunoassay for a diagnostic biomarker of bladder cancer. The CPA correlated well with sandwich ELISA, an established method for measuring biomarkers in bodily fluids. Future work will focus on refinement of the CPA architecture for point-of-care use and subsequent evaluation of analytical performance.
B-289 Acute Kidney Injury Urinary Biomarker, Neutrophil Gelatinase-Associated Lipocalin (NGAL) Analytical Performance Evaluation on Roche Cobas c502 Chemistry Platform
Authors: Samara V , Buggs V , Song L
Published: September 2023

Background

Acute kidney injury (AKI) is a sudden and serious kidney damage condition that affects more than 1.2 million people every year. KDIGO guidelines suggest serum creatinine increase as a diagnostic marker for AKI but elevation of serum creatinine levels can take up to seven days. Neutrophil gelatinase-associated lipocalin (NGAL) has been identified as an early tubular injury biomarker for AKI that is detected within three hours of AKI patients’ urine. In this study, we evaluated technical performance of BioPorto NGAL assay on Roche cobas c502 chemistry analyzer.

Methods

BioPorto’s (Hellerup, Denmark) particle enhanced turbidimetry immunoassay (PETIA) NGAL assay was installed on cobas c502 chemistry analyzer (Roche, Indianapolis, U.S.A) following vendor-specific application parameters with few modifications. Linearity was determined with BioPorto’s linearity material and verified by serial dilutions of a high NGAL patient urine sample. Limit of quantitation was determined in urine samples with values 20, 25 and 35 ng/mL, run in duplicate for 10 days. Precisions were determined by running urine samples containing 100 ng/mL and 400 ng/ml NGAL 20 times for within-run precision and twice a day × 10 days for between-run precision, respectively. To determine the accuracy of the NGAL method on cobas 502, 40 sample results (provided by the vendor) were compared with those measured on Siemens Atellica using BioPorto’s NGAL assay. Reference interval verification was carried out in 40 urine samples collected from healthy adult volunteer-donors.

Results

The linearity was found to be excellent from 0–3000 ng/mL. Limit of quantification was found at 26 ng/mL. Precision results are in the table below.

Method comparison showed a slope of 0.90 and intercept of 32.9. The reference interval was verified to be <50 ng/mL (in accordance with current NGAL reference cut­off for AKI diagnosis).

Conclusion

Performance of BioPorto’s NGAL PETIA assay was validated in urine on cobas c502 chemistry analyzer.