Claus Lohman Brasen

Soluble serum Klotho levels in healthy subjects: Comparison of two different immunoassays

OBJECTIVE Soluble serum Klotho is a new biomarker linked to chronic kidney disease, cardiovascular disease and diabetes. This study describes the evaluation and comparison of two different immunoassays and establishment of assay specific reference intervals in adults. DESIGN AND METHODS Serum Klotho concentrations were determined in 120 healthy adults aged 19-66 years. Blood samples were collected, and stored sera were assayed for Klotho according to age and gender. In addition several other clinical and laboratory characteristics were determined in the cohort and compared to the levels of serum Klotho. RESULTS Serum Klotho levels were significantly higher in time-resolved fluorescence immunoassay (TRF) compared to an ELISA (IBL) and no correlation was found between the assays. No signal was obtained in either assay when the standard curve was switched between the two different immunoassays. The median serum Klotho concentration using TRF was 61 ng/mL (2.5-97.5% reference limits; 11-181 ng/mL) for males and 99 ng/mL (2.5-97.5% reference limits; 19-316 ng/mL) for females while the ELISA gave a mean value of 472 pg/mL (2.5-97.5% reference limits; 204-741 pg/mL) with no difference between genders. Concentrations of serum Klotho were independently associated with estimated glomerular filtration rate (eGFR) and body weight using TRF whereas serum Klotho concentrations were associated with age using the ELISA. CONCLUSION Comparison of two different immunoassays for serum Klotho indicate, that the protein exists in human beings in different forms which may function as independent factors and whose role and potential value as biomarkers needs to be evaluated separately. Reference intervals specific for the different forms recognized by the different assays were calculated in this study.

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.

Familial lipoprotein lipase deficiency: a case of compound heterozygosity of a novel duplication (R44Kfs*4) and a common mutation (N291S) in the lipoprotein lipase gene

Familial lipoprotein lipase (LPL) deficiency (FLLD) is a rare autosomal recessive genetic disorder caused by homozygous or compound heterozygous mutations in the LPL gene. FLLD individuals usually express an impaired or non-functional LPL enzyme with low or absent triglyceride (TG) hydrolysis activity causing severe hypertriglyceridaemia. Here we report a case of FLLD in a 29-year-old man, who initially presented with eruptive cutaneous xanthomata, elevated plasma TG concentration but no other co-morbidities. Subsequent genetic testing of the patient revealed compound heterozygosity of a novel duplication (p.R44Kfs*4) leading to a premature stop codon in exon 2 and a known mutation (N291S) in exon 5 of the LPL gene. Further biochemical analysis of the patients postheparin plasma confirmed a reduction of total lipase activity compared with his heterozygous father carrying the common N291S mutation and to a healthy control. Also the patient showed increased (1.85-fold) activity of hepatic lipase (HL), indicating a functional link between HL and LPL. In summary, we report a case of FLLD caused by compound heterozygosity of a new duplication and a common mutation in the LPL gene, resulting in residual LPL activity. With such mutations, individuals may not receive a diagnosis before classical FLLD symptoms appear later in adulthood. Nevertheless, early diagnosis and lipid-lowering treatment may favour a reduced risk of premature cardiovascular disease or acute pancreatitis in such individuals.

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

Abstract 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.

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.

Standardised Resting Time Prior to Blood Sampling and Diurnal Variation Associated with Risk of Patient Misclassification: Results from Selected Biochemical Components.

Background According to current recommendations, blood samples should be taken in the morning after 15 minutes’ resting time. Some components exhibit diurnal variation and in response to pressures to expand opening hours and reduce waiting time, the aims of this study were to investigate the impact of resting time prior to blood sampling and diurnal variation on biochemical components, including albumin, thyrotropin (TSH), total calcium and sodium in plasma. Methods All patients referred to an outpatient clinic for blood sampling were included in the period Nov 2011 until June 2014 (opening hours: 7am–3pm). Each patient’s arrival time and time of blood sampling were registered. The impact of resting time and the time of day for all components was analysed using simple linear regression. The “maximum allowable bias” was used as quality indicator for the change in reference interval. Results Significant diurnal variation was found for albumin (n = 15,544; p<2×10−16), TSH (n = 20,019; p<2×10−16), calcium (n = 13,588; p = 2.8×10−12) and sodium (n = 51,917; p<2×10−16). Further significant influence for resting time was found for albumin (p = 2.6×10−4), TSH (p = 0.004), calcium (p = 8.9×10−7) and sodium (p = 8.7×10−16). Only TSH and albumin were clinically significantly influenced by diurnal variation. Resting time had no clinically significant effect. Conclusions We found no need for resting 15 minutes prior to blood sampling. However, diurnal variation was found to have a significant and considerable impact on TSH and, to a minor degree, albumin. This has to be taken into account to ensure that reference intervals provided by the laboratory are valid on a 24-hour basis.