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.

BACKGROUND: Cardiac troponin (cTn) is an established marker of myocardial infarction. Pronounced heterogeneity and the minute amounts released into the circulation constitute significant challenges for cTn detection. Recently, autoantibody formation to cTn was shown to be common and to interfere with immunoassay performance. In this study, we investigated cTn autoantibodies and cardiac troponin I (cTnI) in acute coronary syndrome (ACS) patients over a 1-year period after the index event.

The analysis of extracellular vesicles (EVs) typically requires tedious and time-consuming isolation process from bio-fluids. We developed a nanoparticle-based time resolved fluorescence immunoassay (NP-TRFIA) that uses biotinylated antibodies against the proteins of tetraspanin family and tumor-associated antigens for capturing EVs from urine samples and cell culture supernatants without the need for isolation. The captured-EVs were detected either with Eu 3+ -chelate or Eu 3+ -doped nanoparticle-based labels conjugated either to antibodies against the tetraspanins or lectins targeting the glycan moieties on EVs surface. The NP-TRFIA demonstrated specific capturing and detection of EVs by antibodies and lectins. Lectin-nanoparticle based assays showed 2–10 fold higher signal-to-background ratio compared with lectin-chelate assays. The nanoparticle assay concept allowed surface glycosylation profiling of the urine derived-EVs with lectins. It was also applied to establish an assay showing differential expression of tumor-associated proteins on more aggressive (higher ITGA3 on DU145- and PC3-EVs) compared to less aggressive (higher EpCAM on LNCaP-EVs) PCa- cell lines derived-EVs. This NP-TRFIA can be used as a simple tool for analysis and characterization of EVs in urine and cell culture supernatants. Such approach could be useful in identification of disease-specific markers on the surface of patient-derived urinary EVs.

Introduction: The importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine. Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect the earliest perturbations indicative of disease, minimizing therapeutic intervention or even enabling disease reversal. Protein biomarkers are of particular interest given their central role in biochemical pathways. Hence, capabilities to analyze multiple protein biomarkers in one assay are highly interesting for biomedical research. Areas covered: We here review multiple methods that are suitable for robust, high throughput, standardized, and affordable analysis of protein biomarkers in a multiplex format. We describe innovative developments in immunoassays, the vanguard of methods in clinical laboratories, and mass spectrometry, increasingly implemented for protein biomarker analysis. Moreover, emerging techniques are discussed with potentially improved protein capture, separation, and detection that will further boost multiplex analyses. Expert commentary: The development of clinically applied multiplex protein biomarker assays is essential as multi-protein signatures provide more comprehensive information about biological systems than single biomarkers, leading to improved insights in mechanisms of disease, diagnostics, and the effect of personalized medicine.

Background:We recently reported that the pregnancyassociated plasma protein A (PAPP-A) form specifically related to acute coronary syndromes (ACS) is not complexed with the proform of eosinophil major basic protein (proMBP). The aim of this study was to develop rapid point-of-care immunoassays for the measurement of the noncomplexed PAPP-A. Methods: We developed immunofluorometric noncompetitive dry-reagent assays for total PAPP-A with 2 PAPP-A subunit-specific monoclonal antibodies and for PAPP-A/proMBP complex with 1 PAPP-A subunit-specific antibody and 1 proMBP subunit-specific antibody. The concentration of noncomplexed PAPP-A was determined as the difference of the results obtained with the 2 assays. Results: The assays were linear from 0.5 to 300 mIU/L. The analytical detection limit and functional detection limit (CV <20%) were 0.18 mIU/L and 0.27 mIU/L for total PAPP-A assay and 0.23 mIU/L and 0.70 mIU/L for PAPP-A/proMBP assay, respectively. The total assay imprecisions were <10%, and recoveries were 88%-107% for both assays. The mean difference (95% limits of agreement) between the new total PAPP-A assay and a previously reported total PAPP-A assay was ؊3.2% (؊45.7% to 39.3%; n ‫؍‬ 546; P ‫؍‬ 0.0019). In serum samples from 159 non-ACS individuals, median concentrations

No abstract

Background: Pregnancy-associated plasma protein A (PAPP-A) has been suggested as a useful diagnostic and prognostic marker in acute coronary syndromes. Because low molecular weight heparin (LMWH) and unfractionated heparin (UFH) are commonly used in these cases, we analyzed the effects of intravenous administration of these heparins on serum PAPP-A concentrations. Methods: Serum concentrations of total and free PAPP-A were analyzed in 14 patients on chronic hemodialysis and in 10 coronary angiography patients. Ten of the dialysis patients received standard LMWH anticoagulation at the start of dialysis, and 4 were treated with a heparin-free method. Two of the patients on heparin-free hemodialysis received a reduced LMWH bolus 2 h after the start of dialysis. All angiography patients received UFH at the start of the procedure, and 1 patient received 2 extra boluses of UFH. Serum PAPP-A concentrations were analyzed before and during the dialysis session and during the coronary angiography examination. Results: A rapid increase in total PAPP-A (median, 25-fold) was seen in all patients within 5 min of administration for both LMWH and UFH boluses. This response was due to an increase in free PAPP-A in the serum. PAPP-A did not increase significantly in the patients who underwent heparin-free hemodialysis. Repeated heparin boluses induced a new PAPP-A release. In vitro addition of heparins to samples of whole blood did not increase PAPP-A concentrations. Conclusions: Intravenous administration of heparin induces an intense and rapid increase in free PAPP-A in the serum. We recommend that this effect be considered when PAPP-A is assessed as a biomarker in acute coronary syndromes.

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 (&lt;1.27, 1.27–1.74, &gt;1.74 mIU/L) and TPAPP-A (&lt;1.98, 1.98–2.99, &gt;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 &gt;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.