Time-resolved fluorescence based direct two-site apoA-I immunoassays and their clinical application in patients with suspected obstructive coronary artery disease.

Objective: High-density lipoprotein (HDL) is a heterogeneous group of subpopulations differing in protein/lipid composition and in their anti-atherogenic function. There is a lack of assays that can target the functionality of HDL particles related to atherosclerosis. The objective of this study was to construct two-site apolipoprotein A-I (apoA-I) assays and to evaluate their clinical performance in patients with suspected obstructive coronary artery disease (CAD). Approach and results: Direct two-site apoA-I assays (named 109-121 and 110-525) were developed to identify the presence of apoA-I in the HDL of patients with CAD using apoA-I antibodies as a single-chain variable fragment fused with alkaline phosphatase. ApoA-I109-121 and apoA-I110-525 were measured in 197 patients undergoing coronary computed tomography angiography (CTA) and myocardial positron emission tomography perfusion imaging due to suspected obstructive CAD. Among patients not using lipid-lowering medication (LLM, n = 125), the level of apoA-I110-525 was higher in the presence than in the absence of coronary atherosclerosis [21.88 (15.89-27.44) mg/dl vs. 17.66 (13.38-24.48) mg/dl, P = 0.01)], whereas there was no difference in apoA-I109-121, HDL cholesterol, and apoA-I determined using a polyclonal apoA-I antibody. The levels of apoA-I109-121 and apoA-I110-525 were similar in the presence or absence of obstructive CAD. Among patients not using LLM, apoA-I110-525 adjusted for age and sex identified individuals with coronary atherosclerosis with a similar accuracy to traditional risk factors [area under the curve [AUC] (95% CI): 0.75(0.66-0.84) 0.71 (0.62-0.81)]. However, a combination of apoA-I110-525 with risk factors did not improve the accuracy [AUC (95% CI): 0.73 (0.64-0.82)]. Conclusion: Direct two-site apoA-I assays recognizing heterogeneity in reactivity with apoA-I could provide a potential approach to identify individuals at a risk of coronary atherosclerosis. However, their clinical value remains to be studied in larger cohorts.

Cardiac troponin elevations in marathon runners. Role of coronary atherosclerosis and skeletal muscle injury. The MaraCat Study.

BACKGROUND: Marathon running is associated with transient risk of sudden cardiac death and high cardiac troponin levels are common after race. There is limited data whether coronary atherosclerosis or skeletal muscle injury are related to troponin release caused by strenuous exercise. We aimed to assess whether coronary artery calcification (CAC), plaque vulnerability or skeletal muscle injury relate to cardiac troponin T (cTnT) elevations after marathon race. METHODS: In this observational study, 40 male runners participating in Paavo Nurmi 2018 Marathon were recruited with an open email invitation to evaluate the prevalence of post-race cTnT elevations and their predictors. In addition to baseline and post-race laboratory investigations, 28 runners aged >44 years underwent CAC measurement with computed tomography. Coronary plaque vulnerability was evaluated by free pregnancy-associated plasma protein A (fPAPP-A) concentration and skeletal muscle injury by skeletal troponin I (skTnI) measurement. RESULTS: The post-marathon cTnT concentrations rose above the normal reference limit in 38 (95%) participants. A 10-fold increase in skTnI concentrations was observed and elevated post-race values were seen in all participants. The correlation between the post-race cTnT and post-race skTnI (rs = -0.26, p = 0.11) was non-significant. CAC was detected (Agatston score > 0) in 15 (53.6%) participants, with a median score of 2.0 (interquartile range [IQR] 80). There was no correlation between cTnT with CAC score or post-race fPAPP-A levels. CONCLUSIONS: Asymptomatic cardiac troponin elevations are common after prolonged strenuous exercise, but are not related to markers of coronary atherosclerosis, plaque vulnerability or skeletal muscle injury.

Direct Immunoassay for Free Pregnancy-Associated Plasma Protein A (PAPP-A).

BACKGROUND: Pregnancy-associated plasma protein A (PAPP-A), especially in its noncomplexed form (fPAPP-A), is linked to vulnerable atherosclerotic plaques and risk of cardiac events. An assay for sensitive detection of fPAPP-A has been lacking. Our aim was to develop and validate a direct fPAPP-A assay to meet this need. METHODS: Monoclonal antibodies binding exclusively fPAPP-A were produced by immunizing mice with recombinant PAPP-A. In the optimized immunoassay, we used an fPAPP-A-specific capture antibody together with a lanthanide-chelate-labeled monoclonal antibody recognizing all PAPP-A forms. The assay was evaluated with CLSI guidelines and compared to a 2-assay subtractive fPAPP-A approach. Clinical performance was assessed with acute coronary syndrome patients. RESULTS: The limits of detection and quantitation were 0.4 mIU/L and 1.3 mIU/L, respectively, and the assay was linear up to 1000 mIU/L (R2 = 0.999). Both serum and heparin plasma were suitable matrices, and the complexed form of PAPP-A caused no significant interference. Correlation between the developed assay and the 2-assay approach was fair (Pearson's r = 0.819). Median concentration in healthy individuals was 1.0 mIU/L. fPAPP-A concentration was higher in patients who had myocardial infarction or died during the 1-year follow-up period than in those who did not (1.13 mIU/L vs 0.82 mIU/L, P = 0.008, model adjusted with age and sex). fPAPP-A measured with this direct assay predicted this end point as well as (follow-up 1 year) or better (30 days) than the 2-assay fPAPP-A alone or in combination with cTnI. CONCLUSIONS: The new assay enables sensitive and reliable measurement of low cardiac-related fPAPP-A concentrations from blood samples.

Integrated Acoustic Separation, Enrichment, and Microchip Polymerase Chain Reaction Detection of Bacteria from Blood for Rapid Sepsis Diagnostics.

This paper describes an integrated microsystem for rapid separation, enrichment, and detection of bacteria from blood, addressing the unmet clinical need for rapid sepsis diagnostics. The blood sample is first processed in an acoustophoresis chip, where red blood cells are focused to the center of the channel by an acoustic standing wave and sequentially removed. The bacteria-containing plasma proceeds to a glass capillary with a localized acoustic standing wave field where the bacteria are trapped onto suspended polystyrene particles. The trapped bacteria are subsequently washed while held in the acoustic trap and released into a polymer microchip containing dried polymerase chain reaction (PCR) reagents, followed by thermocycling for target sequence amplification. The entire process is completed in less than 2 h. Testing with Pseudomonas putida spiked into whole blood revealed a detection limit of 1000 bacteria/mL for this first-generation analysis system. In samples from septic patients, the system was able to detect Escherichia coli in half of the cases identified by blood culture. This indicates that the current system detects bacteria in patient samples in the upper part of the of clinically relevant bacteria concentration range and that a further developed acoustic sample preparation system may open the door for a new and faster automated method to diagnose sepsis.