Adult and neonate TSH blood spot reference intervals.

Background: Neonatal congenital hypothyroidism screening is performed by measuring thyroid-stimulating hormone (TSH) in a dried blood spot (DBS) sample, whereas acquired hypothyroidism uses serum TSH. There is no established DBS TSH reference interval, but knowing this is useful, as some patients cannot tolerate venepuncture, so DBS collection is seen as an acceptable alternative. The aim of this study was to establish DBS TSH reference intervals in adults and neonates (day 5-8), and determine the relationship between serum and DBS TSH.Methods: Euthyroid adults, not on thyroid medication and with a normal haematocrit, were selected. If they had a paired lithium heparin sample, DBS were prepared by spotting 50 µL of whole blood onto filter paper. Dried blood spot TSH was measured using the PerkinElmer Neonatal hTSH kit on the GSP instrument and serum using the Abbott Architect assay. The relationship between DBS and serum TSH was analysed using Passing-Bablok regression and the adult DBS TSH reference interval calculated using transformed data. The neonatal reference interval was calculated from screening results using the non-parametric method.Results: Overall, 109 adult samples were included in the study (61 female). The Passing-Bablok regression was: DBS TSH = 0.68 × serum TSH + 0.07, and reference interval was 0.49-3.07 mU/L. The neonatal DBS reference interval was 0.40-4.10 mU/L from 8351 results.Conclusion: This study derived adult and neonate TSH DBS reference intervals using the GSP analyser and established the relationship between serum and DBS TSH. Knowing this information will allow for improved interpretation of DBS TSH results.

The potential role of the eGFR in differentiating between true and pseudohyperkalaemia.

BACKGROUND: Differentiating between true and pseudohyperkalaemia is essential for patient management. The common causes of pseudohyperkalaemia include haemolysis, blood cell dyscrasias and EDTA contamination. One approach to differentiate between them is by checking the renal function, as it is believed that true hyperkalaemia is rare with normal function. This is logical, but there is limited published evidence to support it. The aim of this study was to investigate the potential role of the estimated glomerular filtration rate in differentiating true from pseudohyperkalaemia. METHODS: GP serum potassium results >6.0 mmol/L from 1 January 2017 to 31 December 2017, with a repeat within seven days, were included. Entries were retrospectively classified as true or pseudohyperkalaemia based on the potassium reference change value and reference interval. If the initial sample had a full blood count, it was classified as normal/abnormal to remove blood cell dyscrasias. Different estimated glomerular filtration rate cut-points were used to determine the potential in differentiating true from pseudohyperkalaemia. RESULTS: A total of 272 patients were included with potassium results >6.0 mmol/L, with 145 classified as pseudohyperkalaemia. At an estimated glomerular filtration rate of 90 ml/min/1.73 m2, the negative predictive value was 81% (95% CI: 67-90%); this increased to 86% (95% CI: 66-95%) by removing patients with abnormal full blood counts. When only patients with an initial potassium ≥6.5 mmol/L were included (regardless of full blood count), at an estimated glomerular filtration rate of 90 ml/min/1.73 m2, the negative predictive value was 100%. Lower negative predictive values were seen with decreasing estimated glomerular filtration rate cut-points. CONCLUSION: Normal renal function was not associated with true hyperkalaemia, making the estimated glomerular filtration rate a useful tool in predicting true from pseudohyperkalaemia, especially for potassium results ≥6.5 mmol/L.

Sphingosine 1-phosphate activation of ERM contributes to vascular calcification.

Vascular calcification is the deposition of mineral in the artery wall by vascular smooth muscle cells (VSMCs) in response to pathological stimuli. The process is similar to bone formation and is an independent risk factor for cardiovascular disease. Given that ceramide and sphingosine 1-phosphate (S1P) are involved in cardiovascular pathophysiology and biomineralization, their role in VSMC matrix mineralization was investigated. During phosphate-induced VSMC mineralization, endogenous S1P levels increased accompanied by increased sphingosine kinase (SK) activity and increased mRNA expression of SK1 and SK2. Consistent with this, mineralization was increased by exogenous S1P, but decreased by C2-ceramide. Mechanistically, exogenous S1P stimulated ezrin-radixin-moesin (ERM) phosphorylation in VSMCs and ERM phosphorylation was increased concomitantly with endogenous S1P during mineralization. Moreover, inhibition of acid sphingomyelinase and ceramidase with desipramine prevented increased S1P levels, ERM activation, and mineralization. Finally, pharmacological inhibition of ERM phosphorylation with NSC663894 decreased mineralization induced by phosphate and exogenous S1P. Although further studies will be needed to verify these findings in vivo, this study defines a novel role for the SK-S1P-ERM pathways in phosphate-induced VSMC matrix mineralization and shows that blocking these pathways with pharmacological inhibitors reduces mineralization. These results may inform new therapeutic approaches to inhibit or delay vascular calcification.

Regulation of vascular smooth muscle cell calcification by syndecan-4/FGF-2/PKCα signalling and cross-talk with TGFß.

AIMS: Vascular calcification is a major cause of morbidity and mortality. Fibroblast growth factor-2 (FGF-2) plays an instructive role in osteogenesis and bone development, but its role in vascular calcification was unknown. Therefore, we investigated the involvement of FGF-2 in vascular calcification and determined the mechanism by which it regulates this process. METHODS AND RESULTS: We demonstrate that FGF-2 expression is increased in vascular smooth muscle cells (VSMCs) induced to deposit a mineralized matrix by incubation with ß-glycerophosphate. FGF-2 is also localized to sites of calcification within human atherosclerotic plaques. The expression of syndecan-4, a heparan sulfate proteoglycan which regulates FGF-2 signalling, is also increased in mineralizing VSMCs and co-localizes with FGF-2 in human calcified atherosclerotic plaques. Exogenous FGF-2 inhibits VSMC mineralization, and this inhibition is reduced when syndecan-4 expression is knocked-down using siRNA. Biochemical inhibition of FGFR signalling using a pan FGFR inhibitor (BGJ398) or knocking-down syndecan-4 expression in VSMCs using siRNA increases VSMC mineralization. These increases are prevented by inhibiting transforming growth factor-ß (TGFß) signalling with SB431542, suggesting cross-talk between FGF-2 and TGFß signalling is crucial for the regulation of VSMC mineralization. Syndecan-4 can also regulate FGF-2 signalling directly via protein kinase Cα (PKCα) activation. Biochemical inhibition of PKCα activity using Gö6976, or siRNA-mediated suppression of PKCα expression increases VSMC mineralization; this increase is also prevented with SB431542. Finally, the ability of FGF-2 to inhibit VSMC mineralization is reduced when PKCα expression is knocked-down. CONCLUSION: This is the first demonstration that syndecan-4 promotes FGF-2 signalling, and in turn, suppresses VSMC mineralization by down-regulating TGFß signalling. Our discoveries that FGF-2 and syndecan-4 expression is increased in mineralizing VSMCs and that PKCα regulates FGF-2 and TGFß signalling in VSMCs suggests that the syndecan-4/FGF-2/TGFß signalling axis could represent a new therapeutic target for vascular calcification.