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The kidney performs a multitude of essential functions to maintain homeostasis. In clinical medicine, glomerular filtration rate (GFR) provides the best index of overall kidney function, and proteinuria adds additional information on renal and nonrenal prognosis. Several novel biomarkers of kidney injury and function are under investigation.

Plasma creatinine concentration is the most widely used measure for estimation of GFR. Plasma cystatin C and β-trace protein may eventually prove to be superior to creatinine. GFR may be measured directly by use of exogenous filtration markers, although their role is primarily limited to the research setting. Real-time, noninvasive measurement of GFR by using fluorescently labeled markers may be available in the future. Novel biomarkers of tubular injury such as neutrophil gelatinase-associated lipocalin, kidney injury molecule-1, liver-type fatty acid binding protein, N-acetyl-β-(D)-glucosaminidase, and interleukin-18 may enable the early detection of acute kidney injury before or in the absence of a change in GFR.

A variety of methods are available to assist clinicians in the assessment of kidney function and injury. Ongoing investigation will help determine the utility of several new markers and clarify their role in the care of patients with and at risk for kidney disease.

In the search for more powerful tools for diagnoses of endemic diseases in resource-limited settings, we have been analyzing technologies with potential applicability. Increasingly, the process focuses on readily accessible bodily fluids combined with increasingly powerful multiplex capabilities to unambiguously diagnose a condition without resorting to reliance on a sophisticated reference laboratory. Although these technological advances may well have important implications for the sensitive and specific detection of disease, to date their clinical utility has not been demonstrated, especially in resource-limited settings. Furthermore, many emerging technological developments are in fields of physics or engineering, which are not readily available to or intelligible to clinicians or clinical laboratory scientists.

This review provides a look at technology trends that could have applicability to high-sensitivity multiplexed immunoassays in resource-limited settings. Various technologies are explained and assessed according to potential for reaching relevant limits of cost, sensitivity, and multiplex capability. Frequently, such work is reported in technical journals not normally read by clinical scientists, and the authors make enthusiastic claims for the potential of their technology while ignoring potential pitfalls. Thus it is important to draw attention to technical hurdles that authors may not be publicizing.

Immunochromatographic assays, optical methods including those involving waveguides, electrochemical methods, magnetorestrictive methods, and field-effect transistor methods based on nanotubes, nanowires, and nanoribbons reveal possibilities as next-generation technologies.

Noninvasive fetal aneuploidy detection by use of free DNA from maternal plasma has recently been shown to be achievable by whole genome shotgun sequencing. The high-throughput next-generation sequencing platforms previously tested use a PCR step during sample preparation, which results in amplification bias in GC-rich areas of the human genome. To eliminate this bias, and thereby experimental noise, we have used single molecule sequencing as an alternative method.

For noninvasive trisomy 21 detection, we performed single molecule sequencing on the Helicos platform using free DNA isolated from maternal plasma from 9 weeks of gestation onwards. Relative sequence tag density ratios were calculated and results were directly compared to the previously described Illumina GAII platform.

Sequence data generated without an amplification step show no GC bias. Therefore, with the use of single molecule sequencing all trisomy 21 fetuses could be distinguished more clearly from euploid fetuses.

This study shows for the first time that single molecule sequencing is an attractive and easy to use alternative for reliable noninvasive fetal aneuploidy detection in diagnostics. With this approach, previously described experimental noise associated with PCR amplification, such as GC bias, can be overcome.

Single-molecule detection (SMD) technologies are well suited for clinical diagnostic applications by offering the prospect of minimizing precious patient sample requirements while maximizing clinical information content. Not yet available, however, is a universal SMD-based platform technology that permits multiplexed detection of both nucleic acid and protein targets and that is suitable for automation and integration into the clinical laboratory work flow.

We have used a sensitive, specific, quantitative, and cost-effective homogeneous SMD method that has high single-well multiplexing potential and uses alternating-laser excitation (ALEX) fluorescence-aided molecule sorting extended to 4 colors (4c-ALEX). Recognition molecules are tagged with different-color fluorescence dyes, and coincident confocal detection of ≥2 colors constitutes a positive target-detection event. The virtual exclusion of the majority of sources of background noise eliminates washing steps. Sorting molecules with multidimensional probe stoichiometries (S) and single-molecule fluorescence resonance energy transfer efficiencies (E) allows differentiation of numerous targets simultaneously.

We show detection, differentiation, and quantification—in a single well—of (a) 25 different fluorescently labeled DNAs; (b) 8 bacterial genetic markers, including 3 antibiotic drug–resistance determinants found in 11 septicemia-causing Staphylococcus and Enterococcus strains; and (c) 6 tumor markers present in blood.

The results demonstrate assay utility for clinical molecular diagnostic applications by means of multiplexed detection of nucleic acids and proteins and suggest potential uses for early diagnosis of cancer and infectious and other diseases, as well as for personalized medicine. Future integration of additional technology components to minimize preanalytical sample manipulation while maximizing throughput should allow development of a user-friendly ("sample in, answer out") point-of-care platform for next-generation medical diagnostic tests that offer considerable savings in costs and patient sample.

Familial hypercholesterolemia (FH) is an autosomal dominant disorder that affects cholesterol metabolism and is an important risk factor for heart disease. Three different genes were causally linked to this disorder: LDLR (low density lipoprotein receptor), APOB [apolipoprotein B (including Ag(x) antigen)], and PCSK9 (proprotein convertase subtilisin/kexin type 9). We evaluated a new amplicon preparation tool for resequencing these genes on next generation sequencing (NGS) platforms.

For the 3 genes, 38 primer pairs were designed and loaded on the Fluidigm Access Array, a microfluidic array in which a PCR was performed. We amplified 144 DNA samples (73 positive controls and 71 patient samples) and performed 3 sequencing runs on a GS FLX Titanium system from Roche 454, using pyrosequencing. Data were analyzed with the SeqNext module of the Sequence Pilot software.

From the 38 amplicons, 37 were amplified successfully, without any further optimization. Sequencing resulted in a mean coverage of the individual amplicons of 71-fold, 74-fold, and 117-fold for the 3 runs, respectively. In the positive controls, all known mutations were identified. In 29% of the patient samples, a pathogenic point mutation or small deletion/insertion was found. Large rearrangements were not detectable with NGS, but were picked up by multiplex ligation-dependent probe amplification.

Combining a microfluidic amplification system with massive parallel sequencing is an effective method for mutation scanning in FH patients, which can be implemented in diagnostics. For data analysis, we propose a minimum variant frequency threshold of 20% and a minimum coverage of 25-fold.

Warfarin is the most commonly prescribed oral anticoagulant medication but also is the second leading cause of emergency room visits for adverse drug reactions. Genetic testing for warfarin sensitivity may reduce hospitalization rates, but prospective genotyping is impeded in part by the turnaround time and costs of genotyping. Microfluidics-based assays can reduce reagent consumption and analysis time; however, no current assay has integrated multiplexed allele-specific PCR for warfarin genotyping with electrophoretic microfluidics hardware. Ideally, such an assay would use a single PCR reaction and, without further processing, a single microchip electrophoresis (ME) run to determine the 3 single-nucleotide polymorphisms (SNPs) affecting warfarin sensitivity [i.e., CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) *2, CYP2C9 *3, and the VKORC1 (vitamin K epoxide reductase complex 1) A/B haplotype].

We designed and optimized primers for a fully multiplexed assay to examine 3 biallelic SNPs with the tetraprimer amplification refractory mutation system (T-ARMS). The assay was developed with conventional PCR equipment and demonstrated for microfluidic infrared-mediated PCR. Genotypes were determined by ME on the basis of the pattern of PCR products.

Thirty-five samples of human genomic DNA were analyzed with this multiplex T-ARMS assay, and 100% of the genotype determinations agreed with the results obtained by other validated methods. The sample population included several genotypes conferring warfarin sensitivity, with both homozygous and heterozygous genotypes for each SNP. Total analysis times for the PCR and ME were approximately 75 min (1-sample run) and 90 min (12-sample run).

This multiplexed T-ARMS assay coupled with microfluidics hardware constitutes a promising avenue for an inexpensive and rapid platform for warfarin genotyping.

Serum prostate-specific antigen (PSA) concentrations after radical prostatectomy typically become undetectable with the use of current immunometric assay methods. Despite modern surgical techniques, 15%–30% of prostate cancer patients undergoing radical prostatectomy develop a biochemical recurrence during follow-up. Unfortunately, poor analytical sensitivity of standard PSA assays delays biochemical recurrence detection, and because of day-to-day assay imprecision ultrasensitive PSA assays cannot assess PSA kinetics. We developed an immuno-PCR assay for total PSA that has a limit of quantification >10 times lower than current ultrasensitive assays.

The 2-site immunometric assay for total PSA employed 2 monoclonal antibodies, one conjugated to a double-stranded DNA label and the other bound to paramagnetic microparticles. After several washing steps, quantification cycles were determined and values were converted to PSA concentrations. We characterized analytical performance and compared accuracy with a commercially available total PSA assay.

The limit of quantification was 0.65 ng/L and the assay was linear in the range of 0.25–152.0 ng/L. Total imprecision estimates at PSA concentrations of 3.8, 24.1, and 69.1 ng/L were <15.2%, <9.4%, and <10.6%, respectively. Recovery of supplemented PSA ranged from 87.5% to 119.2% (mean 100.3%). Dilution recovery ranged from 96.4% to 115.3% (mean 102.3%). There was no high-dose hook effect up to 50 000 ng/L of PSA. Comparison with the commercial PSA assay showed a regression slope of 1.06 and a correlation coefficient of 0.996.

The analytical characteristics of the assay support the use of this assay for the accurate and precise measurement of serum PSA, even at sub–nanogram-per-liter concentrations.

The clinical differential diagnosis of classic 21-hydroxylase deficiency (C21OHD) and cytochrome P450 oxidoreductase deficiency (PORD) is sometimes difficult, because both deficiencies can have similar phenotypes and high blood concentrations of 17α-hydroxyprogesterone (17OHP). The objective of this study was to identify biochemical markers for the differential diagnosis of C21OHD, PORD, and transient hyper 17α-hydroxyprogesteronemia (TH17OHP) in Japanese newborns. We established a 2-step biochemical differential diagnosis of C21OHD and PORD.

We recruited 29 infants with C21OHD, 9 with PORD, and 67 with TH17OHP, and 1341 control infants. All were Japanese and between 0 and 180 days old; none received glucocorticoid treatment before urine sampling. We measured urinary pregnanetriolone (Ptl), the cortisol metabolites 5α- and 5β-tetrahydrocortisone (sum of these metabolites termed THEs), and metabolites of 3 steroids, namely dehydroepiandrosterone, androstenedione (AD4), and 11β-hydroxyandrostenedione (11OHAD4) by GC-MS.

At a cutoff of 0.020, the ratio of Ptl to THEs differentiated C21OHD and PORD from TH17OHP and controls with no overlap. Among metabolites of DHEA, AD4, and 11OHAD4, only 11β-hydroxyandrosterone (11HA), a metabolite of 11OHAD4, showed no overlap between C21OHD and PORD at a cutoff of 0.35 mg/g creatinine.

A specific cutoff for the ratio of Ptl to THEs can differentiate C21OHD and PORD from TH17OHP and controls. Additionally, the use of a specific cutoff of 11HA can distinguish between C21OHD and PORD.

We measured ⁹-tetrahydrocannabinol (THC), 11-nor-9-carboxy-THC (THCCOOH), cannabidiol (CBD), and cannabinol (CBN) disposition in oral fluid (OF) following controlled cannabis smoking to evaluate whether monitoring multiple cannabinoids in OF improved OF test interpretation.

Cannabis smokers provided written informed consent for this institutional review board–approved study. OF was collected with the Quantisal™ device following ad libitum smoking of one 6.8% THC cigarette. Cannabinoids were quantified by 2-dimensional GC-MS. We evaluated 8 alternative cutoffs based on different drug testing program needs.

10 participants provided 86 OF samples –0.5 h before and 0.25, 0.5, 1, 2, 3, 4, 6, and 22 h after initiation of smoking. Before smoking, OF samples of 4 and 9 participants were positive for THC and THCCOOH, respectively, but none were positive for CBD and CBN. Maximum THC, CBD, and CBN concentrations occurred within 0.5 h, with medians of 644, 30.4, and 49.0 μg/L, respectively. All samples were THC positive at 6 h (2.1–44.4 μg/L), and 4 of 6 were positive at 22 h. CBD and CBN were positive only up to 6 h in 3 (0.6–2.1 μg/L) and 4 (1.0–4.4 μg/L) participants, respectively. The median maximum THCCOOH OF concentration was 115 ng/L, with all samples positive to 6 h (14.8–263 ng/L) and 5 of 6 positive at 22 h.

By quantifying multiple cannabinoids and evaluating different analytical cutoffs after controlled cannabis smoking, we determined windows of drug detection, found suggested markers of recent smoking, and minimized the potential for passive contamination.

New biomarkers are needed to assist clinical decision making in cardiovascular disease. We have recently shown that signal peptides may represent a novel biomarker target in cardiovascular diseases.

We developed a novel immunoassay for the signal peptide of preproANP (ANPsp) and used it to document cardiac tissue levels of ANPsp in explant human hearts (n = 9), circulating venous concentrations of ANPsp in healthy volunteers (n = 65), temporal ANPsp concentrations in patients with ST-elevation myocardial infarction (STEMI) <4 h after chest pain onset (n = 23), and regional plasma ANPsp concentrations in patients undergoing clinically indicated catheterization (n = 10). We analyzed the structure and sequence of circulating ANPsp by tandem mass spectrometry (MS/MS).

ANPsp levels in human heart tissue were 50–1000 times lower than those of ANP/NT-proANP. ANPsp was detectable in control human plasma at concentrations comparable with ANP itself (approximately 20 ng/L). In STEMI patients, plasma concentrations of ANPsp rose to peak values at 5 h after symptom onset, significantly earlier than myoglobin, creatine kinase-MB, and troponin (P < 0.001). There were significant arteriovenous increases in ANPsp concentrations (P < 0.05) across the heart and kidney; arterial and coronary sinus concentrations of ANPsp both negatively correlated with systolic and mean arterial blood pressures (both P < 0.01). MS/MS verified circulating ANPsp to be preproANP(16–25) and preproANP(18–25).

ANPsp is a novel circulating natriuretic peptide with potential to act as a cardiovascular biomarker. The rapid increase of plasma ANPsp in STEMI and its significant relationship with blood pressure encourage further study of its potential clinical utility.

The CDC's Lipid Standardization Program established the chromotropic acid (CA) reference measurement procedure (RMP) as the accuracy base for standardization and metrological traceability for triglyceride testing. The CA RMP has several disadvantages, including lack of ruggedness. It uses obsolete instrumentation and hazardous reagents. To overcome these problems the CDC developed an isotope dilution GC-MS (ID-GC-MS) RMP for total glycerides in serum.

We diluted serum samples with Tris-HCl buffer solution and spiked 200-μL aliquots with [¹³C₃]-glycerol. These samples were incubated and hydrolyzed under basic conditions. The samples were dried, derivatized with acetic anhydride and pyridine, extracted with ethyl acetate, and analyzed by ID-GC-MS. Linearity, imprecision, and accuracy were evaluated by analyzing calibrator solutions, 10 serum pools, and a standard reference material (SRM 1951b).

The calibration response was linear for the range of calibrator concentrations examined (0–1.24 mmol/L) with a slope and intercept of 0.717 (95% CI, 0.7123–0.7225) and 0.3122 (95% CI, 0.3096–0.3140), respectively. The limit of detection was 14.8 μmol/L. The mean %CV for the sample set (serum pools and SRM) was 1.2%. The mean %bias from NIST isotope dilution MS values for SRM 1951b was 0.7%.

This ID-GC-MS RMP has the specificity and ruggedness to accurately quantify total glycerides in the serum pools used in the CDC's Lipid Standardization Program and demonstrates sufficiently acceptable agreement with the NIST primary RMP for total glyceride measurement.

Mass spectrometric assays could potentially replace protein immunoassays in many applications. Previous studies have demonstrated the utility of liquid chromatography–multiple-reaction monitoring–mass spectrometry (LC-MRM/MS) for the quantification of proteins in biological samples, and many examples of the accuracy of these approaches to quantify supplemented analytes have been reported. However, a direct comparison of multiplexed assays that use LC-MRM/MS with established immunoassays to measure endogenous proteins has not been reported.

We purified HDL from the plasma of 30 human donors and used label-free shotgun proteomics approaches to analyze each sample. We then developed 2 different isotope-dilution LC-MRM/MS 6-plex assays (for apoliporoteins A-I, C-II, C-III, E, B, and J): 1 assay used stable isotope-labeled peptides and the other used stable isotope-labeled apolipoprotein A-I (an abundant HDL protein) as an internal standard to control for matrix effects and mass spectrometer performance. The shotgun and LC-MRM/MS assays were then compared with commercially available immunoassays for each of the 6 analytes.

Relative quantification by shotgun proteomics approaches correlated poorly with the 6 protein immunoassays. In contrast, the isotope dilution LC-MRM/MS approaches showed correlations with immunoassays of r = 0.61–0.96. The LC-MRM/MS approaches had acceptable reproducibility (<13% CV) and linearity (r ≥0.99). Strikingly, a single protein internal standard applied to all proteins performed as well as multiple protein-specific peptide internal standards.

Because peak area ratios measured in multiplexed LC-MRM/MS assays correlate well with immunochemical measurements and have acceptable operating characteristics, we propose that LC-MRM/MS could be used to replace immunoassays in a variety of settings.