Polarized P-glycoprotein expression by the immortalised human brain endothelial cell line, hCMEC/D3, restricts apical-to-basolateral permeability to rhodamine 123

P-glycoprotein (P-gp) expression at the blood-brain barrier prevents unwanted blood-borne toxins and signalling molecules from entering the brain. Primary and immortalised human brain endothelial cells (BECs) represent two suitable options for studying P-gp function in vitro. The limited supply of primary human BECs and their instability over passage number makes this choice unattractive for medium/high throughput studies. The aim of this study was to further characterise the expression of P-gp by an immortalised human BEC line, hCMEC/D3, in order to evaluate their use as an in vitro human blood-brain barrier model. P-gp expression was stable over a high passage number (up to passage 38) and was polarised on the apical plasma membrane, consistent with human BECs in vivo. In addition, hCMEC/D3 cell P-gp expression was comparable, albeit slightly lower to that observed in primary isolated human BECs although P-gp function was similar in both cell lines. The P-gp inhibitors tariquidar and vinblastine prevented the efflux of rhodamine 123 (rh123) from hCMEC/D3 cells, indicative of functional P-gp expression. hCMEC/D3 cells also displayed polarised P-gp transport, since both tariquidar and vinblasine selectively increased the apical-to-basolateral permeability of hCMEC/D3 cells to rh123. The results presented here demonstrate that hCMEC/D3 cells are a suitable model to investigate substrate specificity of P-gp in BECs of human origin

Evaluation of European Concerted Action on Anticoagulation lyophilized plasmas for INR derivation using the PT/INR Line

Abstract The prothrombin time/international normalized ratio (PT/INR) Line method based on 5 certified European Concerted Action on Anticoagulation (ECAA) plasmas provides reliable local INR values without conventional World Health Organization international sensitivity index calibrations. The present study investigated the use of different numbers and types of ECAA calibrant plasmas to derive accurate PT/INR Lines and reliable INR values. The numbers ranged from 3 to 10 plasmas in a set with normal or abnormal samples. Sets were selected, and sampling was repeated 1,000 times for each center to derive PT/INR Lines. The lines were selected randomly or from clusters. The INR values of 5 independent “validation” plasmas were compared before and after correction. In 56 calibrations, 5 ECAA plasmas gave better results than did fewer plasmas. Plasmas with wide-ranging INR values gave better results than randomly selected sets, and including a normal plasma was not essential. The INR deviations of validation plasmas from certified values were reduced with sets of human, bovine/combined, and rabbit reagents. Deviations of more than 10% from certified INR values were significantly reduced (P &amp;lt; .001).</jats:p