The Development of a Direct Homologous Radioimmunoassay for Serum Cortisol

Peer Reviewe

Certification of C-reactive Protein in Reference Material ERM-DA472/IFCC

The production and certification of ERM-DA472/IFCC, a new reference material certified for C-reactive protein (CRP), is described. ERM-DA472/IFCC was characterised using the reference material ERM-DA470 as calibrant. This achieved using a value transfer protocol that can be considered as a reference procedure. the principles used to measure the CRP concentration were immunonephelometry and immunoturbidimetry. The measurements were performed with different platform/reagent combinations (Abbott, Beckmann Immage, BN II, different Hitachi instruments, and Olympus AU640). In total 8 laboratories participated in the value assignment. The certified CRP mass concentration is 41.8 mg/L, the expanded uncertainty (k = 2) 2.5 mg/L.JRC.D.2-Reference material

Quantification and Clinical Relevance of Cystatin C

An aging population and increasing rates of diabetes mellitus contribute to a high prevalence of kidney dysfunction – approximately 10 percent of adults in developed countries have chronic kidney disease (CKD). CKD is a progressive loss of kidney function and this remains permanent. Early recognition of this condition is important for prevention or impeding severe adverse cardiac and renal outcomes. Cystatin C is a low molecular weight cysteine protease inhibitor that has emerged as a biomarker of kidney function. The special potential of plasma cystatin C in this setting is related to its independency of muscle mass, which is a remarkable limitation of the traditional marker creatinine. Cystatin C is a sensitive marker in diagnosing mild and moderate CKD, especially in small children, in the elderly and in conditions where muscle mass is affected. Cystatin C is quantified with immunoassays, mainly based on particle-enhanced nephelometry (PENIA) or turbidimetry (PETIA). The aim of this study was to develop a rapid and reliable assay for quantification of human cystatin C in plasma or serum by utilizing time-resolved fluorescence-based immunoassay methods. This was accomplished by utilizing different antibodies, including polyclonal and 7 monoclonal antibodies against cystatin C. Different assay designs were tested and the best assay was further modified to a dry-reagent double monoclonal assay run on an automated immunonalyzer. This assay was evaluated for clinical performance in estimating reduced kidney function and in predicting risk of adverse outcomes in patients with non-ST elevation acute coronary syndrome. Of the tested assay designs, heterogeneous non-competitive assay had the best performace and was chosen to be developed further. As an automated double monoclonal assay, this assay enabled a reliable measurement of clinically relevant cystatin C concentrations. It also showed a stronger concordance with the reference clearance method than the conventional PETIA method in patients with reduced kidney function. Risk of all-cause mortality and combined events, defined by death and myocardial infarction, increased with higher cystatin C and cystatin C remained an independent predictor of death and combined events after adjustment to nonbiochemical baseline factors. In conclusion, the developed dry-reagent double monoclonal assay allows rapid and reliable quantitative measurement of cystatin C. As measured with the developed assay, cystatin C is a potential predictor of adverse outcomes in cardiac patients.Väestön ikääntyminen ja diabeteksen yleistyminen ovat johtaneet munuaisten vajaatoiminnan esiintyvyyden kasvuun. Nykyään arviolta kymmenyksellä kehittyneiden maiden aikuisväestöstä on krooninen munuaisten vajaatoiminta, joka tarkoittaa etenevää ja pysyvää munuaistoiminnan menetystä. Jos munuaisten vajaatoiminta havaitaan ajoissa, vakavia munuaisiin ja sydämeen liittyviä seurauksia on mahdollista estää tai hidastaa. Kystatiini C on pienimolekyylinen kysteiiniproteaasien inhibiittori, joka on lupaava uusi munuaistoiminnan merkkiaine. Kystatiini C:n suurin tunnettu etu perinteiseen munuaismerkkiaineeseen, kreatiniiniin, nähden on, ettei lihasmassan määrä vaikuta sen pitoisuuteen veressä. Kystatiini C:n on osoitettu olevan herkkä merkkiaine lievän ja kohtalaisen munuaisten vajaatoiminnan diagnosoinnissa, erityisesti pikkulapsilla, ikääntyneillä sekä erilaisissa tiloissa, joissa lihasmassan määrä on normaalista poikkeava. Kystatiini C -pitoisuutta mitataan immunomäärityksillä, pääasiallisesti turbidimetrisilla tai nefelometrisilla menetelmillä. Tutkimuksen tavoitteena oli kehittää nopea ja luotettava aikaerotteiseen fluoresenssiin perustuva immunomääritys ihmisen kystatiini C -pitoisuuden mittaamiseen veriplasmasta tai -seerumista. Määrityksen kehittämisessä käytettiin polyklonaalista ja seitsemää eri monoklonaalista vasta-ainetta. Erilaisten määritystyyppien, kuten kilpailevan ja ei-kilpailevan määrityksen, soveltuvuus kystatiini C:n mittaamiseen selvitettiin. Parhainta määritystä kehitettiin edelleen ja se muokattiin kahta monoklonaalista vasta-ainetta käyttäväksi kuivakemiaan perustuvaksi automatisoiduksi määritykseksi. Lopuksi selvitettiin kehitetyn määrityksen kliininen suorituskyky munuaisten vajaatoiminnan arvioinnissa sekä sepelvaltimotautipotilaiden kuoleman ja sydänkohtauksen riskin ennustajana. Testatuista määritystyypeistä ei-kilpaileva määritys toimi parhaiten ja valittiin jatkokehitykseen. Kehitetty automatisoitu kahta monoklonaalista vasta-ainetta käyttävä määritys soveltui kliinisesti merkittävien kystatiini C -pitoisuuksien luotettavaan mittaukseen. Sillä mitatut pitoisuudet vastasivat paremmin munuaistoiminnan tarkimpana pidetyn mittarin eli puhdistumamittauksen tuloksia kuin turbidimetrisella menetelmällä mitatut kystatiini C -pitoisuudet, erityisesti potilailla, joiden munuaistoiminta oli heikentynyt. Kuoleman ja sydänkohtauksen riski oli suurempi korkeammilla kystatiini C -pitoisuuksilla ja kystatiini C oli näiden tapahtumien itsenäinen ennustaja myös sen jälkeen kun ei-biokemiallisten taustatekijöiden vaikutus oli huomioitu riskianalyysissä.Siirretty Doriast

A Facile Graphene Conductive Polymer Paper Based Biosensor for Dopamine, TNF-α, and IL-6 Detection

Paper-based biosensors are a potential paradigm of sensitivity achieved via microporous spreading/microfluidics, simplicity, and affordability. In this paper, we develop decorated paper with graphene and conductive polymer (herein referred to as graphene conductive polymer paper-based sensor or GCPPS) for sensitive detection of biomolecules. Planetary mixing resulted in uniformly dispersed graphene and conductive polymer ink, which was applied to laser-cut Whatman filter paper substrates. Scanning electron microscopy and Raman spectroscopy showed strong attachment of conductive polymer-functionalized graphene to cellulose fibers. The GCPPS detected dopamine and cytokines, such as tumor necrosis factor-alpha (TNF-α), and interleukin 6 (IL-6) in the ranges of 12.5–400 µM, 0.005–50 ng/mL, and 2 pg/mL–2 µg/mL, respectively, using a minute sample volume of 2 µL. The electrodes showed lower detection limits (LODs) of 3.4 µM, 5.97 pg/mL, and 9.55 pg/mL for dopamine, TNF-α, and IL-6 respectively, which are promising for rapid and easy analysis for biomarkers detection. Additionally, these paper-based biosensors were highly selective (no serpin A1 detection with IL-6 antibody) and were able to detect IL-6 antigen in human serum with high sensitivity and hence, the portable, adaptable, point-of-care, quick, minute sample requirement offered by our fabricated biosensor is advantageous to healthcare applications

Measurement of calprotectin (S100A8/S100A9) and S100A12 in serum: method development, analytical validation, and clinical application

A thesis submitted in partial fulfilment of the requirements of the University of Wolverhampton for the degree of Doctor of Biomedical Science.Background Faecal biomarkers of intestinal inflammation, in particular faecal calprotectin and to a lesser extent faecal S100A12, are used to discriminate between inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) and categorise active from inactive disease in established IBD. Faecal biomarkers have limitations including intra–individual and inter–individual variability, spot variability in the same sample and reluctance of some patients to provide stool samples. These issues may be overcome by using serum samples for the measurement of calprotectin and S100A12. This offers the prospect that serum calprotectin and serum S100A12 could replace or supplement faecal calprotectin and faecal S100A12 in the identification and assessment of IBD. The measurement in serum calprotectin and serum S100A12, however, requires method development, validation of assays for serum and evaluation of the validated assays for their diagnostic and prognostic utility in IBD. Method development switches between two processes. It may necessitate adapting an existing method to ensure its suitability for application in a new assay or devising a suitable method by integrating the expertise and experience of the personnel undertaking the task of method development. Assay validation process for commercially available serum immunoassay kits is necessary to underpin assay measurement in order to confirm accuracy of test results, cut costs of undertaking unnecessary and repeat testing procedures, reinforce analytical claim that assay measurement is devoid of uncertainty to justify 'fit for purpose' and confer additional benefit of good reputation to a clinical laboratory. Validation of an assay in serum confirms or disproves kit manufacturer’s analytical claim to robust assay performance characteristics that include accuracy, precision, dilution linearity/parallelism, recovery, sensitivity, interference and stability. Aim/Objectives This project was designed to (1) Develop and analytically validate a faecal S100A12 assay (ImmunodiagnostikTM AG, Stubenwald–Allee 8a, D–64625 Bensheim, Germany) for measurement of S100A12 in serum. Analytically validate serum calprotectin assays provided by Bühlmann (serum BMN®-Cp; Bϋhlmann Laboratories AG, Baselstrasse 55, CH – 4124 Schönenbuch, Switzerland) and ImmunodiagnostikTM (serum IDK®- Cp; ImmunodiagnostikTM AG, Stubenwald–Allee 8a, D–64625 Bensheim, Germany). (2) Assess whether serum BMN®-Cp, serum IDK®-Cp and serum S100A12 could replace or supplement faecal calprotectin and faecal S100A12 in excluding IBD in patients presenting with chronic diarrhoea. (3) Evaluate the utility of serum BMN®-Cp and serum IDK®-Cp in discriminating between active and inactive IBD. (4) Study the effect of the acute phase response (APR) on serum calprotectin determined with two different immunoassays kits (i.e., serum BMN®-Cp and serum IDK®-Cp), and to assess and compare the diagnostic performance of the two assays in APR. Methods (1) ELISA assays for faecal S100A12 were developed and optimised for measurement of S100A12 in serum. The serum BMN®-Cp, serum IDK®-Cp and serum IDK®-A12 were validated by determining analytical sensitivity, functional sensitivity, dilution linearity/parallelism, recovery, precision, and interference. (2) The diagnostic performances of the serum BMN®-Cp, serum IDK®-Cp and serum IDK®-A12 assays were compared against faecal calprotectin, as the diagnostic ‘gold standard’, in 40 patients with IBD and 5 control patients. (3) Serum BMN®-Cp and serum IDK®-Cp and other conventional inflammatory blood biomarkers (serum CRP and platelets) were compared to faecal calprotectin in discriminating between active and inactive disease in a cohort of 175 patients with IBD. (4) The effect of APR, as determined by serum CRP, and serum calprotectin was assessed by measuring serum BMN®-Cp and serum IDK®-Cp before and after elective knee or hip surgery in 30 patients. Results Analytical validation Analytical validation of the assays showed a dynamic working range in serum of 10 to 25000 ng/mL, good precision (%CV for intra– and inter–assay variability for the kits were < 10% respectively, for each assay) and good reproducibility. There was no interference from bilirubin, haemoglobin, or lipid in the assays. There was no significant carryover or cross–reactivity across the assays. Assay kits were stable over 12 months. Analytical sensitivity ranged from 0.673 to 577 ng/mL for limit of the blank (LoB), and 1.119 to 597 ng/mL for lower limit of detection (LLoD). Functional sensitivity or limit of quantitation (LoQ) ranged from 522 to 3615 ng/mL. Measured to Expected ratios for dilution linearity/parallelism and recovery for the kits ranged from 98.4% to 103.7%, and from 82.1% to 126.5% respectively. Method comparison showed 19% positive proportional bias of the BMN®-Cp assay compared to the IDK®-Cp assay. Serum BMN®-Cp, serum IDK®-Cp and serum S100A12 in identifying IBD Using faecal calprotectin as the ‘gold standard’ for identifying IBD, the AUC from ROC curves for serum IDK®-Cp (AUC = 0.793) was greater than that for serum BMN®-Cp (AUC = 0.771) and these were greater than that for serum S100A12 (AUC = 0.700). Faecal calprotectin correlated best with serum IDK®-Cp (r = 0.69), then serum BMN®-Cp (r = 0.66) and least with serum S100A12 (r = 0.44). Serum BMN®-Cp and serum IDK®-Cp in discriminating between active and inactive IBD. The cohort of 175 patients with IBD consisted of 101 (57.7%) patients with Crohns disease (CD), 71 (40.6%) with ulcerative colitis (UC) and 3 (1.7%) inflammatory bowel disease unclassified (IBDU). The clinical classification of disease activity was largely based on faecal calprotectin which indicated that the disease was quiescent in 99 (56.6%) patients, active in 73 (41.7%) patients and in 3 (1.7%) patients were IBDU. Faecal calprotectin was, therefore, higher (p < 0.0001) in active CD than in quiescent CD, and similarly higher (p < 0.0001) in active UC compared to quiescent UC. Serum BMN®-Cp and serum IDK®-Cp in 175 IBD patients were highly correlated (r = 0.97). Serum BMN®-Cp and serum IDK®-Cp were higher (p 0.1) in active and quiescent UC. Serum CRP was higher (p = 0.0095) in active CD compared to quiescent CD but similar (p = 0.0638) in active and quiescent UC. Platelets were similar (p = 0.0579) in active and quiescent CD and similar (p = 0.8055) in active and quiescent UC. Serum BMN®-Cp and serum IDK®-Cp concentrations were higher (p 0.05) in active UC and quiescent UC involving the rectum, distal colon and pancolon. Based on ROC curve analysis, the performance of serum CRP (AUC = 0.699) was marginally superior to that of serum BMN®-Cp (AUC = 0.662) and serum IDK®-Cp (AUC = 0.656), and these were superior to platelets (AUC = 0.547) in all patients with IBD. In patients with CD, none of the blood biomarkers performed well; serum CRP (AUC = 0.585), serum BMN®-Cp (AUC = 0.585), serum IDK®-Cp (AUC = 0.556) and platelets (AUC = 0.609). In patients with UC, the performance of serum CRP (AUC = 0.752) was superior to that of serum BMN®-Cp (AUC = 0.670) and serum IDK®-Cp (AUC = 0.660), and these were superior to platelets (AUC = 0.487). The effect of an APR on serum BMN®-Cp and serum IDK®-Cp Following elective knee and hip surgery in 30 patients, serum CRP, serum BMN®-Cp, serum IDK®-Cp and blood neutrophils increased (p 3900 ng/mL and >3000 ng/mL respectively. Serum BMN®-Cp and serum IDK®-Cp, however, are unlikely to replace faecal calprotectin but may have a role supplementing faecal calprotectin in the identification of IBD. An elevated serum calprotectin in patients with chronic diarrhoea would be an indication for endoscopy since it has a low false positive rate, but a normal serum calprotectin does not exclude IBD. In the cohort of 175 patients with IBD, serum BMN®-Cp and serum IDK®-Cp were significantly associated with disease activity in patients with CD irrespective of site of disease. There was, however, no significant association between serum BMN®-Cp and serum IDK®-Cp and disease activity in patients with UC. ROC curves analyses indicated that serum CRP performed better than serum BMN®-Cp and serum IDK®-Cp in discriminating between active and inactive disease in patients with CD and UC. In this patient cohort, serum calprotectin offers no advantages over serum CRP in discriminating between active and inactive IBD, particularly since serum CRP is easily available and less expensive. Serum calprotectin is a positive acute phase protein, and both the serum BMN®-Cp and serum IDK®-Cp assays perform equally well during an APR elicited by orthopaedic surgery. The increase in serum calprotectin elicited by trauma and previously reported increase in sepsis indicates that serum calprotectin is a non–specific biomarker of inflammation. At two days following an inflammatory insult, serum CRP may be a better discriminatory biomarker of the APR than serum calprotectin based on a much greater incremental response

Methods for the Measurement of Vitamin D Metabolites and Studies on Their Relationships in Health and Disease

The prevalence of vitamin D deficiency in the general population has become a major public health problem. Vitamin D deficiency might have significant consequences not only to bone health but possible to other autoimmune, infectious, cancer and cardiovascular diseases. The explosion of interests in vitamin D had sparked a massive increase in the number of laboratory requests for the measurement of serum 25 hydroxyvitamin D (25(OH)D). This in turn highlighted problems of the methodologies available for measuring vitamin D metabolites. The aim of the study was to develop and fully validate quantitative assays for measuring serum vitamin D metabolites by Liquid Chromatography Tandem Mass Spectrometry (LC/MS-MS). The methods were used to perform analysis on samples collected for vitamin D research studies to establish relationships between the metabolites and determine reference intervals. Using solid phase extraction to remove phospholipids in sample matrix and derivatisation to enhance sensitivity, assays were successfully developed for 25(OH)D3/D2, C3-epi-25(OH)D3/D2, and for the dihydroxyvitamin D 24,25(OH)2D3/D2 and 1,25(OH)2D3/D2. The performance characteristic of the assays satisfied industry standards for method validation. Results showed a high prevalence of C3-epi-25(OH)D3 (87.7%) in paediatric samples that resulted in misclassification of total 25(OH)D status in 10.4% of cases. Serum 25(OH)D showed a significant correlation with 24,25(OH)2D (r2=0.754, p<0.001), but not with 1,25(OH)2D (r2=0.1034). The reference intervals (2.5-97.5 percentile) for 25(OH)D:24,25(OH)2D ratio was established between 7-23. Loess fitting showed an increase in the 25(OH)D:24,25(OH)2D ratio at 25(OH)D <50 nmol/L; evidence of reduced catabolic activity during low vitamin D status. In contrast, when high dose vitamin D3 was supplemented, serum 24,25(OH)2D was found to be grossly elevated to counteract against excessive vitamin D and prevent toxicity. Using the 1,25(OH)2D:24,25(OH)2D/25(OH)D ratio model, this thesis was the first to demonstrate a relationship between the three metabolites, and the association with PTH concentration. This thesis has provided new insights to the vitamin D metabolism that will further our understanding and appreciation of its role in health and pathophysiological conditions. The methods developed have provided an analytical platform for many large scale studies in musculoskeletal research and other areas of science; the publications and citations are a testament to the impact of this research

Accuracy of Molecular Biomarkers in Oral Fluids for Diagnosis of Periodontitis

The diagnosis of periodontitis is a critical element in the success of treatment. Traditional clinical measures have well-known limitations, requiring faster and more specific tools based on quantifiable biomarkers in oral fluids. Interleukins 1alpha, 1beta and 17A in the gingival crevicular fluid are outstanding biomarkers for distinguishing systemically healthy patients with periodontitis from periodontally healthy individuals. Salivary interleukin 1beta has an excellent diagnostic capability when it comes to distinguishing periodontitis from periodontal health, although this discriminatory potential is reduced in smokers. The diagnostic capability of salivary IL1beta remains acceptable for differentiating between untreated and treated periodontitis, especially in smokers. The diagnostic threshold values of these interleukins are lower in smokers than in non-smokers in different clinical settings

Orofacial aspects of juvenile idiopathic arthritis in children

Aims: This thesis, which investigates orofacial aspects and temporomandibular joint (TMJ) involvement in juvenile idiopathic arthritis (JIA), aims to improve knowledge about the variables to include in a clinical examination, radiographic imaging techniques, and whether saliva can be used as a medium for disease monitoring. Material and methods: In a prospective longitudinal study comprising 59 children diagnosed with JIA, clinical and radiological data were collected. Demographic data and data on patient history of localized pain and dysfunction were recorded. Clinical examinations were performed according to the Research Diagnostic Criteria for Temporomandibular Disorders at baseline and repeated after one year and after two years. Radiological examinations were performed with panoramic imaging (PAN) and cone beam computed tomography (CBCT) at baseline and at the two-year follow-up. A classification system for how to grade TMJ morphology on PAN and CBCT was proposed and evaluated using radiological data from this longitudinal study. In a case control study, stimulated whole saliva was collected from 30 children with JIA and 30 healthy age-matched controls. Self-reported orofacial pain was recorded, and saliva flow rate calculated. Saliva samples were analyzed for presence and concentration of 21 immunological active proteins using the Luminex system and customized R&B bead-based immunoassay. Results: The result from the longitudinal study showed a higher proportion of TMJ deformities in children self-reporting TMJ pain and dysfunction. However, self-reported pain was not predictive of change in TMJ status over time. TMJ deformities were associated with a smaller maximum unassisted mouth opening (MUO), palpatory TMJ pain, and TMJ crepitations, but palpatory muscle pain, although common, did not correlate with TMJ deformities. Predictive of finding TMJ deformities was number of years with disease and a smaller MUO. When using the proposed classification system for TMJ morphology, PAN and CBCT recognized presence of TMJ abnormality equally well. The reliability of PAN to distinguish between normal and abnormal TMJ morphology was good, and CBCT was found to be superior for assessing the severity of TMJ abnormality. Regarding presence of immunological biomarkers in saliva, 14 of 21 examined proteins were found in the saliva samples. However, no significant differences in concentrations were found between children with JIA and healthy children. No difference in saliva flow rate was observed between children with JIA and controls, but there was an association between lower salivary flow rate and children reporting orofacial pain regardless of group. Conclusion: In children with JIA, self-reported TMJ pain and dysfunction were common. A high degree of TMJ deformities were found, but clinical variables only showed subtle variations from what is considered normal. No single clinical variable was found to predict or indicate TMJ involvement in JIA. Regarding radiological methods evaluated, the technique that provides diagnostically acceptable information at the lowest radiation dose should be used. The result showed that PANs can be used to determine whether TMJ deformities are present in children with JIA; however, this finding needs to be confirmed in future studies. Furthermore, with the current level of knowledge and based on the results presented, saliva cannot be recommended as a medium for monitoring disease activity in JIA