Point-of-care testing, near-patient testing and patient self-testing: warning points.

Point-of-care testing (POCT), near-patient testing (NPT) and patient self-tests (PST) are diagnostic examinations performed at the time and place of patient care. While POCT and NPT are performed and analyzed by medical professionals, PST are based on samples and parameters directly collected and analyzed by lay users. These tests are spreading both in high income countries and in low to middle income countries as they are expected to improve healthcare efficiency and equity, by saving resources, releasing pressure from hospitals and reducing logistical barriers. However, accurate multidisciplinary assessment is mandatory to ensure that what they promise is real. We reviewed some important ethical aspects, international standards and regulations. The current risks associated with alternative ways of testing are explained by the principles of respect for patient autonomy and non-maleficence. Further evidence from multidisciplinary assessment is needed to evaluate pros and cons in light of the principles of beneficence and justice. Although POCT or NPT need common regulation and accurate provider training to ensure safe and appropriate interpretation of results, PST needs even more attention as they are subject to direct patient use. Randomized controlled trails including patient education should be conducted in order to provide reliable evidence on clinical outcomes, patient acceptance and cost-effectiveness. Mandatory regulation is needed to avoid harm and EU regulation should help different countries maintain a safe use of devices in a global population of producers and users.

The rheumatoid arthritis synovial fluid citrullinome reveals novel citrullinated epitopes in apolipoprotein E, myeloid nuclear differentiation antigen, and ß-actin.

OBJECTIVE: To generate a catalog of citrullinated proteins that are present in the synovia of patients with rheumatoid arthritis (RA) and to elucidate their relevance for the anti-citrullinated protein antibody response in RA. METHODS: Polypeptides isolated from the synovial fluid of patients with RA were identified by mass spectrometry. Three proteins (apolipoprotein E [Apo E], myeloid nuclear differentiation antigen [MNDA], and ß-actin) were studied in more detail, using immunoprecipitation and Western blotting. The presence of autoantibodies to synthetic peptides derived from these proteins in sera from patients with RA, sera from patients with other diseases, and sera from healthy control subjects was studied by enzyme-linked immunosorbent assay (ELISA). RESULTS: RA synovial fluid samples displayed several distinct patterns of citrullinated proteins. Using mass spectrometry, (fragments of) 192 proteins were identified, including 53 citrullinated proteins, some of which contained multiple citrullinated residues. In addition to previously reported citrullinated proteins in RA synovia (e.g., vimentin and fibrinogen), a series of novel citrullinated proteins, including Apo E, MNDA, ß-actin, and cyclophilin A, was identified. Immunoprecipitation experiments confirmed the citrullination of Apo E and MNDA. ELISAs demonstrated the presence of autoreactive citrullinated epitopes in Apo E, MNDA, and ß-actin. CONCLUSION: Synovial fluid samples from the inflamed joints of patients with RA contain many citrullinated proteins. Citrullinated Apo E, MNDA, and ß-actin are novel antigens identified in RA synovial fluid, and only a limited number of their citrullinated epitopes are targeted by the immune system in RA.

Serum amyloid A activation of human coronary artery endothelial cells exhibits a neutrophil promoting molecular profile.

BACKGROUND: Serum amyloid A (SAA) has been shown to be an active participant in atherosclerosis and cardiovascular diseases. SAA-stimulated human coronary artery endothelial cells (HCAEC) were reported to release pro-inflammatory cytokines, chemokines and adhesion molecules; however it remains unclear which putative SAA receptors are present in these cells and how they act. We investigated the effects of inflammatory stimuli on the expression of SAA receptors, signaling pathways and molecular profiles in HCAEC. METHODOLOGY/PRINCIPLE FINDINGS: HCAEC were cultured in vitro and stimulated with SAA (1000nM) or IL-1ß (1000pg/ml). Expression of mRNA was determined by qPCR, and expression and quantification of proteins were assessed by dot array blots and ELISA, respectively. Protein phosphorylation was determined by dot blot arrays and Western blots. We report that all potential SAA receptors tested (FPR2/ALX, RAGE, TANIS, TLR2, TLR4 and CLA-1/hSR-B1) are expressed in HCAEC. Importantly, IL-1ß or SAA significantly increased solely the expression of the innate immune receptor TLR2. SAA upregulated the phosphorylation of ERK1/2, NF-κB (p65, p105) and JNK, as well as expression/release of IL-6, IL-8, G-CSF, GM-CSF, ICAM-1 and VCAM-1, all potent molecules involved in neutrophil-related activities. A TLR2-dependent positive feedback mechanism of SAA expression was found. CONCLUSION/SIGNIFICANCE: SAA stimulated responses in HCAEC target neutrophil rather than monocyte/macrophage activation.

Detection of antiphosphatidylserine/prothrombin antibodies and their potential diagnostic value.

Antiprothrombin antibodies, measured with phosphatidylserine/prothrombin complex (aPS/PT) ELISA, have been reported to be associated with antiphospholipid syndrome (APS). They are currently being evaluated as a potential classification criterion for this autoimmune disease, characterized by thromboses and obstetric complications. Given the present lack of clinically useful tests for the accurate diagnosis of APS, we aimed to evaluate in-house and commercial assays for determination of aPS/PT as a potential serological marker for APS. We screened 156 patients with systemic autoimmune diseases for antibodies against PS/PT, ß2-glycoprotein I, cardiolipin and for lupus anticoagulant activity. We demonstrated a high degree of concordance between the concentrations of aPS/PT measured with the in-house and commercial assays. Both assays performed comparably relating to the clinical manifestations of APS, such as arterial and venous thromboses and obstetric complications. IgG aPS/PT represented the strongest independent risk factor for the presence of obstetric complications, among all tested aPL. Both IgG and IgM aPS/PT were associated with venous thrombosis, but not with arterial thrombosis. Most importantly, the association between the presence of IgG/IgM aPS/PT and lupus anticoagulant activity was highly significant. Taken together, aPS/PT antibodies detected with the in-house or commercial ELISA represent a promising serological marker for APS and its subsets.

Immunochemical properties and pathological relevance of anti-ß2-glycoprotein I antibodies of different avidity.

Despite available treatment, there is still significant morbidity and mortality present among patients with the autoimmune thrombophilic condition termed 'antiphospholipid syndrome' (Espinosa, G. and Cervera, R. 2009. Morbidity and mortality in the antiphospholipid syndrome. Curr. Opin. Pulm. Med. 15:413.). High-avidity (HAv) anti-ß(2)-glycoprotein I (anti-ß(2)GPI) antibodies, shown to correlate with thrombotic events in patients, could represent the much needed improved prognostic marker. By studying their effect on crystalline annexin A5 shield on phospholipid surfaces (one of proposed pathogenic mechanisms), with the use of atomic force microscopy, the pathogenic potential of HAv anti-ß(2)GPI antibodies was confirmed. Furthermore, by using surface plasmon resonance and enzyme-linked immunosorbent assays, unique binding characteristics of HAv antibodies in comparison with low avidity antibodies were established. HAv anti-ß(2)GPI were confirmed to (i) recognize ß(2)-glycoprotein I in a solution, (ii) interact predominantly monovalently (much lower dependency on the antigen density) and (iii) form more stable complexes with the antigen. Since enzyme-linked immunosorbent assays currently used in routine diagnostics detect anti-ß(2)GPI antibodies of unknown avidity, our observations are potentially useful for the development of improved diagnostic tests capable of detecting clinically relevant antibodies.

The use of atomic force microscopy to study the pathologic effects of anti-annexin autoantibodies.

Patients with recurrent pregnancy loss and a history of thrombotic events have often been noted to have autoantibodies directed at annexin A5. However, the relationship of these autoantibodies to immunopathology is still unknown, although it has been proposed that they have a direct effect on the function of annexin A5. Annexin A5 may be a significant immunological target with pathologic implications. Essentially, annexin A5 is an anticoagulant protein that crystallizes over negatively charged phospholipid surfaces and thereby blocks them from availability for coagulation reactions. To address this issue, we have taken advantage of our expertise with atomic force microscopy and studied anti-annexin A5 autoantibodies isolated from patients and focused on the ability of these antibodies to influence annexin A5 crystallization on planar mica-supported phospholipid bilayers. We report herein that such antibodies from patients, but not controls, produced a significant disruption of incomplete annexin A5 crystalline shield on phospholipid bilayer. In addition, the IgG fraction isolated from such patients significantly decreased the velocity of annexin A5 crystallization. Atomic force microscopy is a powerful tool to study the pathologic mechanisms of autoantibodies and the data herein reflect the potential of anti-annexin A5 antibodies that produce pathology in a number of varied but overlapping clinical conditions, including autoimmune thrombosis and antiphospholipid syndrome.

Modified phosphatidylserine-dependent antiprothrombin [corrected] ELISA enables identification of patients negative for other antiphospholipid antibodies and also detects low avidity antibodies.

BACKGROUND: Two approaches for detecting anti-prothrombin antibodies have been described. The first detects antibodies against prothrombin alone and the second, phos-phatidylserine-dependent antiprothrombin antibodies. The latter more often correlate with clinical manifestations of antiphospholipid syndrome and with lupus anticoagulant activity. METHODS: In order to increase the capacity of antibody binding, we modified the previously described phosphatidylser-ine-dependent antiprothrombin ELISA and determined their avidity. We examined 203 patients with systemic autoimmune diseases and 222 blood donors. RESULTS: Our modification resulted in a greater intensity of antibody binding to prothrombin on phosphatidylserine-coated plate surfaces compared to the previously described method. By changing ELISA conditions, we were able to detect with one assay the two, presumably different, populations of antiprothrombin antibodies. Diagnostic specificities of both ELISAs for antiphospholipid syndrome were similar (92.5% vs. 93.1%), while the sensitivity of the modified phosphatidylserine-dependent antiprothrombin ELISA was significantly higher than the anti-prothrombin alone ELISA (59% vs. 25%). Low avidity antiprothrombin antibodies were only detected in the modified phosphatidylserine-dependent antiprothrombin ELISA. Four percent of patients with positive phosphatidylserine-dependent antiprothrombin antibodies, showing clinical manifestations of antiphospholipid syndrome, were negative for all other antiphospholipid antibodies. The risk for antiphospholipid syndrome increased with the number of antiphospholipid antibody positivity. CONCLUSIONS: We conclude that antibodies detected with a modified phosphatidylserine-dependent antiprothrombin ELISA could improve the diagnosis of antiphospholipid syndrome by offering additional information on the risk for thrombosis, especially in patients negative for other antiphospholipid antibodies.

Using phage display in autoimmunity research.

Autoimmune diseases affect approximately 3% of the population and are a significant cause of morbidity and mortality. Therefore, they are the focus of vivid research aimed at the delineation of pathology, more efficient diagnosis and therapy. Phage display is a simple methodology markedly useful in studying protein-protein interactions which are the driving forces of (patho)physiological processes, including autoimmunity. The commercially available phage display peptide libraries allow the characterization and identification of antibody-antigen and receptor-ligand binding sites, thus providing basic insight into the pathological network. Through the phage display of antibody's, receptor's or ligand's protein domains it is possible to produce various self-specificities that have already been proven useful in the therapy of autoimmune diseases. Phage antibody display libraries are able to reprint the in vivo autoimmune response which significantly facilitates structural and functional analyses of auto-reactive antibodies and assessing their role in disease pathology. The aim of this review was an overview of the versatile applications of phage display technology to determine their potential advantages in studying autoimmune diseases and to critically categorize and analyze the usefulness of phage displays towards understanding pathology and towards development of improved diagnostic tools. Above all its focus was on targeted therapy.

Alteration of antibody specificity during isolation and storage.

Isolation buffer characteristics and storage conditions could partially transform natural antibodies. 50 IgG factions were isolated from seven healthy donors' sera using various protein G columns and buffers. PAGE revealed no major antibody cleavages; purity of IgGs eluted at pH 2.4-3.5 was close to homogeneity, independent of buffer composition. Although eluting at pH 2.4 resulted in 3.5- or 17-fold higher antibody yield compared to pH 3.0 or 3.5, respectively, it distorted the antibody molecule. IgGs eluted at pH 2.4 acquired reactivity against diagnostically important autoantigens, confirmed by standardized ELISAs. Preserved antibodies' natural activity is important in further experiments with oxidatively-induced autoantibodies.

Pharmacy Practice and Education in Slovenia.

The PHARMINE ("Pharmacy Education in Europe") project studied pharmacy practice and education in the European Union (EU) member states. The work was carried out using an electronic survey sent to chosen pharmacy representatives. The surveys of the individual member states are now being published as reference documents. This paper presents the results of the PHARMINE survey on pharmacy practice and education in Slovenia. In the light of this, we examine the harmonisation of practice and education in Slovenia with EU norms.

Autoimmune reactions after electro-oxidation of IgG from healthy persons: relevance of electric current and antioxidants.

Proteins, including immunoglobulins, can be modified by oxidation. Extensive oxidation of immunoglobulins leads to denaturation and loss of biological activity, while initial steps of oxidation may change their specificity due to chemical alteration of the paratope. Electro-oxidation of the IgG fraction from healthy persons progress to auto-immunoreactivity, as shown for several autoantibodies including anti-beta2-glycoprotein I. Changes in immunoreactivity of IgG due to oxidative reactions highly depend on electric current and levels of serum antioxidants. Autoimmune reactions, leading to certain autoimmune diseases, may be partially a consequence of unbalanced antioxidative status of an individual.

Diagnostic and Research Aspects of Small Intestinal Disaccharidases in Coeliac Disease.

Disaccharidases (DS) are brush border enzymes embedded in the microvillous membrane of small intestinal enterocytes. In untreated coeliac disease (CD), a general decrease of DS activities is seen. This manuscript reviews different aspects of DS activities in CD: their utility in the diagnosis and their application to in vitro toxicity testing. The latter has never been established in CD research. However, with the recent advances in small intestinal organoid techniques, DS might be employed as a biomarker for in vitro studies. This includes establishment of self-renewing epithelial cells raised from tissue, which express differentiation markers, including the brush border enzymes. Determining duodenal DS activities may provide additional information during the diagnostic workup of CD: (i) quantify the severity of the observed histological lesions, (ii) provide predictive values for the grade of mucosal villous atrophy, and (iii) aid diagnosing CD where minor histological changes are seen. DS can also provide additional information to assess the response to a gluten-free diet as marked increase of their activities occurs four weeks after commencing it. Various endogenous and exogenous factors affecting DS might also be relevant when considering investigating the role of DS in other conditions including noncoeliac gluten sensitivity and DS deficiencies.

Curriculum Mapping of the Master's Program in Pharmacy in Slovenia with the PHAR-QA Competency Framework.

This article presents the results of mapping the Slovenian pharmacy curriculum to evaluate the adequacy of the recently developed and validated European Pharmacy Competences Framework (EPCF). The mapping was carried out and evaluated progressively by seven members of the teaching staff at the University of Ljubljana's Faculty of Pharmacy. Consensus was achieved by using a two-round modified Delphi technique to evaluate the coverage of competences in the current curriculum. The preliminary results of the curriculum mapping showed that all of the competences as defined by the EPCF are covered in Ljubljana's academic program. However, because most EPCF competences cover healthcare-oriented pharmacy practice, a lack of competences was observed for the drug development and production perspectives. Both of these perspectives are important because a pharmacist is (or should be) responsible for the entire process, from the development and production of medicines to pharmaceutical care in contact with patients. Nevertheless, Ljubljana's graduates are employed in both of these pharmaceutical professions in comparable proportions. The Delphi study revealed that the majority of differences in scoring arise from different perspectives on the pharmacy profession (e.g., community, hospital, industrial, etc.). Nevertheless, it can be concluded that curriculum mapping using the EPCF is very useful for evaluating and recognizing weak and strong points of the curriculum. However, the competences of the framework should address various fields of the pharmacist's profession in a more balanced way.

Detection of adalimumab and anti-adalimumab antibodies in patients with rheumatoid arthritis: a comprehensive overview of methodology pitfalls and benefits.

About a third of patients with rheumatoid arthritis treated with adalimumab may develop anti-adalimumab antibodies. Anti-adalimumab antibodies are associated with reduced drug levels, loss of drug efficacy, clinical non-response and an increased risk of adverse effects. In case of suspected drug failure and in order to better define clinical efficacy, adalimumab as well as anti-adalimumab antibodies levels should be monitored. Sandwich or indirect enzyme-linked immunoassay is most commonly used for determining adalimumab, while bridging ELISA and antigen-binding test are most useful for determining anti-adalimumab antibodies. Most current assays cannot detect antibodies complexed with the adalimumab; however, methods for dissociation of the complexes using acid/temperature have been developed. The aim of this review is to report on the latest methodology for detecting adalimumab and anti-ADL antibodies, benefits of their detections in clinical practice, as well as expose problematic issues, such as different analytical sensitivity and specificity, standardization and validation. The main problem in measuring adalimumab or anti-ADL antibodies is high drug sensitivity, which can result in false-negative anti-ADL antibodies. Therefore, drug-tolerant assays have been developed. Cell-based assays, such as the reporter gene assay, are recommended for detection of functionally active adalimumab and their neutralizing anti-ADL antibodies.

Budding, vesiculation and permeabilization of phospholipid membranes-evidence for a feasible physiologic role of beta2-glycoprotein I and pathogenic actions of anti-beta2-glycoprotein I antibodies.

The in vivo physiologic role of beta2-glycoprotein I (beta2GPI) is presumed to be related to its interactions with negatively charged phospholipid membranes. Increased quantities of procoagulant microparticles derived by the vesiculation of blood cells have been detected in patients with antiphospholipid syndrome (APS) frequently associated with antibodies against beta2GPI (anti-beta2GPI). We investigated the influence of beta2GPI and anti-beta2GPI on giant phospholipid vesicles (GPVs). GPVs composed of phosphatidylserine and phosphatidylcholine were formed in an aqueous medium and individually transferred to a compartment containing either beta2GPI, anti-beta2GPI, or beta2GPI along with anti-beta2GPI. Shape changes of a single GPV were observed by a phase contrast microscope. Most GPVs transferred to the solution containing only beta2GPI budded moderately. Upon the transfer of GPVs to the solution containing beta2GPI and anti-beta2GPI either from patient with APS or mouse monoclonal anti-beta2GPI Cof-22, the budding was much more pronounced, generating also daughter vesicles. No such effects were seen when GPV was transferred to the solution containing anti-beta2GPI without beta2GPI. Our results suggest a significant physiologic role of beta2GPI in the budding of phospholipid membranes, which may be explained by the insertion of the C-terminal loop of beta2GPI into membranes, thus increasing the surface of the outer layer of a phospholipid bilayer. Anti-beta2GPI, recognizing domains I to IV of beta2GPI, enhanced the budding and vesiculation of GPVs in the presence of beta2GPI. This might be a novel pathogenic mechanism of anti-beta2GPI, promoting in vivo the expression of proadhesive and procoagulant phospholipid surfaces in APS.

How Do European Pharmacy Students Rank Competences for Practice?

European students (n = 370), academics (n = 241) and community pharmacists (n = 258) ranked 13 clusters of 68 personal and patient care competences for pharmacy practice. The results show that ranking profiles for all three groups as a rule were similar. This was especially true of the comparison between students and community pharmacists concerning patient care competences suggesting that students have a good idea of their future profession. A comparison of first and fifth (final) year students shows more awareness of patient care competences in the final year students. Differences do exist, however, between students and community pharmacists. Students-like academics-ranked competences concerned with industrial pharmacy and the quality aspects of preparing drugs, as well as scientific fundamentals of pharmacy practice, well above the rankings of community pharmacists. There were no substantial differences amongst rankings of students from different countries although some countries have more "medicinal" courses than others. This is to our knowledge the first paper to look at how, within a healthcare sectoral profession such as pharmacy, the views on the relative importance of different competences for practice of those educating the future professionals and their students, are compared to the views of working professionals.

What is a Pharmacist: Opinions of Pharmacy Department Academics and Community Pharmacists on Competences Required for Pharmacy Practice.

This paper looks at the opinions of 241 European academics (who provide pharmacy education), and of 258 European community pharmacists (who apply it), on competences for pharmacy practice. A proposal for competences was generated by a panel of experts using Delphi methodology. Once finalized, the proposal was then submitted to a large, European-wide community of academics and practicing pharmacists in an additional Delphi round. Academics and community pharmacy practitioners recognized the importance of the notion of patient care competences, underlining the nature of the pharmacist as a specialist of medicines. The survey revealed certain discrepancies. Academics placed substantial emphasis on research, pharmaceutical technology, regulatory aspects of quality, etc., but these were ranked much lower by community pharmacists who concentrated more on patient care competences. In a sub-analysis of the data, we evaluated how perceptions may have changed since the 1980s and the introduction of the notions of competence and pharmaceutical care. This was done by splitting both groups into respondents < 40 and > 40 years old. Results for the subgroups were essentially statistically the same but with some different qualitative tendencies. The results are discussed in the light of the different conceptions of the professional identity of the pharmacist.

A Study on How Industrial Pharmacists Rank Competences for Pharmacy Practice: A Case for Industrial Pharmacy Specialization.

This paper looks at the way in which industrial pharmacists rank the fundamental competences for pharmacy practice. European industrial pharmacists (n = 135) ranked 68 competences for practice, arranged into 13 clusters of two types (personal and patient care). Results show that, compared to community pharmacists (n = 258), industrial pharmacists rank competences centering on research, development and production of drugs higher, and those centering on patient care lower. Competences centering on values, communication skills, etc. were ranked similarly by the two groups of pharmacists. These results are discussed in the light of the existence or not of an "industrial pharmacy" specialization.

The Second Round of the PHAR-QA Survey of Competences for Pharmacy Practice.

This paper presents the results of the second European Delphi round on the ranking of competences for pharmacy practice and compares these data to those of the first round already published. A comparison of the numbers of respondents, distribution by age group, country of residence, etc., shows that whilst the student population of respondents changed from Round 1 to 2, the populations of the professional groups (community, hospital and industrial pharmacists, pharmacists in other occupations and academics) were more stable. Results are given for the consensus of ranking and the scores of ranking of 50 competences for pharmacy practice. This two-stage, large-scale Delphi process harmonized and validated the Quality Assurance in European Pharmacy Education and Training (PHAR-QA) framework and ensured the adoption by the pharmacy profession of a framework proposed by the academic pharmacy community. The process of evaluation and validation of ranking of competences by the pharmacy profession is now complete, and the PHAR-QA consortium will now put forward a definitive PHAR-QA framework of competences for pharmacy practice.

Hospital and Community Pharmacists' Perceptions of Which Competences Are Important for Their Practice.

The objective of the PHAR-QA (Quality assurance in European pharmacy education and training) project was to investigate how competence-based learning could be applied to a healthcare, sectoral profession such as pharmacy. This is the first study on evaluation of competences from the pharmacists' perspective using an improved Delphi method with a large number of respondents from all over Europe. This paper looks at the way in which hospital pharmacists rank the fundamental competences for pharmacy practice. European hospital pharmacists (n = 152) ranked 68 competences for pharmacy practice of two types (personal and patient care), arranged into 13 clusters. Results were compared to those obtained from community pharmacists (n = 258). Generally, hospital and community pharmacists rank competences in a similar way. Nevertheless, differences can be detected. The higher focus of hospital pharmacists on knowledge of the different areas of science as well as on laboratory tests reflects the idea of a hospital pharmacy specialisation. The difference is also visible in the field of drug production. This is a necessary competence in hospitals with requests for drugs for rare diseases, as well as paediatric and oncologic drugs. Hospital pharmacists give entrepreneurship a lower score, but cost-effectiveness a higher one than community pharmacists. This reflects the reality of pharmacy practice where community pharmacists have to act as entrepreneurs, and hospital pharmacists are managers staying within drug budgets. The results are discussed in the light of a "hospital pharmacy" specialisation.