Sandwich Immunoluminometric Assay Research Articles

Approach to the Patient: "Utility of the Copeptin Assay".

Copeptin derives from the same precursor peptide preprovasopressin as arginine vasopressin (AVP). The secretion of both peptides is stimulated by similar physiological processes, such as osmotic stimulation, hypovolemia, or stress. AVP is difficult to measure due to complex preanalytical requirements and due to technical difficulties. In the last years, copeptin was found to be a stable, sensitive, and simple to measure surrogate marker of AVP release. Different immunoassays exist to measure copeptin. The 2 assays which have most often be used in clinical studies are the original sandwich immunoluminometric assay and its automated immunofluorescent successor. In addition, various enzyme-linked immunosorbent assay have been developed. With the availability of the copeptin assay, the differential diagnosis of diabetes insipidus was recently revisited. The goal for this article is therefore to first review the physiology of copeptin, and second to describe its use as marker for the differential diagnosis of vasopressin-dependent fluid disorders, mainly diabetes insipidus but also hyper- and hyponatremia. Furthermore, we highlight the role of copeptin as prognostic marker in other acute and chronic diseases.

Temporal Release of High-Sensitivity Cardiac Troponin T and I and Copeptin After Brief Induced Coronary Artery Balloon Occlusion in Humans.

Cardiac troponins (cTns) are the cornerstone of diagnosing acute myocardial infarction. There is limited knowledge on the duration of ischemia necessary to induce a measurable release of cTns or the very-early-release kinetics of cTns after an ischemic event. Copeptin may have a supplementary role in ruling out myocardial infarction early. We investigated the release of cTns and copeptin in the first hours after experimental balloon-induced ischemia in humans. Thirty-four patients (median age, 60 years [interquartile range, 51-64]; 15 men, 43%) with angiographically normal coronary arteries were randomly assigned into 4 groups with different durations of induced myocardial ischemia (0, 30, 60, 90 s). Ischemia was induced by inflating a balloon in the left anterior descending artery between the first and second diagonal branch. Blood was collected before balloon inflation (baseline) every 15 minutes for the first 3 hours, and every 30 minutes for the next 3 hours. The cTns were analyzed by 3 high-sensitivity (hs) cTn assays: hs-cTnT (Roche), hs-cTnI (Siemens), and hs-cTnI (Abbott). Copeptin was analyzed by a sandwich immunoluminometric assay. None of the patients had any complications. Increased cTn concentrations were detected by all 3 assays, and the magnitude of the increase was associated with the duration of ischemia. Increased hs-cTnI (Siemens) concentrations were first detectable 15 minutes after 90-s ischemia (median 43.7% increase) and increased more steeply and had a higher peak than the other assays. Copeptin levels did not significantly change. Using the cTnT, hs-cTnI (Siemens), and hs-cTnI (Abbott) concentrations at 0 and 180 minutes, 1 (11%), 0, and 0 patients from the 60-s ischemia group and 5 (63%), 2 (25%), and 1 (11%) from the 90-s ischemia group, respectively, fulfilled criteria for a biochemical myocardial infarction. This study is the first to report the early-release kinetics of cTn concentrations after different durations of experimental coronary balloon occlusion in humans. All assays detected a cTn increase after only 30 s of ischemia. hs-cTnI (Siemens) rose faster and reached a higher peak. Copeptin levels did not change significantly. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT03203057.

Copeptin as a novel biomarker for detecting early renal dysfunction after TAVI

Abstract Background Acute kidney injury (AKI) is one of the most prevalent (10–30%) complications after transcatheter aortic valve implantation (TAVI). Furthermore, AKI is accompanied by increased mortality, a higher incidence of dialysis and blood transfusion, and a prolonged hospital stay. Although measurement of serum creatinine is the gold standard in diagnosing AKI, changes in serum creatinine may lag behind compromised renal function. Arginine vasopressin (AVP), or antidiuretic hormone, is a nine-amino acid peptide member of the hypothalamo-neurohypophysial axis. Copeptin is the C-terminal moiety of the AVP precursor pre-proAVP that is secreted into the circulation. Recently, copeptin has been suggested to play a role in chronic kidney injury. We evaluated the value of copeptin in the prediction of AKI in patients undergoing TAVI. Methods All patients with severe aortic valve stenosis undergoing TAVI between May 2011 and May 2016 were included in our study. AKI was defined by the VARC-2 definition. Patients with no AKI and stage 1 AKI were compared with patients with stage 2 or 3 AKI. Routine laboratory parameters, including creatinine, were measured immediately after blood draw. Additionally, venous blood samples were collected on admission and after 24, 48, and 72 hours, processed immediately, and stored at −80°C until assay. The copeptin concentration in serum was measured by a sandwich immunoluminometric assay. Results Copeptin levels were available in 642 patients who were treated by TAVI in our centre from 2012–2016. AKI was detected in 113 patients (17.6%), including 61 patients with stage 1 (9.5%), 29 with stage 2 (4.5%), and 23 with stage 3 (3.6%). There were no differences among these patients in baseline measurements, but serum copeptin increased in all patients with AKI 24 h post-procedure according to the AKI stage: no AKI 34.5 (18.0–59.3 pmol/L), AKI stage 1: 68.7 (34.6–130.1 pmol/L); AKI stage 2: 96.0 (48.1–185.1 pmol/L); AKI stage 3: 154.9 (79.5–280.7 pmol/L); ANOVA p<0.001 (Fig. 1). Copeptin showed an earlier and sharper increase than creatinine (Fig. 1), with a negative predictive value of 0.97 to rule out AKI after 24 h. Conclusion AKI subsequent to TAVI is a common and harmful complication that occurred in almost every 5th patient (17.6%) in our cohort. AVP is secreted in response to hypotension, which commonly occurs during TAVI. In our cohort of TAVI patients, those who developed AKI after TAVI showed a rapid increase in copeptin that was earlier than that of creatinine. In light of these observations, copeptin could be a new parameter for detecting early renal dysfunction. Figure 1 Funding Acknowledgement Type of funding source: None

Elevated plasma copeptin levels identify the presence and severity of non-alcoholic fatty liver disease in obesity

IntroductionCopeptin is the stable surrogate marker of vasopressin (VP), which is released in response to elevated plasma osmolality or low blood pressure. Elevated plasma copeptin levels are associated with higher risk of insulin resistance-related disorders, such as type 2 diabetes (T2DM), metabolic syndrome (MS), and cardiovascular disease, and experimental reduction of circulating VP levels is shown to significantly decrease hepatic fat content in obese rats, independently from body adiposity. However, the association between copeptin and non-alcoholic fatty liver disease and steatohepatitis (NAFLD/NASH) in humans has not been explored yet. The aim of this study was to explore the relationship between plasma copeptin and the presence/severity of NAFLD/NASH.MethodsFor this study, we recruited 60 obese patients candidate to bariatric surgery for clinical purposes in which intraoperative liver biopsies were performed for diagnosing NAFLD/NASH. Circulating copeptin levels were also assessed in 60 age- and sex-comparable non-obese individuals without NAFLD at liver ultrasonography. Plasma copeptin was measured by sandwich immunoluminometric assay (Thermo Fisher Scientific).ResultsObese patients with biopsy-proven NAFLD (53%) had significantly higher copeptin levels than both obese individuals without NAFLD and non-obese subjects (ob/NAFLD+ 9.5 ± 4.9; ob/NAFLD− 6.4 ± 2.6; and non-ob/NAFLD− 7.4 ± 5.1 pmol/L; p = 0.004 and p = 0.01 respectively). Plasma copeptin concentration positively correlated with hepatic macro- and micro-vesicular steatosis (r = 0.36, p = 0.026; r = 0.31, p = 0.05), lobular inflammation (r = 0.37, p = 0.024) and significantly increased throughout degrees of NASH severity, as expressed as absence, borderline, and overt NASH at the liver biopsy (r = 0.35, p = 0.01). Greater circulating copeptin predicted the presence of NASH with OR = 1.73 (95% CI = 1.02–2.93) after multivariate adjustment for age, sex, renal function and presence of T2DM and MS components.ConclusionsIncreased plasma copeptin is independently associated with the presence and severity of NAFLD and NASH, pointing to a novel mechanism behind human fatty liver disease potentially modifiable by pharmacological treatment and lifestyle intervention.

Quantification of Relaxin-2 Connecting Peptide (Pro-RLX2) in Human Blood Samples.

The peptide hormone relaxin-2 is implicated in diverse physiological and pathophysiological processes. Several assays are available for quantification of human relaxin-2, but because stability of the mature peptide in serum is limited, measurement of the more stable connecting peptide (pro-RLX2) might be beneficial. Pro-RLX2 was measured in a sandwich immunoluminometric assay using 2 monoclonal antibodies. The concentration of pro-RLX2 was detected in healthy pregnant (n = 100) and healthy male and nonpregnant female (n = 81) subjects and compared with the concentration of mature relaxin-2 in a subset of samples. The pro-RLX2 immunoassay has an analytical and functional assay sensitivity (FAS) of 1.59 pmol/L and 1.7 pmol/L, respectively. The analyte is stable in EDTA plasma samples for 8 days at room temperature, dilutes in a linear fashion, and recovery was 103%. The assay system is not biased by common interfering substances. Measurement of 80% of plasma samples from healthy males and females is below the FAS {median 1.49 pmol/L [interquartile range (IQR) of 0.925-2.14 pmol/L]}, and no concentration difference between male and nonpregnant female plasma samples was observed. The median plasma concentration in healthy pregnant women is increased up to 562 pmol/L (IQR 341-789 pmol/L). During pregnancy, pro-RLX2 concentrations decrease with increasing gestation. The correlation coefficient with the R&D assay for mature relaxin-2 was 0.96 (P < 0.0001). Pro-RLX2 is stable in plasma of healthy individuals. Although samples of pregnant women are reliably measurable, most samples from healthy nonpregnant women and men are below the detection limit. Determination of pro-RLX2 concentrations might indicate rate of synthesis of relaxin-2 during pregnancy and therapeutic application of recombinant relaxin (Serelaxin).

Plasma C-type natriuretic peptide as a predictor for therapeutic response to metoprolol in children with postural tachycardia syndrome.

POTS is a global public-health disease, but predictor for therapeutic response to metoprolol in children with POTS is lacking. This study was designed to investigate predictive value of plasma C-type natriuretic peptide (CNP) in the therapeutic efficacy of metoprolol on postural tachycardia syndrome (POTS) in children. Totally 34 children with POTS and 27 healthy children were included in the study. The head-up test or head-up tilt test was used to check heart rate and blood pressure from supine to upright in subjects. A double antibody (competitive) sandwich immunoluminometric assay was used to detect plasma CNP. Metoprolol was used to treat children with POTS. The difference in plasma concentrations of CNP between responders and non-responders was compared. An ROC curve was used to analyze plasma CNP to predict efficacy of metoprolol on POTS in children. Plasma CNP in children with POTS was significantly higher than that of healthy children [(51.9 ± 31.4) vs. (25.1 ± 19.1) pg/ml, P <0.001]. Plasma CNP in responders to metoprolol was significantly higher than non-responders [(59.1 ± 33.5) vs. (34.8 ± 16.7) pg/ml, P = 0.037] before treatment. The ROC curve showed that area under the curve was 0.821 (95% CI 0.642–0.999). The cut-off value of plasma CNP > 32.55 pg/ml yielded a sensitivity of 95.8% and specificity of 70% in predicting therapeutic efficacy of metoprolol on POTS children. Plasma CNP might serve as a useful predictor for the therapeutic efficacy of metoprolol on POTS in children.

Usefulness of Plasma Copeptin as a Biomarker to Predict the Therapeutic Effectiveness of Metoprolol for Postural Tachycardia Syndrome in Children

Metoprolol is clinically used to treat postural tachycardia syndrome (POTS) in children, but its effectiveness is unsatisfactory. Biomarkers to predict therapeutic efficacy are needed. We aimed to explore changes in the plasma copeptin level for assessing the therapeutic efficacy of metoprolol for POTS in children. We included 49 children with POTS and 25 healthy children as controls. Patients received metoprolol for 1.5 to 3months. The plasma copeptin level was measured by sandwich immunoluminometric assay. The area under the receiver operating characteristic curve was used to explore the predictive value of the plasma copeptin level. The baseline plasma copeptin level was higher in children with POTS than controls (10.524 ± 2.016 vs 8.750 ± 1.419 pmol/L, p <0.001) and was lower for responders than nonresponders to metoprolol (9.377 ± 1.411 vs 12.054 ± 1.662 pmol/L, p= 0.003). The area under the receiver operating characteristic curve was 0.889 (95% confidence interval 0.799 to 0.980). With a baseline plasma copeptin level of 10.225 pmol/L as a cutoff, the sensitivity was 90.5% and specificity 78.6% in predicting the efficacy of metoprolol in children with POTS. In conclusion, the baseline plasma copeptin level can be used as a biomarker to predict the therapeutic effectiveness of metoprolol in children with POTS.

Plasma Copeptin and Therapeutic Effectiveness of Midodrine Hydrochloride on Postural Tachycardia Syndrome in Children

Midodrine hydrochloride is used clinically to treat children with postural tachycardia syndrome (POTS), but it is not effective in all patients. This study was designed to explore the changes in plasma copeptin and its predictive value in assessing the therapeutic efficacy of midodrine hydrochloride in children with POTS. The study included 33 children with POTS and 26 healthy children. The group with POTS received midodrine hydrochloride treatment for 1.5-3 months. The plasma copeptin was measured using a sandwich immunoluminometric assay. A receiver operating characteristic curve was used to explore the predictive value of plasma copeptin. The plasma copeptin in the group with POTS was significantly higher than that of the control group (10.827±2.459 pmol/L vs 8.845±1.471 pmol/L, P<.001). The plasma copeptin in responders to midodrine hydrochloride was significantly higher than that of nonresponders (12.082±1.998 pmol/L vs 9.646±2.301 pmol/L, P=.003). Receiver operating characteristic analysis on the predictive value of plasma copeptin showed that the area under the curve was 0.800 with a 95% CI of 0.640-0.959. Using a plasma copeptin level of 10.482 pmol/L as the cutoff point produced both favorite sensitivity (81.3%) and specificity (76.5%) in predicting the efficacy of midodrine hydrochloride therapy in children with POTS. Plasma copeptin may be considered as a predictive biomarker for the likelihood of successful treatment of children with POTS with midodrine hydrochloride.

Release Kinetics of Copeptin in Patients Undergoing Transcoronary Ablation of Septal Hypertrophy

The release kinetics of copeptin in patients with acute myocardial infarction (AMI) have been difficult to establish. We analyzed the release kinetics of copeptin in patients with hypertrophic obstructive cardiomyopathy undergoing transcoronary ablation of septal hypertrophy (TASH) as a model of AMI. We included 21 consecutive patients who underwent TASH. Blood samples were collected before and at 15, 30, 45, 60, 75, 90, and 105 min, and at 2, 4, 8, and 24 h after TASH. Serum copeptin was quantified by a sandwich immunoluminometric assay. All patients had copeptin concentrations below the 99th percentile at baseline. The median copeptin concentration was significantly increased at 30 min [16.0 pmol/L; interquartile range (IQR), 13.4-20.2 pmol/L], compared with the median baseline concentration (6.6 pmol/L; IQR, 5.3-8.3 pmol/L; P = 0.002). The copeptin concentration peaked 90 min after induction of myocardial infarction and returned to baseline concentrations (median, 8.2 pmol/L; IQR, 6.3-10.1) after 24 h, compared with the above baseline values (P = 0.06). Serum creatine kinase (CK) activities were significantly increased above baseline values by 1 day after TASH [median maximal postprocedural CK activity, 935.0 U/L (IQR, 545.5-1115.0 U/L); median baseline CK activity, 80.0 U/L (IQR, 63.5-109.0 U/L); P < 0.001]. Our results provide additional evidence that early rule-out of suspected AMI is possible by using the copeptin concentration in combination with cardiac troponin T.

Midregional Pro-Adrenomedullin as a Predictor for Therapeutic Response to Midodrine Hydrochloride in Children With Postural Orthostatic Tachycardia Syndrome

This study was designed to explore the predictive value of the midregional fragment of pro-adrenomedullin (MR-proADM) in assessing the therapeutic efficacy of midodrine hydrochloride for children with postural orthostatic tachycardia syndrome (POTS). Midodrine hydrochloride is an important therapeutic option for children with POTS. However, there has not been any method to predict response to the drug. The MR-proADM is produced in equimolar amounts to adrenomedullin (ADM), and directly reflects levels of the rapidly degraded active peptide, ADM. Fifty-seven children with POTS were designated as the POTS group. Twenty healthy children served as the control group. The children in the POTS group received midodrine hydrochloride treatment. The plasma concentration of MR-proADM was measured, using a sandwich immunoluminometric assay. A receiver-operating characteristic curve was used to explore the predictive value of MR-proADM. Plasma levels of MR-proADM were significantly higher in children with POTS (75.0 [62.5 to 96.0] pg/ml) than in the control group (58.5 [50.3 to 69.0] pg/ml). Plasma levels of MR-proADM in responders to midodrine hydrochloride was significantly higher than that of nonresponders (76.0 [66.0 to 91.0] pg/ml vs. 59.0 [54.0 to 65.5] pg/ml, p < 0.01]. A receiver-operating characteristic curve on the predictive value of MR-proADM showed that the area under the curve was 0.879 with a 95% confidence interval of 0.761 to 0.997. Using a cutoff value for MR-proADM of 61.5 pg/ml produced both high sensitivity (100%) and specificity (71.6%) in predicting the efficacy of midodrine hydrochloride therapy for treating POTS. MR-proADM can help guide midodrine hydrochloride therapy in the management of POTS in children.

Cardiac Biomarkers, Mortality, and Post-Traumatic Stress Disorder in Military Veterans

Post-traumatic stress disorder (PTSD) is gaining increasing recognition as a risk factor for morbidity and mortality. The aim of this study was to examine the impact of PTSD and abnormal cardiovascular biomarkers on mortality in military veterans. Eight hundred ninety-one patients presenting for routine echocardiography were enrolled. Baseline clinical data and serum samples for biomarker measurement were obtained and echocardiography was performed at the time of enrollment. Patients were followed for up to 7.5 years for the end point of all-cause mortality. Ninety-one patients had PTSD at the time of enrollment. There were 33 deaths in patients with PTSD and 221 deaths in those without PTSD. Patients with PTSD had a trend toward worse survival on Kaplan-Meier analysis (p = 0.057). Among patients with elevated B-type natriuretic peptide (>60 pg/ml), those with PTSD had significantly increased mortality (p = 0.024). Among patients with PTSD, midregional proadrenomedullin (MR-proADM), creatinine, and C-terminal proendothelin-1 were significant univariate predictors of mortality (p = 0.006, p = 0.024, and p = 0.003, respectively). In a multivariate model, PTSD, B-type natriuretic peptide, and MR-proADM were independent predictors of mortality. In patients with PTSD, MR-proADM was a significant independent predictor of mortality after adjusting for B-type natriuretic peptide, cardiovascular risk factors, cancer, and sleep apnea. Adding MR-proADM to clinical predictors of mortality increased the C-statistic from 0.572 to 0.697 (p = 0.007). In conclusion, this study demonstrates an association among PTSD, abnormal cardiac biomarker levels, and increased mortality.

Immunoluminometric assay for quantification of peroxiredoxin 4 in human serum

Background We describe the validation of a novel assay for the measurement of peroxiredoxin 4 (Prx4), a potentially secreted antioxidant peroxidase, in human serum. Methods A sandwich immunoluminometric assay (ILMA) was set up applying monoclonal antibodies against the amino-terminus of human Prx4. Prx4 levels have been determined in serum of healthy individuals and patients with sepsis and have been correlated to the clinically established sepsis marker procalcitonin (PCT). Results The sandwich ILMA detected Prx4 in a range between 0.5 and 128 arbitrary (arb.) U/L and had a functional assay sensitivity of 0.51 arb. U/L. Serum Prx4 was stable for at least 72 h at 4 °C and 21 °C and circulated in a complex of about 330 kDa as characterized by size-exclusion chromatography. Patients with sepsis ( n = 45; median, 7.7 arb. U/L) showed significantly higher Prx4 serum levels ( P < 0.0001) than healthy controls ( n = 274; median, 0.71 arb. U/L). Serum Prx4 was positively correlated to PCT ( r = 0.63, P < 0.0001). Conclusion The newly developed assay reliably detected Prx4 in human serum of healthy and critically ill subjects. Elevated Prx4 in serum of patients with various diseases might serve as a biomarker reflecting increased oxidative stress.

Incremental Value of Copeptin for Rapid Rule Out of Acute Myocardial Infarction

The purpose of this study was to examine the incremental value of copeptin for rapid rule out of acute myocardial infarction (AMI). The rapid and reliable exclusion of AMI is a major unmet clinical need. Copeptin, the C-terminal part of the vasopressin prohormone, as a marker of acute endogenous stress may be useful in this setting. In 487 consecutive patients presenting to the emergency department with symptoms suggestive of AMI, we measured levels of copeptin at presentation, using a novel sandwich immunoluminometric assay in a blinded fashion. The final diagnosis was adjudicated by 2 independent cardiologists using all available data. The adjudicated final diagnosis was AMI in 81 patients (17%). Copeptin levels were significantly higher in AMI patients compared with those in patients having other diagnoses (median 20.8 pmol/l vs. 6.0 pmol/l, p < 0.001). The combination of troponin T and copeptin at initial presentation resulted in an area under the receiver-operating characteristic curve of 0.97 (95% confidence interval: 0.95 to 0.98), which was significantly higher than the 0.86 (95% confidence interval: 0.80 to 0.92) for troponin T alone (p < 0.001). A copeptin level <14 pmol/l in combination with a troponin T < or =0.01 microg/l correctly ruled out AMI with a sensitivity of 98.8% and a negative predictive value of 99.7%. The additional use of copeptin seems to allow a rapid and reliable rule out of AMI already at presentation and may thereby obviate the need for prolonged monitoring and serial blood sampling in the majority of patients. (Advantageous Predictors of Acute Coronary Syndromes Evaluation [APACE]; NCT00470587).

Adrenomedullin and endothelin-1 are related to inflammation in chronic heart failure

Adrenomedullin (ADM) and endothelin-1 (ET-1) are novel promising peptide biomarkers in chronic heart failure (CHF). According to recent studies among their pleiotropic effect they play roles in the regulation of inflammation. The aim of the study was to measure the above mentioned two vasoactive peptides in parallel in a well characterized population of patients with CHF, and study their associations with inflammatory markers. A total of 186 patients (138 male, 48 female) with <45% left ventricular ejection fraction (LVEF), and without acute inflammatory disease, were enrolled. Plasma midregional-proADM (MR-proADM) and C-terminal-proET-1 (CT-proET-1) were determined by a novel sandwich immunoluminometric assay. Increased MR-proADM and CT-proET-1 plasma levels were measured in patients with severe CHF (NYHA III-IV) as compared to the group of NYHA I-II (p<0.0001). MR-proADM and CT-proET-1 levels showed significant negative correlation with serum albumin and prealbumin levels (p<or=0.0001), while positive correlations were found with levels of CRP, TNF-alpha, soluble TNF receptors and IL-6 (p<or=0.0001). In multiple linear regression models after adjustments for several potential confounders (disease severity [LV-EF, NYHA classes, NT-proBNP], ion and water homeostasis [sodium and presence of peripheral oedema], renal function [serum creatine]) the relationship between ADM and albumin, CRP, soluble TNF receptors and between ET-1 and CRP, TNF receptors and IL-6 remained significant. Vasoregulation and inflammation may be connected in heart failure patients independently of the disease severity. The observed link may contribute to the understanding of the complex pathomechanism in CHF.

Immunoluminometric Assay for Measurement of the C-Terminal Endothelin-1 Precursor Fragment in Human Plasma

Endothelin-1 (ET-1), a potent vasoconstrictor, is difficult to measure because of its instability and its binding to receptors and plasma proteins. We report a rapid, robust way to indirectly quantify ET-1 release by measuring the C-terminal ET-1 precursor fragment (CT-proET-1) without an extraction step. In plasma samples from healthy individuals, patients with congestive heart failure (CHF), and patients with sepsis, we measured CT-proET-1 with a sandwich immunoluminometric assay that uses 2 polyclonal antibodies to amino acids 168-212 of pre-proET-1. We also correlated CT-proET-1 concentrations with bigET-1 measurements. The assay yielded results within 3 h and showed linear dilution with an analytical detection limit of 0.4 pmol/L and an interlaboratory CV <10% for values >10 pmol/L. Ex vivo CT-proET-1 was stable (<10% loss of immunoreactivity) in EDTA-, heparin-, and citrate-plasma for at least 4 h at room temperature, 6 h at 4 degrees C, and in EDTA-plasma for at least 6 months at -20 degrees C. CT-proET-1 values followed a gaussian distribution in healthy individuals (mean, 44.3 pmol/L; range, 10.5-77.4 pmol/L) without significant differences between males and females. The correlation coefficient for CT-proET-1 vs age was 0.25 (P <0.0001). CT-proET1 was significantly (P <0.0001) increased in patients with CHF (median, 104 pmol/L; range, 50.8-315 pmol/L) and patients with sepsis (median, 189 pmol/L; range, 34.6-855 pmol/L). The correlation between CT-proET-1 and bigET-1 for 43 samples was 0.80 (P <0.0001). CT-proET-1 measurement is a rapid and easy method for indirectly assessing the release of ET-1 in critically ill patients.

Assay for the Measurement of Copeptin, a Stable Peptide Derived from the Precursor of Vasopressin

Arginine vasopressin (AVP) is a key regulator of water balance, but its instability makes reliable measurement difficult and precludes routine use. We present a method for quantifying AVP release by use of copeptin, a glycopeptide comprising the C-terminal part of the AVP prohormone. We measured copeptin in 50-microL serum and plasma samples from healthy individuals and from critically ill patients with sepsis. Our sandwich immunoluminometric assay used 2 polyclonal antibodies to amino acids 132-164 of pre-provasopressin. The assay yielded results within 3 h. The analytical detection limit was 1.7 pmol/L, and the interlaboratory CV was <20% for values >2.25 pmol/L. The assay was linear on dilution of the analyte. Ex vivo copeptin stability (<20% loss of analyte) for at least 7 days at room temperature and 14 days at 4 degrees C was shown for serum and EDTA-, heparin-, and citrate plasma. Copeptin (median, 4.2 pmol/L; range, 1-13.8 pmol/L) was detectable in 97.5% of 359 healthy individuals and was not associated with age. Median concentrations were considerably higher in men than women, increased significantly after exercise, and were influenced by fasting and water load. Copeptin was significantly (P <0.001) increased in 60 critically ill patients with sepsis (median, 79.5 pmol/L; range, 10.6-228.0 pmol/L). The correlation between copeptin and AVP for 110 samples was r = 0.78 (P <0.0001). Copeptin is stable for days after blood withdrawal and can be quickly and easily measured. The copeptin assay may be a useful alternative to direct measurement of AVP concentration.

Measurement of Midregional Proadrenomedullin in Plasma with an Immunoluminometric Assay

Adrenomedullin (ADM) is a potent vasodilatory peptide, and circulating concentrations have been described for several disease states, including dysfunction of the cardiovascular system and sepsis. Reliable quantification has been hampered by the short half-life, the existence of a binding protein, and physical properties. Here we report the technical evaluation of an assay for midregional pro-ADM (MR-proADM) that does not have these problems. MR-proADM was measured in a sandwich immunoluminometric assay using 2 polyclonal antibodies to amino acids 45-92 of proADM. The reference interval was defined in EDTA plasma of 264 healthy individuals (117 male, 147 female), and increased MR-proADM concentrations were found in 95 patients with sepsis and 54 patients with cardiovascular disease. The assay has an analytical detection limit of 0.08 nmol/L, and the interassay CV was <20% for values >0.12 nmol/L. The assay was linear on dilution with undisturbed recovery of the analyte. EDTA-, heparin-, and citrate-plasma samples were stable (<20% loss of analyte) for at least 3 days at room temperature, 14 days at 4 degrees C, and 1 year at -20 degrees C. MR-proADM values followed a gaussian distribution in healthy individuals with a mean (SD) of 0.33 (0.07) nmol/L (range, 0.10-0.64 nmol/L), without significant difference between males or females. The correlation coefficient for MR-proADM vs age was 0.50 (P < 0.001). MR-proADM was significantly (P < 0.001) increased in patients with cardiovascular disease [median (range), 0.56 (0.08-3.9) nmol/L] and patients with sepsis [3.7 (0.72-25.4) nmol/L]. MR-proADM is stable in plasma of healthy individuals and patients. MR-proADM measurements may be useful for evaluating patients with sepsis, systemic inflammation, or heart failure.

Serum Proteomic Fingerprinting Discriminates Between Clinical Stages and Predicts Disease Progression in Melanoma Patients

Currently known serum biomarkers do not predict clinical outcome in melanoma. S100-beta is widely established as a reliable prognostic indicator in patients with advanced metastatic disease but is of limited predictive value in tumor-free patients. This study was aimed to determine whether molecular profiling of the serum proteome could discriminate between early- and late-stage melanoma and predict disease progression. Two hundred five serum samples from 101 early-stage (American Joint Committee on Cancer [AJCC] stage I) and 104 advanced stage (AJCC stage IV) melanoma patients were analyzed by matrix-assisted laser desorption/ionisation (MALDI) time-of-flight (ToF; MALDI-ToF) mass spectrometry utilizing protein chip technology and artificial neural networks (ANN). Serum samples from 55 additional patients after complete dissection of regional lymph node metastases (AJCC stage III), with 28 of 55 patients relapsing within the first year of follow-up, were analyzed in an attempt to predict disease recurrence. Serum S100-beta was measured using a sandwich immunoluminometric assay. Analysis of 205 stage I/IV serum samples, utilizing a training set of 94 of 205 and a test set of 15 of 205 samples for 32 different ANN models, revealed correct stage assignment in 84 (88%) of 96 of a blind set of 96 of 205 serum samples. Forty-four (80%) of 55 stage III serum samples could be correctly assigned as progressors or nonprogressors using random sample cross-validation statistical methodologies. Twenty-three (82%) of 28 stage III progressors were correctly identified by MALDI-ToF combined with ANN, whereas only six (21%) of 28 could be detected by S100-beta. Validation of these findings may enable proteomic profiling to become a valuable tool for identifying high-risk melanoma patients eligible for adjuvant therapeutic interventions.