bluuebunny/crossref_metadata_2025 huggingface dataset:
B-015 Magnesium Fractionation using Ultrafiltration System
Authors: Badr H , Anthony O O
Published: September 2023

Background

Total magnesium (tMg) comprises 3 fractions: ionized (iMg), protein-bound (pbMg), and complexed magnesium (cMg). Serum iMg can be measured directly using ion-selective electrode method, but determination of pbMg and cMg has not been addressed. The aim of this study is to examine the efficiency of a newly introduced ultrafiltration system to fractionate tMg and validate proposed its reference intervals for Mg fractions.

Methods

Leftover whole blood samples were collected from 40 subjects. iMg and tMG were measured in whole blood using Stat Profile Prime® ES, and ultrafiltrate using VITROS 5600 Analyzer, respectively. Serum fractionation was done by ultrafiltration system (Centrifree®, Sigma-Aldrich, MA, USA). pbMg and cMG were calculated by subtracting tMg ultrafiltrate from tMg in whole blood, and subtracting iMg from tMg concentrations in the ultrafiltrate, respectively.

Results

Reference intervals of iMg, pbMg, and cMg were proposed as mean± 2 SD of 0.93–1.61 mg/dL, 0–0.85 mg/dL, and 0.07–0.57 mg/dl, respectively. iMg, pbMg, and cMg represented 65%, 20%, and 16% of the tMg concentration in normo-magnesemic subjects with slight variation in hypo- and hyper-magnesemic subjects. iMg showed weak positive correlation with tMg, (r = 0.5, 0.03, 0.005), in normo-magnesemic, hypo- and hyper-magnesemic subjects, respectively. iMg showed weak positive correlation with cMg (r = 0.11) and strongly with pbMg (r = 0.41) in hypermagnesemic subjects. iMg was not dependent on protein concentration or pH, however it correlated positively with blood sugar (r = 0.5 and 0.2) and serum creatinine levels (r = 0.3 and 0.5) in hypo- & hyper-magnesemic subjects, respectively.

Conclusions

The ultrafiltration system was a promising means to assess cMg and pbMg in critical care patients. iMg measurement provides better discrimination in ill patients and was associated with lifestyle factors including alcohol consumption, hypertension and diabetes. Stat Profile Prime® ES point-of-care analyzer provides rapid and precise direct measurement comparable to current tMg standard method. Due to its rapid turnaround time, it can help to efficiently manage workload of high complexity laboratories, and alleviate the burden on the critical care team.
B-037 Comparison of Performance between Siemens Clinitek + Status Analyzer to ARKRAY AUTION MAX AX-4030 for Suspected Urinary Tract Infections
Authors: Ali M , Yang L , Forbush K , Rohr D
Published: September 2023

Background

The purpose of the study is to evaluate the performance and agreement of the rapid point-of-care (POCT) urine dipstick testing in the emergency department (ED) to a laboratory-automated platform.

Methods

A total of 30 urine specimens were collected from the emergency department at our hospital and were simultaneously tested for urinalysis (UA) on the POCT Siemens Clinitek Status+ analyzer using the Multistix 10SG strip. In the laboratory, we performed the tests on the automated ARKRAY Aution MAXTM instrument, using the Aution Sticks 9EB strips. Samples with more than a trace of leukocyte esterase and/or nitrates are considered positive for a urinalysis and further, the samples were reflexed to urine culture. The performance of both platforms was analyzed using EP Evaluator (Data Innovations LLC.).

Results

A total of 30 urine specimens were analyzed for UA using both the POCT and automated laboratory analyzers. The agreement (95% CI) between leukocyte esterase, nitrites, and blood are as follows: 53.3% (36.1 to 69.8%); 96.7% (83.3 to 99.4%), and 70.0% (52.1 to 83.3%) (see Table 1). Furthermore, out of the 30 specimens that were tested on both platforms, there was prominent discordance between the POCT and lab UA test. The 7 urine samples that were negative on POCT were positive on the laboratory UA platform. All 7 were sent for urine culture, out of 7 only 1 sample was positive for culture which was abnormal: >100 000 CFU/ml Escherichia coli. Out of 30 urine samples specimens that were tested on both platforms, 23 urine samples were in concordance with both platforms out of which 8 urine samples tested positive for culture (5 were E. coli, 1 was E. coli combined with Klebsiella pneumoniae, 1 was solely Klebsiella pneumoniae, 1 was Staphylococcus saprophyticus, and 1 was Streptococcus agalactiae). The remaining 15 urine samples resulted in no significant growth of bacteria or suggesting contamination. The sensitivity of POCT UA and laboratory-performed UA was 89% and 100% respectively. Furthermore, the specificity of POCT UA and laboratory-performed UA was 36% and 7% respectively.

Conclusion

Our data shows although POCT UA is rapid, a prominent disparity is observed between the POCT UA and automated lab testing. The lab-performed UA has greater sensitivity and specificity compared to the POCT UA. Clinicians should interpret the urine dipstick test results in a clinical context that includes the patient’s history and symptoms, especially when using POCT in the ED.
B-042 Evaluation of the Analytical Performance of Three Prostate-specific Antigen Immunoassays on the Atellica CI 1900 Analyzer
Authors: Quintanilla M , Thakur K , Clark E , Smith M , Arrode-Bruses G , Rhea-McManus J , Leipold H
Published: September 2023

Background

The Atellica® CI 1900 Analyzer is an automated, mid-throughput, integrated chemistry and immunoassay analyzer employing both Atellica CH and Atellica IM assays. This study was designed to evaluate the analytical performance of the Atellica IM Prostate-Specific Antigen (PSA)*, Complexed Prostate-Specific Antigen (cPSA)*, and Free Prostate-Specific Antigen (fPSA)* assays on the Atellica CI 1900 Analyzer.

Methods

The Atellica CI 1900 PSA, cPSA, and fPSA assays use the same reagents and calibrators as the comparable Atellica IM assays. Limit of quantitation (LoQ), precision, and method comparison (MC) were used as performance indicators for the Atellica CI 1900 Analyzer. LoQ studies were performed in accordance with CLSI EP17-A2 using native and contrived human serum samples. Precision studies were performed according to CLSI EP05-A3 using contrived human serum samples. One aliquot of each sample pool was tested in duplicate in two runs per day >2 hours apart on each analyzer for >20 days. MC studies were performed according to CLSI EP09-A3. Individual native human serum samples were analyzed using the Atellica IM assays on both the Atellica IM and Atellica CI 1900 Analyzers.

Results

LoQ is defined as the lowest amount of analyte in a sample at which the within-laboratory %CV is ≤20%. Representative precision and MC results for each assay across indicated sample ranges are listed in the table. Over the three assays tested, repeatability and within-lab %CVs were ≤8.0% and ≤15.5%, respectively. Slopes determined by the Deming linear regression model were approximately equal to 1.

Conclusion

Evaluation of the Atellica IM PSA, cPSA, and fPSA assays using the Atellica CI 1900 Analyzer demonstrated good precision and equivalent performance compared to the same assays on the Atellica IM Analyzer.

*The products/features mentioned here are not commercially available in all countries. Their future availability cannot be guaranteed. fPSA assay is not available for sale in US.
B-046 Curious Thing, an Artificial Intelligence (AI) Based Conversational Agents in COVID-19 Patient Management
Authors: Chow J S
Published: September 2023

Background

There are no clear guidelines or validated models for artificial intelligence (AI) based approaches in monitoring of COVID-19 patients who were isolated in the community, in order to identify early deterioration of their health symptoms.

Methods

Developed in partnership with Curious Thing (CT), Sydney-based AI conversational technology, a new care robot technology was introduced in South Western Sydney (SWS) in September 2021 to manage the large numbers of low to medium risk patients with a COVID-19 diagnosis and isolated at home. The CT interface made contact with patients via their mobile phone, following a locally produced script to obtain information recording physical condition, wellness and support.

Results

Between 2nd September to 14th December 2021, there were 32 001 conversations (every phone call that resulted in a conversation with a patient) conducted with 6323 unique patients (patients included in call lists identified by phone number) engaged. Over half of the completed calls (n = 13 143, 53.7%) identified that individuals needed no further support (freeing up valuable clinician time). 4.24% (n = 1077) identified as needing urgent support (getting to them faster than a standard clinician dialling pattern). The AI assisted phone calls effectively identified the patients requiring further support, saved clinician time by monitoring less ailing patients remotely and enabled them to spend more time on critically ill patients, thus ensured that service and supply resources could be directed to those at greatest need.

Conclusion

Based on the severity of the COVID-19, different approaches have been adopted by the healthcare professionals for handling the patients in the most effective manner. This paper describes the AI assisted conversational agent which can act as an alternate approach to identify the deteriorating patients requiring further support while they are isolated at home. The findings are intended to inform policy makers and health professionals about the implications of the use of these AI-based technologies for the management of current or any future pandemic.
B-098 Analytical Performance Evaluation of Sigma Strong Clinical Chemistry Assays on the Alinity c System
Authors: Berman M , Bhartia V , Wang X , Cheng P , Kulasingam V
Published: September 2023

Background

The aim of this study was to evaluate the analytical performance of 5 Abbott next generation (Sigma Strong) clinical chemistry assays on the Abbott Alinity c system. Method comparison, precision, linearity, accuracy, and sigma metrics were assessed for Albumin BCG2 (Bromocresol green), Albumin BCP2 (Bromocresol purple), Cholesterol2, Total Protein2 and Amylase2.

Methods

Method comparison between new (second generation) and current on market Alinity assays was performed by measuring 126–138 serum/plasma samples in duplicate. Assessment of imprecision was performed by running 2 levels of quality control material (BioRad; Chemistry UA) and 3 pooled patient samples 5x twice per day for 5 days. 10 replicates of ERM-DA470 k IFCC material for albumin, ERM-AD456 k IFCC material for amylase, NIST SRM 927 material for total protein, and NIST 1951cL1 for cholesterol were run to determine the accuracy and calculated sigma value for each analyte. Linearity testing was performed by running 5–6 levels of commercially available linearity materials in replicates of 3. Bias, acceptable imprecision, and total allowable error were based on Clinical Laboratory Improvement Amendments and Accreditation Canada Diagnostics guidelines. Statistical analysis was performed using EP evaluator.

Results

Data for method comparison (Passing Bablok), precision (Range of total %CV across 5 concentrations), accuracy (% Bias from target value), and linearity (Range of % recovery) for 5 Abbott second generation clinical chemistry assays are shown in Table 1. All assays demonstrated ≥6 Sigma performance.

Conclusion

The Abbott second generation (Sigma Strong) clinical chemistry assays on the Alinity c system showed acceptable performance for precision, accuracy, linearity, and agreement with the on-market Alinity c clinical chemistry assays.
B-102 Screening Food Allergies in Our Health Primary Care Area
Authors: De Haro Romero T , Aldana C , González Cejudo T , de Haro Muñoz T
Published: September 2023

Background

Allergy is the immunological disease with the greatest impact in the world. Food allergy typically manifests with cutaneous, digestive and/or respiratory symptoms, of immediate onset, usually immunoglobulin E-mediated. Doctors from primary health-care providers are often challenged with these patients, becoming the first-line for the recognition and management of food allergies. As a laboratory, our duty is to provide the health care system with the most effective diagnostic tools.

Methods

We recollected all the results from Primary care of our health area analyzed in our laboratory for one year. Screening assay included a panel of specific IgE antibodies to the most common food allergens: egg yolk, egg white, milk, cod, wheat, peanut and soybean. They were detected semi-quantitatively by enzyme-labeled chemiluminescent immunoassay using the Immulite Allergy Food Panel FP5 kit (Siemens Diagnostic Products Corporation, Los Angeles, USA), according to the test procedure. If the assay was positive, all specific IgE allergens contained in the mixture were analyzed separately to determine their responsibility for the positive screening test.

Results

Our laboratory processed 24 125 samples from primary care patients. Of these, 1251 were positive (greater than 0.35 Ku/L), which represented 5.19% positive of the total analyzed. Once all the specific IgE allergens contained in the mixture were analyzed, the results were: 608 positive for egg yolk, 753 positive for egg white, 523 positive for milk, 109 positive for cod, 405 positive for wheat, 597 positive for peanut and 480 positive for soybean.

Conclusion

As with most medical disorders, a thorough medical history is the initial step in diagnosing adverse food reactions. The medical history is extremely useful, especially in acute reactions, but it can be unreliable in chronic disorders such as atopic dermatitis and asthma. In vitro assays help in the diagnosis, but they must be considered as a screening and no diagnosis tool, especially when no symptoms are associated with the positive food allergen result. That’s why in vitro results must be confirmed by prick test, which is considered gold standard for allergy diagnosis. In our study, egg white is the most prevalent allergen detected (60.19%), followed in order by egg yolk (48.60%), peanut (47.72%), milk (41.81%), soybean (38.37%), wheat (32.37%) and being cod the least allergen detected (8.71%).
B-108 T-cell-mediated Immunity against SARS-CoV-2 in Health Care Workers: Head-to-head Comparison of Two Interferon-Gamma Release Assays and Associations between Cellular and Humoral Immunity
Authors: Sabalza M , Martinek J , Tomášková H , Lochmanová A , Zelená H , Motlochová J , Dieckmann K , Grage-Griebenow E , Saschenbrecker S , Zapf D , Herbst V , Ježo E , Janošek J
Published: September 2023

Background

T-cell responses are a crucial constituent in the adaptive immunity to SARS-CoV-2 and can be quantified using interferon-gamma (IFN-γ) release assays (IGRAs). This study aimed at comparing the results of two commercial IGRA systems, the EUROIMMUN Quan-T-Cell SARS-CoV-2 and the Oxford Immunotec T-SPOT.COVID, presenting the first head-to-head comparison of these two assays for the evaluation of SARS-CoV-2-specific T-cell activity.

Methods

Blood samples were collected at the Public Health Institute Ostrava from 90 health care professionals with a history of COVID-19 infection or vaccination. All samples were tested for T-cell-mediated immunity using the two IGRAs as well as for humoral immunity using the EUROIMMUN Anti-SARS-CoV-2 QuantiVac ELISA IgG and an in-house virus neutralization test.

Results

Similar results were obtained by both IGRAs, yet the sensitivity of the Quan-T-Cell appeared to be insignificantly higher than that of the T-SPOT.COVID.

Conclusion

Both assays demonstrated excellent overall agreement with virus neutralization activity and anti-S IgG positivity, with the only exception that 4/6 subjects in the subgroup of unvaccinated Omicron convalescents were devoid of IgG. In these cases, however, T-cell responses were at least borderline positive, indicating that IGRAs provide higher sensitivity in detecting immune responses to SARS-CoV-2 than do IgG-specific assays. This applies particularly to unvaccinated persons who contracted only SARS-CoV-2 Omicron variant infection but is also likely to be relevant to other patient groups.
B-113 Diagnostic Value of Autoantibodies against D-3-phosphoglycerate Dehydrogenase (PGDH) among Patients with Liver Diseases
Authors: Venkataraman I , Feng X , Liu Y , Karl I , Ott A , Scheper T , Meyer W , Yan H
Published: September 2023

Background

We determined serum autoantibodies against D-3-phosphoglycerate dehydrogenase (anti-PGDH) in patients with liver diseases using a well-validated line immunoblot assay, and analysed their diagnostic significance.

Methods

The studied panel comprised serum samples from 383 patients with the following liver diseases: 87 autoimmune hepatitis (AIH), 88 primary biliary cholangitis (PBC), 76 chronic hepatitis B, 79 hepatitis C, 53 drug-induced liver injury as well as sera from 69 patients with autoimmune diseases (systemic lupus erythematosus or rheumatoid arthritis) and 30 healthy blood donors. The line immunoblot EUROLINE Autoimmune Liver Diseases 14 Ag (EUROIMMUN) was used to test for anti-PGDH. The intensities of the bands were automatically evaluated using the EUROLineScan software (EUROIMMUN). Indirect immunofluorescence was used to detect other autoantibodies, including anti-nuclear autoantibodies (ANA) and anti-smooth muscle antibodies (ASMA).

Results

The prevalence of anti-PGDH was 18.3% across all patients with liver diseases (36.8% in AIH; 10.2% in PBC), 1.5% in patients with autoimmune diseases, and 3.3% in blood donors. ANA but not ASMA were detectable in all anti-PGDH positive patients with liver diseases. Patients with anti-PGDH displayed no significant differences in age, liver function, IgG serum level and ANA patterns compared to those without anti-PGDH. Anti-PGDH positivity was observed at various stages of liver disease, including acute and chronic liver injury, liver cirrhosis, and liver cancer.

Conclusion

The anti-PGDH prevalence in AIH patients was highest among patients with liver or autoimmune diseases. Presence of anti-PGDH seems to be a specific marker for AIH and may support the diagnosis of AIH.
B-120 Atellica® Ci 1900 Assay Sigma Metrics Evaluation
Authors: Kolewe K W , Leipold H , Lewisch S A , Snyder J T
Published: September 2023

Background

The Atellica® CI 1900 Analyzer is a stand-alone, integrated clinical chemistry and immunoassay analyzer. Offering the same assay portfolio as the Atellica® Solution, the Atellica® CI 1900 analyzer is designed to complement the Atellica® Solution in hub-and-spoke settings, enabling labs of every size to offer state-of-the-art patient testing. Six Sigma is synonymous with process performance, and Sigma metrics have become the analytical quality benchmark for the medical device industry. In this study, Sigma metrics were calculated for a total of 71 Atellica® assays to provide a simple, translatable value to evaluate a given assay’s performance on the Atellica® CI 1900 Analyzer.

Methods

Allowable total error (ATE) for each assay was defined from the Clinical Laboratory Improvement Amendment (CLIA) table. When an analyte was not present in the CLIA table, alternative sources were consulted, including the RiliBÄK table, Royal College of Pathologists of Australasia (RCPA) table, and Ricos Biological Variability. Precision studies were conducted in accordance with Clinical and Laboratory Standards Institute (CLSI) document EP05-A3 over 20 days (n = 80 replicates) using the Atellica® CI 1900 Analyzer. Method comparison studies conducted on the Atellica® CI and Atellica® CH/IM Analyzers, with at least 100 patient samples spanning the assay’s measuring interval in accordance with CLSI document EP09-A3, were used to establish bias at the selected target precision concentration. Sigma metrics were determined for each assay using the equation Sigma metric = (ATE – %bias)/(Within-lab %coefficient of variation [CV]).

Results

Sigma metrics were determined for a total of 71 Atellica® assays, resulting in 93% of assays performing above Five or Six Sigma and 0 assays performing below 4 Sigma. Of these, 44 of 52 clinical chemistry and 15 of 19 immunoassays performed above the 6 Sigma level.

Conclusions

Sigma metrics that provide a benchmark for laboratory quality classify over 90% of assays on the Atellica® CI 1900 Analyzer with scores of 5 or 6, corresponding to the classification of excellent or world-class performance. Therefore, laboratories can expect high quality assay performance with strong comparability between the Atellica® CI 1900 Analyzer and the Atellica® Solution. **Not available for sale in the US. Product availability varies by country.
B-141 Surprisingly Good Agreement of Siemens and Roche ALT Reference Change Values in Ottawa Hospital Outpatients Distilled from 5 years and 3 years of Siemens and Roche ALT, Respectively
Authors: Cembrowski G , Qiu J , McCudden C
Published: September 2023

Background

Sequential patient data can be transformed into the variable component of the reference change value (RCV) calculation: (CVA2 + CVI2)1/2 (PMID 35137000). We have analyzed sequential healthy and abnormal outpatient ALT to derive RCVs of Ottawa Hospital outpatients (OHOP) who had sequential ALT measured by Siemens or Roche assays.

Methods

We used the American College of Gastroenterology definition of normal ALT with supplementation of P5P as <25 U/L and <33 U/L in females and males, respectively. We defined abnormal as between the upper limit of normal (ULN) and 3xULN. 5 years of Siemens ALT measurements and subsequently 3 years of Roche ALT were abstracted from the Ottawa Hospital EMR. For the 8 patient cohorts, we tabulated consecutive pairs of intrapatient ALT by time intervals of separation: 0–1 weeks, 1–2 weeks, 2–3 weeks . . ., up to 51–52 weeks. For each interval, we determined the standard deviation of duplicates (SDD) between the paired intrapatient results. SDD was graphed against the midpoints of the weekly interval. Linear regression was used to determine the y-intercept (yo). The 95% RCVs of the male and female, normal and abnormal populations were calculated for the Siemens (with P5P) and Roche (non P5P) assays.

Results

The Table describes each cohort, yo and RCV. According to the Roche/Siemens cross-over study, Roche ALT values were 10% less than Siemens. This Roche bias is reflected in the normal outpatient ALT means and less so in those with elevated ALT. Also presented are the RCV of low ALT (normal) patients as documented by Ricos and Carobene.

Conclusion

Our RCVs are realistic: normal ALT patients demonstrate lower RCVs, close to Ricos’ but much larger than Carobene’s which are unachievable due to diurnal variation (over 40% of OHOP ALT are drawn at different times of the day). Furthermore, Carobene’s CVA is tiny.
B-147 Evaluation of Laboratory Database: Is there a Causal Relationship between COVID-19 and Type 1 Diabetes
Authors: Castelo M C , Cruz M , Bandeira S P , Lopes A C , Cavalcante L L
Published: September 2023

Background

Type 1 diabetes mellitus (T1DM) constitutes about 5% of all diagnosed cases of diabetes. The global incidence is increasing over the past few years. To date, the pathophysiology of the disease is not fully understood. with descriptions of activation of the immune system by some viral agents. SARS-CoV-2 has been suggested as a potential inducer of new-onset type 1 diabetes mellitus (T1DM). DASA has one of the largestData and Analytics areas in Diagnostic Medicine. The use of laboratory data can represent an important tool in broader knowledge of this relationship. This study aimed to evaluate the frequency of new-onset T1DM in the years 2019, 2020 and 2021.

Methods

This study was done through an observational, retrospective big data laboratory approach with a network that extends over a large part of Brazil to analyze whether there was an increase in T1DM cases. Individuals aged 18 years or younger were evaluated from January 2019 to December 2021. The subjects were characterized with possible new-onset T1DM through the presence of glycated hemoglobin equal to or greater than 6.5% in the presence of at least one disease-related antibody. The antibodies evaluated were: anti-GAD, anti-islet, anti-insulin and anti-zinc transporter type 8. For data evaluation, the means and standard deviations and the chi-square test were used. The results of patients who underwent RT PCR COVID19 were evaluated.

Results

A total of 431 060 subjects (<18 years) were evaluated, with 229 201 female (53.17%). Table 1 shows the distribution of data over the years evaluated, the number of patients with glycated levels greater than 6.5% and the number of probable new cases of DM1 from the positivity of at least 1 antibody. In the years evaluated, there was no statistical difference in the incidence of the disease between the sexes (2019 - 51.2%; P:0.92, 2020 - 50.3%; P:0.99 and 2021 - 48.9%; P: 0.67). The mean age with standard deviation in the years 2019, 2020 and 2021, respectively, was: 12.4% ± 4.4; 12.6 ± 4.3 and 12.5% ± 4.3. The mean values of glycated hemoglobin of patients with a value ≥6.5% with standard deviation in the years 2019, 2020 and 2021, respectively, were: 9.3% ± 2.1; 9.2 ± 2.5 and 9.3% ± 3.4. When comparing the incidence of the disease between the years, there was a significant increase in cases when comparing the years 2021 with 2020 and 2019 (P: 0.01 and P < 0.01, respectively). Of the total number of patients in 2020, only 5 underwent RT PCR COVID 19, with no positive result, and in 2021 a total of 32, with 7 positive, corresponding to 21.8% of cases.

Conclusion

The present study, using a large database, seems to demonstrate an increase in the incidence of DM1 among children diagnosed with diabetes. Prospective studies that evaluate the possible relationship of the disease with the SARS-CoV-2 virus may show this agent as a trigger of disease-related automunity.
B-183 The Impact of 3-EPI-25-Hydroxyvitamin D on 25-Hydroxyvitamin D Measurement by Liquid Chromatography/Mass Spectrometry
Authors: Turner K A , Radecki T W , Boughner E L , Bolhuis K D
Published: September 2023

Background

25-hydroxyvitamin D is the recommended target to evaluate vitamin D status. The accurate measurement of 25-hydroxyvitamin D by liquid chromatography/mass spectrometry (LC/MS/MS) is impacted by the presence of the biologically inactive epimer of 25-hydroxyvitamin D2 and D3, 3-EPI-25-hydroxyvitamin D2 and D3. The epimers are isomers and share the same mass and fragment transitions as 25-hydroxyvitamin D2 and D3. For an accurate measurement the epimer should be chromatographically separated from 25-hydroxyvitamin D. Herein we assess the significance of 3-EPI-25-hydroxyvitamin D in our patient population.

Methods

A LC/MS/MS method was developed to chromatographically separate 25-hydroxyvitamin D2 and D3 from 3-EPI-25-hydroxyvitamin D2 and D3 on a Waters Xevo TQ-S Micro with an Acquity UPLC System using a 2.6 μm Pentafluorophenyl (PFP) column (Phenomenex). There were 231 residual serum samples analyzed.

Results

The cohort tested consisted of 16 infant samples (<1 years), 62 pediatric samples (1–17 years), adult samples (≥18 years), and 20 pregnant samples. 3-EPI-25-hydroxyvitamin D2 was not detected in any sample analyzed (<1 ng/mL). The prevalence and concentration of 3-EPI-25-hydroxyvitamin D3 was variable (Fig. 1).

Conclusion

The specificity of 25-hydroxyvitamin D measurement by LC/MS/MS is not affected by 3-EPI-25-hydroxyvitamin D2 as it is not present in our patient population. However, 3-EPI-25-hydroxyvitamin D3 is a significant interference. The presence of 3-EPI-25-hydroxyvitamin D3 is variable with the highest prevalence and concentration in infants. The prevalence and concentration decrease with age and is minimal in pregnancy. For infant and pediatric samples, an assay that chromatographically separates 3-EPI-25-hydroxyvitamin D3 from 25-hydroxyvitamin D3 is needed. For adult samples, it may be appropriate not to separate 25-hydroxyvitamin D3 from the epimer. For this patient population a balance between accuracy and throughput must be established.
B-204 Determination of Lyso-GB3 in Dried Blood Spots: A Useful Biomarker for Fabry Disease
Authors: Paulo B F , Novo E M , Zauli D A , Serra A P
Published: September 2023

Background

Fabry disease, an X-linked inborn error of metabolism, results from pathogenic mutations in the α-galactosidase A gene (GLA). These mutations reduce or abolish the α-galactosidase A activity, which result in accumulation of glycosphingolipids in the lysosomes, including globotriaosyl-sphingosinein (lyso-GB3), throughout the body, in particular the kidney, heart, and brain. Lyso-GB3 may be elevated in symptomatic patients and supports a diagnosis of Fabry disease. It may also be helpful as a tool for monitoring disease progression, as well as determining treatment response in known patients. Moreover, measurement of Lyso-GB3, may provide additional diagnostic information in the evaluation of uncertain cases, such as in asymptomatic heterozygous female patients, individuals with novel GLA variants of unclear clinical significance, as well as asymptomatic patients identified by family screening. This study aimed to determine Lyso-GB3 in dried blood spot (DBS) samples with a simple, rapid and sensitive LC-MS/MS method, and identify its reference interval in the Brazilian population.

Methods

All experiments were performed on the Waters TQ-S Micro LC-MS/MS system. The gradient chromatographic separation was carried out using a Waters BEH C18 column in 3.5 min. One DBS punch of 3.1 mm diameter was used for analysis in a microtiter plate and then, a solvent extraction containing stable isotope analogue of Lyso-GB3 was added. After incubation time, the supernatant was directly injected into LC-MS/MS system. The method was validated for linearity, carryover, intra and inter-day precision and the limit of quantification was determined. A reference interval was obtained by analyzing 94 healthy volunteers (49 women and 45 men). The difference of Lyso-GB3 concentration between men and women was investigated (Wilcoxon paired test). The reference interval was determined by the superior limit of the confidence interval (90% CI) of a normal distribution of the results.

Results

The method was linear between 0.2–100.0 ng.mL−1. The recovery was between 81.0 and 139.7%, which was acceptable considering CLSI guidelines for DBS validation (CLSI NBS04-A). The intra and inter-day precision were below 12.1%, and the limit of quantification was 0.2 ng.mL−1. The reference interval determined was below 0.8 ng/mL.

Conclusion

In conclusion, the LC-MS/MS method for Lyso-GB3 determination in DBS samples was successfully validated. The sample preparation is simple and the chromatographic method is rapid for routine laboratory. The cutoff determined is comparable with what is available in the literature and adequate for analysis in clinical routine.
B-206 The Determination of Vanillylmandelic Acid (VMA), Homovanillic Acid (HVA) and 5-Hydroxyindolacetic Acid (5-HIAA) by LC-Ms/MS Assay in Urine Samples for Diagnosis of Neuroendocrynes Tumors
Authors: Junior L F , Paulo B F , Zauli D A
Published: September 2023

Background

The catecholamines dopamine, norepinephrine and epinephrine, as well the neurotransmitter serotonin are biogenic amines that were formed by enzymatic descarboxylation of aminoacids in the body. They realize an important function in central nervous system. Vanillylmandelic acid (VMA) and Homovanillic Acid (HVA) are degradation products of the catecholamines, and 5-Hydroxyindolacetic Acid (5-HIAA) is a serotonine degradation product. They are important to the diagnosis of neuroendocrynes tumors, that produce catecholamines and/or serotonine in excess, resulting in elevated levels of VMA, HVA, and/or 5-HIAA in 24 h collection urine.

Objective

This study aimed to validate a simple LC-MS/MS method for quantification of VMA, HVA and 5-HIAA in isolated and 24 h urine samples for the diagnosis of neuroendocrines tumors.

Methods

Using a deep-well microtiter plate, 20 µL of isolated or 24 h urine was diluted in 540 µL of 0.05% of formic acid solution. Then, 20 µL of an internal standard solution containing stable isotopes of the analytes were added (VMA-D3, HVA-13C6,18O and 5-HIAA-13C6). The plate was sealed, homogenized for 30 s at 2500 rpm, and centrifuged for 15 min at 4000 rpm and ambient temperature. Then, 5.0 µL of the supernatant was injected into the LC-MS/MS system. Chromatographic separation was performed on the Waters ACQUITY UPLC system equipped with Kinetex XB-C18 1.7 µm X 50 mm X 2.1 mm column (Phenomenex), and methanol and formic acid 0.2% solution as mobile phase in 3.5 min of gradient mode separation. The detection was performed on a Waters XEVO TQ-S Micro mass spectrometer with electrospray ionization (ESI+). The current method was validated for carryover, linearity, precision, recovery, and limit of quantification, and it was evaluated by comparing the analysis in the developed method and a commercial kit by HCPL-ECD, of 15 real samples from volunteers in the laboratory.

Results

The method was successfully validated for all the parameters. The linearity was between 0.5–100.0 mg/L. The precision was less than 6.3%, the recovery was between 85.4%–103.4% and the quantification limit was 0.5 mg/L. The comparison with the reference method showed good coefficient correlations (R2 > 0.92) for the three analytes.

Conclusion

We validate a simple LC-MS/MS method to quantify three metabolites to help the diagnosis of neuroendocrynes tumors. The method represents a simplification of the extraction compared with the commercial kit employing HPLC-ECD, and an important reduction of the per sample production cost, without loss of quality.
B-217 The study of association of FokI Polymorphism of Vitamin D Receptor (VDR) Gene, Vitamin D and Parathyroid Hormone Levels in Stage 4–5 Chronic Kidney Disease Patients: A Cross-sectional Study
Authors: Yadav R , Naagar S , Datta S K , Ahirwar A K , Mahajan S , Chopra P , Arulselvi S
Published: September 2023

Background

Chronic Kidney Disease (CKD) is an important public health problem globally and almost 35% patients of CKD have deficiency in vitamin D which progresses to 80% in stage 4–5 CKD patients. Active vitamin D binds to Vitamin D Receptor (VDR) to regulate target gene transcription. Various genetic variations (Single Nucleotide Polymorphism) such as FokI polymorphism, which is a T/C transition polymorphism (ACG to ATG) may affect the VDR gene expression and thus may play an important role in pathogenesis of CKD. The aim of the study is to study association between FokI Polymorphism of Vitamin D Receptor (VDR) Gene, serum Vitamin D and PTH levels in Stage 4–5 CKD patients.

Methods

A total of 150 patients, aged 25–60 years, attending the Department of Nephrology AIIMS New Delhi with chronic kidney disease (CKD) stage 4–5, were enrolled for the study. FokI polymorphism was analyzed by using polymerase chain reaction-restriction fragment length Polymorphism (PCR-RFLP) in study subjects. Following DNA isolation and PCR amplification, restriction digestion was done using FokI restriction endonuclease and visualised on 1.5 % agarose gel electrophoresis. The prevalence of different genotypes and allelic frequency distribution was compared between CKD patients and healthy controls (HC). Levels of PTH, Vitamin D were estimated by eCLIA.

Results

No significant differences in genotype and in allelic frequencies between CKD patients and HC were observed as shown in Table 1. No significant differences in biochemical parameters based on genotypic variations in CKD patients and in HC were observed.

Conclusion

The present study reveals no association of VDR FokI polymorphism with CKD. Furthermore, no significant differences in biochemical parameters were observed in FF, Ff and ff genotypic subgroups in CKD or HC groups. Further analysis revealed no significant differences in biochemical parameters based on genotypic variations in CKD patients and in HC. Table 1.Genotype frequency and allelic frequency of VDR FOK1 gene polymorphism in CKD patients and HC.Genotype(FOK1)Genotype frequency (%)P-valueX2 (chi2)Allelic frequencyCKD (n = 150)HC (n = 150)Total study group (n = 300)0.7700.5220CKD (n = 150)HC (n = 150)FF53.33 (n = 80)57.33 (n = 86)55.33 (n = 166)FfFfFf41.33 (n = 62)37.33 (n = 56)39.33 (n = 118)0.740.260.760.24ff5.33 (n = 8)5.33 (n = 8)5.33 (n = 16)
B-226 Clinical Evaluation of ElsiQ™ STI-12 Detection Kit 3/4 for Detection of Human Sexual Transmitted Infection Pathogens in Human Vaginal Swab and/or Urine Specimens
Authors: Ahn J , Kim D , Lee W , Yoon S , Cho S
Published: September 2023

Background

Nucleic acid amplification tests are a highly sensitive method for detecting sexual transmitted infection (STI) pathogens, and multiplex molecular assays are even more advantageous in screening and diagnosing multiple STI pathogens simultaneously. The ElsiQ™ STI-12 Detection Kit 3/4 (ElsiQ) is in vitro diagnostic marked multiplex real-time PCR assay that targets 6 STI pathogens, including human herpes simplex virus 1 (HSV1), human herpes simplex virus 2 (HSV2), Gardnerella vaginalis (GV), Treponema pallidum (TP), Candida albicans (CA), and Ureaplasma parvum (UP) in human vaginal swab and/or urine specimens.

Methods

In this retrospective study, 575 clinical specimens suspected of having STI pathogen were enrolled; 280 genital swabs and 295 urine samples from April to July 2021. The comparison tests were performed using both the ElsiQ and PANA RealTyper™ STD Kit (PANA). Clinical sensitivity and clinical specificity were evaluated by comparing the patient’s clinical features.

Results

The comparison results between ElsiQ and PANA were all consistency in 5 strains (HSV1, HSV2, GV, TP, UP), and 96.4% positive percent agreement value with 0.97 kappa value in CA. (Table 1). The clinical sensitivity and clinical specificity showed 100% in 5 strains (HSV1, HSV2, GV, TP, UP). Only CA has 97.0% sensitivity, and 100% specificity.

Conclusion

The ElsiQ assay demonstrated excellent analytical performance for detecting six STD pathogens, with high sensitivity and specificity. The results of this study support the use of the ElsiQ assay as a reliable and efficient tool for the simultaneous diagnosis of multiple STIs.
B-230 Monitoring SARS-CoV-2 Subvariants for Evaluation of the Diagnostic Kit’s Annealing Site Using Nanopore Sequencing
Authors: Kim Y , Noh T , Lee S , Kim Y , Prakash D , Lee K
Published: September 2023

Background

Currently, various diagnostic kits are being developed to aid in the rapid diagnosis or detection of SARS-CoV-2, but viruses continue to evolve through mutations resulting in phenotypic changes such as increased infectivity and pathogenicity, or vaccine evasion. Over time, such alterations in sequence may also occur at the annealing sites of the primers and probes in the diagnostic kit, leading to false-negative results. To prevent this, a continuous mutation monitoring is required. The authors performed SARS-CoV-2 Whole Genome Sequencing (WGS) and then compared and monitored the difference between actual experimental and public data from the National Center for Biotechnology Information (NCBI) datasets for each mutant strain.

Methods

WGS was performed by using GridION (Oxford Nanopore Technologies, UK) on 1500 nasopharyngeal swab SARS-CoV-2 positive samples requested for testing at SCL from January 2020 to December 2022. Experimental data were aligned through epi2me and analyzed using the Nextclade platform (Fig. 1A). For public data, 119 sequences reported in Korea (Fig. 1B) were retrieved from the NCBI database, reanalyzed using Nextclade platform, and compared.

Results

In numerous samples tested, mutation, insertion, and deletion were found to occur over time. In particular, in the case of Omicron subvariants, a common Gap (region 1; Gap: 11288–11296, region 2; Gap: 28362–28370) were identified in orf1ab (RdRP), the target gene for the majority of existing diagnostic kits, and in N gene (Fig. 1). Therefore, while designing a diagnostic kit, this region should not be included in the annealing site.

Conclusion

In order to maintain an effective diagnosis and quarantine system for COVID-19, mutations affecting the annealing site of primers and probes must be identified through a continuous genome monitoring. The results obtained from the continuous genome monitoring can be utilized to upgrade the existing commercially available kits as well as to develop new kits.
B-236 Portable 2 Hour Sample-to-Answer Multi-pathogen Phenotypic Antibiotic Susceptibility Test (AST)
Authors: Roegner M E
Published: September 2023

Background

Antimicrobial Resistance is a rapidly growing issue worldwide, arising from the overuse and misuse of antibiotics. The strongest tool the medical community has available to combat AMR is rapid, correct application of antibiotics to identified bacterial infections. Clinics and physician offices seek on-site rapid solutions to speed the pathway to timely and appropriate antibiotic therapies. GeneCapture is prototyping a rapid, sample-to-answer product to provide antibiotic susceptibility results directly from infected urine or wound samples. The test provides a susceptible vs non-susceptible call in under two hours and can be applied to samples with single or mixed pathogens. With support from the Defense Health Agency, a portable table-top instrument and disposable cartridge are being engineered to run this test at the point of care, allowing same-day prescriptions. The process is currently operational on the lab bench.

Methods

Fluorescent labeling of bacterial species, followed by optical counting, determines population growth with/without a suite of antibiotics. An on-board algorithm instantly converts raw cell numbers into a differential call of Susceptible or Non-susceptible for each bug/drug pair. A disposable cartridge with on-board antibiotics has been designed and is being finalized for fabrication. The optical counting is operating in a desktop device.

Results

Testing to date includes 124 strains from 7 of the most common Urinary Tract Infection and wound pathogens against a panel of six antibiotics. A total of 541 antibiotic/bacterial strain pairs in single or multiple pathogen antibiotic susceptibility testing (AST), are producing an initial 88.7% agreement to gold standard culture results. These results are all generated in two hours or less of assay time and represent a strong preliminary data set.

Conclusion

This 2-hour portable, novel, phenotypic AST profiling will produce informed and effective treatment options for medics, clinics and physician offices long before existing AST methods could return results.
B-250 Simultaneous Detection of Twelve Pathogens of Sexually Transmitted Infections Using an Innovative All-in-One PCR Instrument
Authors: Nam S , Lee J , Song M , Heo H , Jeong Y , Kim D , Park H
Published: September 2023

Background

Every day, nearly 1 million people are infected with sexually transmitted infections (STI) that are transmitted through sexual contact. Early diagnosis and treatment of STI are critical in reducing their transmission, and PCR is the most sensitive method among diagnostic tests. To address this need, we have developed a new STI Real-Time PCR-based diagnostic kit, which is capable of detecting twelve STI pathogens simultaneously in up to 94 specimens at once. To evaluate the analytical performance, we used the ExistationTM FA System, which automatically performs from decapping primary sample tubes followed by nucleic acid extraction to PCR analysis within 2 h. Twelve (12) pathogens of STI are described below: Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Ureaplasma urealyticum (UU), Mycoplasma genitalium (MG), Trichomonas vaginalis (TV), Mycoplasma hominis (MH), Herpes simplex virus type1 (HSV1), Herpes simplex virus type2 (HSV2), Ureaplasma parvum (UP), Treponema pallidum (TP), Candida albicans (CA), Gardnerella vaginalis (GV), from male urine and female vaginal swabs.

Methods

The analytical performance evaluation was performed using the new STI diagnostic kit to detect twelve pathogens of STI. The analysis was performed using culture fluids (CT, NG, UU, MG, TV, MH, HSV1, HSV2, UP, CA, GV; ATCC, USA) and TP genomic DNA (Vircell, Spain), which were spiked in urine and vaginal swab media. The limit of detection (LoD) was determined by testing multiple replicates of serial dilutions of the spiked sample around the expected LoD. The LoD was defined as the lowest dilution at which the assay detected ≥95% of 24 replicates. To evaluate precision, each sample was tested 2 times a day in duplicate per run for 5 days. To assess cross-reactivity, twenty-two bacteria and viruses related to STI were used.

Results

The LoD was determined 60.26 IFU/mL, 35.48 cfu/mL, 467.74 copy/mL, 45.71 cell/mL, 19.5 cells/mL, 316.23 copy/mL, 199.53 TCID50/mL, 13.18 TCID50/mL, 588.84 copy/mL, 993.25 copy/ml, 660.69 copy/mL, 389.05 copy/mL in Urine and 47.86 IFU/mL, 27.54 cfu/mL, 363.08 copy/mL, 38.90 cell/mL, 15.14 cells/mL, 245.47 copy/mL, 181.97 TCID50/mL, 8.71 TCID50/mL, 575.44 copy/mL, 741.31 copy/ml, 524.81 copy/mL, 288.40 copy/mL in vaginal swab media for CT, NG, UU, MG, TV, MH, HSV1, HSV2, UP, CA, GV, and TP, respectively. For repeatability assay, the 12 STI samples showed similar Ct value in each run, with all standard deviation (SD) were less than 2.0. In terms of cross-reactivity, there were no positive results obtained from testing bacteria and viruses using the new STI diagnostic kit.

Conclusions

The ExistationTM FA system is highly convenient platform for Real-Time PCR-based diagnostics. Particularly, our new STI diagnostic kit provides an accurate and efficient method for simultaneous detection of 12 STI pathogens.
B-267 Validation of a New Molecular Test to Quantify Plasma Cytomegalovirus Load
Authors: Dehesa-García B , García-Murillo A , Cortés-Portero I , Pérez-Grijalba V , García-Manrique B
Published: September 2023

Background

Cytomegalovirus (CMV) is a DNA virus that often causes severe disease in immunosuppressed or immunocompromised patients. With the expanding indications for immune-modulating agents and transplant patients, the number of patients at risk for developing CMV disease is increasing. Test results are used to determine active CMV disease, risk of developing active disease, response to therapy, risk of relapsed infection, and/or appropriate time to discontinue therapy. For this purpose, molecular assays are attractive because of their broad linear range and low limits of detection. CerTest Biotec has developed a quantitative real time PCR assay for the detection and quantification of cytomegalovirus in plasma samples.

Methods

Plasma-EDTA samples characterized as positive or negative and quantified by IU/ml were selected. The selected samples were collected from both male and female adults (except 1 sample of an infant) between July 2016 and December 2020. Two CE-marked molecular reference assays were used for the comparative analysis: RealStar® CMV PCR Kit 1.0 (Altona), using the CFX96™ Real-Time PCR Detection System (Bio-Rad) and extracted with the QIAamp® MinElute® Virus Spin Kit (QIAGEN); and cobas® CMV (Roche Diagnostics), using the cobas® 8800 system. VIASURE analysis was performed using the following workflow: the automated extraction method MagDEA® Dx SV with magLEAD® 12gC and CFX96™ Real-Time PCR Detection System (Bio-Rad).

Results

A total of 273 EDTA-plasma clinical samples were analysed, 120 samples were reported as CMV positive samples and quantified and 153 samples were reported as CMV negative samples. Comparative analysis determined >99% sensitivity and specificity. Regarding quantification, all methods obtained a correlation value of R2 ≥ 0.77.

Conclusion

With this study it was possible to observe that the VIASURE CMV q Real Time PCR Detection Kit present good clinical sensitivity and specificity compared to reference molecular assays results. No false positive results were observed. However, all variables employed on the workflow contribute to final quantification results and therefore should be taken into account as accumulative error in the calculation.