bluuebunny/crossref_metadata_2025 huggingface dataset:
A-246 Desialylation Profiles of Monocytes and Macrophages Upon LPS Stimulation
Authors: Aljohani M A , Sun X A , Zhao Y A
Published: September 2023

Background

Cell surface expresses a dense layer of glycans terminated with sialic acids (Sias), known as sialylation, which often represent different cellular statuses. Meanwhile, the removal of Sias from the glycans catalyzed by sialidase (desialylation) is also involved in many biological processes. Previous studies have established that lipopolysaccharide (LPS) can induce the expression of endogenous sialidases in monocytes and macrophages, leading to desialylation of the LPS receptor TLR4. This desialylation of TLR4 has been shown to impact the LPS/TLR4 signaling pathway. In this study, we conduct a profiling of desialylation in THP-1 monocytes and THP-1 macrophages in response to LPS stimulation. This will provide insights on the intricate connection between sialylation, desialylation, and LPS-induced signaling.

Methods

The activity of sialidases was assessed by utilizing 4-methylumbelliferyl-N-acetylneuraminic acid (4-MU-NANA) and ganglioside GM3 as substrates. The total sialic acid in the THP1 was quantified using liquid chromatography tandem mass spectrometry (LC-MS/MS). Flow cytometry was used to analyze the exposure of galactose on THP-1 monocytes and macrophages after LPS stimulation (100 ng/mL LPS for 24 h), using PNA-FITC staining. The expression level of Neu1 and Neu3 sialidases in cell lysates of THP-1 monocytes and macrophages were assessed by western blot technique after LPS stimulation. ELISA was used to measure the levels of sialidases in the cell supernatant of THP-1 monocytes and macrophages upon LPS stimulation.

Result

We found a significant reduction in total sialic acid levels in both THP-1 monocytes and macrophages upon LPS stimulation. This was further confirmed by the significant increase in total sialidase activities which measured by both 4-MU-Neu5Ac and GM3 substrates in THP-1 monocytes and macrophages upon LPS stimulation. Surprisingly, we also found that sialidase activities significantly increased in the cell supernatant of both THP-1 monocytes and macrophages, indicting sialidases secretion upon LPS stimulation. Western blot analysis further confirmed the high expression and secretion of both Neu1 and Neu3 sialidases from THP-1 monocytes and macrophages after LPS stimulation.

Conclusion

The present study conducted the first systematic profiling of desialylation and sialidase expression in monocytes and macrophages upon LPS stimulation. This results demonstrate that endogenous sialidase expression and secretion play a role in the LPS/TLR4 signaling pathway and subsequent cellular processes and functions in the context of inflammation and immune response.
A-250 Rapid molecular Identification of Herpes Simplex, Varicella-zoster, and Enteroviruses from Cerebrospinal Fluid
Authors: Bialas J , Joki L , Hermanowski M , Brea-Sellheim K , Leibhan H , Jacobs M , Lång M , Vainio J , Martelin J
Published: September 2023

Background

Accurate and efficient molecular testing of viral meningitis/encephalitis is crucial for proper patient care. Here we describe the clinical evaluation of the GenomEra® HSV-1/2, VZV + EV Assay Kit (Uniogen, Finland) allowing simultaneous detection of four key viruses responsible for causing meningitis/encephalitis, namely HSV-1 and HSV-2, VZV, and enteroviruses.

Methods

The study was carried out at three European institutions in 2022, with a total of 193 frozen, anonymized leftover cerebrospinal fluid (CSF) specimens analyzed retrospectively. For the GenomEra HSV-1/2, VZV + EV Assay Kit, 50 µL of CSF was mixed with lysis buffer in a 1:1 ratio, allowed to incubate for 1 min at room temperature, and then filtered by a 1-min centrifugation through an Extraction Column utilizing size-exclusion chromatography. A 35-µL sample of the flow-through was applied into the Test Chip and analyzed using the GenomEra CDX System. The cotemporally performed comparison tests included the FilmArray Meningitis/Encephalitis (ME) panel (bioMérieux), Xpert® EV Assay (Cepheid), artus VZV assay (Qiagen), and a well-established, validated non-commercial real-time RT-PCR assay for the detection of HSV-1/2, VZV, and EV.

Results

The GenomEra HSV-1/2, VZV + EV Assay Kit produced results in 70 min with a sensitivity and specificity of 92.2% (95% CI: 82.7–97.4%) and 99.9% (95% CI: 99.2–100%), respectively, as compared to the reference tests. Of the discordant results, five tested negative with the GenomEra assay, whereas the reference tests produced low positive results (Ct values ≥ 35). In addition, one specimen yielded a positive signal for VZV while remaining negative with the reference tests.

Conclusion

The GenomEra HSV-1/2, VZV + EV Assay Kit showed a high overall specificity and a high level of sensitivity particularly for herpes simplex and varicella-zoster viruses. For enteroviruses, while the specificity reached 100%, the sensitivity was not as high as with the reference methods. The high clinical performance, together with the simple sample preparation protocol and short turnaround time make the GenomEra assay a competitive molecular test especially for small and medium-sized laboratories that may not have a specialist in molecular biology. In addition, the GenomEra assay only requires a small amount of sample, conserving the valuable specimens for additional testing.
A-252 Differentiation of SARS-CoV-2, Influenza A and B, and Respiratory Syncytial Virus Using a Single Swab: A Clinical Performance Evaluation of the BD MAX System Multiplex PCR Assay
Authors: Faron M , Ledeboer N , Van Der Pol B , Davis T , Lockamy E , Paradis S , Andrews J
Published: September 2023

Background

Some shared clinical features of SARS-CoV-2, influenza (flu), and respiratory syncytial virus (RSV) infection often pose a diagnostic challenge for clinicians. In that context, the use of molecular diagnostic systems multiplex assays, using a nasopharyngeal or nasal swab, facilitates the detection of the causing virus by differentiating between the RNA of each disease. Our study evaluated the clinical accuracy of one such multiplex PCR assay, the BD Respiratory Viral Panel for BD MAX™ System (BD MAX RVP).

Methods

Symptomatic patients that had a nasopharyngeal and nasal specimen prospectively collected from 6 geographical locations in the US were enrolled in the study between January and April 2022 along with nasopharyngeal swabs retrospectively collected between December 2019 and January 2022. All specimen testing was performed using the BD MAX System. Hologic® Flu A/B/RSV assay on Panther Fusion™ System was the comparator for flu A, flu B, and RSV while a composite of two out of three assays authorized under FDA Emergency Use Authorization (Roche cobas® SARS-CoV-2 on cobas® 6800 System, Hologic® Aptima® SARS-CoV-2 on Panther® System, and Quidel Lyra® SARS-CoV-2 on Applied Biosystems® 7500 Fast Dx) was used as comparator for SARS-CoV-2. Positive and negative percentage agreements (PPA and NPA, respectively) were calculated to determine performance. Reproducibility and precision assessments were also performed as part of the study.

Results

Tested specimens included 252 nasopharyngeal and 254 nasal swabs, prospectively collected, and 240 retrospectively acquired nasopharyngeal specimens. For prospectively collected SARS-CoV-2 nasopharyngeal specimens, PPA of 98.8% (95% CI 93.6, 99.8) and NPA of 98.2% (95% CI 94.9, 99.4) were observed while for flu A, PPA was 100% (95% CI 61.0, 100) and NPA 99.6% (95% CI 97.7, 99.9). PPA and NPA for prospectively collected SARS-CoV-2 anterior nasal specimens were 98.8% (95% CI 93.3, 99.8) and 98.3 (95% CI 95.1, 99.4), respectively. For flu A, PPA of 100% (95% CI 61.0, 100) and NPA of 99.6% (95% CI 97.8, 99.9) were observed. Lack of enrolled positive patients with flu B or RSV precluded the PPA calculation for those viruses. However, NPA for both flu B and RSV was 100% for the two specimen types. In retrospectively obtained nasopharyngeal specimens, PPA was 100% (95% CI 93.7, 100) for flu A, 100% (95% CI 93.8, 100) for flu B, and 98.4% (95% CI 91.5, 99.7) for RSV while NPA was 98.9% (95% CI 96.1, 99.7) for flu A, 98.9% (95% CI 96.1, 99.7) for flu B, and 100% (95% CI 97.9, 100) for RSV.

Conclusion

The BD MAX RVP assay showed a strong clinical performance in detecting and differentiating between the RNA of SARS-CoV-2, flu A and B, and RSV.
A-259 Detecting Human RNase P Gene Prevents False-Negative of SARS-CoV-2
Authors: Chuang F
Published: September 2023

Background

Real-time Reverse transcriptase—polymerase chain reaction (RT-PCR) is a regular technique for detecting SARS-CoV-2 infection. Human RNase P sequence is using as internal control for PCR assay usually. This article discusses the correlation between SARS-CoV-2 PCR result and human RNase P gene result.

Methods

Nasopharyngeal swabs were collected from two patients within three consecutive days. We detected for SARS-CoV-2 target gene and human RNase P gene by using real-time RT-PCR assay, which SARS-CoV-2 target gene includes E gene, Rarp gene and N gene . And the threshold cycle values (Ct) were determined by cobas® z 480. Evaluate the results of SARS-CoV-2 and RNase P gene detection. All samples were also analysis for SARS-CoV-2 infection in Taiwan Centers for Disease Control.

Results

Results of SARS-CoV-2 PCR assay were consistent in both laboratories. SARS-CoV-2 target genes were detected from specimens of both patients on day1. Ct values of E gene were 29.63 and 29.50. Ct values of Rdrp gene were 32.23 and 32.50. Ct values of N gene were 33.09 and 32.68. Ct values of RNase P gene were 21.21 and 21.22. On day 2 and 3, all specimens were negative detected for SARS-CoV-2 PCR. Ct values of RNase P gene were 25.73 and 26.16 on day 2 and 29.82 and 30.28 on day 3.

Conclusion

The Ct values of human RNase P gene detection can be correlated with the numbers of collected cells. Comparing the results of two patients for three consecutive days, the Ct value of human RNase P gene related to SARS-CoV-2 PCR result. Collection technique can be a factor to affect Ct value of human RNase P gene detection. Develop acceptance range for Ct value of RNase P gene detection can be a quality standard for sample collection to prevent false-negative of SARS-CoV-2.
A-307 Spread Dynamic of SARS-CoV-2 Variants and Related Alterations in Transmission Rates: A National Scale Study
Authors: Silva J D , de Lima A B , Alvim L B , Malta F S , Mendonça C P , Fonseca P L , de Souza R P , de Aguiar R S , Zauli D A
Published: September 2023

Background

In late 2019, the world was surprised by the new coronavirus (SARS-CoV-2), responsible for the COVID-19 pandemic and the ability to develop and share knowledge about SARS-CoV-2 was essential in combat of the pandemic. Genomic surveillance has played a fundamental role in identifying new mutations, detecting emerging variants, and monitoring viral evolution. Although next-generation sequencing (NGS) remains the gold standard for the identification of new variants, this technology is still restricted due to the high cost. Thus, the use of accessible methods for SARS-CoV-2 monitoring is necessary. This study aimed to collect and share monitoring data of COVID-19 in Brazil and to evaluate the dynamics of SARS-CoV-2 variants during the two periods of changes in the epidemiological scenario (second and third pandemic waves) and the transmissibility rates among circulating VOCs.

Methods

The study evaluated 27 003 samples from all federative units in Brazil between April/2021 and January/2022. Specific primers and probes were used for the mutations K417T/N, L452R, E484K/Q, N501Y, P681R (TaqMan SARS-CoV-2 Mutation Panel), and the SGTF (S-gene target failure) by the TaqPath COVID-19 CE-IVD RT-PCR Kit according to the frequencies of the Gamma, Delta, and Omicron VOCs in the evaluated time. Samples showing an unexpected mutation profile in RT-qPCR genotyping were submitted to NGS. Data for viral targets of about 227 661 positive samples were collected to assess the period of predominance of each VOC as an inference of transmissibility potential.

Results

Samples were collected in all 27 Brazilian federative units. The replacement of the Gamma by Delta variant occurred in about 5 months, between June and September 2021, when the Delta variant reached 100% of infections. The transition of predominance among the Delta to Omicron variant was only 2 months, demonstrated by the progressive increase of Omicron in November (3.4%), December (67.5%), and January (97%), accompanied by the consequent growth of COVID-19 positive cases in the country. These results reinforce the higher transmissibility of Omicron compared to other variants identified in Brazil. The adopted strategy was able to identify the Gamma, Delta, and Omicron VOCs as well as the presence of other variants in 99.2% of the samples. Only 0.8% of the samples were not characterized and it was submitted to NGS.

Conclusion

We described a methodology that was able to effectively follow the fast establishment of Omicron VOC related to its higher transmission rates and genomic mutations that explain the highest peak of COVID-19 in our country. This study comprises the largest national-scale of genomic surveillance of SARS-CoV-2, providing information about the virus spread in Brazil.
A-338 TSH Reference Range in the First Trimester in a Iodine Sufficient Population of Pregnant Women Attended by the Public Health System in Curitiba South of Brazil
Authors: Paula R F , Felchner P C , Telles T M , Júnior C O , Valdati K D , Panizza T M , Moraes T P , Carlos G B
Published: September 2023

Background

Thyroid diseases are common among women of reproductive age, and the serum concentration of thyroid-stimulating hormone (TSH) is the most used index to assess thyroid function during pregnancy. Changes in thyroid function during pregnancy can have a clinical impact on a woman's health and pregnancy outcome, as well as interfere with fetal health and child development. An adequate thyroid function is essential for normal intrauterine development. When comparing the levels of TSH between pregnant and non-pregnant women, TSH levels are lower in pregnancy, mainly in the first trimester, with gradual increase during pregnancy. Although some guidelines propose fixed TSH reference values in pregnancy according to the gestational trimester, the main recommendation is to carry out a study of their local population due to the significant variation in TSH reference range levels in different populations and different assays. Therefore, it is necessary to determine values of a reference interval for TSH for the local population of pregnant women. This study aims to determine the normal reference range of TSH in the first trimester of gestation in the public health system pregnant women from Curitiba.

Methods

This is an ongoing prospective cohort study, with the population sample from the pregnant women using the public health system, with age >18 years old in Curitiba, South of Brazil. 383 pregnant women were invited to participate. After evaluate the exclusion criteria (gestational age more than 13 weeks, twin pregnancy, thyroid disease, iodine ingestion, identification of the samples in the laboratory), 225 pregnant women had the TSH measured by chemiluminescent immunoassay on Atellica IM Analyzer (Siemens Healthineers), as well as the TPO-ab presence, FreeT4 and Total T4. The 2.5, 50 end 97,5 TSH percentiles were estimated for the normal pregnant population.

Results

The mean gestational age was 8,6 weeks (standard deviation 2.33). From 225 pregnant women, 22 (9.8%) had TPO-ab positive. The 2.5 and 97.5 percentiles of TSH were 0.12 and 5.28 μIU/mL after using de exclusion criteria. The populational iodine status was sufficient. In the TPO-ab positive population was found a higher level of TSH. The proportion of TPO-ab positive was significantly higher in the population with TSH >2.5 μIU/mL.

Conclusions

This preliminary data shows the TSH reference range with levels higher than other regions, reinforcing to do not use fixed values of TSH from other populations to evaluate the thyroid function in pregnancy. Comparing to the laboratory reference for non-pregnant adult (0.48 to 5.60 μIU/mL), the recommended reduction of 0.5 μIU/mL on upper limit and 0.4 μIU/mL on the lower limit found similar values to our findings.
A-363 Evaluating the Performance of Emerging STI Point of Care Assays Using Novel, Room Temperature Stable CT/NG/TV/MG Positive Swab Quality Control Materials
Authors: Rivers S , Zhelev P , Thakur M , Alagic A , Li J , Randall C , Luscher M , Hughes K
Published: September 2023

Background

The emergence of Point of Care tests that detect Sexually Transmitted Infections (STIs) is critical for screening and diagnostic programs in low-resource settings. Point of Care tests that are designed to simultaneously detect Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV), and Mycoplasma genitalium (MG) promote rapid diagnosis and early treatment of the most common etiological agents causing STIs. Along with the roll-out and use of these tests, there is a growing need for complex multi-analyte quality control materials that are stable at room temperature and challenge test performance by identifying result deviations, lot-to-lot variability, and routine laboratory workflow errors. Microbix developed a whole-workflow multiplex quality control desiccated on a Copan FLOQSwab® that is stable at 2–30°C and contains inactivated whole-genome target pathogens. The objective of this study is to evaluate sample performance with multiple Point of Care diagnostic platforms that are currently in development and commercial IVD tests that are routinely used in the laboratory.

Methods

Microbix designed an inactivated STI whole-workflow control that contains whole-genome CT, NG, TV, MG, and human cells to satisfy sample adequacy control requirements. The formulation is desiccated on a Copan FLOQSwab® to mimic patient specimen formats and ensure sample compatibility with all assay workflows and elution buffers. Sample performance was evaluated with Cepheid Xpert® CT/NG assay, Cepheid Xpert® TV assay, Abbott Alinity m STI assay, BD MAX™ CT/GC/TV assay, Seegene Novaplex™ CT/NG/TV/MG assay, EliTechGroup STI PLUS ELITe MGB®Kit, and commercial Point of Care assays currently in development.

Results

The STI positive swab formulation demonstrated acceptable performance when tested with various nucleic acid amplification tests that detect CT, NG, TV, and MG targets. The formulations also behaved as useful quality management tools by highlighting design limitations for commercial Point of Care assays that are currently in development.

Conclusion

CT/NG/TV/MG whole-genome multiplex formulation desiccated on a Copan FLOQSwab® is an advantageous prospective quality control material that is stable at room temperature and supports the clinical use and accuracy of emerging STI assays, including the most challenging Point of Care test formats. The formulations showed acceptable performance on multiple platforms, demonstrating excellent commutability. Additionally, the dry swab format offers great practicality for monitoring assay performance in low-resource settings.
A-379 Frequency of Reassignment of Blood Gas Sample Source at a Large Tertiary Care Hospital
Authors: Kim R , Altura M , Gantt K , Hashim I A
Published: September 2023

Introduction

Arterial blood gases analysis are central to the assessment and management of the critical patient acid-base status and its collection requires expertise and is often collected by trained respiratory therapists, nursing, and medical staff. Venous blood gases, arterialized venous blood gases, and capillary samples are however easier to collect. In this retrospective, study we examined the frequency of reassignment of blood gases results to a different sample type.

Method

Retrospective review of blood gases results requiring sample type reassignment collected during the last three years from January 2020 to December 2022 were obtained. Results were analyzed for concordance with sampling site and workflow analysis.

Results

Over the three years study period (2020 to 2022), a total of 196 samples for blood gases analysis required reassignment to a different collection site representing 0.12% of all blood gases testing. Among the 123 initial arterial collections, 72 were reassigned as venous, 40 reassigned to mixed venous sample, and one to capillary collection. Whereas, among the initial 24 venous samples collections 16 were reassigned as arterial and 8 to mixed venous collections. Additionally, among the 25 initial mixed venous collections, 16 were reassigned as arterial, 9 reassigned as venous and 3 were reassigned as capillary collections. The frequency of reassignment from arterial to venous was significantly higher compared to venous to arterial (P < 0.05) or for the reassignment in the mixed venous collections. Although a smaller number of samples were amended to capillary, capillary blood gases reflect arterial samples.There was no significant difference in the frequency of sample type reassignment between the three years of collection with the exception of a slight drop in year 2020 where the majorly of patients were of COVID-19 infection and that collections were likely performed by the same assigned medical personnel.

Conclusion

Common errors in blood gases collection are failure to obtain an arterial sample and accidental collection of venous samples instead often due to close proximity is common. In this retrospective review, the common reassignment is that of arterial to venous followed by arterial to mixed venous and venous to arterial sample types. Artifactual causes that may lead to misclassification of sample type include sample integrity as exposure to room air falsely reduces pCO2 and elevates pO2 giving a false impression of an arterial collection, similarly, dilution of arterial blood samples by heparin is common (inadequate blood fill of syringe) falsely lowers pCO2 both raising doubt as to the sample type.The impact of reassignment of change of sample type on outcomes and on interpretation requires analysis.
B-014 Impact of Centrifugation Time Reduction in Laboratory Automation System
Authors: Choosongsang P
Published: September 2023

Background

The centrifugation of specimens is an important preanalytical process which will be impact the most on turnaround time of the laboratory results. To verify the automation systems, we tested different combinations of tube and centrifugation settings to determine the most efficient for routine chemistry procedure. The outcomes of each combination were then compared to the benchmark to find out the best setting to reduce turnaround time for laboratory results.

Methods

In this study, we evaluated a total of 40 leftover blood samples that were collected on 4 mL plastic Vacutainer® (Becton Dickinson) lithium heparin tubes and 2 mL plastic Vacuette® (Greiner bio-one) lithium heparin tubes. All tubes were centrifuged on Abbott Automation GLP systems. The first set of samples was centrifuged according to the manufacturer’s guideline (10 min at 1300 × g, room temperature for Vacutainer® tubes and 10 min at 2200 × g, room temperature for Vacuette® tubes). The second set was centrifuged 7 min at 2700 × g, room temperature. And the third set was centrifuged 5 min at 2700 × g, room temperature. Then, analyzed all samples with 22 routine chemistry parameters on Alinity ci analyzers. The allowable total error and correlation coefficient (R) were used to determine the significance of the differences between 3 protocols of the centrifugation time.

Results

The study showed that the centrifugation times of 7 min and 5 min were not significantly different from the manufacturer’s protocol (10 min) after testing with 22 routine chemistry parameters (albumin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, direct bilirubin, total bilirubin, calcium, chloride, cholesterol, creatinine, low-density lipoprotein-cholesterol, iron, potassium, lactate dehydrogenase, sodium, phosphorus, total protein, triglycerides, unsaturated iron-binding capacity, high-density lipoprotein-cholesterol, urea nitrogen and uric acid) in both 4 mL plastic Vacutainer® lithium heparin tubes and 2 mL plastic Vacuette® lithium heparin tubes. Overall, the results of the study indicate that shorter centrifugation times of 7 min and 5 min at 2700 × g able to use for routine chemistry.

Conclusion

The study concluded that the centrifugation time did not affect the analytical results of 22 routine chemistry parameters. This suggests that shorter centrifugation times able to use with no impact on accuracy or reliability. The results of the study will support laboratory to have efficiency improvements by shorten the turnaround time on the results release.
B-029 Validation of an Automated Analyzer for Urine Microscopic Examination
Authors: Majoy L , Riedy H , Chappel M , Nakashima M O
Published: September 2023

Background

Microscopic examination of urine sediment can be time-consuming and labor intensive. Automated analyzers can help to standardize resulting and reduce manual work. Urinalysis has traditionally utilized semi-quantitative grading scales, eg. “0–3/high power field”. The Sysmex UF 5000 (Symex, Kobe, JP) uses flow cytometry to enumerate microscopic particles including white and red blood cells (WBC, RBC), bacteria, casts, and epithelial cells (EC). We sought to validate the counting of these microscopic particles against our reference manual method, and also determine if the research parameters from the analyzer could be used to accurately measure other particles in urine (eg. crystals).

Methods

Residual urine samples were analyzed for microscopic particles both by the UF 5000 and manually using KOVA tubes (KOVA International, Garden Grove, CA). Particles were enumerated using the laboratory’s current semi-quantitative grading system. Exact agreement and agreement +/- one level were calculated.

Results

Conclusion

The analyzer results were comparable to manual resulting; in discrepant cases, the UF result was typically higher than manual, which may reflect the increased sensitivity of this method. Additional studies are ongoing to determine if the UF 5000 research parameters are accurate for semi-quantitative reporting of pathologic casts, crystals, and yeast.
B-145 An R Shiny App for Automated Peak Deconvolution, Interpretation, and Quantitation of Monoclonal Proteins Using Capillary Electrophoresis Immunotyping Data
Authors: Cotten S
Published: September 2023

Background

Machine learning methods have been utilized to train algorithms for interpretation of immunofixation electrophoresis. This work developed an R Shiny app for immunotyping (IT) data capable of deconvoluting discrete peaks in overlapping backgrounds and automatic quantitation of monoclonal proteins. Unlike machine learning which obfuscates decision making, each step of this deconvolution and quantitation process is displayed to the end user.

Methods

Individual traces for 1315 previously interpreted IT results were used in the study. A modified Levenberg-Marquardt algorithm using 50 sets of randomized start parameters was used for deconvolution. Primitive peak features such as height, FWHM, AUC and retention time were used to match heavy and light chain deconvoluted peaks for interpretation. The matched peak with the largest AUC was compared to reviewer interpretation and peak quantitation. Repeatability of quantitation was evaluated through 10 consecutive rounds of deconvolution. Code was written for end user visualization in Shiny or batch processing in base R.

Results

Deconvolution was successful for 97.2% (1279/1315) of specimens. Agreement between the software and reviewer interpretation was 90.6% (11 927/13 150) across all deconvolutions and was consistent for 84.7% of samples in all 10 rounds of deconvolution. Discordant interpretation in 15.3% of samples was often attributed to splitting of large peaks with sharp maximums, large percentage (but small absolute) changes in calculated AUC for monoclonal proteins under 0.5 g/dL, or drifting baseline signal between channels. Mean and median percent CV for consecutive rounds of monoclonal quantitation was 5.9% and 1.16% respectively (protein range 0.02–4.5 g/dL). Deming regression of reviewer vs software quantitation had a calculated slope of 0.728 and Pearson’s r coefficient of 0.933.

Conclusion

Deconvolution and automatic quantitation of monoclonal protein peaks is possible using the Levenberg-Marquardt algorithm for immunotyping data. Future work aims to improve interpretation agreement with reviewers using more advanced ranking of matched peaks.
B-161 Mass Spectrometric Assay for the Early Diagnosis of Barth Syndrome
Authors: Byeon S , Vernon H , Oglesbee D , Pandey A
Published: September 2023

Background

Barth syndrome (BTHS) is an inborn error of phospholipid metabolism caused by pathogenic variants in the TAFAZZIN gene locus on a chromosome X. TAFAZZIN encodes a phospholipid transacylase enzyme, tafazzin, that promotes cardiolipin (CL) remodeling. Deficiency of tafazzin activity in BTHS results in accumulation of monolysocardiolipin (MLCL) and decreased abundance of mature CL, making an increased ratio of MLCL to CL as a diagnostic marker for BTHS. 52:2-MLCL to 72:8-CL in dried-blood spot (DBS) has been used for provisional identification BTHS using LC-MS/MS. A development of multiplexed method that targets several species of MLCL and CL can enhance the diagnosis of BTHS.

Methods

A targeted LC-MS/MS with scheduled multiple reaction monitoring (MRM) mode in negative electrospray ionization was developed using a triple quadrupole MS. DBS from patients with BTHS were obtained at Johns Hopkins University and provided by Barth Syndrome Foundation while control DBS were provided by Biochemical Genetics Laboratory at Mayo Clinic. Internal standards and chloroform:methanol (1:1, v/v) were added to DBS (1/8″ diameter) and sonicated. Supernatants were collected and dried under nitrogen gas. Followed by reconstitution in methanol, samples were subjected to LC-MS/MS analysis.

Results

More than 20 species of MLCL and CL species having various fatty acyl chains were detected and quantified. Remodeled CL species were decreased while MLCL and nascent CL species were found to be increased in known patients of BTHS (n = 8) compared to controls (n = 8). Notably, several ratios of lipids including 52:2-MLCL/72:8-CL and 54:2-MLCL/70:6-CL showed >300-fold elevation in the patients.

Conclusion

Various species of MLCL and CL that can be used to diagnose BTHS can be measured from an easily accessible specimen such as DBS using this method.
B-218 Role of Delta Tocotrienol and Resveratrol Supplementation in Regulation of Micro RNAs in Patients With Metabolic Syndrome: A Randomized Controlled Trial
Authors: Khan D A , Fatima S
Published: September 2023

Background

Micro-RNAs (miRNAs) are involve in the regulation of central obesity, insulin resistance, inflammation and dyslipidemia in development of MetS. δ-tocotrienol and resveratrol improve the insulin resistance, inflammation and clinico-biochemical parameters in the patients with MetS.

Objective

To determine the effect of δ-tocotrienol and Resveratrol Mixture (TRM) supplementation for 24 weeks in comparison with placebo, on the relative expression of metabolic related miR-130b, miR-221, miR-15b-, miRNA-122, and miR-376b in the patients with MetS.

Methods

This randomized placebo-controlled trial was conducted at AFIP, NUMS, Pakistan. A total of 82 adult MetS patients, aged 18 to 60 years of both genders, were enrolled and randomly grouped into TRM (n = 41) and Placebo (n = 41). The TRM group was given 400 mg capsules of (δ-tocotrienol 250 mg; Resveratrol 150 mg), and placebo received 400 mg cellulose capsules twice daily for 24 weeks. miRNA was extracted by Trizole L S reagent with cel-miR-39 (Spike-In control). The cDNA was prepared by Reverse Transcriptase Kit. miRNAs expression profile was done by using qRT-PCR Kit (Qiagen) protocol on the Rotor-Gene.

Results

The TRM supplementation significantly (P < 0.001) upregulated miR-130b-5p (3.05fold), miR-221-5p (2.45fold), and downregulated miR-122-5p by 2.22fold as compared to placebo after 24 weeks. No significant difference was observed in miR-15b-5p and miR-376b-5p.

Furthermore, patients in the TRM group had significant (P < 0.05) correlation in improvement of MetS biomarkers and miRNAs including hs-CRP, miR-130b; miR-221, TNF-α; miR-221, fasting plasma glucose, miR-122, triglyceride and miR-130b with waist circumference. No significant side effect was reported.

Conclusion

TRM daily supplementation in addition to dietary restriction, is effective in the improvement of the metabolic syndrome components by upregulation of miR-130b-5p involve in central obesity, inflammation; miR-221-5p by decreasing insulin resistance and downregulation of miRNA 122 to improve dyslipidemia in the patients with MetS.
B-241 A Novel Approach to Develop Multiplex Loop Mediated Amplification (LAMP) Assays
Authors: Chander Y , Mead D
Published: September 2023

Background

Loop mediated amplification (LAMP) technology is a sensitive, accurate and rapid method for point-of-care (POC) molecular diagnostic detection. One disadvantage is that current LAMP methods can detect only one target per reaction whereas in a standard real-time PCR assay, multiple targets (3–4) can be detected in a single reaction. This is because current methods of detection for amplification products in a LAMP assay are not sequence-specific. Common detection methods include turbidity, use of color-changing dye or intercalating dye in the reaction mix, gel electrophoresis, etc. These methods are not sequence-specific and, thus, unable to differentiate between targets if amplified in a single tube, with the exception of lateral flow devices or melt analysis, which add time, complexity, and cost. To overcome this challenge, we have developed a novel method which simplifies development of multiplex LAMP without use of any additional primers/probes/reagents or steps.

Methods

Triplex RT-LAMP assay was developed for rapid detection of foot-and-mouth disease virus (FMDV) at POC. For this, LAMP primers targeting two different regions of 3D pol gene of FMDV were designed. For use as internal control (IC), LAMP primers were also designed targeting a conserved region of the bovine 18S rRNA gene. For multiplexing, best performing primer sets for each target were labelled with a unique fluorophore at 5′ end and a quencher (BHQ-1) at 3′ end. FMDV-1 primers were labeled with Cy5 fluorophore, FMDV-2 primers were labelled with Texas Red (TxR) fluorophore, and 18S rRNA with FAM fluorophore. Triplex RT-LAMP assay was performed using a specially formulated Multiplex Isothermal Master Mix but without any intercalating dye added to the mix. All 3 primer mixes were added to the Master Mix and reactions were carried out in a real-time thermocycler at 72°C for 30 min, with data collected every 30 seconds. Reaction kinetics were monitored as an increase in fluorescence associated with the accumulation of double-stranded DNA in respective channels (FAM for 18S rRNA, Cy5 for FMDV-1, and TxR for FMDV-2).

Assay sensitivity was determined by testing 10-fold serial dilution of respective template, individually as well as mixed together.

Results

Results obtained showed feasibility of triplex RT-LAMP assay for simultaneous detection of 3 targets in a single reaction. Sensitivity and specificity of triplex RT-LAMP was comparable to control reactions (RT-LAMP assay with intercalating dye) for each target. No cross reactivity or non-specific amplification was observed with any of the primer designs.

Conclusions

This newly developed method allows detection of up to 3 targets in a single reaction without affecting the assay performance. This method is much easier to develop and optimize than previously reported methods as it uses standard LAMP primers without need of any additional primers/probes.
B-248 Rapid PCR Method for Sexually Transmitted Infection (STI) Multiplex Testing Based on Novel Microfluidic Chip
Authors: Lee S , Park J , Koo H , Kim D , Park J , Lee D
Published: September 2023

Background

qPCR is being widely used for STI (Sexually Transmitted Infection) testing as the most sensitive and accurate diagnostic method. Genesystem developed a novel qPCR-based diagnostic platform, called Genoplex chip, which enables the simultaneous detection of multiple sexually transmitted pathogens with a single test. Genoplex chip realized high multiplex detection (up to 14 pathogens by a single pipetting), making it easy and fast to process more targets than conventional qPCR. In this study, Genoplex chip was used for diagnosing sexually transmitted infection(STI) to verify the usefulness in diagnostics.

Methods

Genoplex chip is a flat-shaped microfluidic chip where target-specific primers and probes are prelabeled free from contamination. This chip has 1 well for negative control and 9 connected wells for target detection. By a single pipetting into the inlet hole, samples are loaded and spread into every well of this microfluidic chip since all the wells are connected to each other. We completed performance validation of this new chip with STI diagnosis. Primers and probes for identifying STI pathogens are prelabeled on the inner surface of the Genoplex chip. We can detect and identify 12 major pathogens of STI, which are Chlamydia trachomatis (CT), Ureaplasma urealyticum (UU), Mycoplasma hominis (MH), Mycoplasma genitalium (MG), Neisseria gonorrhoeae (NG), Trichomanas vaginalis (TV), Ureaplasma parvum (UP), Gardnerella vaginalis (GV), Candida albicans (CA), Treponema pallidum (TP), Herpes simplex virus type 1 (HSV1) and Herpes simplex virus type 2 (HSV2) by using these probes. Amplification of the target genes was performed by Genechecker UF-300 PCR System.

Results

We estimated 50 copies of 12 STI pathogens by qPCR process in 40 min. We also confirmed that there was no contamination and no interference in 12 targets.

Conclusion

With Genoplex chip, various targets can be quantitatively detected in one sample and more accurate results can be provided in efficient way. This novel system gives an opportunity to overcome the limitations of current molecular diagnostics by innovative multiplex detection technology. Therefore, it is expected that the Genoplex chip system will attract attention in the molecular diagnostics market.
B-249 Usefulness of Simultaneous Detection of Germline and Somatic Variants From Different Specimen Types by Next-generation Sequencing
Authors: Han M , Kim J
Published: September 2023

Background

Genetic variants detection by next-generation sequencing (NGS) from tumor specimen only is considered limited in diagnosis due to difficulties in discriminating somatic variants from germline ones. It will be even more helpful in diagnosing patients with cancer if we perform NGS analysis from specimen of both tumor and blood. This study shows the usefulness of simultaneous detection of germline and somatic variants from different specimen types by NGS.

Methods

NGS panel assay designed for solid tumor composed of 80 genes was conducted for detection of both somatic and germline variants. DNA samples extracted from both buffy coat of whole blood and formalin-fixed paraffin-embedded (FFPE) specimen from 4 patients with renal cancer and 3 patients with ovarian cancer were used in assay. We compared results from both specimen and finalized somatic and germline variants detected.

Results

In 4 patients specimen with renal cell carcinoma, we were able to rule out germline variants from somatic variants by analysis of NGS assay from both specimen types. Genes ruled out for germline variants of uncertain significance (tier 3) or higher levels are ARID1B: c.1031C>T, p.A344V, ERBB2: c.3430G>C, p.D1144H, BRCA2: c.3220A>T, p.S1074C, POLE: c.2974G>A, p.A992T, EP300: c.1666A>G, p.M556V, EP300: c.2539C>T, p.P847S, ERBB3: c.1115C>G, p.P372R, BAP1: c.1124A>T, p.E375V. In 3 patients with ovarian cancer, genes ruled out are EGFR: c.739G>C, p.D247H, MET: c.4172A>T, p.D1391V, PBRM1: c.2678A>G, p.Y893C, SETD2: c.1208G>A, p.S403N, AKT1: c.1373T>C, p.M458T, FGFR3: c.160G>A, p.G54R, KMT2C: c.9530G>A, p.R3177H, SMARCB1: c.1116G>A, p.T372T. All variants confirmed are tier 3 level of clinical significance.

Conclusion

According to the results of this study, we were able to exclude germline variants form somatic ones in NGS assay reports for cancer patients. This study shows the usefulness of simultaneous detection of germline and somatic variants from different specimen types by NGS.
B-269 Plasmodium Detection: Sera, Blood, Do They All Work?
Authors: Franco-Marín E , Ulloa G , Dehesa-García B , Santolalla M , Lescano A , García-Manrique B , Mayor P , Mayor A , Alonso H
Published: September 2023

Background

When diagnosing malaria, testing employs blood samples using either microscopy, the current gold standard or molecular diagnostic tests (more sensitive but significantly more expensive). In Plasmodium spp. screening programs in at-risk populations where there are not many resources available, dried blood on filter paper is used to ease transport, storage and preservation of the sample. Although Plasmodium detection studies using blood serum as a sample are available, it isn´t always as sensitive as using whole blood-based sampling, as the concentration of DNA in plasma is proportional to the level of parasitaemia. This study aims to confirm the differences in sensitivity using both samples, demonstrating the importance of choosing the right type of sample to use for diagnosis.

Methods

The study was carried out using the VIASURE Malaria Real Time PCR Detection Kit with sera and blood samples from 94 inhabitants from Nueva Esperanza community in the Peruvian Amazon collected between 2007 and 2020, establishing a comparative analysis between matrixes. Nucleic acid extraction was performed using the QIAamp DNA Mini Kit from QIAGEN and the VIASURE assay was performed using QuantStudio®6 Flex Real-Time PCR System thermocycler (Thermo Fisher Scientific) and the BioRad CFX96TM Real-Time PCR Detection System for analysing peripheral blood samples and the BioRad CFX96TM Real-Time PCR Detection System for analysing sera.

Results

From the total of samples, only 5 sera (5.31%) showed amplification of Plasmodium spp., whereas from blood samples, 30 resulted to be Plasmodium spp-positive. Therefore, 26.59% of detection is lost when serum is used as a sample, as opposed to blood.

Conclusion

After this analysis it could be concluded that malaria detection sensitivity is appreciably higher for blood samples than for sera samples. Thus, serum samples aren´t the best choice for Plasmodium spp. testing and whole blood-based matrixes are preferable for this diagnosis. These results reinforce the idea of the importance of the sample type when diagnosing different pathogens, as well as the relevance of a confirming comparative analysis when similar sample types are involved.
B-284 Evaluating the Range of Serum Neurofiament Light Chain at Different Ages in Chinese
Authors: Guo S , Meng Z , Tang M , Ren Y , Ying B
Published: September 2023

Background

Neurofilament light chain (NFL) has recently been identified as a sensitive biomarker for diagnosis and prognosis in many neurological diseases. However, studies focused on NFL levels ranges in healthy individuals remain few, especially in the Chinese population. The clarification of the reference range among healthy individuals at different ages would be useful for further clinical utility.

Objectives

To evaluate the serum neurofilament light chain range at different ages in healthy Chinese and analyze the potential contributing factors for NFL levels.

Methods

Healthy individuals undergoing rigorous cognitive and neuroimaging detection were enrolled. Serum samples and plasma samples of different anticoagulants were collected for NFL levels detection by the ultra-sensitive Simoa HD-1 platform. The tendency of NFL levels to change with age and potential influence factors were analyzed.

Results

We enrolled three hundred and ninety-nine healthy participants. The positive correlation between serum NFL levels and age was observed and the regression formula was further established. There were no statistical differences between the <20 years group and the 20–29 years group (P = 0.2512), as well as the 60–69 years group and the 70–79 years group (P = 0.0528) and differences,were statistically significant among the remaining pairwise groups. We further observed the positive correlation between serum tNFL levels and age was 0.76 in total (P < 0.0001). The NFL levels between the serum and heparin plasma are statistically different and there was no statistical difference between the serum and plasma of other anticoagulants.

Conclusions

The age-related change pattern was uncovered, providing important assistance for the clinical use of NFL to monitor the potential neurological alterations for people of different ages.
B-359 Bap1 Overexpression Enhances Epithelial-mesenchymal Transition in Cholangiocarcinoma
Authors: You H , Liao W , Huang W
Published: September 2023

Background

BRCA1-associated protein 1 (BAP1) is a nuclear protein that considered as a tumor suppressor by its involvement in DNA double-strand-break repair. However, BAP1 overexpression was observed in many kinds of cancer, such as intrahepatic cholangiocarcinoma (iCCA).

Methods and Results

Formalin-fixed paraffin-embedded cancer tissues were acquired from the archive of the Department of Pathology, Chang Gung Memorial Hospital at Kaohsiung, Taiwan. In this study, we identified 23.8% cytoplasmic BAP1 overexpression and 20.8% nuclear BAP1 overexpression cases in 101 iCCA patients. There were significant correlations between cytoplasmic expression of BAP1 with tumor growth pattern (P = 0.005), T classification (P = 0.006), and clinical AJCC seventh staging (P < 0.001). In cell models (iCCA cell line RBE), overexpressed BAP1 were accumulated in cytoplasm. The translatome profile suggested that BAP1 overexpression may promote the migratory and invasive capacities of iCCA cells. In-vitro assays validated that BAP1 overexpression enhanced cell migration and invasion. Furthermore, we found that BAP1 overexpression induced epithelial-mesenchymal transition (EMT) in iCCA cells, showing E-cadherin and fibronection down-regulation and vimentin up-regulation.

Conclusion

These findings indicated that cytoplasmic BAP1 overexpression increased migratory and invasive capacities in iCCA cells, at least in part, via enhanced EMT regulations. Based on these findings, cytoplasmic BAP1 expression could be a candidate predictor for metastasis of iCCA.