Content: This article examines whether a recent successful Netflix-sponsored competition using mathematical analysis to develop a prediction model for movie ratings of individual subscribers can serve to improve studies of markers in the field of proteomics. Netflix developed a database of movie preferences of individual subscribers using a longitudinal cohort research design. Groups of researchers then competed to develop better ways to analyze the data. Against this background, the strengths and weaknesses of research design are reviewed, contrasting the Netflix design with that of studies of biomarkers to detect cancer. Such biomarker studies generally have less-strong design, lower numbers of outcomes, and greater difficulty in even just measuring predictors and outcomes, so the fundamental data that will be used in mathematical analysis tend to be much weaker than in other kinds of research.

Conclusions: If the fundamental data that will be analyzed are not strong, then better analytic methods have limited use in improving the situation. Recognition of this situation is an important first step toward improving the quality of clinical research about markers to detect cancer.

Content: We provide an overview of antibody-based multiplexed immunoassay platforms and discuss technical and operational challenges. Multiplexed immunoassays use traditional immunoassay principles in which high-affinity capture ligands are immobilized in parallel arrays in either planar format or on microspheres in suspension. Development of multiplexed immunoassays requires rigorous validation of assay configuration and analytical performance to minimize assay imprecision and inaccuracy. Challenges associated with multiplex configuration include selection and immobilization of capture ligands, calibration, interference between antibodies and proteins and assay diluents, and compatibility of assay limits of quantification. We discuss potential solutions to these challenges. Criteria for assessing analytical multiplex assay performance include the range of linearity, analytical specificity, recovery, and comparison to a quality reference method. Quality control materials are not well developed for multiplexed protein immunoassays, and algorithms for interpreting multiplex quality control data are needed.

Summary: Technical and operational challenges have hindered implementation of multiplexed assays in clinical settings. Formal procedures that guide multiplex assay configuration, analytical validation, and quality control are needed before broad application of multiplexed arrays can occur in the in vitro diagnostic market.

Content: To address unmet needs in disease biomarkers, investigators have turned to proteomics approaches. We describe advances in proteomics discovery technologies based on liquid chromatography–tandem mass spectrometry that facilitate the unbiased analysis of low-abundance blood proteins. We detail the development of emerging techniques to enhance the biomarker verification process, such as accurate inclusion mass screening, stable isotope dilution–multiple reaction monitoring–mass spectrometry (SID-MRM-MS), and stable isotope standards with capture by antipeptide antibodies, which combines the advantages of specific immunoaffinity enrichment of a target peptide with the structural specificity and quantitative capabilities of SID-MRM-MS. We highlight new assays incorporating these techniques for troponin I, a representative low-abundance cardiac biomarker, and interleukin-33, an emerging novel marker of myocardial stress for which no existing ELISA exists. We demonstrate that troponin I and interleukin-33 peptides have a linear, dynamic range spanning 4 orders of magnitude and limits of detection of approximately 0.5 µg/L back-calculated to the protein concentration.

Conclusions: There remain important unmet diagnostic and prognostic needs in cardiology. Advances in technology may allow proteomics to play a vital role in the discovery and validation of novel biomarkers to help fill those needs.

Content: We describe and explore the implications of microheterogeneity using the exemplar of human vitamin D binding protein (Gc-Globulin) as observed in cohorts of 400 individuals. Our investigations yielded: (a) population frequency data comparable to genotyping; (b) population frequency data for protein variants, with and without genotype linkage; (c) reference values for the different protein variants per cohort and genotype; and (d) associations between variant, frequency, relative abundance, and diseases.

Summary: With the exception of the genotype frequency, such population data are unique and illustrate a need to more fully understand the exact full-length qualitative and quantitative idiosyncrasies of individual proteins in relation to health and disease as part of the standardized biomarker development and clinical proteomic investigation of human proteins.

Content: Mass spectrometry (MS), and in particular multiple reaction monitoring (MRM)-MS, has emerged as an alternative technology to immunoassays for quantification of target proteins. Novel biomarkers are expected to be present in serum in the low (µg/L–ng/L) range, but analysis of complex serum or plasma digests by MS has yielded milligram per liter limits of detection at best. The coupling of prior sample purification strategies such as enrichment of target analytes, depletion of high-abundance proteins, and prefractionation, has enabled reliable penetration into the low microgram per liter range. This review highlights prospects for candidate verification through MS-based methods. We first outline the biomarker discovery pipeline and its existing bottleneck; we then discuss various MRM-based strategies for targeted protein quantification, the applicability of such methods for candidate verification, and points of concern.

Summary: Although it is unlikely that MS-based protein quantification will replace immunoassays in the near future, with the expected improvements in limits of detection and specificity in instrumentation, MRM-based approaches show great promise for alleviating the existing bottleneck to discovery.

Content: In this article, we survey clinical tests that target carbohydrate modifications for diagnosing and treating cancer. We present the biological relevance of glycosylation to disease progression by highlighting the role these structures play in adhesion, signaling, and metastasis and then address current methodological approaches to biomarker discovery that capitalize on selectively capturing tumor-associated glycoforms to enrich and identify disease-related candidate analytes. Finally, we discuss emerging technologies—multiple reaction monitoring and lectin-antibody arrays—as potential tools for biomarker validation studies in pursuit of clinically useful tests.

Summary: The future of carbohydrate-based biomarker studies has arrived. At all stages, from discovery through verification and deployment into clinics, glycosylation should be considered a primary readout or a way of increasing the sensitivity and specificity of protein-based analyses.

Methods: From January 2001 to January 2007, we assessed Aβ42, Tau, and pTau by commercial ELISAs in CSF from 248 consecutive AD patients and 131 patients with subjective memory complaints attending our outpatient memory clinic. Diagnoses were made blind to the results of the biomarker assays. We assessed sensitivity and specificity and analyzed trends over time.

Results: Interassay CVs from analysis of pools of surplus CSF specimens were mean 11.3% (SD 4.9%) for Aβ42; 9.3% (1.5%) for Tau, and 9.4% (2.5%) for pTau, respectively (n = 7–18). To achieve 85% sensitivity, cutoff values were 550 (95% CI 531–570) ng/L for Aβ42; 375 (325–405) ng/L for Tau, and 52 (48–56) ng/L for pTau. Corresponding specificities were 83% (95% CI 76%–89%) for Aβ42, 78% (70%–85%) for Tau, and 68% (60%–77%) for pTau. Logistic regression to investigate the simultaneous impact of the 3 CSF biomarkers on the diagnosis yielded a sensitivity of 93.5% and specificity of 82.7%, at a discrimination line of Aβ42 = 373 + 0.82 x Tau. The area under the ROC curves of Tau and pTau showed significant fluctuation over time.

Conclusions: CSF biomarkers Aβ42 and Tau can be used as a diagnostic aid in AD. pTau did not have additional value over these 2 markers. Cutoff values, sensitivities, specificities, and discrimination lines depend on the patient groups studied and laboratory experience.

Methods: Validation included testing of analytical sensitivity, specificity, interferences, and precision. We established the 99th percentile cutoff from healthy reference populations (n = 616). In addition, we studied differences in time to a positive result when using serial measurements of hs-cTnT vs cTnT in patients with a confirmed diagnosis of non-ST elevation myocardial infarction (non-STEMI).

Results: The hs-cTnT assay had an analytical range from 3 to 10 000 ng/L. At the 99th percentile value of 13.5 ng/L, the CV was 9% using the Elecsys® 2010 analyzer. The assay was specific for cTnT without interferences from human cTnI or cTnC, skeletal muscle TnT, or hemoglobin concentrations up to 1000 mg/L, above which falsely lower values would be expected. When the assay was evaluated clinically, a hs-cTnT higher than the 99th percentile concentration identified a significantly higher number of patients with non-STEMI on presentation (45 vs 20 patients, P = 0.0004) compared with cTnT, and a final diagnosis of non-STEMI was made in 9 additional patients (55 vs 46 patients, P = 0.23) after serial sampling. Time to diagnosis was significantly shorter using hs-cTnT compared with cTnT [mean 71.5 (SD 108.7) min vs 246.9 (82.0) min, respectively; P < 0.01].

Conclusions: The analytical performance of hs-cTnT complies with the ESC-ACCF-AHA-WHF Global Task Force recommendations for use in the diagnosis of MI.

Methods: We subjected identically collected and processed serum samples from healthy volunteers and patients to automated polypeptide extraction on octadecylsilane-coated magnetic beads and separately on ZipTips before MALDI-TOF MS profiling at 2 centers. The 2 platforms were compared and case control profiling data analyzed to find altered MS peak intensities. We tested models built from training datasets for both methods for their ability to classify a blinded test set.

Results: Both profiling platforms had CVs of approximately 15% and could be applied for high-throughput analysis of clinical samples. The 2 methods generated overlapping peptide profiles, with some differences in peak intensity in different mass regions. In cross-validation, models from training data gave diagnostic accuracies up to 87% for discriminating malignant ovarian cancer from healthy controls and up to 81% for discriminating malignant from benign samples. Diagnostic accuracies up to 71% (malignant vs healthy) and up to 65% (malignant vs benign) were obtained when the models were validated on the blinded test set.

Conclusions: For ovarian cancer, altered MALDI-TOF MS peptide profiles alone cannot be used for accurate diagnoses.

Methods: The synthetic reporter peptides that are cleaved by tumor-associated endopeptidases were systematically optimized with regard to flanking affinity tags, linkers, and stabilizing elements. Serum specimens were incubated with reporter peptides under standardized conditions and the peptides subsequently extracted with affinity chromatography before MS. In a pilot study an optimized reporter peptide with the cleavage motif WKPYDAADL was added to serum specimens from colorectal tumor patients (n = 50) and healthy controls (n = 50). This reporter peptide comprised a known cleavage site for the cysteine-endopeptidase "cancer procoagulant."

Results: Serial affinity chromatography using biotin- and 6xHis tags was superior to the single affinity enrichment using only 6xHis tags. Furthermore, protease-resistant stop elements ensured signal accumulation after prolonged incubation. In contrast, signals from reporter peptides without stop elements vanished completely after prolonged incubation owing to their total degradation. Reporter-peptide spiking showed good reproducibility, and the difference in proteolytic activity between serum specimens from cancer patients and controls was highly significant (P < 0.001).

Conclusions: The introduction of a few structural key elements (affinity tags, linkers, d-amino acids) into synthetic reporter peptides increases the diagnostic sensitivity for MS-based protease profiling of serum specimens. This new approach might lead to functional MS-based protease profiling for improved disease classification.

Methods: Preparation of mass spectrometric immunoassay pipettor tips and MALDI-TOF mass spectrometric analysis were carried out as previously described. We used novel software to develop SRM assays on a triple-quadrupole mass spectrometer. Heavy isotope-labeled versions of target peptides were used as internal standards.

Results: Top-down analysis of samples from healthy individuals and renal failure patients revealed numerous PTH variants, including previously unidentified aa28–84, aa48–84, aa34–77, aa37–77, and aa38–77. Quantitative SRM assays were developed for PTH1–84, PTH7–84, and variant aa34–84. Peptides exhibited linear responses (R² = 0.90–0.99) relative to recombinant human PTH concentration limits of detection for intact PTH of 8 ng/L and limits of quantification of 16–31 ng/L depending on the peptide. Standard error of analysis for all triplicate measurements was 3%–12% for all peptides, with <5% chromatographic drift between replicates. The CVs of integrated areas under the curve for 54 separate measurements of heavy peptides were 5%–9%.

Conclusions: Mass spectrometric immunoassays identified new clinical variants of PTH and provided a quantitative assay for these and previously identified forms of PTH.

Methods: The algorithm objectively evaluates MRM-MS data with 2 orthogonal approaches. First, it compares the relative product ion intensities of the analyte peptide to those of the SIS peptide and uses a t-test to determine if they are significantly different. A CV is then calculated from the ratio of the analyte peak area to the SIS peak area from the sample replicates.

Results: The algorithm identified problematic transitions and achieved accuracies of 94%–100%, with a sensitivity and specificity of 83%–100% for correct identification of errant transitions. The algorithm was robust when challenged with multiple types of interferences and problematic transitions.

Conclusions: This algorithm for automated detection of inaccurate and imprecise transitions (AuDIT) in MRM-MS data reduces the time required for manual and subjective inspection of data, improves the overall accuracy of data analysis, and is easily implemented into the standard data-analysis work flow. AuDIT currently works with results exported from MRM-MS data-processing software packages and may be implemented as an analysis tool within such software.

Methods: We isolated 1–84 PTH from 1 mL serum by immunocapture on a 6.5-mm polystyrene bead. The immobilized PTH was digested in situ and analyzed by LC-MS/MS. For quantification, we used the selected reaction monitoring response from the N-terminal tryptic peptide 1–13 PTH (¹SVSEIQLMHNLGK¹³).

Results: The linear range of the assay was 39.1–4560 ng/L, and the limit of detection and limit of quantification were 14.5 ng/L and 39.1 ng/L, respectively. The intraassay CVs ranged from 6% to 11%, and the interassay CVs ranged from 7% to 17%. Interference by PTH fragments 1–44 PTH, 7–84 PTH, 43–68 PTH, 52–84 PTH, 64–84 PTH, and PTH-related protein (PTHrP) was ≤1% to ≤0.001%. Method comparison of LC-MS/MS vs the Roche Cobas® immunoassay yielded Deming fit of LC-MS/MS = 1.01x immunoassay – 13.21. The mean bias by Bland–Altman plot was –9.4%.

Conclusions: In patients with hyperparathyroidism, the immunocapture in situ digestion LC-MS/MS method can provide accurate and precise PTH results compared with immunoassay.

Methods: Assay platforms MULTI-ARRAY (Meso Scale Discovery), Bio-Plex (Bio-Rad Laboratories), A² (Beckman Coulter), FAST Quant (Whatman Schleicher & Schuell BioScience), and FlowCytomix (Bender MedSystems) were selected as representative examples of technologies currently used for high-throughput immunoanalysis. All assays were performed according to protocols specified by the manufacturers and with the reagents (diluents, calibrators, blocking reagents, and detecting-antibody mixtures) included with their kits.

Results: The quantifiable interval determined for each assay and antigen was based on precision (CV < 25%) and percentage recovery (measured concentration within 20% of the actual concentration). The MULTI-ARRAY and Bio-Plex assays had the best performance with the lowest limits of detection, and the MULTI-ARRAY system had the most linear signal output over the widest concentration range (10⁵ to 10⁶). Cytokine concentrations in unspiked and cytokine-spiked serum samples from healthy individuals were further investigated with the MULTI-ARRAY and Bio-Plex assays.

Conclusions: The MULTI-ARRAY and Bio-Plex multiplex immunoassay systems are the most suitable for biomarker analysis or quantification.

Content: We discuss the current classification, diagnosis, and treatment of stroke, focusing on use of novel biomarkers (either solitary markers or multiple markers within a panel) that have been studied in a variety of clinical settings.

Summary: The current diagnosis of stroke remains hampered and delayed due to lack of a suitable mechanism for rapid (ideally point-of-care), accurate, and analytically sensitive biomarker-based testing. There is a clear need for further development and translational research in this area. Potential biomarkers identified need to be transitioned quickly into clinical validation testing for further evaluation in an acute stroke setting; to do so would impact and improve patient outcomes and quality of life.

Methods: In the Physicians’ Health Study, we prospectively examined the association of prediagnostic plasma concentrations of adiponectin and leptin with risk of developing incident prostate cancer (654 cases diagnosed 1982–2000 and 644 age-matched controls) and, among cases, risk of dying from prostate cancer by 2007.

Results: Adiponectin concentrations were not associated with risk of overall prostate cancer. However, men with higher adiponectin concentrations had lower risk of developing high-grade or lethal cancer (metastatic or fatal disease). The relative risk (95% CI) comparing the highest quintile to the lowest (Q5 vs Q1) was 0.25 (95% CI 0.07–0.87; P_trend = 0.02) for lethal cancer. Among all the cases, higher adiponectin concentrations predicted lower prostate cancer–specific mortality [hazard ratio (HR)_{Q5 vs Q1}= 0.39; 95% CI 0.17–0.85; P_trend = 0.02], independent of body mass index (BMI), plasma C-peptide (a marker of insulin secretion), leptin, clinical stage, and tumor grade. This inverse association was apparent mainly among men with a BMI ≥25 kg/m² (HR_{Q5 vs Q1}= 0.10; 95% CI 0.01–0.78; P_trend = 0.02), but not among men of normal weight (P_trend = 0.51). Although the correlation of leptin concentrations with BMI (r = 0.58, P < 0.001) was stronger than that of adiponectin (r = –0.17, P < 0.001), leptin was unrelated to prostate cancer risk or mortality.

Conclusions: Higher prediagnostic adiponectin (but not leptin) concentrations predispose men to a lower risk of developing high-grade prostate cancer and a lower risk of subsequently dying from the cancer, suggesting a mechanistic link between obesity and poor prostate cancer outcome.

Methods: The CLSI protocols EP-10, EP-5, and EP-9 were applied to investigate imprecision, accuracy, and bias. We assessed bias using 3 certified secondary reference measurement procedures and the mean of the 3 reference methods. Assay conformance with the National Glycohemoglobin Standardization Program (NGSP) certification criteria, as calculated from analyses with 2 different reagent lot numbers for each Hb A_1c method, was also evaluated.

Results: Because of disappointing EP-10 results, 2 of the 8 manufacturers decided not to continue the evaluation. The total CVs from EP-5 evaluations for the different instruments with a low and high Hb A_1c value were: In2it 4.9% and 3.3%, DCA Vantage 1.8% and 3.7%, Clover 4.0% and 3.5%, InnovaStar 3.2% and 3.9%, Nycocard 4.8% and 5.2%, and Afinion 2.4% and 1.8%. Only the Afinion and the DCA Vantage passed the NGSP criteria with 2 different reagent lot numbers.

Conclusions: Only the Afinion and the DCA Vantage met the acceptance criteria of having a total CV <3% in the clinically relevant range. The EP-9 results and the calculations of the NGSP certification showed significant differences in analytical performance between different reagent lot numbers for all Hb A_1c POC instruments.

Methods: Denaturing gradient gel electrophoresis was used to genotype the BRCA1 c.591C>T variant in 685 index cases of B(O)C families, 326 sporadic breast cancer cases, and 450 healthy controls from Spain. In silico tools were used to predict the effect of the c.591C>T variant on splicing. In vitro splicing analysis was performed in 7 c.591C>T carriers and 10 noncarriers. cDNAs were PCR-amplified with primers designed to detect BRCA1 alternative splicing isoforms. The products were analyzed by capillary electrophoresis. Peak areas were used to quantify the relative abundance of each isoform. Sequencing through exonic single-nucleotide polymorphisms (SNPs) enabled us to discriminate wild-type and variant transcripts.

Results: c.591C>T was detected in B(O)C families (1.5%), breast cancer cases (0.3%), and controls (0.9%). c.591C>T induced BRCA1 exon 9 skipping and modified the relative expression of (9,10), (9,10,11B), 11B, and full-length isoforms. The mean ratio of (9,10) to the full-length isoform increased from 0.25 in noncarriers to 1.5 in carriers. The mean (9,10,11B)/11B ratio increased from 0.2 to 4. Overall expression levels of c.591C>T and wild-type alleles were similar.

Conclusions: Our data support a nonpathogenic role for the BRCA1 c.591C>T variant. Naturally occurring alternative splicing isoforms need to be considered when assessing the role of BRCA1 UVs on splicing.

Methods: In this study, we investigated a process for enriching DNA samples that uses a customized high-density oligonucleotide microarray to enrich a targeted 280-kb region of the NF1 (neurofibromin 1) gene. The captured DNA was sequenced with the Roche/454 GS FLX system. Two NF1 samples (CN1 and CN2) with known genotypes were tested with this protocol.

Results: Targeted microarray capture may also capture sequences from nontargeted regions in the genome. The capture specificity estimated for the targeted NF1 region was approximately 60%. The de novo Alu insertion was partially detected in sample CN1 by additional de novo assembly with 50% base-match stringency; the single-base deletion in sample CN2 was successfully detected by reference mapping. Interferences by pseudogene sequences were removed by means of dual-mode reference-mapping analysis, which reduced the risk of generating false-positive data. The risk of generating false-negative data was minimized with higher sequence coverage (>30x).

Conclusions: We used a clinically relevant complex genomic target to evaluate a microarray-based sample-enrichment process and an NGS instrument for clinical resequencing purposes. The results allowed us to develop a systematic data-analysis strategy and algorithm to fit potential clinical applications.

Methods: We studied maternal plasma samples from 153 pregnant women carrying euploid and trisomy 21 fetuses. For the samples in which the fetuses were heterozygous for the studied PLAC4 single-nucleotide polymorphism (SNP), we measured the ratio between 2 alleles of the SNP in maternal plasma PLAC4 mRNA (RNA-SNP) by mass spectrometric (MS) and digital PCR methods. For pregnancies involving fetuses homozygous for the SNP, we quantified the total PLAC4 mRNA concentration in maternal plasma by real-time PCR and digital PCR.

Results: For the RNA-SNP approach, we achieved a diagnostic sensitivity and specificity of 100% (95% CI, 40.2%–100%) and 89.7% (95% CI, 78.8%–96.1%), respectively, for both the MS and the digital PCR methods. For the mRNA-quantification approach, the areas under the ROC curves were 0.859 (95% CI, 0.741–0.903) and 0.833 (95% CI, 0.770–0.923) for plasma PLAC4 mRNA concentrations measured by the real-time PCR and the digital PCR methods, respectively.

Conclusions: For prenatal screening of trisomy 21, the quantification of the total PLAC4 mRNA concentration can be used in a synergistic manner with the RNA-SNP allelic ratio approach to increase the population coverage of cases in which diagnostic information can be obtained.

Methods: We genotyped ALB mRNA molecules in samples of plasma and whole blood from liver and bone marrow transplant recipients by RNA single-nucleotide polymorphism analysis. Plasma and whole blood ALB mRNA genotypes were compared with the DNA genotypes of the recipients and donors. A reverse-transcription quantitative real-time PCR assay was used to measure plasma ALB mRNA concentrations in 107 patients [hepatocellular carcinoma (HCC), cirrhosis, or chronic hepatitis B (CHB)] and 207 healthy controls.

Results: The RNA genotype data revealed ALB mRNA in plasma to be liver derived, whereas tissue compartments other than the liver also contributed to the ALB mRNA detected in whole blood. Statistically significant increases in plasma ALB mRNA concentrations were observed for HCC, cirrhosis, and active CHB, compared with controls. A cutoff of 835 copies/mL of plasma ALB mRNA identified by ROC curve analysis showed 85.5% diagnostic sensitivity and 92.8% diagnostic specificity for the detection of liver pathologies. Only 21.5% of patients with liver pathologies had increased alanine aminotransferase (ALT) activities, whereas 73.8% had increased plasma ALB mRNA concentrations. Only 48.6% of the HCC patients had increased serum -fetoprotein concentrations, whereas 91.4% had increased plasma ALB mRNA concentrations.

Conclusions: ALB mRNA is liver specific in plasma, but not in whole blood. Plasma ALB mRNA is increased in some liver pathologies and may be more diagnostically sensitive than -fetoprotein and ALT.

Methods: We used combined bisulfite restriction analysis to search for fetal DNA markers on chromosome 21 that were differentially methylated in the placenta and maternal blood cells and confirmed any target locus with bisulfite sequencing. We then used methylation-sensitive restriction endonuclease digestion followed by microfluidics digital PCR analysis to investigate the identified marker. Chromosome-dosage analysis was performed by comparing the dosage of this epigenetic marker with that of the ZFY (zinc finger protein, Y-linked) gene on chromosome Y.

Results: The putative promoter of the HLCS (holocarboxylase synthetase) gene was hypermethylated in the placenta and hypomethylated in maternal blood cells. A chromosome-dosage comparison of the hypermethylated HLCS and ZFY loci could distinguish samples of T21 and euploid placental DNA. Twenty-four maternal plasma samples from euploid pregnancies and 5 maternal plasma samples from T21 pregnancies were analyzed. All but 1 of the euploid samples were correctly classified.

Conclusions: The epigenetic–genetic chromosome-dosage approach is a new method for noninvasive prenatal detection of T21. The epigenetic part of the analysis can be applied to all pregnancies. Because the genetic part of the analysis uses paternally inherited, fetal-specific genetic markers that are abundant in the genome, broad population coverage should be readily achievable. This approach has the potential to become a generally usable technique for noninvasive prenatal diagnosis.

Methods: We used horseradish peroxidase (HRP)-conjugated oligonucleotides to couple in situ PLA with enzymatic visualization of the localized detection event.

Results: We demonstrate the detection of protein complexes, both in cells and in tissue sections, and show that we can quantify the complexes with image-analysis software specially developed for recognizing HRP signals in bright-field microscopy images. We show that fluorescence and HRP signals produce equivalent results, both in cultured cells and in tissue samples.

Conclusions: The combination of in situ PLA with bright-field detection and automated image analysis allows the signals present to be counted in an automated fashion and thus provides a sensitive and specific method for quantification of proteins and protein complexes with bright-field microscopy. With this approach, in situ PLA can be used without the requirement for expensive fluorescence microscopes, thereby avoiding problems with nonspecific fluorescence while maintaining compatibility with conventional histologic staining.

Methods: We performed a cross-sectional study of 980 healthy, older (age 60–72 years) individuals of different ethnicities living in the San Francisco Bay area and analyzed ADMA plasma concentrations and their relationship to other cardiovascular risk factors. Plasma ADMA concentrations were measured using a recently developed, highly sensitive ELISA.

Results: In our entire sample, we were able to define a reference interval for ADMA plasma concentrations of 0.47 (90% CI 0.46–0.48) µmol/L to 0.85 (0.84–0.89) µmol/L. The mean ADMA concentration was 0.63 (SD 0.11) µmol/L (median 0.61 µmol/L). Mean ADMA concentrations were significantly lower in African Americans (0.60 µmol/L; P < 0.01) and mixed non-Hispanics (0.60 µmol/L; P < 0.05) compared with whites (0.63 µmol/L). ADMA was positively correlated with cystatin-C in both men ( = 0.29) and women ( = 0.37), and median plasma ADMA concentrations increased across cystatin-C quintiles.

Conclusions: ADMA varies nearly 2-fold across a healthy sample of older men and women, correlates with age, body mass index, and renal function, and is different across ethnic groups. Additional studies in a wider age range and including larger ethnic subgroups would be useful.

Methods: In this prospective study we included 181 patients with chronic systolic HF after an episode of hospitalization for worsening HF. Subsequently, HF therapy was gradually increased in the outpatient setting until optimized. We measured copeptin, midregional proadrenomedullin, C-terminal endothelin-1 precursor fragment, midregional proatrial natriuretic peptide, and B-type natriuretic peptide before and after optimization of HF therapy. The primary endpoint was all-cause mortality at 24 months.

Results: Angiotensin-converting enzyme/angiotensin receptor blocker and β-blockers were increased significantly during the 3-month titration period (P < 0.0001 for both). In a stepwise Cox regression analysis adjusted for age, sex, glomerular filtration rate, diabetes mellitus, and ischemic HF, baseline and follow-up neurohormone concentrations were predictors of the primary endpoint as follows (baseline hazard ratios): copeptin 1.92, 95% CI 1.233–3.007, P = 0.004; midregional proadrenomedullin 2.79, 95% CI 1.297–5.995, P = 0.009; midregional proatrial natriuretic peptide 2.05, 95% CI 1.136–3.686, P = 0.017; C-terminal endothelin-1 precursor fragment 2.24, 95% CI 1.133–4.425, P = 0.025; B-type natriuretic peptide 1.46, 95% CI 1.039–2.050, P = 0.029.

Conclusions: In pharmacologically unstable chronic HF patients, baseline values and follow-up measures of copeptin, midregional proadrenomedullin, C-terminal endothelin-1 precursor fragment, midregional proatrial natriuretic peptide, and B-type natriuretic peptide were equally predictive of all-cause mortality. Relative change of neurohormone values was noncontributory.

Methods: Two-dimensional fluorescence difference gel electrophoresis was used to compare proteomic markers in 3 individuals before and after 12 weeks of TCC exercise. The different protein spots were identified by mass spectrometry and validated in an additional 20 individuals by western blot analysis.

Results: We identified 39 protein spots for 18 proteins with a noticeable increase or decrease after TCC exercise. Validation of the differentially displayed proteins with 20 paired pre- and postexercise samples revealed a significant increase in complement factor H (P = 0.0034) associated with decreases in C1 esterase inhibitor (P = 0.0038) and complement factor B (P = 0.0029).

Conclusions: In this first study of proteomic biomarkers of TCC exercise, we found an increase in complement factor H associated with a decrease in complement factor B. Complement factor H is involved in protection from microangiopathy and macular degeneration and may represent a useful marker of the health effects of exercise.

Methods: We recruited 5248 participants free from overt CVD and acute infection [mean age 53.5 (SD 12.4) years, 55.5% women] from the Scottish Health Survey, a representative sample of community-dwelling adults. hsCRP and other conventional risk factors were measured at baseline.

Results: Over an average of 7 years’ follow-up, there were a total of 259 incident CVD events (including myocardial infarction, coronary artery bypass, percutaneous coronary angioplasty, stroke, heart failure) and 357 all-cause deaths. Very highly increased hsCRP was associated with CVD events after adjustment for Framingham risk score (FRS), body mass index (BMI), central obesity, and hormone replacement therapy (HRT) (hazard ratio 2.40, 95% CI 1.51–3.81) and also with all-cause death (hazard ratio 3.64, 95% CI 2.57–5.15). With the addition of CRP scores to the conventional Framingham model, 7.4% of participants were reclassified into a high-risk (>20% FRS) CVD category. Very highly increased hsCRP was also associated with several modifiable risk factors, including smoking, HDL cholesterol, and central obesity.

Conclusions: hsCRP >10 mg/L was a stronger predictor of clinical events than a conventional cut point of 3 mg/L. Very highly increased hsCRP may provide clinically meaningful prognostic information.

Method: We developed a novel universal-template (UT) real-time qPCR assay that was specific for the DYS14 sequence on Y chromosome and had a short amplicon size of 50 bp. We examined this "short" assay with 50 maternal plasma samples and compared the results with those for a conventional real-time qPCR assay of the same locus but with a longer amplicon (84 bp).

Results: Qualitatively, both assays detected male cell-free fetal DNA with the same specificity and detection capability. Quantitatively, however, the new UT real-time qPCR assay for shorter amplicons detected, on average, almost 1.6-fold more cell-free fetal DNA than the conventional real-time qPCR assay with longer amplicons.

Conclusions: The use of short PCR amplicons improves the detection of cell-free fetal DNA. This feature may prove useful in attempts to detect cell-free fetal DNA under conditions in which the amount of template is low, such as in samples obtained early in pregnancy.