Qiu-Ping Qin

Circulating Pregnancy-Associated Plasma Protein A Predicts Outcome in Patients With Acute Coronary Syndrome but No Troponin I Elevation

Background—Risk stratification in troponin (cTn)-negative acute coronary syndrome (ACS) remains a clinical challenge. We investigated the predictive value of circulating pregnancy-associated plasma protein A (PAPP-A), a novel marker of atherosclerotic plaque activity, in these patients. Methods and Results—Two hundred consecutive hospitalized ACS patients were included, of whom 136 (69 men and 67 women; mean±SD age, 66±16 years) remained cTnI-negative for up to 24 hours. PAPP-A was measured at admission, 6 to 12 hours, and 24 hours. During 6-month follow-up, 26 (19.1%) of the cTnI-negative patients reached a primary end point (cardiovascular death, myocardial infarction, or revascularization). At a cutoff level of 2.9 mIU/L, elevated PAPP-A was an independent predictor of adverse outcome (adjusted risk ratio [RR], 4.6; 95% confidence interval, 1.8 to 11.8; P =0.002). Another independent predictor was admission CRP >2.0 mg/L (RR, 2.6; P =0.03). Conclusions—Measurement of plasma PAPP-A, a zinc-binding matrix metalloproteinase, is a strong independent predictor of ischemic cardiac events and need of revascularization in patients who present with suspected myocardial infarction but remain troponin negative.

Release Patterns of Pregnancy Associated Plasma Protein A (PAPP-A) in Patients with Acute Coronary Syndromes

Objective : Pregnancy associated plasma protein A (PAPP-A) has recently been shown to be associated with acute coronary syndromes (ACS). The goal of this study was to investigate its release patterns in patients with ACS. Design : PAPP-A concentrations in plasma samples serially collected after admissions from 15 patients with ACS were measured. The levels of PAPP-A were compared with a reference range determined from 80 normal subjects. The associations between PAPP-A and myoglobin (Mb), C-reactive protein (CRP), fatty-acid-binding protein (FABP) and creatine kinase MB (CK-MB) were determined. Results : Various release patterns were observed with 2-10-fold changes of PAPP-A in the different patients. Increases in PAPP-A levels above the reference range could appear early at 2 h or late at 30 h after onset of chest pain. Only in 4 of the 15 cases were significantly elevated PAPP-A levels detected before 6 h after onset. Elevations early after admission showed rapid decline whereas later elevations were more persistent. No associations between PAPP-A and Mb, CRP, FABP and CK-MB were found. However, a weak but significant association to cardiac troponin I (cTn I) was found. Conclusion : PAPP-A is an additional marker for ACS, but does not seem to be a useful early marker for acute myocardial infarction (AMI). The possible clinical utility of PAPP-A calls for extensive studies of chest pain patients using serial sampling combined with short- and long-term outcome studies.

Time-resolved Fluorescence Resonance Energy Transfer Assay for Point-of-Care Testing of Urinary Albumin

BACKGROUND Microalbuminuria is an established early marker of diabetic nephropathy and an important cardiovascular risk factor in diabetes and hypertension. We aimed to develop a rapid point-of-care assay for the measurement of urine albumin. METHODS The competitive homogeneous assay used an albumin-specific monoclonal antibody labeled with a stable fluorescent europium chelate as donor and an albumin labeled with cyanine 5 (Cy5) as acceptor. The assay was performed at room temperature in single microtitration wells that contained all the required dry-form reagents. The close proximity between the two labels in the immune complex allowed fluorescence resonance energy to be transferred from the pulse-excited europium chelate to the acceptor Cy5. The emission of long-lived energy transfer signal from the sensitized Cy5 was measured at 665 nm with time-resolved fluorometry that eliminated short-lived background. RESULTS The assay procedure required 12 min for a 10- micro L urine sample. The working range was from 10 to approximately 320 mg/L, and the lower limit of detection was 5.5 mg/L. The within- and between-run CVs were 6.9-10% and 7.5-13%, respectively. Recovery was 103-122%. The assay correlated well (r(2) = 0.98; n = 37) with a laboratory-based immunoassay, although mean (SD) results were 7 (29)% lower. CONCLUSIONS The speed and ease of performance of this assay recommend it for near-patient use. The assay is the first to combine a fluorescence resonance energy transfer-type rapid competitive assay with an all-in-one dry reagent.

Pregnancy‐associated plasma protein A: A biomarker in acute ST‐elevation myocardial infarction (STEMI)

Background. Elevated circulating levels of pregnancy‐associated plasma protein A (PAPP‐A), a novel marker of atherosclerotic plaque instability, are associated with increased risk of future cardiac events in patients with acute coronary syndromes (ACS). However, little is known of the kinetics or clinical significance of circulating PAPP‐A after plaque rupture in acute ST‐elevation myocardial infarction (STEMI). Aim. To evaluate the 48‐hour release of pregnancy‐associated plasma protein A (PAPP‐A) and its association with 12‐month outcome in patients with acute ST‐elevation myocardial infarction (STEMI). Methods. Sixty‐two consecutive STEMI patients were included (40 men and 22 women, median age 67.5 years (range 34–84)), of whom 54 (87.1%) received reperfusion therapy. PAPP‐A was measured at admission and 6–12, 24 and 48 hours thereafter. In 14 patients, samples were obtained also at 1, 2 and 4 hours. Results. There was an early peak of circulating PAPP‐A during the first 12 hours from symptom onset, followed by rapid normalization. A second, late PAPP‐A elevation was noticed in 20/62 patients (32.3%). Admission PAPP‐A >10.0 mIU/L (highest tertile) was associated (P = 0.049) with increased 12‐month risk of cardiovascular death or non‐fatal myocardial infarction. Moreover, the combination of failed early reperfusion together with late PAPP‐A elevation was strongly (7/13 versus 10/49 patients, P = 0.016) associated with adverse outcome. Admission PAPP‐A did not correlate with admission C‐reactive protein or cardiac troponin I. Conclusions. PAPP‐A is elevated early in STEMI and then declines rapidly, a pattern consistent with release from the ruptured plaque. The variability of PAPP‐A kinetics at 48 hours reflects the success of reperfusion. This study also shows that PAPP‐A may have prognostic value in STEMI.

Dual-label time-resolved immunofluorometric assay for simultaneous determination of pregnancy-associated plasma protein A and free β-subunit of human chorionic gonadotrophin

Using time-resolved fluorometry, a simple one-step dual-label immunometric assay has been developed, which allows simultaneous determination of pregnancy-associated plasma protein A (PAPP-A) and free beta-subunit of human chorionic gonadotrophin (beta-hCG) in first-trimester maternal serum samples. Two monoclonal antibodies were biotinylated and immobilized onto the surface of streptavidin-coated microtitration plates, and used to capture PAPP-A and beta-hCG. respectively. Europium (Eu) and Samarium (Sm) chelates were conjugated to two additional monoclonal antibodies acting as detection antibodies for PAPP-A and beta-hCG. The assay was performed using a 4-h one-step format. The within-run precision with buffer-based calibrators was below 8% over the working range of PAPP-A (40-10000 mIU/l) and beta-hCG (7.3-525 micrograms/l) and no hook effect was observed. The intra- and inter-assay coefficients of variation were below 7.1% for serum samples. PAPP-A and beta-hCG concentrations measured by the dual assay in 39 first-trimester serum samples correlated excellently with those obtained by DELFIA single-label PAPP-A (r = 0.997) and the beta-hCG part (r = 0.993) of the DELFIA AFP/beta hCG dual-label assay.

Free vs Total Pregnancy-Associated Plasma Protein A (PAPP-A) as a Predictor of 1-Year Outcome in Patients Presenting with Non-ST-Elevation Acute Coronary Syndrome

BACKGROUND The free fraction of pregnancy-associated plasma protein A (FPAPP-A) was found to be the PAPP-A form released to the circulation in acute coronary syndrome (ACS). We estimated the prognostic value of FPAPP-A vs total PAPP-A (TPAPP-A) concentrations in forecasting death and nonfatal myocardial infarction (combined endpoint) in patients with non-ST-elevation ACS. METHODS We recruited 267 patients hospitalized for symptoms consistent with non-ST-elevation ACS and followed them for 12 months. FPAPP-A, TPAPP-A, C-reactive protein (CRP), and cardiac troponin I (cTnI) were measured at admission; cTnI was also measured at 6-12 h and 24 h. Because of the recently shown interaction between PAPP-A and heparin, we excluded patients treated with any heparin preparations before the admission blood sampling. RESULTS During the follow-up, 57 (21.3%) patients met the endpoint (22 deaths and 35 nonfatal myocardial infarctions). According to FPAPP-A (<1.27, 1.27-1.74, >1.74 mIU/L) and TPAPP-A (<1.98, 1.98-2.99, >2.99 mIU/L) tertiles, this endpoint was met by 12 (13.5%), 18 (20.2%), 27 (30.3%) (P = 0.02), and 17 (19.1%), 17 (19.1%), 23 (25.8%) (P = 0.54) patients, respectively. After adjusting for age, sex, diabetes, previous myocardial infarction, and ischemic electrocardiogram (ECG) findings, FPAPP-A >1.74 mIU/L [risk ratio (RR) 2.0; 95% CI 1.0-4.1, P = 0.053), increased cTnI, and CRP >/=2.0 mg/L were independent predictors of an endpoint. The prognostic performance of TPAPP-A was inferior to that of FPAPP-A. CONCLUSIONS FPAPP-A seems to be superior as a prognostic marker compared to TPAPP-A, giving independent and additive prognostic information when measured at the time of admission in patients hospitalized for non-ST-elevation ACS.

Studies on the effects of heparin products on pregnancy-associated plasma protein A

BACKGROUND Intravenous low molecular weight (LMWH) and unfractionated heparin (UFH) increase the circulating concentrations of pregnancy-associated plasma protein A (PAPP-A), a novel cardiac risk marker, in haemodialysis and coronary angiography patients. METHODS To further investigate the mechanisms of heparin effects, free PAPP-A was analysed in serial serum samples collected during haemodialysis (intravenous LMWH), carotid endarterectomy or abdominal aortic aneurysm surgery (intravenous UFH), treatment at intensive care unit (subcutaneous LMWH), and coronary angiography (intravenous bivalirudin). PAPP-A was extracted from plaque tissue samples of endarterectomy and aneurysm patients. The interaction between heparin products and free PAPP-A was studied with gel filtration. RESULTS After intravenous UFH and LMWH free PAPP-A increased significantly but bivalirudin had no effect. After LMWH bolus in haemodialysis patients 85% of free PAPP-A was cleared with a half-life of 13.1 min and the rest with a half-life of 96.6 min. Subcutaneous LMWH led to lower and slower free PAPP-A elevation. PAPP-A extracted from plaque tissues was in free form and extraction was strongly enhanced by LMWH. Heparin products increased the molecular size of free PAPP-A. CONCLUSIONS The heparin-induced PAPP-A elevation is seen in various patients and should be taken into account when PAPP-A is studied as a biomarker.

Time-resolved immunofluorometric assay of pregnancy-associated plasma protein A in maternal serum screening for Down's syndrome in first trimester of pregnancy

A low maternal serum concentration of pregnancy associated plasma protein-A (MS-PAPP-A) in the first trimester has been suggested as a marker for the presence of a Downs syndrome (DS) fetus. We developed a time-resolved immunofluorometric assay (TrIFMA) for PAPP-A with a sensitivity < 3.9 mIU/l. In the 7-12 gestational weeks interval the median multiples of the median (MoM) was 0.57 (95%-confidence interval; 0.47-0.99) in DS pregnancies (n = 29) and lower than in controls (n = 223) (P < 0.005). The efficiency of MS-PAPP-A alone was evaluated using empirical receiver-operator-characteristics (ROC) and a sensitivity of about 25% was found for a false-positive rate of about 10% in the 7-12 gestational weeks interval. In parameterized ROC analysis a sensitivity of 9% was found for a false-positive rate of 5%. The TrIFMA PAPP-A assay seems to fulfil the quality criteria for an assay to be used in large-scale serum screening for Downs syndrome.

Dry-Reagent Double-Monoclonal Assay for Cystatin C

BACKGROUND Cystatin C is a low molecular weight cysteine proteinase inhibitor whose plasma or serum concentrations have been shown to be better correlated with glomerular filtration rate than serum creatinine concentrations. Routine assays for cystatin C are based on use of polyclonal antibodies and immunoturbidimetric and nephelometric designs. This study aimed to develop a double-monoclonal immunoassay for cystatin C. METHODS We tested functionality of 42 2-site antibody combinations involving 7 monoclonal antibodies with recombinant and plasma cystatin C. We developed a heterogeneous assay using 2 antibodies selected to give the best analytical performance. The assay used a dilution step and was based on a dry-reagent, all-in-one immunoassay concept with time-resolved fluorometry. The assay was performed on an automated immunoanalyzer in single wells that contained all the required assay components. We used heparin-derived plasma samples for methodological evaluation of the assay. RESULTS From a relative epitope map involving 7 cystatin C-specific antibodies, we selected a pair of antibodies for a 2-site sandwich-type dry-reagent assay. Total assay time was 15 min, and 10 microL of a 100-fold diluted sample was used. The analytical detection limit (background + 3SD) and functional detection limit (CV 20%) were 0.01 mg/L and 0.02 mg/L, respectively. Within-run and total assay imprecision were <4.7% and <5.6% (at 0.84-3.2 mg/L), respectively, and plasma recoveries of added cystatin C were 94%-110%. Regression analysis with the Roche particle-enhanced immunoturbidimetric method yielded the following (SD): slope, 1.391 (0.029); y-intercept, -0.152 (0.045) mg/L; S(y logical or, bar belowx)=0.294 mg/L (n=131). CONCLUSIONS The developed assay enables rapid and reliable measurement of cystatin C.

Performance of immunofluorometric point-of-care assays for free pregnancy-associated plasma protein A detection in whole blood samples.

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