Free PAPP-A: a Novel Marker in Acute Coronary Syndrome Patients

In recent years, one important objective of cardiovascular research has been to find new markers that would improve the risk stratification and diagnosis of patients presenting with symptoms of acute coronary syndrome (ACS). Pregnancy-associated plasma protein A (PAPP-A) is a large metalloproteinase involved in insulin-like growth factor signalling. It is expressed in various tissues and seems to be involved in many physiological and pathological processes, such as folliculogenesis, bone formation, wound healing, pregnancy and atherosclerosis. The aim of this thesis was to investigate PAPP-A in ACS patients. Circulating concentrations of PAPP-A had been previously shown to be elevated in ACS. In this study it was revealed that the form of PAPP-A causing this elevation was the free noncomplexed PAPP-A. Thus, the form of PAPP-A in the circulation of ACS patients differed from the complexed PAPP-A form abundantly present in the circulation during pregnancy. A point-of-care method based on time-resolved immunofluorometric assays was developed, which enabled the rapid detection of free PAPP-A. The method was found to perform well with serum and heparin plasma samples as well as with heparinized whole blood samples. With this method the concentrations of free PAPP-A in healthy individuals were shown to be negligible. When the clinical performance of the method was evaluated with serum samples from ACS patients, it was shown that the free PAPP-A concentration in the admission sample was an independent predictor of myocardial infarction and death. Moreover, as a prognostic marker, free PAPP-A was revealed to be superior to total PAPPA, i.e. the combination of free and complexed PAPP-A, which has been measured by the other groups in this field. As heparin products are widely used as medication in ACS patients, the effect of heparin products on free PAPP-A molecule and circulating concentrations were also investigated in this study. It was shown that intravenous administration of low molecular weight or unfractionated heparin elicits a rapid release of free PAPP-A into the circulation in haemodialysis patients and patients undergoing angiography. Moreover, the interaction between PAPP-A and heparin was confirmed in gel filtration studies. Importantly, the patients included in the clinical evaluation of the free PAPP-A detection method developed had not received any heparin product medication before the admission sample and thus the results were not affected by the heparin effect. In conclusion, free PAPP-A was identified as a novel marker associated with ACS. The point-of-care methods developed enable rapid detection of this molecule which predicts adverse outcome when measured in the admission sample of ACS patients. However, the effect revealed of heparin products on circulating PAPP-A concentrations should be acknowledged when further studies are conducted related to free or total PAPP-A in ACS.

Sensitive Troponin Assays: Diagnostic and Prognostic Use in Cardiology

Cardiac troponins (cTns) are the recommended biochemical markers in the diagnosis of myocardial infarction (MI). They are very sensitive and tissue-specific but are limited by their delayed appearance in the circulation. Biochemical markers with more rapid release kinetics, e.g. myoglobin and especially heart-type fatty acid-binding protein (H-FABP), have been used to enhance the early identification of MI. The implementation of cTns into clinical practice has shown that cardiomyocyte injury occurs in many other clinical conditions than MI. The aim of this study was to evaluate the impact of modern and highly sensitive cTnI assays on the early diagnosis of MI. In a patient cohort with suspected MI, such a sensitive cTnI assay enhanced the early diagnostic accuracy when compared to a less sensitive cTnI assay and to myoglobin. When compared to H-FABP during the early hours after symptom onset, the sensitive cTnI assay showed at least similar and, after 6 hours, superior diagnostic accuracy. A positive cTnI test result had superior prognostic value when compared to H-FABP, even among early presenters. The prognostic value of cTn in acute heart failure (AHF) was evaluated in 364 patients who participated in the FINN-AKVA study. The patients presented with AHF but no acute coronary syndrome (ACS). Up to half of the patients had elevated cTn levels which were associated with higher 6-month mortality. The magnitude of cTn elevation was directly proportional to mortality. Finally, the clinical spectrum of cTnI elevations was evaluated in 991 cTnI positive emergency department (ED) patients. 83% of the patients had MI and 17% had cTnI elevation due to other clinical conditions. The latter patient group was characterized by lower absolute cTnI levels and – importantly – higher in-hospital mortality when compared to the MI patients. In conclusion, the use of a highly sensitive cTnI assay enhances the early diagnostic accuracy and risk stratification in suspected MI patients. Cardiac troponin elevations are highly prevalent also in other acute clinical conditions and indicate an adverse outcome of these patients.

Effects of Socioeconomic Status and Sociodemographic Features on Cardiovascular Disease. Mortality and Morbidity in Finland

Despite declining trends in morbidity and mortality, cardiovascular diseases have a considerable impact on Finnish public health. A goal in Finnish health policy is to reduce inequalities in health and mortality among population groups. The aim of this study was to assess inequalities in cardiovascular diseases according to socioeconomic status (SES), language groups and other sociodemographic characteristics. The main data source was generated from events in 35-99 year-old men and women registered in the population-based FINMONICA and FINAMI myocardial infarction registers during the years ranging from 1988-2002. Information on population group characteristics was obtained from Statistics Finland. Additional data were derived from the FINMONICA and FINSTROKE stroke registers and the FINRISK Study. SES, measured by income level, was a major determinant of acute coronary syndrome (ACS) mortality. Among middle-aged men, the 28-day mortality rate of the lowest group of six income groups was 5.2 times and incidence 2.7 times as high when compared to the highest income group. Among women, the differences were even larger. Among the unmarried, the incidence of ACS was approximately 1.6 times as high and their prognosis was significantly worse than among married persons - both in men and women and independent of age. Higher age-standardized attack rates of ACS and stroke were found among Finnish-speaking compared to Swedish-speaking men in Turku and these differences could not be completely explained by SES. In these language groups, modest differences were found in traditional risk factor levels possibly explaining part of the found morbidity and mortality inequality. In conclusion, there are considerable differences in the morbidity and mortality of ACS and stroke between socioeconomic and sociodemographic groups, in Finland. Focusing measures to reduce the excess morbidity and mortality, in groups at high risk, could decrease the economic burden of cardiovascular diseases and thus be an important public health goal in Finland.

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.

Cardiac Troponin Specific Autoantibodies: Analytical Tools for Exploring Their Impact on Cardiac Troponin I Testing

Cardiac troponin (cTn) I and T are the recommended biomarkers for the diagnosis and risk stratification of patients with suspected acute coronary syndrome (ACS), a major cause of cardiovascular death and disability worldwide. It has recently been demonstrated that cTn-specific autoantibodies (cTnAAb) can negatively interfere with cTnI detection by immunoassays to the extent that cTnAAb-positive patients may be falsely designated as cTnI-negative. The aim of this thesis was to develop and optimize immunoassays for the detection of both cTnI and cTnAAb, which would eventually enable exploring the clinical impact of these autoantibodies on cTnI testing and subsequent patient management. The extent of cTnAAb interference in different cTnI assay configurations and the molecular characteristics of cTnAAbs were investigated in publications I and II, respectively. The findings showed that cTnI midfragment targeting immunoassays used predominantly in clinical practice are affected by cTnAAb interference which can be circumvented by using a novel 3+1-type assay design with three capture antibodies against the N-terminus, midfragment and C-terminus and one tracer antibody against the C-terminus. The use of this assay configuration was further supported by the epitope specificity study, which showed that although the midfragment is most commonly targeted by cTnAAbs, the interference basically encompasses the whole molecule, and there may be remarkable individual variation at the affected sites. In publications III and IV, all the data obtained in previous studies were utilized to develop an improved version of an existing cTnAAb assay and a sensitive cTnI assay free of this specific analytical interference. The results of the thesis showed that approximately one in 10 patients with suspected ACS have detectable amounts of cTnAAbs in their circulation and that cTnAAbs can inhibit cTnI determination when targeted against the binding sites of assay antibodies used in its immunological detection. In the light of these observations, the risk of clinical misclassification caused by the presence of cTnAAbs remains a valid and reasonable concern. Because the titers, affinities and epitope specificities of cTnAAbs and the concentration of endogenous cTnI determine the final effect of circulating cTnAAbs, appropriately sized studies on their clinical significance are warranted. The new cTnI and cTnAAb assays could serve as analytical tools for establishing the impact of cTnAAbs on cTnI testing and also for unraveling the etiology of cTn-related autoimmune responses.

Nanoparticle-Assisted Immunoassays for Point-of-Care Testing - With Specific Interest in Minimally Interference-Prone Assays for Cardiac Troponin I

Cardiac troponins (cTn) I and T are the current golden standard biochemical markers in the diagnosis and risk stratification of patients with suspected acute coronary syndrome. During the past few years, novel assays capable of detecting cTn‐concentrations in >50% of apparently healthy individuals have become readily available. With the emerging of these high sensitivity cTn assays, reductions in the assay specificity have caused elevations in the measured cTn levels that do not correlate with the clinical picture of the patient. The increased assay sensitivity may reveal that various analytical interference mechanisms exist. This doctoral thesis focused on developing nanoparticle‐assisted immunometric assays that could possibly be applied to an automated point‐of‐care system. The main objective was to develop minimally interference‐prone assays for cTnI by employing recombinant antibody fragments. Fast 5‐ and 15‐minute assays for cTnI and D‐dimer, a degradation product of fibrin, based on intrinsically fluorescent nanoparticles were introduced, thus highlighting the versatility of nanoparticles as universally applicable labels. The utilization of antibody fragments in different versions of the developed cTnI‐assay enabled decreases in the used antibody amounts without sacrificing assay sensitivity. In addition, the utilization of recombinant antibody fragments was shown to significantly decrease the measured cTnI concentrations in an apparently healthy population, as well as in samples containing known amounts of potentially interfering factors: triglycerides, bilirubin, rheumatoid factors, or human anti‐mouse antibodies. When determining the specificity of four commercially available antibodies for cTnI, two out of the four cross‐reacted with skeletal troponin I, but caused crossreactivity issues in patient samples only when paired together. In conclusion, the results of this thesis emphasize the importance of careful antibody selection when developing cTnI assays. The results with different recombinant antibody fragments suggest that the utilization of antibody fragments should strongly be encouraged in the immunoassay field, especially with analytes such as cTnI that require highly sensitive assay approaches.

Translational models of coronary artery disease, myocardial infarction and heart failure. Development, validation and in vivo imaging studies using positron emission tomography

Coronary artery disease is an atherosclerotic disease, which leads to narrowing of coronary arteries, deteriorated myocardial blood flow and myocardial ischaemia. In acute myocardial infarction, a prolonged period of myocardial ischaemia leads to myocardial necrosis. Necrotic myocardium is replaced with scar tissue. Myocardial infarction results in various changes in cardiac structure and function over time that results in “adverse remodelling”. This remodelling may result in a progressive worsening of cardiac function and development of chronic heart failure. In this thesis, we developed and validated three different large animal models of coronary artery disease, myocardial ischaemia and infarction for translational studies. In the first study the coronary artery disease model had both induced diabetes and hypercholesterolemia. In the second study myocardial ischaemia and infarction were caused by a surgical method and in the third study by catheterisation. For model characterisation, we used non-invasive positron emission tomography (PET) methods for measurement of myocardial perfusion, oxidative metabolism and glucose utilisation. Additionally, cardiac function was measured by echocardiography and computed tomography. To study the metabolic changes that occur during atherosclerosis, a hypercholesterolemic and diabetic model was used with [18F] fluorodeoxyglucose ([18F]FDG) PET-imaging technology. Coronary occlusion models were used to evaluate metabolic and structural changes in the heart and the cardioprotective effects of levosimendan during post-infarction cardiac remodelling. Large animal models were used in testing of novel radiopharmaceuticals for myocardial perfusion imaging. In the coronary artery disease model, we observed atherosclerotic lesions that were associated with focally increased [18F]FDG uptake. In heart failure models, chronic myocardial infarction led to the worsening of systolic function, cardiac remodelling and decreased efficiency of cardiac pumping function. Levosimendan therapy reduced post-infarction myocardial infarct size and improved cardiac function. The novel 68Ga-labeled radiopharmaceuticals tested in this study were not successful for the determination of myocardial blood flow. In conclusion, diabetes and hypercholesterolemia lead to the development of early phase atherosclerotic lesions. Coronary artery occlusion produced considerable myocardial ischaemia and later infarction following myocardial remodelling. The experimental models evaluated in these studies will enable further studies concerning disease mechanisms, new radiopharmaceuticals and interventions in coronary artery disease and heart failure.