Development of an optimized multimarker strategy for early risk assessment of patients with acute coronary syndromes.

BACKGROUND: A multitude of biomarkers have been suggested for early risk-assessment in patients admitted to the emergency department with suspected acute coronary syndromes. We used logistic regression synergistically with classification and regression tree (CART) analysis to define a multimarker strategy and the cut-off values and sequencing needed to optimize risk stratification in a low to moderate risk population of the emergency department. METHODS: 432 unselected patients (59.7+/-14.5 y, 60.4% male) admitted to the emergency department (ED) with acute coronary syndromes (ACS) were enrolled. Cardiac troponin I (cTnI), N-terminal pro-B-Type natriuretic peptide (NT-proBNP), high sensitivity C-reactive protein (hsCRP), placental growth factor (PlGF), lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) and D-dimers were measured by immunoassay and whole blood choline (WBCHO) and plasma choline (PLCHO) were measured using LC/MS from baseline samples. Logistic regression and CART analysis were used to define the importance of the various biomarkers tested and to define their hierarchy with respect to the prediction of major adverse cardiac events (MACE; cardiac death, non-fatal MI, unstable angina, CHF requiring admission, urgent PCI and CABG) over the 42-day follow-up period. RESULTS: A combination of NT-proBNP, WBCHO and Lp-PLA2 with cutoffs identified by CART-analysis was optimal for risk-stratification and superior to all other possible combinations of markers. Increased concentrations of both NT-proBNP (>1400 ng/l) and WBCHO (>21 micromol/l) identified patients with very high risk (RR=2.4, 39% primary endpoint) while low concentrations of NT-proBNP (< or = 1400 ng/l), WBCHO (< or = 17 micromol/l) and LP-PLA2 (< or = 210 microg/l) indicated very low risk (0% primary endpoint). WBCHO > 17 micromol/l additionally identified a subgroup with intermediate risk (RR=3.0, 13.5% primary endpoint) in patients with NT-proBNP concentrations < or = 1400 ng/l. Troponin when increased was highly prognostic but was not often positive in this early cohort. CONCLUSIONS: A multimarker strategy defined synergistically by logistic regression and by classification and regression tree (CART) analysis can stratify patients into risk groups ranging from very low risk (0% MACE) to very high risk (39.5% MACE) based on admission values.

Whole blood choline and plasma choline in acute coronary syndromes: prognostic and pathophysiological implications.

BACKGROUND: Whole blood choline (WBCHO) and plasma choline (PLCHO) concentrations increase rapidly after stimulation of phospholipase D in acute coronary syndromes (ACS). Early risk-stratification was analyzed in 217 patients with suspected ACS and a negative admission troponin T (<0.03 microg/L). METHODS: WBCHO and PLCHO were measured using high-performance-liquid-chromatography mass spectrometry. Major cardiac events (MACE) were defined as cardiac death/arrest, coronary intervention or myocardial infarction (MI). RESULTS: WBCHO (> or = 28.2 micromol/L) was predictive for MACE (hazard ratio [HR] 2.7; p<0.001), cardiac death/arrest (HR 4.2; p=0.015), heart failure (HR 2.8; p=0.003), coronary intervention (HR 2.1; p=0.01) and MI (HR 8.4; p=0.002) after 30 days. PLCHO (> or = 25.0 micromol/L) was predictive for MACE (HR 2.6; p=0.005), cardiac death/arrest (HR 15.7; p<0.001), heart failure (HR 6.0; p<0.001) but not for coronary intervention and MI. WBCHO and PLCHO were predictive for MACE in multivariate analysis (Odds ratio [OR] 2.7, p=0.009 and OR 3.3, p=0.03) independently of age, gender, prior MI, coronary risk factors and ECG. CONCLUSIONS: WBCHO and PLCHO are significant and independent predictors of major cardiac events in admission troponin T negative acute coronary syndromes. Both are predictive for events related to tissue ischemia and WBCHO is capable of detecting risks associated with coronary plaque instability.

Prognostic implications of elevated whole blood choline levels in acute coronary syndromes.

Troponins I and T represent the current biomarker standard for diagnosis of myocardial infarction. Even small increases of cardiac troponins have prognostic implications, but not all patients at risk are correctly classified, particularly at admission. We identified elevated whole-blood choline as a promising marker and performed a prospective study of 327 patients with a suspected acute coronary syndrome that focused on the analysis of troponin-negative patients. Diagnostic classification of patients and the definition of troponin cutoffs were performed according to the new European Society of Cardiology/American College of Cardiology criteria. Blood was sampled serially and choline was measured using high-performance liquid chromatography mass spectrometry in whole blood. Patients were followed for 30 days. In patients with negative troponin I test results at admission (n = 250), choline was a predictor of cardiac death and nonfatal cardiac arrest (hazard ratio 6.0, p = 0.003), life-threatening arrhythmias (hazard ratio 3.75, p = 0.004), heart failure (hazard ratio 2.87, p = 0.002), and coronary angioplasty (hazard ratio 2.57, p = 0.001). In multivariate analysis of troponin-negative patients, choline was the strongest predictor of cardiac death or arrest (odds ratio 6.05, p = 0.01). Choline was not a marker for myocardial necrosis but indicated high-risk unstable angina in patients without acute myocardial infarction (sensitivity 86.4%, specificity 86.2%). Thus, an increased concentration of choline at hospital admission is a predictor of adverse cardiac events in patients with suspected acute coronary syndromes. Whole blood choline may be useful for early risk stratification of these patients, particularly if troponin results are negative on admission.

Serial plasma choline measurements after cardiac arrest in patients undergoing mild therapeutic hypothermia: a prospective observational pilot trial.

OBJECTIVE: Choline is related to phospholipid metabolism and is a marker for global ischaemia with a small reference range in healthy volunteers. The aim of our study was to characterize the early kinetics of plasma free choline in patients after cardiac arrest. Additionally, we investigated the potential of plasma free choline to predict neurological outcome. METHODS: Twenty patients admitted to our medical intensive care unit were included in this prospective, observational trial. All patients were enrolled between May 2010 and May 2011. They received post cardiac arrest treatment including mild therapeutic hypothermia which was initiated with a combination of cold fluid and a feedback surface cooling device according to current guidelines. Sixteen blood samples per patient were analysed for plasma free choline levels within the first week after resuscitation. Choline was detected by liquid chromatography-tandem mass spectrometry. RESULTS: Most patients showed elevated choline levels on admission (median 14.8 µmol/L; interquartile range; IQR 9.9-20.1) which subsequently decreased. 48 hours after cardiac arrest choline levels in all patients reached subnormal levels at a median of 4.0 µmol/L (IQR 3-4.9; p = 0.001). Subsequently, choline levels normalized within seven days. There was no significant difference in choline levels when groups were analyzed in relation to neurological outcome. CONCLUSIONS: Our data indicate a choline deficiency in the early postresucitation phase. This could potentially result in impaired cell membrane recovery. The detailed characterization of the early choline time course may aid in planning of choline supplementation trials. In a limited number of patients, choline was not promising as a biomarker for outcome prediction.

The role of myeloperoxidase (MPO) for prognostic evaluation in sensitive cardiac troponin I negative chest pain patients in the emergency department.

BACKGROUND: The diagnostic work-up of patients with acute chest pain in the emergency department (ED) is a challenging task. Serial troponin testing is required to rule-out acute myocardial infarction. OBJECTIVE: To evaluate the value of myeloperoxidase (MPO) testing in sensitive cardiac troponin I (cTnI) negative patients with suspected acute coronary syndromes (ACS) in the routine setting of an ED. METHODS: MPO was assessed in 432 consecutive patients presenting to the ED with ACS. In 266 patients, serial blood samples were available. After 6 weeks, major adverse cardiac events (MACE) were assessed. MPO and cTnI were measured in all available samples. For cTnI, a sensitive assay was used. Cut-off values were derived from an independent sample of 300 healthy volunteers. RESULTS: Incidence of MACE in our population was 13%. MPO levels revealed sensitivity (Sens) of 82.1% and specificity (Spec) of 37.2% for MACE compared with 60.7% Sens and 61.4% Spec for sensitive cTnI. In serial sensitive cTnI negative patients (n=218), MACE incidence was 6.4%. MPO continued to demonstrate significant discriminatory power for the prognosis of MACE. Multivariate analyses confirmed these findings. CONCLUSION: MPO has an independent prognostic value overall and most notably in patients tested negative with a higher sensitive cardiac troponin I assay. MPO could be a promising biomarker for the initial evaluation of patients in chest pain units and is worth further investigation.

Choline in acute coronary syndrome: an emerging biomarker with implications for the integrated assessment of plaque vulnerability.

Whole-blood choline, plasma choline and serum choline are emerging biomarkers in acute coronary syndrome related to coronary plaque instability with platelet thrombus formation and ischemia. Whole-blood choline is an early predictor for cardiac events, which adds to troponins, natriuretic peptides and inflammatory markers. Serum choline is highly predictive for myocardial infarction and discriminates high- from low-risk subgroups in troponin-positive patients. Choline is a candidate marker to aid decision making in the emergency room in the upcoming era of sensitive troponin tests and the growing need to differentiate between ischemic and nonischemic etiologies of troponin elevations. The integrated approach of in vitro choline measurement in combination with advanced techniques of in vivo choline imaging represents a novel future strategy for detecting vulnerable plaques. This paper provides an up-to-date review of choline in acute coronary syndrome including key aspects of pathophysiology, analytical methods, clinical studies and implications for the integrated assessment of plaque vulnerability.

Choline in whole blood and plasma: sample preparation and stability.

BACKGROUND: Choline is critical for a variety of biological functions and has been investigated as a biomarker for various pathological conditions including acute coronary syndrome. METHODS: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used to quantify choline in whole blood and plasma in freshly collected samples prepared with ultrafiltration or protein precipitation. We investigated the effects of preanalytical variables including types of anticoagulants and storage temperature and time. RESULTS: We observed no significant differences in whole-blood choline concentration in EDTA-anticoagulated vs heparin-anticoagulated samples: mean (SD) difference 0.9% (3.2%), P = 0.80. For plasma, choline concentrations with heparin in 5 of 12 volunteers were >10% higher than with EDTA, P = 0.01. One freeze-thaw cycle led to significant mean (SD) increases in choline concentrations in heparin whole blood, 19.3% (11.4%), P <0.01, and the effect was not significant for other sample types studied (P >0.33). For freshly collected samples stored at ambient temperature, choline concentrations in all types of samples increased with storage time. For EDTA whole blood, EDTA plasma, and heparin plasma, the choline concentration increased for the first 60 min and then stabilized. For heparin whole blood, the choline concentration continued to increase linearly with storage time for >4 h, at which time the choline concentrations were increased by approximately 50%. CONCLUSIONS: Sample collection, storage, and sample preparation procedures are critical for clinical measurements of choline in whole blood and plasma.

Effect of collection tube type and preanalytical handling on myeloperoxidase concentrations.

BACKGROUND: Myeloperoxidase (MPO) has shown potential as a marker for cardiovascular disease. Limited studies have been published with a variety of sample types, resulting in a wide range of MPO values. Little is known or understood about the impact of collection tube type and preanalytical handling of specimens for MPO determination. METHOD: MPO concentration was determined by use of the ARCHITECT(R) MPO research use assay, which is currently under development. Samples were collected into multiple anticoagulant collection tubes from donors and patients presenting to the emergency department with symptoms of acute coronary syndromes. Whole blood was stored on ice or at room temperature for predetermined time periods. We also evaluated serum and plasma after centrifugation followed by storage at room temperature, 2-8 degrees C, and below -10 degrees C. RESULTS: Baseline sample concentrations were dependent on collection tube type as well as handling conditions. MPO concentrations were consistently higher in samples collected in serum and heparin plasma tubes than in samples in EDTA or citrate tubes. Spike recovery was acceptable in all sera and plasma tested, indicating that the increased MPO concentrations were not due directly to an anticoagulant interference. CONCLUSIONS: The collection tube type and preanalytical handling are critical for accurate and consistent MPO measurement. The preferred anticoagulant and tubes are the EDTA or EDTA plasma preparation tube. MPO concentrations in samples collected in these tubes are stable before centrifugation as whole blood as well as plasma after processing.

Future biomarkers for detection of ischemia and risk stratification in acute coronary syndrome.

BACKGROUND: Evaluation of patients who present to the hospital with a complaint of chest pain or other signs or symptoms suggestive of acute coronary syndrome (ACS) is time-consuming, expensive, and problematic. Recent investigations have indicated that increases in biomarkers upstream from biomarkers of necrosis (cardiac troponins I and T), such as inflammatory cytokines, cellular adhesion molecules, acute-phase reactants, plaque destabilization and rupture biomarkers, biomarkers of ischemia, and biomarkers of myocardial stretch may provide earlier assessment of overall patient risk and aid in identifying patients with higher risk of an adverse event. APPROACH AND CONTENT: The purpose of this review is to provide an overview of the pathophysiology and clinical and analytical characteristics of several biomarkers that may have potential clinical utility to identify ACS patients. These biomarkers (myeloperoxidase, metalloproteinase-9, soluble CD40 ligand, pregnancy-associated plasma protein A, choline, ischemia-modified albumin, unbound free fatty acids, glycogen phosphorylase isoenzyme BB, and placental growth factor) have demonstrated promise and need to be more thoroughly evaluated for commercial development for implementation into routine clinical and laboratory practice. SUMMARY: Specifications that have been addressed for cardiac troponins and natriuretic peptides will need to be addressed with the same scrutiny for the biomarkers discussed in this review. They include validating analytical imprecision and detection limits, calibrator characterization, assay specificity and standardization, pre-analytical issues, and appropriate reference interval studies. Crossing boundaries from research to clinical application will require replication in multiple settings and experimental evidence supporting a pathophysiologic role and, ideally, interventional trials demonstrating that monitoring single or multiple biomarkers improves outcomes.