Content: This review outlines OF testing advantages and limitations, and the progress in OF that has occurred during the last 5 years in collection, screening, confirmation, and interpretation of cannabinoids, opioids, amphetamines, cocaine, and benzodiazepines. We examine controlled drug administration studies, immunoassay and chromatographic methods, collection devices, point-of-collection testing device performance, and recent applications of OF testing.

Summary: Substance Abuse and Mental Health Services Administration approval of OF testing was delayed because questions about drug OF disposition were not yet resolved, and collection device performance and testing assays required improvement. Here, we document the many advances achieved in the use of OF. Additional research is needed to identify new biomarkers, determine drug detection windows, characterize OF adulteration techniques, and evaluate analyte stability. Nevertheless, there is no doubt that OF offers multiple advantages as an alternative matrix for drug monitoring and has an important role in DUID, treatment, workplace, and criminal justice programs.

Content: This report presents a review of the role of cystatin C as a predictor of cardiovascular risk.

Summary: Patients with higher circulating cystatin C concentrations appear to have an increased cardiovascular risk profile, i.e., they are older and have a higher prevalence of systemic hypertension, dyslipidemia, documented CVD, increased body mass index, and increased concentrations of C-reactive protein. Prospective studies have shown, in various clinical scenarios, that patients with increased cystatin C are at a higher risk of developing both CVD and CKD. Importantly, cystatin C appears to be a useful marker for identifying individuals at a higher risk for cardiovascular events among patients belonging to a relatively low-risk category as assessed by both creatinine and estimated glomerular filtration rate values. Of interest, elastolytic proteases and their inhibitors, in particular cystatin C, have been shown to be directly involved in the atherosclerotic process. Increased concentrations of cystatin C appear to be indicative of preclinical kidney disease associated with adverse outcomes. Clinical studies involving direct glomerular filtration rate measurements are required to ascertain both the true role of this promising marker in renal disease and whether atherogenic factors like inflammation can account for increases in cystatin C concentrations, thus explaining its predictive value in CVD.

Methods: We used isoproterenol-induced myocardial injury in rats as a model and miRNA array analyses to identify candidate miRNAs specifically produced in the ventricles of the heart. Individual miRNA concentrations were measured by real-time reverse-transcription PCR. Plasma cardiac troponin I (cTnI) concentrations were measured with an ELISA.

Results: Array analyses revealed miR-208 to be produced exclusively in the heart, and we selected this miRNA as a possible biomarker of myocardial injury. Plasma concentrations of miR-208 increased significantly (P < 0.0001) after isoproterenol-induced myocardial injury and showed a similar time course to the concentration of cTnI, a classic biomarker of myocardial injury.

Conclusions: The plasma concentration of miR-208 may be a useful indicator of myocardial injury. Our results suggest that profiling of circulating miRNAs may help identify promising biomarkers of various pathologic conditions.

Methods: We conducted a systematic review to examine whether Arg702Trp, Gly908Arg, and Leu1007fsinsC are equally important risk factors for Crohn disease. In addition, we used studies for which combined information from all genotypes was available to compare risks in simple heterozygotes, compound heterozygotes, and homozygotes. PubMed, EMBASE, and Web of Science were searched. Seventy-five articles (18 727 cases and 17 102 controls) met the inclusion criteria and contributed data to the metaanalyses.

Results: The odds ratios per allele for Crohn disease were 2.2 (95% CI, 2.0–2.5) for Arg702Trp, 2.6 (2.2–2.9) for Gly908Arg, and 3.8 (3.4–4.3) for Leu1007fsinsC (z-test results: Arg702Trp vs Gly908Arg, P = 0.03; Arg702Trp vs Leu1007fsinsC, P < 0.001; Gly908Arg vs Leu1007fsinsC, P < 0.001). When all 3 genotypes were combined, odds ratios for Crohn disease were 2.4 (95% CI, 2.0–2.8) for simple heterozygotes, 9.0 (6.0–13.5) for compound heterozygotes, and 6.7 (4.1–10.9) for homozygotes, compared with noncarriers (z-test results: simple heterozygotes vs compound heterozygotes, P < 0.001; simple heterozygotes vs homozygotes, P < 0.001; compound heterozygotes vs homozygotes, P = 0.18).

Conclusions: The per-allele risk of Crohn disease was markedly higher for Leu1007fsinsC than for Arg702Trp and Gly908Arg. Combining all genotypes revealed the risks of Crohn disease for compound heterozygotes and homozygotes to be similar and markedly higher than for simple heterozygotes.

Methods: Plasma and urine samples from 100 renal transplant recipients were obtained during the first 3 months after transplantation. tCF-DNA and ddCF-DNA were analyzed by quantitative PCR for the HBB (hemoglobin, beta) and the TSPY1 (testis specific protein, Y-linked 1) genes, respectively. We observed 19 episodes of AR, as well as other complications, such as acute tubular necrosis, nephrotoxicity, and infections.

Results: Plasma tCF-DNA concentrations increased markedly during AR episodes, often before clinical diagnosis, and returned to reference values after antirejection treatment. A cutoff plasma tCF-DNA concentration of 12 000 genome equivalents/mL correctly classified AR and non-AR episodes in 86% of posttransplantation complications (diagnostic sensitivity, 89%; specificity, 85%). Although similar increases were observed during severe posttransplantation infections, use of the combination of plasma tCF-DNA and procalcitonin (PCT), a specific marker of sepsis, significantly improved the diagnostic specificity (to 98%; 95% CI, 92%–100%), with 97% of the episodes being correctly classified. Use of transrenal DNA and ddCF-DNA concentrations did not add relevant information.

Conclusions: Given that renal biopsy is the gold standard for detecting AR, analysis of both plasma tCF-DNA and PCT could permit a more selective use of this invasive procedure.

Methods: We quantified cfsRNA by reverse-transcription quantitative real-time PCR (RT-qPCR) targeting of the 5' region of the ACTB (actin, beta) transcript. cfsRNA integrity was analyzed by microcapillary electrophoresis and by amplification of full-length ACTB and DDX4 [DEAD (Asp-Glu-Ala-Asp) box polypeptide 4] transcripts, including measurement of the relative amounts of different regions of ACTB and DDX4 transcripts. Stability of cfsRNA was measured by time-course analysis of different regions of ACTB and DDX4 transcripts. To investigate whether cfsRNA was protected in complexed forms, we processed seminal plasma in 2 ways: filtration through pores of different sizes and Triton X-100 treatment before RNA recovery.

Results: cfsRNA concentrations varied from 0.87–3.64 mg/L [mean (SD), 1.75 mg/L (0.92 mg/L)]. Most cfsRNA was present in partially degraded forms, with smaller amounts of middle and 3' amplicons compared with 5' amplicons. Although the 3' region of the DDX4 transcript was degraded completely by 90 min, the 5' regions of ACTB and DDX4 transcripts were stable up to 24 h. Filtration through 0.22-µm pores reduced ACTB and DDX4 mRNA concentrations by 72% and 61%, respectively. Nearly all seminal ACTB and DDX4 mRNA disappeared after Triton X-100 treatment.

Conclusions: Although cfsRNA was partially degraded, it represented diverse transcript species and was abundant, fairly stable, and associated with particles in healthy individuals. cfsRNA may represent a potential noninvasive biomarker of the male reproductive system and of germline epigenetics.

Methods: We investigated the use of liver-, muscle- and brain-specific miRNAs as circulating biomarkers of tissue injury. We used a highly sensitive quantitative PCR assay to measure specific miRNAs (miR-122, miR-133a, and miR-124) in plasma samples from rats treated with liver or muscle toxicants and from a rat surgical model of stroke.

Results: We observed increases in plasma concentrations of miR-122, miR-133a, and miR-124 corresponding to injuries in liver, muscle, and brain, respectively. miR-122 and miR-133a illustrated specificity for liver and muscle toxicity, respectively, because they were not detectable in the plasma of animals with toxicity to the other organ. This result contrasted with the results for alanine aminotransferase (ALT) and aspartate aminotransferase, which were both increased with either organ toxicity. Furthermore, miR-122 exhibited a diagnostic sensitivity superior to that of ALT when the results were correlated to the liver histopathologic results. The miR-124 concentration increased in the plasma of rats 8 h after surgery to produce brain injury and peaked at 24 h, while the miR-122 and miR-133a concentrations remained at baseline values.

Conclusions: These results demonstrate that tissue-specific miRNAs may serve as diagnostically sensitive plasma biomarkers of tissue injury.

Methods: We developed a method based on the traceable quantification of tryptic peptides released from the protein by isotope dilution mass spectrometry to compare 2 standard preparations of somatropin (recombinant human growth hormone), WHO 98/574 and Ph.Eur.CRS S0947000. Relative quantification using isotope-coded affinity tagging, isobaric tagging for relative and absolute quantification, and standard additions were also performed to validate the digestion method used and to determine whether any modifications were present.

Results: The total somatropin content in both materials was determined and an uncertainty estimation undertaken [WHO 2.19 ± 0.21) mg/vial, European Pharmacopeia 2.06 ± 0.21 mg/vial]. Each uncertainty in this paper is a fully estimated uncertainty, with 95% CI (k = 2). Isotope coded affinity tag and standard addition results fully validated the robustness of the digestion method used. In addition, iTRAQ (isobaric tagging for relative and absolute quantification analysis) identified 2 modifications, neither of which impacted the quantification.

Conclusions: An independent method that does not rely on a preexisting protein standard has been developed and validated for the traceable value-assignment of total somatropin. The methods reported here address the amount of substance (mass fraction) of the standard materials but address neither biological activity nor other characteristics that may be important in assessing suitability for use as a calibrator.

Methods: We quantified urinary albumin in patient samples by using 3 commercially available reagent systems: DiaSorin SPQ^TM and Beckman Coulter LX® 20 (immunoturbidimetric), and Siemens Immulite® (competitive immunoassay). Results were compared to values obtained by protein-cleavage liquid chromatography–tandem mass spectrometry (LC-MS/MS).

Results: In general, results from the 3 immunoassays agreed with results from LC-MS/MS. However, the SPQ results showed a negative bias across all ranges of albuminuria [(0–200 mg/L, y = 0.91x – 3.74 (CI 0.86–0.96); > 200 mg/L, y = 0.88x – 40.30 (CI 0.76–1.00)], whereas the LX 20 showed minimal bias in the 0–200 mg/L range [y = 0.97x – 88 (CI 0.92–1.02)] and the Immulite assay showed positive bias in the 0–200 mg/L range [y = 1.15x – 4.38 (CI 1.09–1.20)].

Conclusions: These results showed a reasonable quantification of urinary albumin by representative polyclonal and monoclonal immunoassays compared to an LC-MS/MS assay. In addition, the results do not suggest the presence of nonimmunoreactive albumin in urine. However, differences in analytic performance between assays support the need for a reference calibration material and reference method to standardize clinical laboratory measurements of urinary albumin.

Methods: We developed an approach for sensitively and reliably measuring mRNA abundances in FFPE tissue samples. This approach involves automated RNA extractions, direct hybridization of extracted RNA to immobilized capture probes, antibody-mediated labeling, and readout with an instrument applying the principle of planar waveguides (PWG). A 14-gene multiplex assay conducted with RNA isolated from 20 FFPE blocks was correlated to an analysis of the same with reverse-transcription quantitative real-time PCR (RT-qPCR).

Results: The assay sensitivity for gene expression analysis obtained for the PWG microarray platform was <10 fmol/L, eliminating the need for target preamplification. We observed a correlation coefficient of 0.87 to state-of-the-art RT-qPCR technology with RNA isolated from FFPE tissue, despite a compressed dynamic range for the PWG system (a 2.9-log dynamic range for PWG in our test system vs 5.0 logs for RT-qPCR). The precision of the PWG platform was comparable to RT-qPCR (Pearson correlation coefficient of 0.9851 for PWG vs 0.9896 for RT-qPCR) for technical replicates.

Conclusions: The presented PWG platform demonstrated excellent sensitivity and precision and is especially well suited for any application for which fast, simple, and robust multiplex assays of RNA in FFPE tissue are required.

Method: Solid-phase extraction was performed with a Gilson ASPEC XL4 system equipped with Bond Elut Certify sample cartridges. OF samples (200 mg) diluted with 5 mL of ammonium acetate/methanol (vol/vol 90:10) buffer were applied to the columns and eluted with 3 mL of acetonitrile with aqueous ammonium hydroxide. Target drugs were quantified by use of a Waters ACQUITY UPLC system coupled to a Waters Quattro Premier XE triple quadrupole (positive electrospray ionization mode, multiple reaction monitoring mode).

Results: Extraction recoveries were 36%–114% for all analytes, including -9-tetrahydrocannabinol and benzoylecgonine. The lower limit of quantification was 0.5 µg/kg for all analytes. Total imprecision (CV) was 5.9%–19.4%. With the use of deuterated internal standards for most compounds, the performance of the method was not influenced by matrix effects. A preliminary account of OF samples collected at the roadside showed the presence of amphetamine, cocaine, codeine, -9-tetrahydrocannabinol, tramadol, and zopiclone.

Conclusions: The UPLC-MS/MS method makes it possible to detect all 29 analytes in 1 chromatographic run (15 min), including -9-tetrahydrocannabinol and benzoylecgonine, which previously have been difficult to incorporate into multicomponent methods.

Methods: FT₄ and TSH results of 871 healthy individuals [361 women and 510 men, 18–40 years old, without history of thyroid-related disease or medication, negative for anti–thyroid peroxidase (anti-TPO) antibody] were transformed to standard normal variables by logarithmic transformation with correction for skewness and subsequent normalization. We established a 95% reference interval of the distance of each FT₄/TSH pair of values to the center of the 2-dimensional probability distribution.

Results: The bivariate 95% reference interval is enclosed by a circular profile with radius 2.45 SD. By contrast, conventional reference intervals comprise a square with the boundaries of –1.96 and +1.96 SD for both FT₄ and TSH that enclose only 90% of all data. Compared with the ±1.96 SD square, the bivariate reference interval classified 4% fewer of 3651 healthy individuals older than 40 years as subclinically hyperthyroid and 14% fewer of 712 anti-TPO–positive healthy individuals as subclinically hypothyroid.

Conclusions: Conventional application of separate cutoff values for FT₄ and TSH leads to overestimation of the incidence of subclinical thyroid disease. Application of a composite overall reference interval is recommended.

Methods: We conducted a community-based prospective cohort study comprising 7220 participants (mean age 37 years; 73.8% men) in the Qingdao Port Health and Nutrition Examination Survey in China, who were free from hypertension at study entry in 1999–2000. During 4-year follow-up, 1370 men (19.0%) and 208 women (11.0%) had developed hypertension.

Results: After adjustment for age, body mass index, and other covariates, the relative risks (RRs) of developing hypertension comparing the highest and lowest uric acid quartiles were 1.55 (95% CI 1.10–2.19; P for trend <0.001) for men and 1.91 (1.12–3.25; P for trend <0.001) for women. After additional adjustment for abdominal obesity, the RRs comparing the participants in the highest and lowest quartiles of uric acid were 1.39 (1.16–1.68; P for trend 0.003) for men and 1.85 (1.06–3.24; P for trend 0.006) for women. In joint analysis, compared with those in the lowest uric acid quartile and without abdominal obesity, participants who were in the highest quartile and also had abdominal obesity had a 3.0- and 3.4-fold greater risk of incident hypertension (1.56–3.97 for men and 2.10–3.81 for women, respectively).

Conclusions: These data suggest a positive association between plasma uric acid and incidence of hypertension during short-term follow-up in a Chinese population. The association between hyperuricemia and hypertension was partly mediated by abdominal obesity.

Methods: We analyzed UCP using a routine electrochemiluminescence immunoassay in samples from 21 healthy volunteers. We investigated the stability of UCP with different preservatives and storage conditions and compared the reproducibility of urinary C-peptide/creatinine ratio (UCPCR) in first- and second-void fasting urines, then assessed correlations with 24-h collections.

Results: UCPCR was unchanged at room temperature for 24 h and at 4 °C for 72 h even in the absence of preservative. UCPCR collected in boric acid was stable at room temperature for 72 h. UCPCR remained stable after 7 freeze-thaw cycles but decreased with freezer storage time and dropped to 82%–84% of baseline by 90 days at –20 °C. Second-void fasting UCPCRs were lower than first-void (median 0.78 vs 1.31, P = 0.0003) and showed less variation (CV 33% vs 52%), as second-void UCPCRs were not influenced by evening food-related insulin secretion. Second-void fasting UCPCR was highly correlated with 24-h UCP (r = 0.8, P = 0.00006).

Conclusions: Second-void fasting UCPCR is a reproducible measure that correlates well with 24-h UCP in normal samples. The 3-day stability of UCPCR at room temperature greatly increases its potential clinical utility.

Methods: In 94 subjects undergoing clinically indicated cardiac catheterization, we collected blood samples from arterial and multiple venous sites to measure transorgan gradients of plasma UII immunoreactivity.

Results: Net UII release occurred (in descending order of proportional transorgan gradient) across the heart, kidney, head and neck, liver, lower limb, and pulmonary circulations (P < 0.01). Although no specific clearance site was localized, the absence of an overall subdiaphragmatic aorto-caval peptide gradient indicated that there were lower body segment sites of UII clearance as well as secretion. The proportional increase in UII immunoreactivity was significantly correlated across all sites of net peptide release within an individual (P ≤ 0.05). In univariate analyses, mixed venous UII concentrations were correlated with diagnosis of acute coronary syndrome and femoral artery oxygen tension and inversely with systolic blood pressure and body mass index. Diagnosis of acute coronary syndrome and body mass index were independent predictors of mixed venous UII immunoreactivity in multivariate analysis. No correlates of net cardiac UII release were identified.

Conclusions: UII is secreted from the heart and multiple other tissues into the circulation. Related increments in UII immunoreactivity across multiple tissue sites suggest that peptide release occurs via a shared mechanism. Increased UII immunoreactivity is observed in subjects with acute coronary syndrome.

Methods: We used GC-MS to study the kinetics of LDL apo B-100 after intravenous administration of deuterated leucine and analyzed the data by compartmental modeling. The plasma PCSK9 concentration was measured by ELISA.

Results: Univariate regression analysis revealed the plasma PCSK9 concentration to be significantly and positively correlated with cholesterol (r = 0.543; P = 0.011), LDL cholesterol (r = 0.543; P = 0.011), apo B-100 (r = 0.548; P = 0.010), and LDL apo B-100 concentrations (r = 0.514; P = 0.023), and inversely correlated with the LDL apo B-100 fractional catabolic rate (FCR) (r = –0.456; P = 0.038). The association between plasma PCSK9 concentration and the LDL apo B-100 FCR remained statistically significant after adjusting for age, obesity, plasma insulin, homeostasis model assessment score, and dietary energy; however, this association had borderline significance after adjusting for plasma lathosterol.

Conclusions: In men, variation in plasma PCSK9 concentration influences the catabolism of LDL apo B-100. This finding appears to be independent of obesity, insulin resistance, energy intake, and age.