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To present the process used to develop the low back pain (LBP) assessment tool including evaluation of the initial content validity of the tool. The development process comprised the elements: definition of construct and content, literature search, item generation, needs assessment, piloting, adaptations, design, and technical production. The LBP assessment tool was developed to assess the construct “functioning and disability” as defined by the International Classification of Functioning, Disability and Health (ICF). Involvement of patients and health professionals was essential. The elements were collapsed into five steps. In total, 18 patients and 12 health professionals contributed to the content and the design of the tool. The LBP assessment tool covered all ICF components shared among 63 ICF categories. This study presents the process used to develop the LBP assessment tool, which is the first tool to address all ICF components and integrate biopsychosocial perspectives provided by patients and health professionals in the same tool. Initial evaluation of content validity showed adequate reflection of the construct “functioning and disability”. Further work on the way will evaluate comprehensiveness, acceptability, and degree of implementation of the LBP assessment tool to strengthen its use for clinical practice.Implications for RehabilitationA biopsychosocial and patients-centred approach is a strong foundation for identifying the many relevant aspects related to low back pain (LBP).Responding to a lack of tools to support a biopsychosocial and patients-centred approach the LBP assessment tool was developed using a robust, multi-step process with involvement of patients and health professionals.The LBP assessment tool is a strong candidate for a user-friendly tool to facilitate use of the International Classification of Functioning, Disability and Health in routine clinical practice. A biopsychosocial and patients-centred approach is a strong foundation for identifying the many relevant aspects related to low back pain (LBP). Responding to a lack of tools to support a biopsychosocial and patients-centred approach the LBP assessment tool was developed using a robust, multi-step process with involvement of patients and health professionals. The LBP assessment tool is a strong candidate for a user-friendly tool to facilitate use of the International Classification of Functioning, Disability and Health in routine clinical practice.

To evaluate comprehensiveness and acceptability of the patient-reported outcome instrument (PRO-LBP) and the clinician-reported outcome instrument (ClinRO-LBP) included in the low back pain (LBP) assessment tool. Second, to assess degree of implementation after three months. Feasibility-testing, training of health professionals, field-testing, and a feedback meeting was undertaken. Field-testing provided data to evaluate comprehensiveness, acceptability, and degree of implementation. Feasibility-testing and training of health professionals revealed that the LBP assessment tool was usable and ready for field-testing. In total, 152 patients participated in the field-testing of whom 95% considered the PRO-LBP comprehensive and 59% found it acceptable. Health professionals found the ClinRO-LBP comprehensive and acceptable. The feedback meeting revealed that the LBP assessment tool broadened the health professionals’ approach to functioning and facilitated a consultation based on the patient perspective. The degree of implementation reached 79%. The PRO-LBP and the ClinRO-LBP covered key concepts of LBP and were found acceptable by patients and health professionals. Despite the reduced degree of implementation after three months the LBP assessment tool allowed the health professionals to apply a biopsychosocial and patient-centred approach. Future research should investigate whether the LBP assessment can enhance patient-centred care.Implications for rehabilitationThe low back pain (LBP) assessment tool is the first evidence-based tangible tool to cover biopsychosocial aspects related to LBP as defined by the International Classification of Functioning, Disability and Health (ICF).The LBP assessment tool allowed health professionals to apply a biopsychosocial and patients-centred approach and has the potential to be used in rehabilitation planning.Awareness to continuous facilitation and training of health professionals is important to facilitate and maintain implementation of new procedures into routine clinical practice. The low back pain (LBP) assessment tool is the first evidence-based tangible tool to cover biopsychosocial aspects related to LBP as defined by the International Classification of Functioning, Disability and Health (ICF). The LBP assessment tool allowed health professionals to apply a biopsychosocial and patients-centred approach and has the potential to be used in rehabilitation planning. Awareness to continuous facilitation and training of health professionals is important to facilitate and maintain implementation of new procedures into routine clinical practice.

BACKGROUND: Patient-reported outcomes are expected to play an important role in patient-centred health care. To capture patients' perspectives, patient involvement in the development of patient-reported outcome (PRO) instruments is essential, but often lacking. This qualitative study explored the perspectives of patients with low back pain, to gain an understanding of how to qualify a patient-centred consultation by using PROs. This was done by exploring patients' perspectives regarding the assessment of functioning and disability as part of the development of a new PRO instrument based on the International Classification of Functioning, Disability and Health Core Set.METHODS: Semi-structured focus group interviews with seven patients with low back pain were conducted. Data were analysed by drawing on the Interpretive Description methodology.RESULTS: The analysis revealed three core themes: simplicity, individuality and application. Simplicity represented keeping items to a minimum and avoiding overlaps; individuality implied the need for individualized answers; and application signified that PROs should be utilized during the consultation, and that they can provide useful information for clinical decision-making.CONCLUSIONS: The study provides essential knowledge about elements of importance to patients with low back pain when aiming for a patient-centred consultation using PROs. Furthermore, it underlines the importance of involving patients in PRO development because their perspectives improved the new PRO instrument.

This review presents recent findings from the literature on the challenges that researchers may face, when patients get involved as partners in research: which patients to recruit, how to clarify and set boundaries to the roles of the patient and the researcher, and how to evaluate the outcome of involvement. Patient involvement may challenge the professional identity of a researcher. Researchers are often uncertain about how to establish a meaningful collaboration with partners whose knowledge comes from the lived experience of being a patient.

Background Low back pain (LBP) is the leading cause to years lived with disability. 10–20% of patients with LBP experience radicular pain (lumbar radiculopathy). Patient-reported outcomes (PROs) play an important role in advancing patient-centered health care. Although patient involvement is essential to develop valid patient-centred PRO instruments patients are not always involved. The International Classification of Functioning, Disability and Health (ICF) are proposed to facilitate consistent description and measurement of LBP related disability. PurposeTo involve patients with lumbar radiculopathy in the development of a PRO instrument based on ICF (ICF-PRO), by exploring patients' perspectives on the instrument's ability to describe functioning and provide information for the clinical decision-making process.MethodsThe scientific fundament for the development is ICF Core Set for LBP and ICF Rehabilitation Set. Items in the ICF-PRO are developed within methods and terminology of The Patient-Reported Outcomes Measurement Information System (PROMIS®). The development process contains five phases (figure 1). ResultsThis poster presents preliminary results from phase 1-3. 89% of the PROMIS items were linked to ICF (table 1). PROMIS items covered 34% of the two core sets, resulting in development of 44 new items. Focus group interviews with patients (n=7) revealed that ICF-PRO sufficiently reflecting how the patients everyday life was affected by lumbar radiculopathy. Further the focus group identified three themes: 'Simplicity', 'Application' and 'Individuality' representing elements of most importance for the patients toward a patient centered consultation (figure 2). ConclusionsWe found that ‘Simplicity’, ‘Application’ and ‘Individuality’ was essential to patients to lead towards a patient centered consultation. The development process has reached phase 3 and next step is piloting (n=15) before testing in clinical practice among patients with lumbar radiculopathy (n=250). RecommendationsThis study contributes with important insight into engaging patients in development of a PRO instrument and the use of patients´ perspective in clinical decision making. It is expected that the ICF-PRO will facilitate a patient centered clinical consultation and enhance consistent description and measurement of functioning in patients with lumbar radiculopathy by utilising ICF-related data.

Title: Development of a patient-reported outcome instrument for patients with lumbar radicular pain - STUDY PROTOCOLObjective: To develop and pilot test a patient-reported outcome (PRO) instrument based on the International Classification of Functioning, Disability and Health (ICF) to assess functioning in patients with lumbar radicular pain. Methods: The PRO will be developed with direct input from patients with lumbar radicular pain (n=10) and clinicians with extensive experience assessing and treating the population (n=10). The development of the PRO will be based on ICF Comprehensive Core Set for Low Back Pain and Rehabilitation Set. The development process will be divided into three steps. First, patients and clinicians select meaningful categories from the two ICF sets. A three round web based consensus process will be used, and specified criteria for consensus will determine which ICF categories should be included in the PRO. Secondly, items from the PROMIS® Physical Functioning item bank will be linked to ICF, and items corresponding to the included categories will be identified. Thirdly, patients and clinicians will select suitable PROMIS® items to be included in the PRO by means of another web based consensus process. The PRO will then be pilot tested (Alfa test) among the participating patients and clinicians. Adjustments will be done due to results from the Alfa test, and a Beta test among patients in the target population will be performed (n=100). In both Alfa and Beta test the patients have to respond additional questions to test face validity, construct- and content validity.The development process starts in august 2016, with completion December 2017. Results: The development process and preliminary results will be presented at the conference.Conclusions: The study develops a PRO, based on ICF and PROMIS® Physical Functioning item bank, to be used in clinical practise in order to systemize and qualify the description of functioning among patients with lumbar radicular pain.

Concentrations of triglycerides and cholesterol in serum and the distribution of plasma lipoprotein fractions have been studied in 25 patients with chronic non‐nephrotic renal failure. Hyperlipoproteinemia was demonstrated in 17 of them (type II 1 case, type III 1 case, type IV 13 cases, type V 2 cases). None of the recognized causes of secondary hyperlipoproteinemia appeared to be responsible for this high incidence. In 19 patients the i.v. glucose tolerance test showed abnormal responses. For patients with 24‐hour urinary losses of protein exceeding 3.5 g a statistically significant (inter‐patient) correlation was found between the concentration of triglyceride in serum and the rate of renal protein excretion. Including patients with milder degrees of proteinuria this relationship was obscured, and no further correlation could be established between serum lipid values and proteinuria, renal function or glucose tolerance. 1972 Association for the Publication of the Journal of Internal Medicine

We have recently developed a simple mutation screening assay based on the denaturing gradient gel electrophoresis (DGGE) technique for detection of mutations in the coding and regulatory regions of the low density lipoprotein receptor (LDLR) gene and the codon 3500 region of the apolipoprotein (apo) B-100 gene leading to familial hypercholesterolemia (FH) and familial defective apo B-100 (FDB), respectively. To evaluate the assay, 14 Danish families suspected of FH were studied. In ten families, the DGGE assay detected seven different point mutations, including mutations undescribed prior to establishing the assay. In addition, in one of these ten families and in one of the remaining four families, Southern blotting detected the FH-DK3 exon 5 deletion. Based on segregation analysis and clinical data, the FH diagnosis was dubious in the remaining three families without DGGE or Southern blotting detectable mutations. In conclusion, a simple DGGE based mutation screening assay may detect underlying mutations in most FH/FDB families, thus allowing its routine use in genetic counselling of FH-families.

The lipid disorder familial hypercholesterolemia (FH) predisposes to cardiovascular disease. With a prevalence of approximately one in 500 in the general Caucasian population, FH is one of the most frequent single-gene disorders. As the mutational spectra vary between populations, it is crucial to identify the mutations in a given population in order to implement a molecular genetic screening strategy. A total of 1053 referred individuals with clinical signs of FH were investigated, and mutations were identified in 425 individuals. Fifty-four different mutations were identified, of which 13 are novel. The five most frequent mutations accounted for 56.3% of all disease-causing mutations. The majority of the remaining mutations were of a private nature only encountered in single families. In this study, a reliable molecular genetic screening protocol was established, and the relevance of performing presymptomatic genetic analysis as part of a preventive strategy was documented. We have acquired knowledge of the mutational spectra in the Danish population and thus will be able to trace mutations in their relatives through our index cases.

Defects in the enzyme porphobilinogen deaminase (PBG-D) are associated with acute intermittent porphyria (AIP). Human PBG-D is transcribed into a housekeeping or an erythroid form as a result of differential promoter usage and splicing. In addition, three pairs of isoallelic forms have been described. However, whether the enzymatic properties of housekeeping and erythroid forms differ is unknown. In this study the two isoallelic forms, K210 and E210, were cloned and expressed in Escherichia coli together with three mutations associated with a clinical AIP phenotype. The mutations were introduced in the K210 isoallelic background and expressed as both the housekeeping and the erythroid form. The proteins were expressed as GST fusions and purified to homogeneity. Initial experiments revealed that the GST-PBG-D fusions and the purified PBG-D obtained by proteolytic removal of the GST moiety had enzymatic properties that were indistinguishable. Consequently, all analyses with mutant PBG-D were performed on the GST-fusion proteins. Comparison of the wild-type proteins revealed a significant difference in Km between isoalleles with a Km of 9 microM for K210 and 7 microM for E210, whereas no significant difference in activity or kinetics between the housekeeping and the erythroid isoforms was observed. The mutant proteins showed 0.3-1.0% wild-type activity, depending on mutation. There was a clear correlation between yield of recombinant protein and CRIM status of patients. Furthermore, co-expression of the mutant proteins with the bacterial chaperone GroESL did not affect protein yield or function to any significant extent, supporting the view that the investigated mutations primarily influence structure and function and not folding of the proteins.

Hereditary haemorrhagic telangiectasia (HHT) is a rare disorder with one per 6000-10,000 affected individuals in the general Caucasian population. HHT is genetically heterogeneous, involving at least two loci HHT1 mapping to chromosome 9q34.1 and HHT2 mapping to chromosome 12q31. The loci have been identified as endoglin (ENG) and activin receptor-like kinase 1 (ALK1). In order to gain knowledge of the genotype distribution and prevalence in the Danish population and to establish a reproducible and sensitive molecular genetic test method, we developed a denaturating gradient gel electrophoresis protocol for mutation scanning of the two loci. Twenty-five Danish HHT families were tested. A total of eight new as well as seven previously reported mutations were identified. A founder mutation was characterized present in seven families and possibly introduced around 350 years ago. In one individual, a presumed spontaneous mutation was characterized. The method developed proved to be very sensitive for mutation detection in both ENG and ALK1. Genetic screening in HHT families facilitates an early treatment strategy for silent HHT manifestations in first degree relatives.

Background: We describe the preparation of a lyophilised reference material containing purified human adenosine deaminase 1 and the certification of its catalytic concentration. Methods: The enzyme was purified from human erythrocytes. Results: The enzyme was >99% pure on polyacrylamide gel electrophoresis. Only trace amounts (

Background: Thrombolytic therapy often fails to obtain reperfusion in acute myocardial infarction (AMI). To allow further intervention, various methods for non-invasive reperfusion assessment, e.g. release kinetics of biochemical markers and ST segment recovery analysis on the ECG, have been proposed. Methods and Results: In 85 thrombolysed AMI patients and relative increases (RIs) of the markers were calculated. Moreover, the relative decrease in total sum of ST elevation (ST recovery0–90) on the ECG was calculated. A slope0–90 or RI0–90 below previously validated threshold values or an ST recovery Conclusions: Dependency on infarct size seems to limit the usefulness of biochemical markers for reperfusion assessment. Correction for expected infarct size might improve performance in the future. At present, routine use of biochemical markers for reperfusion assessment is not justified.

Aims CYP2D6 and CYP2C19 are polymorphically expressed enzymes that show marked interindividual and interethnic variation. The aim of this study was to determine the frequency of the defective alleles in CYP2D6 and CYP2C19 in Africans and to test whether the genotype for CYP2C19 is better correlated with the proguanil/cylcoguanil ratio than the mephenytoin S/R ratio. Methods Two hundred and sixteen black Tanzanians were phenotyped for CYP2D6 with the use of sparteine, and for CYP2C19 with the use of mephenytoin and proguanil. Of these 196 subjects were also genotyped for CYP2D6 (including the CYP2D6*1, CYP2D6*3 and CYP2D6*4 alleles) and 195 were genotyped for CYP2C19 (including the CYP2C19*1, CYP2C19*2 and the CYP2C19*3 alleles). Furthermore 100 subjects were examined for the allele duplication in CYP2D6, leading to ultrarapid metabolism, with long PCR. Results The sparteine metabolic ratio (MR) was statistically significantly higher in the Tanzanian group of homozygous, extensive metabolizers compared to a historical control group of white Danish extensive metabolizers. Only one poor metabolizer for CYP2D6 (MR=124 and genotype CYP2D6*1/CYP2D6*4 ) was found. The gene frequencies were 0.96 for the CYP2D6*1 allele and 0.04 for the CYP2D6*4 allele. No CYP2D6*3 alleles were found. Nine subjects had an allele duplication in CYP2D6 (9%). For CYP2C19 there were seven subjects (3.6%) who were phenotyped as poor metabolizers, but only three subjects (1.5%) had a genotype (CYP2C19*2/CYP2C19*2 ) indicative of poor metabolism. The gene frequencies were 0.90 for the CYP2C19*1 allele and 0.10 for the CYP2C19*2 allele. No CYP2C19*3 alleles were found. The mephenytoin S/R ratios were not bimodally distributed. Conclusions Both the genotyping and phenotyping results show that there is a substantial difference between an African black population and a Caucasian population in the capacity to metabolize drugs via CYP2D6 and CYP2C19.

A BsmI restriction enzyme polymorphism in the vitamin D receptor (VDR) gene has been reported to be associated with bone mineral density (BMD) and bone turnover. However, findings in other studies suggest the presence of considerable interaction by race, body size, and environmental factors. Therefore, we VDR BsmI genotyped 200 healthy perimenopausal Danish white women (mean age 50.8 years, mean calcium intake 900 mg/day) in a comprehensive, longitudinal, community-based population study. Bone loss was assessed by dual-energy X-ray absorptiometry (DXA) using cross-calibrated Hologic QDR-1000 W and QDR-2000 densitometers, with a mean follow-up period of 4 years (range 1-5 years). Despite a distribution of genotypes similar to that of other white populations (28% bb, 49% Bb, 23% BB), VDR genotypes were not associated with lumbar or femoral baseline BMD, subsequent bone loss rates, or biochemical markers of bone metabolism (bone-specific alkaline phosphatase, urinary hydroxyproline, and serum osteocalcin). Controlling for body size, calcium intake, and serum levels of 25-hydroxyvitamin D3 [25(OH)D3] did not alter this finding. The possible existence of a threshold effect was subsequently investigated by restricting analysis to women with low serum 25(OH)D3 levels or low calcium intake, VDR BsmI genotypes showed no significant impact on bone density or bone loss in healthy Danish early postmenopausal women, even when allowance was made for calcium intake, serum 25(OH)D3, and body size.

Quality specifications for analytical imprecision and bias based on ‘the state of the art’, ‘biology’ and ‘analysis of clinical situations’ have been proposed by several scientists. Most interesting is the assessment of ‘diagnostic misclassifications’ based on direct evaluation of the consequences of analytical bias on the percentage of false positives and false negatives from a clinical decision situation, or based on the percentage of healthy individuals outside each reference limit when common reference intervals are used. With use of graphical or computer simulations assuming increasing (positive or negative) analytical bias, the expected percentage of misclassifications can be estimated — and, for the error for which the outcome (the fraction of misclassifications) is considered unacceptable, the maximum allowable analytical bias can be defined. An overview is given of previous proposals for specification of allowable analytical bias, and new examples are presented: (i) for S-transferrin, an analytical bias of + 10% will increase the percentage of healthy individuals with measured concentration values above the upper reference limit from 2.5 to 10%; (ii) the percentage of healthy men with concentration values for S-cholesterol above 6.2 mmol/1 (240 mg/dl) will vary between 25 and 85% for analytical bias from − 1.0 to + 1.0 mmol/l (± 16%); (iii) for glycated haemoglobin, two examples are given which illustrate the effect of analytical bias on the risk of retinopathy and so-called ‘microalbuminuria’ for measured values identical to the target 7.5% and 10.1% glycated haemoglobin, respectively. It is concluded that analytical bias may have significant impact on diagnostic performance, better standardization is needed, and quality specifications for allowable analytical bias should be based on medical usefulness criteria or, if such data are not available, on biological criteria.

Using a simple, standardized denaturing gradient gel electrophoresis (DGGE) based mutation screening technique, a novel G-to-A mutation in the last base of the intron 12 splice acceptor site of the LDL receptor gene was found in 2 Danish families with familial hypercholesterolemia (FH). The mutation is shown to result in 2 mRNA splice variants, both leading to truncated LDLR proteins, containing only the first 594 of the normal 839 amino acids. In one of the FH-families harbouring the mutation, a striking difference in the clinical picture amongst biochemically diagnosed FH patients was clarified when genetic analysis showed that 2 hypercholesterolemic family members, who despite advanced age had no atherosclerotic disease, had not inherited the family LDLR mutation. DGGE analyses of the LDLR exons, LDLR promoter, and apolipoprotein B codon 3456-3553 as well as Southern blotting of the LDLR gene were without signs of other mutations in the non-atherosclerotic hypercholesterolemics of the family. Availability of the clinically applicable mutation screening assay for FH may thus aid in defining reasons for phenotypic differences in FH families and potentially supply information allowing a more differentiated therapeutic approach to individual members of FH families.

Familial hypercholesterolaemia (FH) is an autosomal dominant hereditary disease of lipid metabolism that in most families is caused by mutations in the low density lipoprotein receptor (LDLR) gene. Though more than 150 mutations are known, the clinical picture associated with most of these is not known. Genetic FH diagnosis may soon become routine in the setting of genetic counselling, and therefore thorough information on the phenotype-genotype relationship of different mutations is now important. In this study, index patients from each of 14 Danish FH families were screened for mutations in exon 2 of the LDLR gene using a denaturing gradient gel electrophoresis (DGGE)-based mutation screening assay. A deviating DGGE pattern identified two index patients, where subsequent sequencing revealed heterozygosity for the FH Cincinnati type 5 Trp23-to-Stop LDLR mutation. Data from three generations of the families allowed the first clinical and biochemical description of this mutation. Evidence that genetic analysis adds independent diagnostic information compared to traditional clinical/biochemical FH diagnosis was documented by demonstrating the presence of the FH Cincinnati mutation in a family member with a completely normal lipid profile. By comparison to non-FH family members, it was documented that carrier status for the FH Cincinnati mutation is associated with a significant risk of cardiovascular disease. Thus, genetic analysis may improve diagnostic precision and help to define more precisely which of the members of FH families are in need of preventive interventions and may aid in establishing phenotype-genotype relationships allowing more refined genetic counselling in FH.

Familial defective apolipoprotein B (FDB) is a clinical condition resembling familial hypercholesterolemia. The underlying genetic defects are mutations in the apolipoprotein B-100 (apo B-100) gene. Two mutations (Arg3500 --> Gln and Arg3531 --> Cys) are known to date. We designed a denaturing gradient-gel electrophoresis (DGGE) technique to detect sequence variations in codons 3456-3553 of the apo B-100 gene. In 46 heterozygous FDB patients with the predominant codon 3500 mutation, a uniform four-band DGGE pattern was seen, whereas 57 non-FDB patients showed the uniform single-band pattern expected in normal homozygotes. The recently described codon 3531 mutation and a previously unpublished Arg --> Pro mutation in codon 3480 showed unique DGGE patterns, allowing unambiguous differentiation of the three mutations. The DGGE method thus both detects known FDB mutations and screens for other mutations in codons 3456-3553 of the low-density lipoprotein receptor binding region of apo B-100; it can be used as a first-line screening method for FDB.

Objectives. We sought to determine the incidence and independent prognostic value of increased serum levels of sensitive serologic markers in patients in whom a conventionally diagnosed acute myocardial infarction had been ruled out. Background. Increased serum levels of creatine kinase (CK) isoenzyme MB mass and cardiac troponin T in patients with unstable angina pectoris are associated with a poor prognosis. Methods. We analyzed data from 196 consecutive patients with suspected acute myocardial infarction, which was later ruled out in 124. Increased serum levels of CK-MB mass, troponin T and myosin light chains were compared with clinical findings, ST-T wave abnormalities and presence of arrhythmias. Results. Of the patients in the noninfarction group, 28% had serum CK-MB mass ≥ μ g/liter, 20% had troponin T ≥0.20 μ g/liter, and 26% had myosin light chains ≥0.4 μ g/liter (discrimination limits). The cardiac event rate (cardiac death, nonfatal acute myocardial infarction) within 28 months was significantly higher in patients in the noninfarction group with elevated marker levels (range 22% to 24%) than in patients with values below these discriminators (range 3% to 5%) but was not significantly different from that in patients with a definite diagnosis of acute myocardial infarction (29%). Further, significant predictors of cardiac events were previous myocardial infarction; myocardial infarction or angina pectoris, or both; previous congestive heart failure; ST-T wave abnormalities on admission; a transient ST-T wave shift on serial electrocardiograms (ECGs); recurrent chest pain; and occurrence of supraventricular or ventricular tachycardia, or both, during the 1st 48 h after admission. It was found that all three biochemical markers, in the main, convey independent prognostic information with respect to clinical findings and presence of arrhythmias but not ST-T wave abnormalities on admission or a transient ST-T wave shift on serial ECGs. Conclusions. Increased serum levels of CK-MB mass, troponin T and myosin light chains all detect a subgroup of 25% of patients without acute myocardial infarction who have as poor a prognosis as that of patients with a definite diagnosis of acute myocardial infarction. All three biochemical markers provide similar important independent prognostic information with regard to clinical findings and arrhythmias but add no additional prognostic information once ECG ST-T wave changes are considered.

A new nonradioactive oligonucleotide ligation assay for the detection of a common point mutation in the CYP2D6 gene is presented. The assay takes advantage of simultaneous time-resolved fluorescence measurements of lanthanide-labeled probes and the specificity of T4-DNA ligase in combination with the polymerase chain reaction. This strategy makes it possible to perform the assay using both the wild-type-specific and mutant-specific probes simultaneously, securing an internal control in each reaction. We show that the allele-specific ligation part of the assay can be performed with great accuracy over a wide range of temperatures, salt concentrations, and T4-DNA ligase concentrations. This eliminates the risk of false-positive or false-negative reactions due to variations in these factors. Because the assay is simple to perform, is very reliable, and can be partly automated, we conclude that it is well-suited for analysis in a routine laboratory.

A model for reliable estimation of variance components for biological within- and between-subject variation as well as for analytical variation when both the quantity and the reagents are unstable has been established. This model was applied to the alpha-interferon receptor on human leucocytes which involves two major problems. First, the receptor has to be quantified within a few hours after blood sampling, and second, the reagents for the measurement procedure must be used within 2 weeks. For the number of receptors per cell the biological estimates of coefficients of variation were 14% for within-subject variation and 20% for between-subject variation, respectively. For the dissociation constant both estimates were zero as expected. The model is robust and applicable to other systems with unstable quantities and reagents.

We performed a screening of exon 9 of the low density lipoprotein receptor (LDLR) gene in 14 Danish families with familial hypercholesterolemia (FH) using the denaturing gradient gel electrophoresis (DGGE) technique. In one of the probands from these families an abnormal band pattern in the gradient gel was detected. Subsequent DGGE analysis of the family of this index patient revealed that the DGGE pattern cosegregated with the disease in this family. Sequencing of the exon showed a deletion of a C in codon 424 of the LDLR gene resulting in a frame shift with the introduction of a stop codon 5 codons further downstream. The mutation is referred to as FH-Odense. The predicted truncated receptor protein consists of the 428 amino terminal amino acids. Consequently, the cytosolic and membrane spanning parts of the mature LDL receptor, which normally secure the receptor in the plasma membrane, are missing. The FH-Odense mutation results in severe premature coronary atherosclerosis as shown by the clinical expression in 5 generations of the affected family.

Creatine kinase (CK; EC 2.7.3.2) catalytic activity in serum is widely measured in clinical chemistry practice and provides information for diagnosis and follow-up in many pathological conditions affecting heart, muscle, and brain. Depending on the organ involved, the predominant CK isoenzyme in serum varies. However, routine methods measure total CK catalytic activity, and standardized methods for doing so have been recommended by the International Federation of Clinical Chemistry and by several national scientific societies. Many commercial kits for those methods are now available. With use of a reference material for CK, commercial reagents can be compared with standardized methods, improving confidence in the results. Here we present a reference preparation of CK consisting of the BB isoenzyme purified from human placentae. We describe the procedure of purification and the properties of the lyophilized preparation of CK-BB, which has been certified by the Community Bureau of Reference of the Commission of the European Communities under the designation CRM 299. The preparation can be used to calibrate assays of the catalytic activity of CK-MM and CK-MB, as well as CK-BB.

SUMMARY 2,5 AS is induced by interferon-alpha, -beta (IFN-α, -β) and polymerizes adenosine triphosphate (ATP) into pppA (2′ p5′ A) oligomers, leading to inhibition of protein synthesis and virus growth. The gene coding for the IFN-α, -β receptor is localized lo chromosome 21. In trisomy 21 cells from patients with Down's syndrome a 50% increase in gene dosage is present for the genes on this chromosome. Corresponding increase of gene products has been shown for several of these genes, e.g. The IFN-α, -β receptor molecules. Here, we show a direct proportionality between the number of IFN-α, -β receptors and both the basal activity of 2,5AS and the sensitivity of this enzyme for IFN-α, -β. The relative increase of 2,5AS activity (the reactivity) becomes reduced consistent with the increased basal 2,5AS activity in trisomy 21 cells compared with control cells.

To evaluate myocardial cell damage in relation to spontaneous and exercise-induced ischaemia, release of myoglobin, creatine kinase (CK) and its isoenzyme MB (CK-MB) into the serum was estimated in 10 patients with severe stable angina. All patients had a positive exercise test, significant stenosis of one or more of the main coronary arteries and more than five ischaemic attacks per week. ST-segment monitoring was performed for 36 h. During the last 24 h of that period (period A) serial blood samples were analysed for myoglobin, CK and CK-MB using sensitive assays. Three days later (period B) the patients performed an exercise test at 0815 h, with ST-segment monitoring and blood sampling carried out as described for period A. During period A, 47 ischaemic episodes (100% silent) with a total duration of 599 min were noted in four patients. Forty-seven ischaemic episodes (94% silent) with a total duration of 804 min, were observed in seven patients during period B. Release of myoglobin, CK, and CK-MB did not increase in relation either to spontaneous or exercise-induced ischaemia. Thus even frequent and prolonged episodes of transient myocardial ischaemia (symptomatic or asymptomatic) in patients with severe stable angina pectoris does not seem to cause irreversible myocardial damage.

This prospective study was an evolution of a new sensitive creatine kinase (CK) isoenzyme MB immunoassay in 156 patients, admitted consecutively to the coronary care unit, suspected of having acute my

Individuals with the most common form of the porphyrias, porphyria cutanea tarda (PCT), are believed to be genetically predisposed to development of clinically overt disease through mutations and polymorphisms in genes associated with known precipitating factors. In this study, we have examined a group of Danish patients with PCT for the presence of the C/A polymorphism in intron 1 of CYP1A2. The results demonstrate that the frequency of the highly inducible A/A genotype is increased in both familial and sporadic PCT. This suggests that inheritance of this genotype is a susceptibility factor in development of PCT.

Hereditary haemochromatosis is an autosomal recessive disease that is genetically expressed by excessive accumulation of iron in the tissues, resulting in cirrhosis, diabetes mellitus, cardiomyopathy and hypogonadism. As the disease may be diagnosed before the appearance of symptoms, and prevented by repeated phlebotomies, there are strong implications for adoption of a screening procedure. Determinations of transferrin saturation (TS) and serum ferritin concentration (SF) were used to screen 4302 blood donors, who were selected for follow-up studies if they fulfilled any of the following three criteria: (i) TS greater than or equal to 0.7; (ii) TS greater than or equal to 0.5 together with SF greater than or equal to 150 micrograms l-1; (iii) SF greater than or equal to 300 micrograms l-1. A total of 58 subjects who fulfilled at least one of these criteria were reinvestigated, after which 18 individuals still fulfilled at least one criterion. Fifteen subjects having SF greater than or equal to 300 micrograms l-1 were offered liver biopsy and thirteen of these accepted. In one individual, no stainable iron was detected, and two subjects did not fulfil the previously established diagnostic criteria for the diagnosis of hereditary haemochromatosis. Ten subjects who had a high TS and liver iron grade 2-4 according to Bassett were classified accordingly as homozygotes. On the basis of these results, the prevalence of haemochromatosis in Denmark was estimated to be 0.0037-0.0046.

Magnesium is an essential ion for the structural and functional integrity of cells. We have examined whether a low extracellular magnesium concentration contributes to cell damage induced by ATP depletion in a model system of quiescent fibroblasts. Cell damage assessed by the release of lactate dehydrogenase was slightly, but significantly increased in the absence of Mg2+. The increased cell damage may be caused by an increased Mg2+ loss during ATP depletion, by an altered cell membrane permeability for potassium and sodium, or by a disturbed balance between Mg2+ and Ca2+.

Inflammation and endothelial activation are associated with an increased risk of CVD and epidemiological evidence suggests an association between levels of markers of inflammation or endothelial activation and the intake of fruit. Also, vitamin E, a fat-soluble antioxidant, has anti-inflammatory properties. We performed a randomised 2 × 2 factorial, crossover trial to determine the effect of orange and blackcurrant juice (500 ml/d) and vitamin E (15 mg RRR-α-tocopherol/d) supplementation on markers of inflammation and endothelial activation in forty-eight patients with peripheral arterial disease. Patients were randomly allocated to two dietary supplements from the four possible combinations of juice and vitamin E: juice+vitamin E; juice+placebo; reference beverage (sugar drink)+vitamin E; and reference beverage+placebo. The supplementations were given for 28 d, separated by a 4-week wash-out period. Analysis of main effects showed that juice decreased C-reactive protein (CRP) by 11 % and fibrinogen by 3 % while the reference drink increased CRP by 13 % and fibrinogen by 2 % (P P

Without a consensus on either a reference method or a single glycated haemoglobin standard, the individual laboratory has to establish and secure its own assay method. Prior to selection of a method for glycated haemoglobin we used an assessment model and defined our clinical goals. Based on clinical goals it was possible to set up goals of analytical quality and evaluate available literature concerning the performance characteristics of different assay methods. We wanted a method that measured HbA]c, without measuring the labile intermediate pre-HbA1c, and that provided separate detection of haemoglobin variants. Low imprecision was required to secure the measured, non-diabetic reference interval, and to minimize random analytical error. We found the isoelectric focusing method the only one able to meet our goals, and a method evaluation was carried out. Clinical goals of analytical quality should always be defined before method selection and implementation. Using this model, we were able to establish a precise isoelectric focusing method that fulfilled our clinical goals by measuring HbA1c with a total coefficient of variation of 2.1% and a non-diabetic reference interval from 5.2% to 6.8% HbA1c.

Udgivelsesdato: 2007-Sep BACKGROUND: Paraoxonase-1 (PON1) is an antioxidative enzyme associated with HDL and its serum activity is associated with risk of cardiovascular disease. The interindividual variation in PON1 activity is partly determined by genetic factors, such as polymorphisms in the PON1 gene, but also by dietary factors like the antioxidants. AIM OF THE STUDY: We examined the effect of antioxidant-rich orange and blackcurrant juices and vitamin E supplement on PON1 activity in patients with peripheral arterial disease. Furthermore, we studied whether genetic polymorphisms in the PON1 gene predicted the change in PON1 activity. METHODS: The study was designed as a cross-over trial with 48 participants who received two of the four possible treatments: (1) 250 ml orange juice and 250 ml blackcurrant juice; (2) 15 mg vitamin E; (3) 250 ml orange juice and 250 ml blackcurrant juice and 15 mg vitamin E; or (4) control/placebo (energy-equivalent sugar-containing beverage). The treatments were given for 28 days, separated by a 4-week wash-out period. RESULTS: The PON1 activity was not affected by juice or vitamin E supplement neither was there evidence of synergetic effects. However, a statistically significant interaction was observed between treatment and PON1 genotype, such that PON1 activity increased after juice alone in patients carrying the PON1 L55-allele. Results need to be interpreted with care since the study population was relatively small. CONCLUSION: Consumption of orange and blackcurrant juice and vitamin E supplement does not affect the activity of PON1 in patients with peripheral arterial disease. However, a gene-diet interaction may be present.

Acute intermittent porphyria (AIP) is a dominantly inherited disorder of heme metabolism caused by a 50% decrease in the activity of porphobilinogen deaminase (EC 4.3.1.8.). The estimated prevalence of the disease is 1:1000 to 1.5:100 000, making it the most common of the neurologic porphyrias (1). Although its penetrance is only 10–20% (2), acute attacks of AIP are disabling and can be life-threatening. Prevention of acute attacks is the most important principle in managing the disease; however, it requires prior identification of individuals at risk. Because AIP has a genetic basis, molecular analysis for this disease is ultimately the most reliable way to identify gene carriers. More than 130 mutations in the porphobilinogen deaminase gene have been characterized to date (3). The majority have been restricted to a single family, or at most a few families, and no mutation accounts for more than a small percentage of all cases. With the exception of selected geographic areas where single mutations predominate (4)(5), testing for known mutations in a patient who has not previously been investigated is likely to be negative. Molecular analysis therefore requires an efficient strategy for finding the causative mutation. Denaturing gradient gel electrophoresis (DGGE) has been used by several groups to screen the exons and flanking intronic segments of the porphobilinogen deaminase gene for variations in the wild-type sequence (6)(7)(8)(9)(10)(11)(12)(13). This work has been carried out in European laboratories, and the mutations that have been discovered reflect the molecular defects within that population. By contrast, relatively few mutations have been described …

Familial defective apolipoprotein B-100 (FDB) is commonly attributable to mutation of glutamine to arginine in codon 3500 of the apolipoprotein B (APOB) gene. APOB, the protein component of low-density lipoprotein (LDL) acts as the ligand for the LDL receptor (LDLR), mediating the clearance of LDL from plasma. This mutation causes hypercholesterolaemia and consequent coronary artery disease phenotypically similar to familial hypercholesterolaemia (FH) attributable to LDLR gene defects. APOB gene mutation R3500Q is prevalent in Western Europe, attributable to a single founder mutation, and occurring at up to one in 1000 in the general population and 1%-2% in apparent FH collections. Many studies applying direct assay of patient groups for R3500Q have been undertaken, but there are indications that other ligand defects exist, R3531C and R3500W having been described. Different regions or countries may display different mutational spectra which can be instructive for research and useful for establishing genetic diagnostic assays. Codon region 3456-3553 of the APOB gene contains the mutations so far identified, and therefore is a strong candidate for a functional role in receptor binding. We have applied denaturing gradient gel electrophoresis (DGGE), a sensitive de novo mutation scanning technique, to this region in 106 apparent FH index cases from the South of England.

Objectives:Health Technology Assessment (HTA) is defined as a policy research approach that examines the short- and long-term social consequences of the application or use of technology. Internationally different institutions have translated this definition to local contexts. In Denmark, HTA is comprehensive with focus on four aspects of the problem in question (technology [clinical evidence], economy, patient, and organization). The objective of this study is to study how the application of HTA differs across leading countries and to study the extent to which Danish HTA reports differ from foreign HTAs.Methods:A sample of 433 HTA reports published in the period 1989–2002 by eleven leading institutions or agencies in Denmark and eight other countries were reviewed. We looked at the characteristics of the HTA with respect to focus on the four main aspects and the manner in which each aspect has been approached.Results:The study shows health technology procedures to be the most common type of health technology assessed in HTAs and literature review to be the most often used method of analysis. Policy recommendations are only present in approximately half of the HTA reports.Conclusions:In the HTAs one generally sees a great focus on the clinical aspect of health technologies, leaving the economic, the patient-related, and the organizational aspect much more unanalyzed. The Danish HTAs generally have a wider scope than HTAs produced in other countries and tend to focus more frequently on patient-related and organizational dimensions.

The functional consequence of six uroporphyrinogen decarboxylase (UROD) gene mutations found in Danish patients with familial porphyria cutanea tarda was investigated. Wild-type UROD and the 6 mutants (3 missense, 1 nonsense and 2 frameshift mutants) were cloned and expressed using the prokaryotic gGEX-6P system, in which the protein is produced in fusion with glutathione S-transferase (GST). Enzymatic activity of the purified recombinant mutant fusion proteins ranged from undetectable to less than 12% of the recombinant wild-type protein. Mutant proteins cleaved from the GST part did not retain any catalytic activity. These observations can be ascribed to the structure/function relationships of the enzyme, and the fact that the enzyme is a dimer in its active form. Although the clinical manifestation of familial porphyria cutanea tarda is complex, the findings support the notion that different mutations may affect individuals differently.

The manifestation of porphyria cutanea tarda reflects genetic and environmental factors. Mutations in the uroporphyrinogen decarboxylase gene, located at chromosome 1p34, discriminate familial porphyria cutanea tarda from sporadic cases. Furthermore, mutations in the haemochromatosis gene may be involved in the aetiology. In this study 53 unrelated Danish patients with porphyria cutanea tarda were classified according to uroporphyrinogen decarboxylase and haemochromatosis gene mutations and the genotype related to the clinical and biochemical data. Thirteen patients (25%) had familial porphyria cutanea tarda. The results signify the advantage of DNA diagnostics for identification of familial cases, as anamnestic data are doubtful and erythrocyte uroporphyrinogen decarboxylase activity measurements insufficient for correct classification. Eight patients with porphyria cutanea tarda (15%) were homozygous for the haemochromatosis gene C282Y mutation and 8 patients were heterozygous. Patients homozygous for the haemochromatosis related mutation showed biochemical evidence of excessive iron storage as well as increased urine porphyrin excretion levels. This seems to confirm a relationship between porphyria cutanea tarda and haemochromatosis. No differences were found between patients with sporadic and familial porphyria cutanea tarda regarding age of onset, clinical severity, sex distribution, liver function tests and iron storage parameters. However, daily alcohol intake and use of oestrogens were reported more frequently in the group of sporadic patients. It was found that women were over-represented in our study.