The Diagnostic Accuracy of Procalcitonin, Soluble Urokinase-Type Plasminogen Activator Receptors, and C-Reactive Protein in Diagnosing Urinary Tract Infections in the Emergency Department-A Diagnostic Accuracy Study.

Background: Urinary tract infections (UTIs) are a leading bacterial infection in the emergency department (ED). Diagnosing UTIs in the ED can be challenging due to the heterogeneous presentation; therefore, fast and precise tests are needed. We aimed to evaluate the diagnostic precision of procalcitonin (PCT), soluble urokinase plasminogen activator receptors (suPARs), and C-reactive protein (CRP) in diagnosing UTIs, grading the severity of UTIs, and ruling out bacteremia. Methods: We recruited adults admitted to three Danish EDs with suspected UTIs. PCT, suPAR, and CRP were used in index tests, while blood cultures, expert panel diagnosis, and severity grading were used in the reference tests. Logistic regression and area under the receiver operator characteristic curves (AUROCs) were utilized to evaluate the models and determine the optimal cut-offs. Results: We enrolled 229 patients. PCT diagnosed UTI with an AUROC of 0.612, detected severe disease with an AUROC of 0.712, and ruled out bacteremia with an AUROC of 0.777. SuPAR had AUROCs of 0.480, 0.638, and 0.605, while CRP had AUROCs of 0.599, 0.778, and 0.646. Conclusions: The diagnostic performance of PCT, suPAR, or CRP for UTIs or to rule out severe disease was poor. However, PCT can safely rule out bacteremia in clinically relevant numbers in ED patients suspected of UTI.

Monitoring performance of clinical artificial intelligence: a scoping review protocol.

OBJECTIVE: The objective of this scoping review is to describe the scope and nature of research on the monitoring of clinical artificial intelligence (AI) systems. The review will identify the various methodologies used to monitor clinical AI, while also mapping the factors that influence the selection of monitoring approaches. INTRODUCTION: AI is being used in clinical decision-making at an increasing rate. While much attention has been directed toward the development and validation of AI for clinical applications, the practical implementation aspects, notably the establishment of rational monitoring/quality assurance systems, has received comparatively limited scientific interest. Given the scarcity of evidence and the heterogeneity of methodologies used in this domain, there is a compelling rationale for conducting a scoping review on this subject. INCLUSION CRITERIA: This scoping review will include any publications that describe systematic, continuous, or repeated initiatives that evaluate or predict clinical performance of AI models with direct implications for the management of patients in any segment of the health care system. METHODS: Publications will be identified through searches of the MEDLINE (Ovid), Embase (Ovid), and Scopus databases. Additionally, backward and forward citation searches, as well as a thorough investigation of gray literature, will be conducted. Title and abstract screening, full-text evaluation, and data extraction will be performed by 2 or more independent reviewers. Data will be extracted using a tool developed by the authors. The results will be presented graphically and narratively. REVIEW REGISTRATION: Open Science Framework https://osf.io/afkrn.

Establishing reference intervals for soluble urokinase plasminogen activator receptor in Northern European adults.

Objectives: Soluble urokinase plasminogen activator receptor (suPAR) may have untapped potential in clinical diagnostics. Previous studies determined reference intervals using an enzyme-linked immunoassay, but there is a need for reference intervals using a faster assay if the analysis is to be used in emergency medicine. The current study aims to determine reference intervals for suPAR using a fully automated particle-enhanced turbidimetric immunoassay (PETIA) according to the Clinical and Laboratory Standards Institute guideline A28-A3c. Design and methods: Blood samples were prospectively collected from Danish blood donors. Plasma suPAR was analyzed on the cobas 8000 module c502 in an open channel using a PETIA. Sex-partitioned reference intervals were determined using a parametric quantile approach. Results: The study included 241 participants-123 females and 118 males. The common reference interval for suPAR was 1.56-4.11 ng/mL (95% confidence intervals (CI) for the lower and upper limits were 1.56-1.63 and 3.81-4.47, respectively). The reference interval for females was 1.59-4.65 ng/mL (95% CIs 1.48-1.70 and 4.09-5.48, respectively) and for males, 1.56-3.59 ng/mL (95% CIs 1.47-1.65 and 3.31-3.93, respectively). Conclusions: Our results support using sex-partitioned reference intervals for suPAR and provide a basis for future studies using the PETIA method.

Urine Flow Cytometry and Dipstick Analysis in Diagnosing Bacteriuria and Urinary Tract Infections among Adults in the Emergency Department-A Diagnostic Accuracy Trial.

Urinary tract infections (UTIs) are a leading infectious cause of emergency department admission. Early UTI diagnosis is challenging, and a faster, preferably point-of-care urine analysis is necessary. We aimed to evaluate the diagnostic accuracy of urine flow cytometry (UFC) and urine dipstick analysis (UDA) in identifying bacteriuria and UTIs. This study included adults suspected of an infection admitted to three Danish emergency departments. UFC and UDA were the index tests, and urine culture and an expert panel diagnosis were the reference tests. We used logistic regression and receiver operator characteristics curves to find each test's optimal model and cut-off. We enrolled 966 patients and performed urine cultures on 786. Urine culture was positive in 337, and 200 patients were diagnosed with a UTI. The UFC model ruled out bacteriuria in 10.9% with a negative predictive value (NPV) of 94.6% and ruled out UTI in 38.6% with an NPV of 97.0%. UDA ruled out bacteriuria in 52.1% with an NPV of 79.2% and UTI in 52.8% with an NPV of 93.9%. Neither UFC nor UDA performed well in ruling out bacteriuria in our population. In contrast, both tests ruled out UTI safely and in clinically relevant numbers.

Establishing Sex-Dependent Reference Intervals for KL-6 in Danish Adults.

Krebs von den Lungen-6 (KL-6) is a promising biomarker for the diagnosis and prognosis of interstitial lung disease. However, reference intervals in Northern Europeans remain to be established using a latex-particle-enhanced turbidimetric immunoassay. The participants were Danish blood donors subjected to strict health requirements. Analyses were performed using the Nanopia KL-6 reagent on the cobas 8000 module c502. Sex-partitioned reference intervals were determined using a parametric quantile approach according to the Clinical and Laboratory Standards Institute guideline EP28-A3c. The study included 240 participants-121 females and 119 males. The common reference interval was 59.4-398.5 U/mL (95% confidence intervals (CI) for the lower and upper limits were 47.3-71.9 and 369.5-430.1, respectively). For females, the reference interval was 56.8-324.0 U/mL (95% CIs for the lower and upper limits were 36.1-77.6 and 303.3-344.7, respectively). For males, the reference interval was 51.5-448.7 U/mL (95% CIs for the lower and upper limits were 32.8-71.2 and 397.3-508.1, respectively). These results emphasize the importance of sex partitioning when evaluating KL-6 reference intervals. The reference intervals increase the clinical applicability of the KL-6 biomarker and provide a basis for future scientific studies of its utility in patient management.

Rest reduces venous lactate levels significantly in patients in outpatient clinic.

Lactate is produced in the human body during physical activity and elimination takes time with a half-life of approximately 18 min. We, therefore, investigated the potential impact of resting time (RT) duration on lactate concentration in our outpatient venipuncture clinic for all lactate requests during a 4½-year period. All samples drawn for venous lactate analysis during a 4½-year period in our hospital outpatient venipuncture clinics were included in this study. RT was reported electronically at each visit. Results from a total of 831 samples were obtained for further analysis. We found varying lactate concentrations across resting time <15min (median 1.6 mmol/L, IQR[1.2-2.1] mmol/L), between <15 min and >30 min (median 1.4 mmol/L, IQR[1.0-1.9] mmol/L) and for >30 min (median 1.3 mmol/L, IQR[1.0-1.7] mmol/L). There was a significant difference between <15 min versus 15-30 min (p = 0.015), which gives a 17.7% higher lactate from 15-30 min to <15 min. There was a significant 28.3% increase in mean lactate concentration from >30min to <15min (p < 0.0001) when corrected for age. We found that lactate concentration was dependent on RT in the outpatient clinic. The difference was clinically significant. Based on the results of this study, we, therefore, conclude that a 15 min waiting time before venipuncture for lactate sampling in an outpatient clinic is of clinical importance.

Adult patient diagnosed with NADSYN1 associated congenital NAD deficiency and analysis of NAD levels to be published in: European Journal of Medical Genetics.

INTRODUCTION: Nicotinamide adenine dinucleotide (NAD) is an essential cosubstrate/coenzyme in multiple cellular redox processes and a substrate in several non-redox reactions. NADSYN1 encodes NAD synthetase 1, an enzyme in the NAD de novo synthesis pathway and the Preiss-Handler pathway, and biallelic pathogenic variants causes NAD deficiency associated with vertebral, cardiac, renal and limb defects. Szot et al. and Kortbawi et al. have reported a total of seven patients with NADSYN1 associated congenital NAD deficiency disorder with the oldest patient being seven years old. PATIENT DATA: We present a male patient age 30 with a height of 130 cm and numerous skeletal malformations including segmentation defects of the spine, rib anomalies and unequal leg length as well as bilateral ptosis, cleft palate and asymmetric dysmorphic facial features. The patient underwent surgery for an aortic stenosis due to a bicuspid valve. No malformations of the kidneys or urinary tract were identified. RESULTS: Trio exome sequencing revealed a homozygous missense variant in NADSYN1 c.1717G > A (p.Ala573Thr). Both parents were unaffected carriers of the variant. Analysis of NAD levels showed that the patient had a lower NAD pool compared to his unaffected siblings. The NAD pool rose approximately 25% after supplementation with nicotinamide, a NAD precursor for the salvage pathway. CONCLUSION: The variant was previously reported in four patients and functional analyses by Szot et al. support the pathogenicity of the variant. We report an adult patient with NADSYN1 associated congenital NAD deficiency disorder and expand the phenotypic spectrum. We also present analysis of the NAD levels before and after supplementation with nicotinamide.

POCT urine dipstick versus central laboratory analyses: Diagnostic performance and logistics in the medical emergency department.

OBJECTIVES: The aim of this study was to evaluate the logistics and diagnostic performances of dipstick analyses compared to their counterpart central laboratory analyses for detection of bacteriuria, proteinuria, hyperglycemia, ketosis and hematuria. DESIGN AND METHODS: Urine dipstick results, urine culture results, flow cytometric cell counts, U-albumin-to-creatinine ratio, P-glucose and P-beta-hydroxybutyrate were retrospectively reviewed in a cohort of consecutive patients admitted to the medical emergency departments of two Danish hospitals. Sensitivity, specificity and predictive values of traditional dipstick analysis were estimated and dipstick was compared to flow cytometry for detection of significant bacteriuria using logistic regression. Turn-around-time for central laboratory analyses were assessed. RESULTS: For each comparison, 1,997 patients or more were included. Traditional dipstick analyses for proteinuria, bacteriuria and ketosis reached sensitivities of up to 90%, while sensitivity for hyperglycemia was 59%. Flow cytometry outperformed traditional dipstick analysis for detection of bacteriuria with a difference in the area under the ROC-curve of 0.07. Turn-around-times for 95% delivery of central laboratory analysis results ranged from approximately 1½ to 2 h. CONCLUSIONS: For the detection of bacteriuria and albuminuria, central laboratory analyses reach better performance than dipstick analysis while achieving acceptable turn-around-times and are thus viable alternatives to dipstick analysis. For detection of ketosis and hyperglycemia, dipstick analysis does not perform adequately, but as very short turn-around-time is often required, these conditions may be best diagnosed by point-of-care blood test rather than dipstick or central laboratory analyses. Dipstick hemoglobin analysis, flow cytometry and microscopic evaluation may serve each their distinct purposes, and thus are relevant in the emergency department.

Hypophosphatasia as a plausible cause of vitamin B6 associated mouth pain: a case-report.

BACKGROUND: Mouth pain has been associated with abnormal vitamin B6 levels. Hypophosphatasia is a rare genetic disease, which causes imbalances between B6 vitamers. We report the case of a patient with hypophosphatasia and burning mouth pain. CASE PRESENTATION: A 39-year old Caucasian male with chronic burning mouth pain underwent extensive investigations with no cause of the pain being found. During the course of the investigation, an elevated vitamin B6 (pyridoxal phosphate) level was detected, which led to the diagnosis of hypophosphatasia. We hypothesize that the patient's mouth pain stems from hypophosphatasia through a B6 dependent mechanism. CONCLUSIONS: Mouth pain may, in some cases, be a symptom of hypophosphatasia and when investigating B6 in relation to mouth pain, attention should be paid to the exact B6 vitamer measured. The case underlines the importance of low alkaline phosphatase results, especially in patients with unexplained pain, as this should prompt suspicion of hypophosphatasia.

A collection of multiregistry data on patients at high risk of lung cancer-a Danish retrospective cohort study of nearly 40,000 patients.

Background: Lung cancer (LC) is the leading cause of cancer related deaths, and several countries are implementing screening programs. Risk models have been introduced to refine the LC screening criteria, but the use of real-world data for this task demands a robust data infrastructure and quality. In this retrospective cohort study, we aim to address the different relevant risk factors in terms of data sources, descriptive statistics, completeness and quality. Methods: Data on comorbidity, prescription medication, smoking history, consultations, symptoms, familial predispositions, exposures, laboratory data among others were collected for all patients examined on a risk of LC over a 10-year period in the Region of Southern Denmark. Data were delivered from the regional data warehouse as well as the Danish Lung Cancer Registry. Associations between LC and non-LC groups were examined through Chi-squared test (categorical variables) and Wilcoxon signed-rank test (continuous variables that were non-parametric). These associations were investigated on both the original datasets and the subset of patients with complete data. Results: The number of examined individuals increased over the study period and more patients were diagnosed with LC in stage I-II, from 18% in 2009 to 31% in 2018. LC patients were more likely to be older, smoker, with a registered prescription of the included medication. They also exhibited differences in laboratory analysis indicating inflammation and hyponatremia. Weight loss, fatigue and pain were more prevalent in the LC group, while hemoptysis and fever were more common among the non-LC patients. Advanced-stage LC patients experienced a higher rate of symptoms compared to those in the low stages. Within the sub-cohort with complete dataset results, most observed trends persisted, although data on comorbidities were susceptibility to change. Conclusions: This study provides key insights into LC risk assessment using a robust dataset of patients examined for suspected LC. A consistent positive trend in early-stage LC diagnosis was observed throughout the study period. LC patients exhibited distinct smoking behaviors, medication patterns, variations in lab results, and specific symptoms. These discoveries have the potential to enhance discrimination in machine learning-based prediction models, particularly those capable of handling complex distributions. Serving as a detailed account of real-world data collection and processing, the study establishes a foundation for future development of prediction models aimed at facilitating the early referral of LC patients.

Vitamin K supplementation and arterial calcification in dialysis: results of the double-blind, randomized, placebo-controlled RenaKvit trial.

BACKGROUND: Arterial calcification is associated with cardiovascular mortality in dialysis patients. Active matrix Gla protein (MGP) is a vitamin K-dependent inhibitor of arterial calcification. Elevated plasma concentrations of inactive MGP, i.e. dephosphorylated-uncarboxylated MGP (dp-ucMGP), are prevalent in dialysis patients. MGP inactivity might contribute to arterial calcification. We investigated whether vitamin K supplementation had an effect on arterial calcification in chronic dialysis patients. METHODS: In a 2-year, double-blind, placebo-controlled intervention trial, 48 dialysis patients were randomized to vitamin K [menaquinone-7 (MK-7), 360 µg daily] or placebo. MK-7 in serum and dp-ucMGP in plasma were used to assess vitamin K status. Carotid-femoral pulse wave velocity (cfPWV) and scores of coronary arterial calcification (CAC) and abdominal aortic calcification (AAC) were used to assess arterial calcification. RESULTS: Thirty-seven participants completed Year 1, and 21 completed Year 2. At Year 2, serum MK-7 was 40-fold higher, and plasma dp-ucMGP 40% lower after vitamin K supplementation compared with placebo {mean dp-ucMGP difference: -1380 pmol/L [95% confidence interval (CI) -2029 to -730]}. There was no significant effect of vitamin K supplementation on cfPWV [mean difference at Year 2: 1.2 m/s (95% CI -0.1 to 2.4)]. CAC Agatston score increased significantly in vitamin K supplemented participants, but was not significantly different from placebo [mean difference at Year 2: 664 (95% CI -554 to 1881)]. AAC scores increased in both groups, significantly so within the placebo group at Year 1, but with no significant between-group differences. CONCLUSIONS: Vitamin K supplementation improved vitamin K status, but did not hinder or modify the progression of arterial calcification in dialysis patients.

Daily monitoring of viral load measured as SARS-CoV-2 antigen and RNA in blood, IL-6, CRP and complement C3d predicts outcome in patients hospitalized with COVID-19.

OBJECTIVES: We hypothesized that the amount of antigen produced in the body during a COVID-19 infection might differ between patients, and that maximum concentrations would predict the degree of both inflammation and outcome for patients. METHODS: Eighty-four hospitalized and SARS-CoV-2 PCR swab-positive patients, were followed with blood sampling every day until discharge or death. A total of 444 serial EDTA plasma samples were analyzed for a range of biomarkers: SARS-CoV-2 nuclear antigen and RNA concentration, complement activation as well as several inflammatory markers, and KL-6 as a lung marker. The patients were divided into outcome groups depending on need of respiratory support and death/survival. RESULTS: Circulating SARS-CoV-2 nuclear antigen levels were above the detection limit in blood in 65 out of 84 COVID-19 PCR swab-positive patients on day one of hospitalization, as was viral RNA in plasma in 30 out of 84. In all patients, complete antigen clearance was observed within 24 days. There were definite statistically significant differences between the groups depending on their biomarkers, showing that the concentrations of virus RNA and antigen were correlated to the inflammatory biomarker levels, respiratory treatment and death. CONCLUSIONS: Viral antigen is cleared in parallel with the virus RNA levels. The levels of antigens and SARS-CoV-2 RNA in the blood correlates with the level of IL-6, inflammation, respiratory failure and death. We propose that the antigens levels together with RNA in blood can be used to predict the severity of disease, outcome, and the clearance of the virus from the body.

In Search of Normality for Vitamin K1: Establishing Age-Dependent Reference Intervals in the Danish Population.

BACKGROUND: A growing body of evidence suggests that vitamin K has beneficial effects on human health, especially cardiovascular and bone health. Vitamin K1 (phylloquinone), the predominant form of vitamin K in blood, is regarded as an indicator of vitamin K status, but to our knowledge no reference intervals (RIs) have been established for vitamin K1. METHODS: In this population-based study, vitamin K1 was measured in serum from 3808 Caucasian individuals without diabetes from 26 to 78 years of age. The need for gender- and age-partitioned vitamin K1 reference intervals was evaluated using Lahti's method, and exclusion criteria were defined to obtain as healthy a study group as possible. The excluded subgroups were tested for differences in mean serum vitamin K1 levels. Serum vitamin K1 levels were quantified using an in-house newly developed, validated, and highly sensitive online SPE-LC-MS/MS method with a limit of quantitation of (LOQ) 0.05 nmol/L. RESULTS: The reference interval for serum vitamin K1 was 0.22 to 3.95 nmol/L for individuals aged 26 to 44 years and 0.35 to 3.70 nmol/L for individuals aged 45 to 78. Similar age-specific reference intervals were established for vitamin K1-triglyceride ratio being 0.20 to 3.16 and 0.31 to 3.44, respectively. No significant difference was found between genders. Serum vitamin K1 was detectable in all serum samples. Individuals with known comorbidity were found to have significantly lower serum vitamin K1 compared to those without comorbidity. Current smokers had lower serum vitamin K1 compared to nonsmokers. CONCLUSION: Age-dependent reference intervals were established for serum vitamin K1 and vitamin K1-triglyceride ratio in a well-defined, healthy Caucasian population. Lower serum vitamin K1 levels were found in individuals with known comorbidity, suggesting an association between serum vitamin K1 and disease status. Further studies are needed to determine an optimal serum vitamin K1 level.

Serum vitamin K1 associated to microangiopathy and/or macroangiopathy in individuals with and without diabetes.

OBJECTIVE: Vitamin K has proposed beneficial effects on cardiovascular health. We investigated whether serum vitamin K1 was associated with prevalence of microangiopathy and/or macroangiopathy. RESEARCH DESIGN AND METHODS: Serum vitamin K was quantified in 3239 individuals with and 3808 without diabetes enrolled in Vejle Diabetes Biobank (2007-2010). Each individual was assessed for microangiography and macroangiopathy at enrollment based on registered diagnoses in the Danish National Patient Registry according to the International Classification of Disease 8 (1977-1993) and 10 (since 1994). Using multinomial logistic regression, relative risk ratios (RRRs) were calculated within each group of individuals with and without diabetes. RRRs were estimated for microangiopathic/macroangiopathic status compared with individuals without complications as a function of 1 nmol/L increments in K1. Adjustment for potential confounders was also performed. RESULTS: Vitamin K1 (median) varied 0.86-0.95 nmol/L depending on diabetes, microangiopathic and macroangiopathic status. In individuals with diabetes, the crude RRR for only having microangiopathy was 1.05 (95% CI 0.98 to 1.12) and was found significant when adjusting 1.10 (95% CI 1.01 to 1.19). RRR for having only macroangiopathy was 0.89 (95% CI 0.77 to 1.03) and was again significant when adjusting 0.79 (95% CI 0.66 to 0.96). In individuals without diabetes, adjustments again led to similar estimates that was not significant. The adjusted RRR for having only macroangiopathy was 1.08 (95% CI 0.98 to 1.19). CONCLUSIONS: Serum vitamin K1 levels were associated with microangiopathic and macroangiopathic status in individuals with diabetes, but considered of no clinical relevance. The clinical value of other candidate markers for vitamin K status needs to be evaluated in future studies.

Quantification of microRNA in plasma using probe based TaqMan assays: is microRNA purification required?

OBJECTIVE: Circulating microRNAs are promising diagnostics and prognostics biomarkers in a wide variety of diseases. However, there is a critical reproducibility challenge, which in part may be due to preanalytical factors. MicroRNA purification has been identified as the major contributor to the total intra assay variation, thus we found great interest in recent papers describing methods for direct quantification of circulating microRNAs without the purification step. With one exception, all the studies we identified where a direct quantification of circulating microRNAs had been performed were using SYBR Green chemistry. In our laboratory we use platelet-poor plasma and TaqMan assays for microRNA analysis, and thus we investigated whether we could adapt the procedures for the direct reverse transcription described by these studies to be used with our TaqMan assays. RESULTS: We did not achieve valid results by direct quantification of selected microRNAs (miR-92a, miR-16 and miR-126) in platelet-poor plasma using TaqMan assays.

Identification of aspirin resistance using a PDW-miR92a-score: Validation in an intermittent claudication cohort.

OBJECTIVE: Aspirin is a widely used platelet inhibitor to prevent thrombotic events. However, in 25% of patients the antiplatelet effect is insufficient. The current study aimed to validate a newly developed PDW-miR92a-score as a biomarker of the individual response to aspirin enabling targeted antithrombotic therapy. METHODS: Blood samples were collected from 209 patients with intermittent claudication on daily aspirin therapy. Based on results from the arachidonic acid stimulated aggregation test, patients were defined as aspirin resistant (n = 92) or responders (n = 117). Using the cut-off values for platelet distribution width (PDW) and plasma levels of microRNA-92a (miR-92a) defined in our pilot study, we investigated the performance of the combined PDW-miR92a-score in the validation study. Furthermore, receiver operating characteristic curve analysis was performed in the validation cohort in order to optimize the cut-off values of the two score parameters. RESULTS: PDW and miR-92a levels were significantly higher in aspirin resistant compared to responding patients. When using the predefined cut-off values for PDW and miR-92a the combined PDW-miR92a score showed high specificity (93.1%) but poor sensitivity (19.8%) for aspirin resistance. By recalculation using new cut-off values identified in the validation cohort, a score with a specificity of 75% and a sensitivity of 54.9% was obtained. CONCLUSION: Both PDW and plasma levels of miR-92a were confirmed to be significantly higher in aspirin resistant compared to responding patients in our validation cohort. We were, however, unable to confirm the high sensitivity of the combined PDW-miR92a-score previously published by our group in a pilot study.

Quantification of microRNA levels in plasma - Impact of preanalytical and analytical conditions.

Numerous studies have reported a potential role for circulating microRNAs as biomarkers in a wide variety of diseases. However, there is a critical reproducibility challenge some of which might be due to differences in preanalytical and/or analytical factors. Thus, in the current study we systematically investigated the impact of selected preanalytical and analytical variables on the measured microRNA levels in plasma. Similar levels of microRNA were found in platelet-poor plasma obtained by dual compared to prolonged single centrifugation. In contrast, poor correlation was observed between measurements in standard plasma compared to platelet-poor plasma. The correlation between quantitative real-time PCR and droplet digital PCR was found to be good, contrary to TaqMan Low Density Array and single TaqMan assays where no correlation could be demonstrated. Dependent on the specific microRNA measured and the normalization strategy used, the intra- and inter-assay variation of quantitative real-time PCR were found to be 4.2-6.8% and 10.5-31.4%, respectively. Using droplet digital PCR the intra-assay variation was 4.4-20.1%, and the inter-assay variation 5.7-26.7%. Plasma preparation and microRNA purification were found to account for 39-73% of the total intra-assay variation, dependent on the microRNA measured and the normalization strategy used. In conclusion, our study highlighted the importance of reporting comprehensive methodological information when publishing, allowing others to perform validation studies where preanalytical and analytical variables as causes for divergent results can be minimized. Furthermore, if microRNAs are to become routinely used diagnostic or prognostic biomarkers, the differences in plasma microRNA levels between health and diseased subjects must exceed the high preanalytical and analytical variability.

Combination of real-time PCR and sequencing to detect multiple clinically relevant genetic variations in the lactase gene.

BACKGROUND: Lactase persistence is an autosomal dominant trait commonly distributed in Europe as well as some parts of east Africa and the Arabian Peninsula. Using real-time PCR to detect the -13910C > T variant common in the European population is a reliable analysis although other variants in the probe-binding site may cause errors in analysis. The aim of this study was to determine the prevalence of the variants in a Danish cohort examined for lactose intolerance as well as to improve the real-time PCR analysis for detection of the different variants. METHODS: We genotyped 3395 routine samples using real-time PCR for the -13910C > T-variant. All consecutive samples identified as -13910CC were sequenced using Sanger Sequencing. Using the SDS software we examined various quality value settings to improve on the genetic analysis. RESULTS: Using real-time PCR resulted in 100% successful genotyping of the -13910C > T variant. By using a quality value of 99% and sequencing the undetermined samples we improved the ability of the assay to identify variants other than -13910C > T. This resulted in a reduction of the diagnostic error rate by a factor of 2.4 while increasing the expenses only 3%. CONCLUSIONS: We conclude that using a quality value of 99% in the SDS software significantly improves the diagnostic efficiency of the real-time PCR assay for detecting variants associated to lactase persistence.

Aspirin resistance may be identified by miR-92a in plasma combined with platelet distribution width.

OBJECTIVE: Aspirin is a widely used drug for prevention of thrombotic events in cardiovascular patients, but approximately 25% of patients experience insufficient platelet inhibition due to aspirin, and remain in risk of cardiovascular events. This study aimed to investigate the value of circulating miR-92a and platelet size as biomarkers of the individual response to aspirin therapy. METHODS: Blood samples were collected from 50 healthy blood donors without antithrombotic medication and 50 patients with intermittent claudication on daily aspirin therapy. Based on results from the arachidonic acid stimulated aggregation test on Multiplate®analyzer (ASPItest), patients were defined as aspirin resistant (n=10) or aspirin responders (n=40). Plasma levels of miR-92a were evaluated by RT-qPCR analysis and platelet distribution width (PDW) was used to assess platelet size variability. Receiver operating characteristic curves for miR-92a levels and PDW were used to set cut-off values for discrimination between aspirin responding and aspirin resistant patients. RESULTS: When defining aspirin resistance as an ASPItest ≥30U, the optimal cut-off values for discrimination of aspirin responders and aspirin resistant patients were found to be PDW>11.8fL and a relative expression level of miR-92a>4.5. Using these cut-off values we could define a PDW/miR-92a-score with a specificity of 97.5% and a sensitivity of 80.0% in relation to detect aspirin resistance. The corresponding positive and negative predictive values were found to be 88.9% and 95.1%, respectively. CONCLUSION: Aspirin resistance can potentially be identified by miR-92a levels in plasma combined with PDW.

Pre-storage centrifugation conditions have significant impact on measured microRNA levels in biobanked EDTA plasma samples.

BACKGROUND: In the past few years, an increasing number of studies have reported the potential use of microRNAs (miRNA) as circulating biomarkers for diagnosis or prognosis of a wide variety of diseases. There is, however, a lack of reproducibility between studies. Due to the high miRNA content in platelets this may partly be explained by residual platelets in the plasma samples used. When collecting fresh plasma samples, it is possible to produce cell-free/platelet-poor plasma by centrifugation. In this study, we systematically investigated whether biobanked EDTA plasma samples could be processed to be suitable for miRNA analysis. MATERIALS AND METHODS: Blood samples were collected from ten healthy volunteers and centrifuged to produce platelet-poor-plasma (PPP) and standard biobank plasma. After one week at -80 °C the biobanked EDTA plasma was re-centrifuged by different steps to remove residual platelets. Using RT-qPCR the levels of 14 miRNAs in the different plasma preparations were compared to that of PPP. RESULTS: We were able to remove residual platelets from biobanked EDTA plasma by re-centrifugation of the thawed samples. Nevertheless, for most of the investigated miRNAs, the miRNA level was significantly higher in the re-centrifuged biobanked plasma compared to PPP, even when the platelet count was reduced to 0-1×109/L. CONCLUSION: We found, that pre-storage centrifugation conditions have a significant impact on the measured EDTA plasma level of miRNAs known to be present in platelets. Even for the miRNAs found to be less effected, we showed that a 1.5-3 fold change in plasma levels may possible be caused by or easily overseen due to sample preparation and/or storage.