Una Ørvim Sølvik

Human serum-induced expression of E-selectin on porcine aortic endothelial cells in vitro is totally complement mediated.

BACKGROUND Whereas complement is a key mediator of hyperacute xenograft rejection, its role in acute vascular rejection (AVR) is a matter of controversy. AVR is associated with de novo synthesis of endothelial cell-derived inflammatory mediators, including the leukocyte-recruiting adhesion molecule E-selectin. Here we investigate the role and mechanism of complement in human serum-induced porcine endothelial cell activation. METHODS An in vitro xenotransplantation method was designed using porcine aortic endothelial cells stimulated with human serum in microculture wells. E-selectin expression was measured by cell-enzyme immunoassay. Complement inhibitors acting at different levels in the cascade were investigated for their effect on E-selectin expression. RESULTS E-selectin was strongly induced by normal human serum but not by heat-inactivated serum. Compstatin, a synthetic C3 inhibitor, markedly reduced human serum-induced E-selectin expression. Purified C1-inhibitor suppressed E-selectin induction completely, indicating activation through the classical or lectin pathway. Furthermore, a monoclonal antibody (mAb) that inhibits cleavage of C5 or another mAb that blocks the function of C7, completely inhibited the expression of serum-induced E-selectin, consistent with the terminal C5b-9 complement complex being the mediator of the endothelial cell activation. Inhibition of the alternative pathway using a novel antifactor D mAb did not reduce E-selectin expression. CONCLUSION Human serum-induced expression of porcine E-selectin is totally complement dependent, induced by a C1-inhibitor regulated pathway and mediated through the terminal complement complex. The data may have implications for therapeutic strategies, particularly of C1-inhibitor and anti-C5 mAb, to protect against endothelial cell activation and subsequent AVR of porcine xenografts.

Diagnosing Diabetes Mellitus: Performance of Hemoglobin A1c Point-of-Care Instruments in General Practice Offices

BACKGROUND Hemoglobin A1c (Hb A1c) measurement by hospital laboratory instruments, but not by point-of-care (POC) instruments, has been recommended for use to diagnose diabetes mellitus. We evaluated results from 13 Hb A1c external quality assurance (EQA) surveys over a 6-year period in Norway, from both POC instruments used in general practice (GP) offices and instruments in hospital laboratories, against the analytical quality specifications recommended for use of Hb A1c to diagnose diabetes mellitus. METHODS All GP offices (n = 1288) and hospital laboratories (n = 52) measuring Hb A1c in Norway participated in the EQA survey. The percentage of participants that performed measurements within the quality specifications was calculated. Pooled within-laboratory CVs were estimated for the Afinion, DCA 2000, DCA 2000+, DCA Vantage(TM), and Nycocard Hb A1c Reader instruments and for hospital laboratory instruments. RESULTS Between 60% to 90% of Afinion and DCA users and hospital laboratories performed Hb A1c measurements within the quality specifications for both trueness (6.0%) and imprecision (CV ≤2.0%) at 2 levels in each EQA survey. The pooled within-laboratory CVs for the Afinion and DCA instruments and hospital laboratories were below the recommended limit of 2.0% for most of the surveys. CONCLUSIONS A large proportion of GP offices using Afinion and DCA POC instruments to measure Hb A1c fulfill the analytical quality specifications for diagnosing diabetes mellitus, and these instruments demonstrate analytical quality comparable to that of hospital laboratory instruments. When GP offices participate in a stringent quality assurance program and generate Hb A1c measurements that meet analytical quality specifications, these measurements can be recommended for use to diagnose diabetes mellitus.

Discrepancies in International Normalized Ratio Results between Instruments: A Model to Split the Variation into Subcomponents

BACKGROUND Observed differences between results obtained from comparison of instruments used to measure international normalized ratio (INR) have been higher than expected from the imprecision of the instruments. In this study the variation of these differences was divided into subcomponents, and each of the subcomponents was estimated. METHODS Blood samples were collected at 4 different patient visits from each of 36 outpatients who were receiving warfarin treatment and were included in the study. INR was determined on 1 laboratory instrument (STA Compact®) and 3 point-of-care instruments (Simple Simon®PT, CoaguChek®XS, and INRatio™). All 4 INR instruments were compared in pairs. Linear regression was used to correct for systematic deviations. The remaining variation of the differences was subdivided into between-subject, within-subject, and analytical variation in an ANOVA nested design. RESULTS The mean difference between instruments varied between 1.0% and 14.3%. Between-subject variation of the differences (expressed as CV) varied between 3.3% and 7.4%, whereas within-subject variation of the differences was approximately 5% for all 6 comparisons. The analytical imprecision of the differences varied between 3.8% and 8.6%. CONCLUSIONS The differences in INR between instruments were subdivided into calibration differences, between- and within-subject variation, and analytical imprecision. The magnitude of each subcomponent was estimated. Within results for individual patients the difference in INR between 2 instruments varied over time. The reasons for the between- and within-subject variations of the differences can probably be ascribed to different patient-specific effects in the patient plasma. To minimize this variation in a monitoring situation, each site and patient should use results from only 1 type of instrument.

Sample collections from healthy volunteers for biological variation estimates' update: a new project undertaken by the Working Group on Biological Variation established by the European Federation of Clinical Chemistry and Laboratory Medicine.

Abstract Background: Biological variation (BV) data have many fundamental applications in laboratory medicine. At the 1st Strategic Conference of the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) the reliability and limitations of current BV data were discussed. The EFLM Working Group on Biological Variation is working to increase the quality of BV data by developing a European project to establish a biobank of samples from healthy subjects to be used to produce high quality BV data. Methods: The project involved six European laboratories (Milan, Italy; Bergen, Norway; Madrid, Spain; Padua, Italy; Istanbul, Turkey; Assen, The Netherlands). Blood samples were collected from 97 volunteers (44 men, aged 20–60 years; 43 women, aged 20–50 years; 10 women, aged 55–69 years). Initial subject inclusion required that participants completed an enrolment questionnaire to verify their health status. The volunteers provided blood specimens once per week for 10 weeks. A short questionnaire was completed and some laboratory tests were performed at each sampling consisting of blood collected under controlled conditions to provide serum, K2EDTA-plasma and citrated-plasma samples. Results: Samples from six out of the 97 enroled subjects were discarded as a consequence of abnormal laboratory measurements. A biobank of 18,000 aliquots was established consisting of 120 aliquots of serum, 40 of EDTA-plasma, and 40 of citrated-plasma from each subject. The samples were stored at –80 °C. Conclusions: A biobank of well-characterised samples collected under controlled conditions has been established delivering a European resource to enable production of contemporary BV data.

External quality assessment of prothrombin time: The split‐sample model compared with external quality assessment with commercial control material

Objective. CoaguChek S is a point‐of‐care, whole‐blood, prothrombin time monitor. The purpose of this study was to compare two different methods for external quality assessments of CoaguChek S. Material and methods. In the traditional external quality assessment scheme, commercial control material was sent to office laboratories and the results were compared with a method‐specific target value. In the alternative external quality assessment (the split‐sample survey) patient samples were analyzed on CoaguChek S at office laboratories, and venous blood samples from the same patients were analyzed at a hospital laboratory using an assigned comparison method. To obtain comparable performance criteria for the two methods, the limits for “good”, “acceptable” and “poor” performance evaluation in the split‐sample survey had to be expanded because of uncertainties in preanalytical factors and the comparison method. Results. In the traditional external quality assessment the total imprecision (between‐office and within‐office) was 8.0 % at the low level (1.6 INR (International Normalized Ratio)) and 10.5 % at the therapeutic level (3.4 INR). In the split‐sample survey the total imprecision was 12.3 % at the low level (2.1 INR) and 10.7 % at the high level (3.0 INR). Seventy‐five percent of the participating office laboratories were characterized as “good” with the traditional external quality assessments, whereas the corresponding number was 73 % using the split‐sample model. Conclusions. Available commercial control material for CoaguChek S is different from patient samples. This study demonstrates that split‐sample survey is achievable, and is an acceptable alternative to traditional external quality assessment for point‐of‐care prothrombin time monitors where appropriate control material is difficult to obtain.

Inhalation of nitric oxide inhibits ADP-induced platelet aggregation and alpha-granule release

To gather further information about the effects on blood platelet activation of in vivo exposure to nitric oxide (NO), platelet reactivity was studied in blood from healthy, non-smoking male volunteers before and after 30 min inhalation of 40 ppm NO. Whole blood was stimulated in vitro with adenosine diphosphate or thrombin receptor activation peptide (TRAP-6). In an ex vivo perfusion model, non-anticoagulated blood was exposed to immobilised collagen at arterial blood flow conditions (2600 s(-1)). Blood samples from both the in vitro and ex vivo experiments were stained with fluorochrome-labelled Annexin-V and antibodies against CD42a, CD45, CD49b, CD61, CD62P and fibrinogen, and analysed with a three-colour flow cytometry technique. NO inhalation reduced the platelet activation response to adenosine diphosphate (ADP) stimulation by decreasing platelet-platelet aggregation, alpha-granule release and platelet-leukocyte conjugate formation. TRAP-stimulated platelet activation, collagen-induced platelet activation and thrombus growth was unaffected by NO inhalation. We therefore suggest an ADP receptor inhibitor mode of action of inhaled NO, selective on the newly suggested G protein- and phospholipase C-coupled P2Y1 receptor. Our results demonstrate that blood platelet activation in healthy subjects is modulated by inhalation of NO in therapeutically relevant doses, although the clinical impact of our findings remains unclear.

Biological Variation Estimates Obtained from 91 Healthy Study Participants for 9 Enzymes in Serum

BACKGROUND We sought to develop estimates of biological variation (BV) for 9 enzymes in blood serum as part of the European Biological Variation Study. METHODS Ninety-one healthy study participants (38 male and 53 female, 21-69 years old) were phlebotomized in each of 10 consecutive weeks at 6 European laboratories. The same preanalytical sample-handling protocol was followed at each center before transport to San Raffaele Hospital, Milan, Italy, for analysis. Sera were stored at -80 °C before analysis in duplicate within a single run on an ADVIA 2400 Clinical Chemistry System (Siemens Healthcare) following a protocol designed to minimize analytical imprecision. Assay traceability was established using frozen sera with target values assigned by reference methods. The results were subjected to outlier analysis before CV-ANOVA to deliver valid BV estimates. Results for 9 enzymes were subsequently partitioned for graphical display allowing visual assessment of the effects of country of origin, sex, and age on BV estimates. RESULTS We found no effect of country upon the observed variation, but overall sex-related differences were evident for alanine amino transferase (ALT), γ-glutamyl transferase (GGT), and creatine kinase (CK). The following estimates for within-subject BV (CVI) and between-subject BV (CVG), respectively, were obtained: ALT: 9.3%, 28.2%; aspartate aminotransferase: 9.5%, 20.3%; GGT: 8.9%, 41.7%; alkaline phosphatase : 5.3%, 24.9%; lactate dehydrogenase: 5.2%, 12.6%; CK: 14.5%, 31.5%; amylase: 6.8%, 30.4%; pancreatic α-amylase: 6.3%, 24.9%; and lipase (LIP): 7.7%, 23.8%. CONCLUSIONS All CVI and some CVG estimates were lower than those reported in the online BV 2014 updated database. Analytical performance specifications derived from BV can be applied internationally.

The EuBIVAS Project: Within–and Between-Subject Biological Variation Data for Serum Creatinine Using Enzymatic and Alkaline Picrate Methods and Implications for Monitoring

BACKGROUND The European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) European Biological Variation Study (EuBIVAS) has been established to deliver rigorously determined biological variation (BV) indices. EuBIVAS determined BV for serum creatinine using the enzymatic and alkaline picrate measurement methods. METHOD In total, 91 healthy individuals (38 males, 53 females; age range, 21-69 years) were bled for 10 consecutive weeks at 6 European laboratories. An equivalent protocol was followed at each center. Sera were stored at -80 °C before analysis. Analyses for each patient were performed in duplicate within a single run on an ADVIA 2400 system (San Raffaele Hospital, Milan). The data were subjected to outlier and homogeneity analysis before performing CV-ANOVA to determine BV and analytical variation (CVA) estimates with confidence intervals (CI). RESULTS The within-subject BV estimates [CVI (95% CI)] were similar for enzymatic [4.4% (4.2-4.7)] and alkaline picrate [4.7% (4.4-4.9)] methods and lower than the estimate presently available online (CVI = 5.9%). No significant male/female BV differences were found. Significant differences were observed in mean creatinine values between men and women and between Turkish individuals and those of other nationalities. Between-subject BV (CVG) estimates, stratified accordingly, produced CVG values similar to historical BV data. CVA was 1.1% for the enzymatic and 4.4% for alkaline picrate methods, indicating that alkaline picrate methods fail to fulfill analytical performance specifications for imprecision (CVAPS). CONCLUSIONS The serum creatinine CVI obtained by EuBIVAS specifies a more stringent CVAPS than previously identified. The alkaline picrate method failed to meet this CVAPS, raising questions regarding its future use.

Effect of coagulation factors on discrepancies in International Normalized Ratio results between instruments.

Abstract Background: The reasons for discrepancies between International Normalized Ratio (INR) results determined by point-of-care-instruments and laboratory measurements are not fully understood. In this study we investigated whether different levels of coagulation factors in the plasma of patients can explain some of the systematic and/or random parts of the difference in INR between the instruments. Methods: Blood samples were collected at four different patient visits from each of 34 outpatients on warfarin treatment. INR was determined on a laboratory instrument (STA Compact®) and on three point-of-care instruments (Simple Simon®PT, CoaguChek®XS and INRatio™). In addition, the level of fibrinogen, coagulation factors II, V, VII and X was determined. INR instruments were compared in pairs. Simple linear regressions as well as multiple linear regressions and nested ANOVA analyses were used to examine the data. Results: The coagulation factors, especially fibrinogen, factors II and VII, could explain between 16% and 45% of the total variance of the differences in INR between instruments dependent on instruments compared. After correction for factors no systematic difference was seen for four of the six comparisons and the between- and within-subject variation of the differences were reduced by up to 69% and 52%, respectively. Conclusions: By correcting for the appropriate coagulation factors, especially the systematic differences, but also the between- and within-subject variation of the differences between instruments, were reduced. This indicates that different levels of coagulation factors in the plasma of the patients play an important role in explaining discrepancies between INR instruments.

Human serum-induced porcine endothelial cell E-selectin expression is associated with IgG3 and IgM anti-Gal antibodies.

Naturally occurring anti‐Galα1‐3Gal (anti‐Gal) antibodies and complement induce hyperacute rejection (HAR) of porcine organs transplanted to primates. If the hyperacute reaction is prevented, an acute vascular rejection (AVR) occurs within hours to few days. Antibodies are important for the development of AVR, whereas the role of complement is still not clarified. AVR is characterized by protein synthesis‐dependent endothelial cell (EC) activation. In the present study we investigated the relation between EC activation as measured by E‐selectin expression, and the concentrations of anti‐Gal antibodies of IgM, IgG and IgG subclasses in sera from 80 healthy blood donors selected on the basis of sex and age. There was a significant correlation between E‐selectin expression and the concentration of IgG3 anti‐Gal (r=0.39; P=0.019), which was not seen for the other IgG subclasses or for total IgG anti‐Gal. A modest, but significant correlation was found between the concentration of IgM anti‐Gal and E‐selectin expression (r=0.38; P=0.040), but not between IgM and IgG3 anti‐Gal. There was a large interindividual variation in anti‐Gal antibodies, 50‐fold for IgM and 70‐fold for IgG. Females had significantly higher concentrations of IgM anti‐Gal than males (P=0.0006), which was explained by a substantial increase in IgM anti‐Gal concentration in younger women. The concentration of IgG anti‐Gal and the degree of E‐selectin expression did not differ between sex or age groups. In conclusion, the close correlation between anti‐Gal antibodies of the potent complement activating IgG3 subclass and porcine EC activation, may imply that these antibodies play a role in EC activation characteristic of AVR.