Cumulative risk assessment in unstable angina: clinical, electrocardiographic, autonomic, and biochemical markers.

OBJECTIVES: To determine the incremental value of clinical data, troponin T, ST segment monitoring, and heart rate variability for predicting outcome in patients with non-ST elevation acute coronary syndromes. METHODS: Prospective cohort study of 304 consecutive patients. Baseline clinical and electrocardiographic data were recorded, serial blood samples were obtained for troponin T assay, and 48 hour Holter monitoring was performed for ST segment and heart rate variability analysis. End points were cardiac death and non-fatal myocardial infarction during 12 months' follow up. RESULTS: After 12 months, 7 patients had died and 21 had had non-fatal myocardial infarction. The risk of an event was increased by troponin T > 0.1 microg/l, T wave inversion on the presenting ECG, Holter ST shift, and a decrease in the standard deviation of 5 minute mean RR intervals. Positive predictive values of individual multivariate risk were low; however, analysis of all multivariate risk markers permitted calculation of a cumulative risk score, which increased the positive predictive value to 46.9% while retaining a negative predictive value of 96.9%. CONCLUSION: A cumulative approach to risk stratification in non-ST elevation coronary syndromes successfully identifies a group in whom the risk of cardiac death or non-fatal myocardial infarction approaches 50%.

Circulating MMP9, vitamin D and variation in the TIMP-1 response with VDR genotype: mechanisms for inflammatory damage in chronic disorders?

BACKGROUND: Vitamin-D deficiency and vitamin-D receptor genotype (VDR) are risk factors for several disorders with inflammatory components, including coronary heart disease (CHD) and diabetes, though the mechanisms involved are unclear. AIM: To examine the hypothesis that vitamin D status modulates the matrix metalloproteinase (MMP) system in a population with a high prevalence of vitamin D deficiency, a situation affecting susceptibility to CHD and diabetes. DESIGN: Prospective cross-sectional, interventional and embedded studies. METHODS: Circulating MMP2,9, the inhibitor TIMP-1 and C-reactive protein (CRP) were measured during studies of vitamin-D deficiency as a risk factor for type 2 diabetes and CHD in 171 healthy British Bangladeshi adults, free of known diabetes or major illness. Vitamin D status, VDR genotype, body-build, blood pressure, lipid and insulin profiles, glucose tolerance, fibrinogen, PAI-1, folate and homocysteine were measured. Vitamin-D-deficient subjects were re-assessed after 1 years' supplementation. MMP, TIMP-1 and CRP levels were measured in 41 subjects halfway through 5-year follow-up. Independent determinants of circulating concentrations of MMP9, TIMP-1 and CRP were assessed by multiple regression analysis. RESULTS: Vitamin D status was the sole determinant of circulating MMP9 (inversely) and an independent determinant of CRP (inversely). Determinants of TIMP-1 were MMP9, systolic blood-pressure (directly) and VDR genotype (TaqI). Significant reductions in MMP9 (-68%), TIMP-1 (-38%) and CRP (-23%) concentrations followed vitamin-D supplementation. DISCUSSION: Vitamin-D insufficiency is associated with increased circulating MMP2,9 and CRP, correctable by supplementation. This finding provides a possible mechanism for tissue damage in chronic inflammatory conditions, including CHD and diabetes.

Interaction between smoking and the glycoprotein IIIa P1(A2) polymorphism in non-ST-elevation acute coronary syndromes.

OBJECTIVES: The goal of this study was to determine the interaction between smoking and the glycoprotein IIIa P1(A2) polymorphism in patients admitted with non-ST-elevation acute coronary syndromes (ACS). BACKGROUND: An increased incidence of the P1(A2) polymorphism in smokers presenting with ST-elevation acute myocardial infarction (AMI) has recently been reported. We, therefore, postulated that, as a consequence of this interaction, fewer smokers with the P1(A2) polymorphism would present with non-ST-elevation ACS. METHODS: We performed a prospective cohort analysis of 220 white Caucasoid patients admitted with non-ST-elevation ACS fulfilling Braunwald class IIIb criteria for unstable angina who were stratified by smoking status. RESULTS: There were twice as many nonsmokers as smokers. Nonsmokers compared with smokers were older (mean [SD]; 63.9 [11.2] vs. 57.6 [10.3]; p < 0.0001), more likely to have had a previous admission with unstable angina (24.3% vs. 13.2%; p = 0.051) and AMI (45.8% vs. 30.3%; p < 0.026), more likely to have undergone revascularization (24.3% vs. 1.8%; p = 0.028) and were more likely to be on aspirin on admission (60.4% vs. 44.7%; p = 0.026). The proportion of nonsmokers positive for the P1(A2) polymorphism was equivalent to that expected for this population but was significantly reduced in smokers (28.7% vs. 10%; Pearson chi-square = 9.09, p = 0.0026). In a logistic regression model, the odds ratio (OR) for being positive for the P1(A2) polymorphism was significantly reduced by smoking (OR [interquartile range]: 0.26 [0.11 to 0.62]; p = 0.0026). CONCLUSIONS: There is a significant reduction in the P1(A2) polymorphism in smokers admitted with non-ST-elevation ACS compared with nonsmokers, which suggests an interaction between smoking and this polymorphism.

Angiotensin-converting enzyme inhibition is associated with reduced troponin release in non-ST-elevation acute coronary syndromes.

OBJECTIVES: This study was done to determine the effects of angiotensin-converting enzyme (ACE) inhibition and other clinical factors on troponin release in non-ST-elevation acute coronary syndrome (ACS). BACKGROUND: Troponin is now widely used as a marker of risk in ACS, but determinants of its release have not been defined. METHODS: This was a prospective cohort study of 301 consecutive patients admitted with non-ST-elevation ACS. Baseline clinical data were recorded, ACE gene polymorphism was determined and serial blood samples were obtained for troponin-I assay. RESULTS: Significant troponin-I release (>0.1 microg/l) was detected in 93 (31%) patients. Pretreatment with ACE inhibitors, recorded in 53 patients (17.6%), independently reduced the odds of troponin-I release (odds ratio 0.25; 95% confidence intervals 0.10 to 0.64) and was associated with lower maximum troponin-I concentrations (median [interquartile range]) compared with patients not pretreated with ACE inhibitors (0.44 microg/l [0.19 to 2.65 microg/l] vs. 4.18 microg/l [0.91 to 12.41 microg/l], p = 0.01). Pretreatment with aspirin, recorded in 173 patients (57.5%), did not significantly reduce the odds of troponin-I release after adjustment but was associated with lower maximum troponin-I concentrations compared with patients not pretreated with aspirin (2.31 microg/l [0.72 to 8.02 microg/l] vs. 5.85 microg/l [1.19 to 12.79 microg/l], p = 0.05). The ACE genotyping (n = 268) showed 81 patients (30%) DD homozygous and 77 (29%) II homozygous. There was no association between ACE genotype and troponin release. CONCLUSIONS: We conclude that ACE inhibition reduces troponin release in non-ST-elevation ACS. This is likely to be mediated by the beneficial effects of treatment on vascular reactivity and the coagulation system.

Effect of poly(ethylene glycol), tetramethylammonium hydroxide, and other surfactants on enhancing performance in a latex particle immunoassay of C-reactive protein.

The influence of a variety and combination of both ionic surfactants and different chain lengths of the polyelectrolyte poly(ethylene glycol) (PEG) on the performance characteristics (with particular reference to signal response) of a homogeneous, latex agglutination immunoassay was investigated. The test analyte was human serum C-reactive protein (CRP), and the antibody reagent consisted of a sheep polyclonal anti-CRP IgG fraction covalently coupled to 50-nm-sized latex including a glycine-capped chloromethylstyrene shell. The amount and rate of immunoagglutination was monitored turbidimetrically after sample addition. It was found that 2.5 mmol/L concentrations of the small cationic surfactant tetramethylammonium hydroxide (TMH), when present alone, substantially increased both reaction rates and sensitivity in the lower clinical ranges of CRP concentration when compared to normally used assay conditions containing PEG and the anionic detergent Gafac. The nonspecific binding (NSB) was also found to be unchanged. Evidence is presented that the TMH enhances the actual antibody-antigen interaction as opposed to the known effects of other surfactants in immunocomplex dissociation or in maintenance of colloidal stability. We suggest that the enhancement seen with TMH could be an alternative to PEG and may provide a new means of further extending detection limits. The utility of this type of immunoassay technology could therefore be increased whenever clinically required.

Effects of the ionic environment, charge, and particle surface chemistry for enhancing a latex homogeneous immunoassay of C-reactive protein.

The role of the solution environment for a light-scattering, latex-particle-enhanced, homogeneous immunoassay of C-reactive protein (CRP) has been investigated in order to assess and optimize the immunoagglutination response. Latex particles of 50-170-nm sizes were covalently coupled with an IgG polyclonal antibody and subjected to an extensive optimization regime. This consisted of conditions responsible, in different degrees, for the principal attractive/repulsive forces affecting both colloidal stability and the antibody/antigen interaction: particle size, antibody concentration, ionic strength and species, pH, and amino acid chemistry of the particle surface. Careful control of these parameters was found to be necessary to achieve the desired effects of balancing high colloidal stability in the absence of antigen but promoting a rapid, sensitive, and dose-dependent agglutination with pathological serum samples. In addition, the estimation of fundamental properties governing intermolecular interaction (i.e. the "Hamaker" constant and critical coagulation concentration) was attempted to order to investigate a simple, practical means of defining a colloidal/immunoassay system under "real conditions" as well as "real time". It is concluded that because each antibody system is unique, a similar optimization should be performed in diagnostic immunoassays of this type to maximize their clinical utility.

Serum cystatin C in patients with myeloma.

BACKGROUND: Cystatin C is a low molecular weight protein thought to be synthesised by all nucleated cells and freely filtered by the kidney. It has been proposed as a marker for GFR; however, it has been suggested that there may be limitations to its use, because it may be over-expressed in some tumour cells and the abnormal tissue growth may also lead to an increased circulating level. METHODS: We investigated the serum cystatin C levels in 60 patients with myeloma, comparing results with those for serum creatinine, beta(2)-microglobulin and the paraprotein concentration. RESULTS: We found no correlation between cystatin C and the paraprotein concentration in these patients. CONCLUSION: These results suggest that disease burden does not correlate to the circulating level of cystatin C in patients with myeloma.

Point-of-care testing. Impact on medical outcomes.

There is now clear evidence that POCT has a positive benefit on morbidity and mortality. In addition, there are other tangible benefits that may themselves influence morbidity and mortality, e.g., reduced blood sample requirement in pediatrics, reduced length of stay, and greater doctor and patient satisfaction. These benefits accrue from the ability to make decisions and implement the appropriate intervention more quickly. It has also been demonstrated that POCT can facilitate improved patient motivation and satisfaction and thereby compliance with a prescribed disease management strategy. Improvement in health outcome, morbidity, and mortality can only be achieved, however, when the diagnostic and therapeutic interventions operate in concert. A review of the literature on medical outcomes of POCT has demonstrated the complexity of establishing evidence and the paucity of robust literature that exists at the present time. It is hoped, however, that the reader will appreciate how important it is to stress the role of a diagnostic test in decision making, to ensure that decisions are made and that benefits will be achieved. From a more pragmatic standpoint, however, it is hoped the reader will see the potential value of the arguments put forward in favor of implementing POCT when submitting a business case to a funding authority; the fact that the cost of POCT may be greater should not be a deterrent.

The effect of aspirin on C-reactive protein as a marker of risk in unstable angina.

OBJECTIVES: This study was designed to assess the interaction between aspirin and C-reactive protein (CRP) release in unstable angina. BACKGROUND: C-reactive protein release in acute coronary syndromes may be a response to myocardial necrosis or may reflect the inflammatory process that drives atherogenesis. Aspirin has the potential to influence CRP release, either by its anti-inflammatory activity or by reducing myocardial necrosis. The clinical significance of this potential interaction has not previously been tested. METHODS: We conducted a prospective cohort study of 304 consecutive patients admitted with non-ST-elevation acute coronary syndromes. Serial blood samples were obtained for CRP and troponin I assay. End points were cardiac death and nonfatal myocardial infarction during follow-up for 12 months. RESULTS: A total of 174 patients (57%) were taking aspirin before admission. Patients taking aspirin had lower troponin I concentrations throughout the sampling period, only 45 (26.0%) having concentrations >0.1 mg/l compared with 48 (37.8%) patients not taking aspirin (p = 0.03). Maximum CRP concentrations were also lower in patients taking aspirin (8.16 mg/l [3.24 to 24.5]) than in patients not taking aspirin (11.3 mg/l [4.15 to 26.1]), although the difference was not significant. However, there was significant interaction (p = 0.04) between prior aspirin therapy and the predictive value of CRP concentrations for death and myocardial infarction at 12 months. Thus, odds ratios (95% confidence intervals) for events associated with an increase of 1 standard deviation in maximum CRP concentration were 2.64 (1.22-5.72) in patients not pretreated with aspirin compared with 0.98 (0.60-1.62) in patients pretreated with aspirin. CONCLUSIONS: The association between CRP and cardiac events in patients with unstable angina is influenced by pretreatment with aspirin. Modification of the acute-phase inflammatory responses to myocardial injury is the major mechanism of this interaction.

Development of a charged-coupled device-based light-scattering instrument for the detection of C-reactive protein using particle-enhanced immunoassay.

A novel light-scattering instrument has been developed for rapid detection of immunoreactions in test latex particle-enhanced immunoassays. The detector consists of a flat-field grating and a charge-coupled device mounted on a rotating platform, and the detector measures a continuous spectrum from 350 nm to 735 nm at 440 polar angles with a resolution of 0.5 degrees. Optimal detection for rates of immunoreaction were determined by intensity of scattered light at different angles. Instrumental precisions were all shown to fall within 5% of the target relative standard deviation limits. The accuracy of the instrument was confirmed using monodispersed latex particles of known size and shape. The initial results showed the possibility of a sensitive and accurate detection of C-reactive protein throughout the range of clinical interest, thus demonstrating a significant potential for biomedical applications.

Recommendations and opinions for the use of point-of-care testing for hospitals and primary care: summary of a 1999 symposium.

As part of a symposium on laboratory medicine, a colloquium on point-of-care testing was held in June 1999 where four experts were invited to produce recommendations and opinions on the use of point-of-care testing under various clinical venues. Each commented on costs for providing POCT services. A total of eleven recommendations and four opinions were rendered and discussed in an open forum. While one expert concluded that some forms of POCT are less expensive than central laboratory testing if entire laboratory workstations are eliminated, another expert suggested that POCT offered little advantage if rapid transport systems are available. A recommendation was made that POCT be considered for analytes that have a required reporting turnaround time of <30 min, and that the goals for precision and accuracy should be dictated by the clinical need and not by analytical limitations. Recommendations for POCT in specific clinical situations include use of glycated hemoglobin and urine albumin testing with personal glucose monitoring at the time of consultation, use of glycated albumin for gestational diabetes, leukocyte esterase and nitrite testing in urine to screen for urinary tract infections, coagulation tests for monitoring patients on oral anticoagulant therapy and in the operating room, testing for H. pylori for patients with dyspepsia, and cardiac markers and urine drugs-of-abuse testing in the emergency department.

Evidence-based laboratory medicine: supporting decision-making.

There is an implicit acceptance that an evidence-based culture underpins the practice of laboratory medicine, in part because it is perceived as the scientific foundation of medicine. However, several reviews of specific test procedures or technologies have shown that the evidence base is limited and in many cases flawed. One of the key deficiencies in the scientific literature on diagnostic tests often is the absence of an explicit statement of the clinical need, i.e., the clinical or operational question that the use of the test is seeking to answer. Several reviews of the literature on specific procedures have also demonstrated that the experimental methodology used is flawed with, in some cases, significant bias being introduced. Despite these limitations it is recognized that a more evidence-based approach will help in the education and training of health professionals, in the creation of a research agenda, in the production of guidelines, in the support of clinical decision-making, and in resource allocation. Furthermore, as knowledge and technologies continue to be developed, an evidence-based strategy will be critical to harnessing these developments.

Plasma preparation from whole blood using ultrasound.

A technique to efficiently separate plasma from human whole blood is described. Essentially, 3-mL samples are held on the axis of a tubular transducer and exposed for 5.7 min to an ultrasonic standing wave. The cells concentrate into clumps at radial separations of half wavelength. The clumps grow in size and sediment under gravity. A distinct plasma/cell interface forms as the cells sediment. The volume of clarified plasma increases with time. The separation efficiencies of transducers of 29-mm and 23-mm internal diameters driven, by test equipment, at radial resonances of 3.4 and 1.5 MHz, respectively, were compared. The average efficiency of separation was 99.6% at 1.5 MHz and 99.4% with the 3.4-MHz system. The cleared plasma constituted 30% of the sample volume at 1.5 MHz and 25% at 3. 4 MHz. There was no measurable release of haemoglobin or potassium into the suspending phase, indicating that there was no mechanical damage to cells at either frequency. A total of 114 samples from volunteers and patients were subsequently clarified in a 1.5-MHz system driven by an integrated generator. The average efficiency of clarification of blood was 99.76% for the latter samples. The clarification achieved is a significant improvement on that previously reported (98.5%) for whole blood exposed to a planar ultrasonic standing wave field (Peterson et al. 1986). We have, therefore, now achieved a six-fold reduction of cells in plasma compared to previous results.

Clarification of plasma from whole human blood using ultrasound.

There has been interest for a number of years in the possibility of separating blood into cells and plasma by methods other than centrifugation, so that the plasma can be analysed on-line. Cells in whole blood normally occupy about 45% of the suspension volume. It has been shown with a number of different cell types, such as yeast and bacteria, that for concentrations of this order the cells are not as efficiently harvested by ultrasound as those for lower concentrations. In this study, removal of cells from 3-4 ml whole blood volumes has been examined in ultrasonic standing wave fields from tubular transducers driven at a frequency of 1.6 MHz. Samples of whole human blood (n = 11) from two volunteers have been processed by three tubular transducers where high levels of cell removal, 99.7% on average, have been demonstrated with high reproducibility between samples as well as for different transducers.

Developments in the assessment of glomerular filtration rate.

The assessment of the glomerular filtration rate (GFR) is the most commonly used test of renal function. The accepted reference procedure employs an exogenous clearance marker whilst the most popular test is that of serum or plasma creatinine. All of these tests have limitations, although the surrogate endogenous markers are the most practical. Cystatin C, a low molecular weight protein which can be measured by light scattering immunoassay, possesses many of the attributes required of the ideal GFR marker. Data on reference ranges indicate that circulating cystatin C levels reflect the variation in GFR throughout life and the marker demonstrates a better correlation with the reference procedure than serum creatinine.