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On this page considered biochemistry journals:
Clinical Chemistry - published by
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... is the leading forum for peer-reviewed, original research on innovative practices in today s clinical laboratory.
Clinical Chemistry and Laboratory Medicine - published by
deGruyter -
CCLM keeps you up-to-date with the latest developments in the clinical laboratory sciences. It reports on progress in fundamental and applied research. Areas covered include: clinical biochemistry, molecular medicine, hematology, immunology, microbiology, virology, drug measurement, genetic epidemiology, evaluation of diagnostic markers, new reagents and systems, reference materials, and reference values.
Journal of Laboratory Medicine - published by
deGruyter -
JLM reports on the latest developments in the various disciplines of laboratory medicine.
Current research articles of the mentioned
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BACKGROUND: In the workup of patients with suspected hereditary nonpolyposis colorectal cancer (HNPCC), detection of loss of heterozygosity (LOH) could help pinpoint the DNA in the mismatch-repair (MMR) gene carrying the germline mutation, but analysis of microsatellite markers has proved unreliable for this purpose. We developed a simple, low-cost method based on single-nucleotide polymorphism (SNP) genotyping and capillary electrophoresis for the assessment of LOH at 2 MMR loci simultaneously.
METHODS: We used the Applied Biosystems SNaPshot® Multiplex Kit with meticulously selected primers to assess 14 common SNPs in MLH1 [mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli)] and MSH2 [mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli)] and optimized the protocol for DNA isolated from peripheral blood and fresh/frozen or archival microsatellite-unstable tumors from patients with confirmed (n = 42) or suspected (n = 25) HNPCC. The 42 tumors from patients with confirmed MLH1 or MSH2 germline mutations were used to validate the method's diagnostic accuracy against results obtained with DNA sequencing or multiplex ligation-dependent probe amplification.
RESULTS: The SNaPshot assay provided better detection of certain SNPs than DNA sequencing. The MLH1 and MSH2 SNP marker sets were informative in 82% and 76% of the 67 cases analyzed, respectively. The new assay displayed 100% specificity for detecting LOH and predicted the location of the germline mutation in 40% of the cases (54% of those involving MLH1, 22% in MSH2).
CONCLUSIONS: Our SNP-based method for detecting LOH in MLH1 and MSH2 is simple to perform with instruments available in most clinical genetics laboratories. It can be a valuable addition to protocols now used to guide mutational screening of patients with suspected HNPCC.
BACKGROUND: Although the methodological quality of therapeutic guidelines (GLs) has been criticized, little is known regarding the quality of GLs that make diagnostic recommendations. Therefore, we assessed the methodological quality of GLs providing diagnostic recommendations for managing diabetes mellitus (DM) and explored several reasons for differences in quality across these GLs.
METHODS: After systematic searches of published and electronic resources dated between 1999 and 2007, 26 DM GLs, published in English, were selected and scored for methodological quality using the AGREE Instrument. Subgroup analyses were performed based on the source, scope, length, origin, and date and type of publication of GLs. Using a checklist, we collected laboratory-specific items within GLs thought to be important for interpretation of test results.
RESULTS: The 26 diagnostic GLs had significant shortcomings in methodological quality according to the AGREE criteria. GLs from agencies that had clear procedures for GL development, were longer than 50 pages, or were published in electronic databases were of higher quality. Diagnostic GLs contained more preanalytical or analytical information than combined (i.e., diagnostic and therapeutic) recommendations, but the overall quality was not significantly different. The quality of GLs did not show much improvement over the time period investigated.
CONCLUSIONS: The methodological shortcomings of diagnostic GLs in DM raise questions regarding the validity of recommendations in these documents that may affect their implementation in practice. Our results suggest the need for standardization of GL terminology and for higher-quality, systematically developed recommendations based on explicit guideline development and reporting standards in laboratory medicine.
Clinical Chemistry and Laboratory Medicine, Volume 0, Issue 0, Page -, Ahead of Print.
Abstract We present a practical protocol for the assignment of values to serum proteins in a Target Material using a Reference Material. This protocol is based on the model of Direct Value Transfer between serum matrices and is intended to improve the ...
Clinical Chemistry and Laboratory Medicine, Volume 0, Issue 0, Page -, Ahead of Print.
Abstract Standardization of cardiac troponin I (cTnI) measurement is important because of the central role for diagnosis of myocardial infarction. In blood, cTnI is present as a heterogeneous mixture of different molecular species. The analytical problem ...
Clinical Chemistry and Laboratory Medicine, Volume 0, Issue 0, Page -, Ahead of Print.
Abstract Background: Oxcarbazepine is an antiepileptic drug (AED) used to treat partial seizures as a monotherapy or adjunctive therapy. Oxcarbazepine is metabolized in the liver to its active metabolite, 10,11-dihydro-10-hydroxy-carbamazepine (MHD). ...
Clinical Chemistry and Laboratory Medicine, Volume 0, Issue 0, Page -, Ahead of Print.
Abstract Background: Myeloperoxidase (MPO) neutrophils have been considered an important pathophysiological factor in oxidative stress. Mainly through generation of hypochlorous acid in the phagosome, unchecked activity may lead to inactivation of ...
BACKGROUND: Saliva, which offers a noninvasive and stress-free alternative to plasma and serum, is a widely accepted sample source for analysis of steroids and also of certain amines and peptides. In recent years, numerous publications have described the use of salivary hormone analysis in many fields of clinical and basic research.
CONTENT: This review provides an overview of the current applications of salivary hormone analysis. A description of the different modes of hormone entry into saliva is followed by a detailed description of analytical methods and approaches for reliable collection of saliva, including several interesting applications in diverse fields including psychiatry, stress research, clinical endocrinology, sports medicine, and veterinary medicine.
SUMMARY: Although saliva has not yet become a mainstream sample source for hormone analysis, it has proven to be reliable and, in some cases, even superior to other body fluids. Nevertheless much effort will be required for this approach to receive acceptance over the long term, especially by clinicians. Such effort includes the development of specific and standardized analytical tools, the establishment of defined reference intervals, and implementation of round-robin trials. One major problem, the lack of compliance sometimes seen in outpatient saliva donors, requires strict standardization of both collection and analysis methods to achieve better comparability and assessment of published salivary hormone data.
BACKGROUND: Environmental conditions during sample processing, shipping, and storage are often suboptimal, particularly in less developed countries. We used samples from US volunteers to investigate the effects of delayed whole blood (WB) processing and delayed freezing of serum on selected nutritional indicators.
METHODS: WB tubes (n = 35) were either stored at 32 °C for up to 3 days before serum separation or centrifuged within 2 h of collection; serum samples were stored at 11 °C for up to 14 days to simulate delayed shipping. We assessed analyte stability by comparing results with data from optimally prepared/stored serum samples (<2 h on the clot, frozen at -70 °C) and by using clinical-acceptability criteria based on combined analytical imprecision and intraindividual biologic variability.
RESULTS: Clinically acceptable changes in concentration varied from 3%–15%. Delayed WB processing did not unacceptably affect concentrations of carotenoids and vitamins B12, D, and E; however, we obtained clinically unacceptable changes for ferritin (+9%), soluble transferrin receptor (sTfR) (+5%), and folate (-30%) after 1 day, and for vitamin A (-10%) after 3 days. Delayed freezing of serum did not affect concentrations of ferritin, sTfR, carotenoids, and vitamins A, B12, and E; however, we obtained clinically unacceptable changes for vitamins C (-20%) and D (+7%) after 7 days and for folate after 14 days (-22%).
CONCLUSIONS: Despite substantial delays in WB processing or in the freezing of serum samples, most nutritional indicators showed remarkable stability. This information is important for both the design of field studies and the use of residual samples subjected to suboptimal preanalytical factors.
Background: Sophisticated methods of cardiac imaging have the potential to revolutionize the care of patients with cardiovascular disease. The benefits of these state-of-the art imaging techniques can be enhanced by their use in combination with new cardiac biomarkers. This review addresses potentially useful interactions between imaging and biomarkers.
Content: Areas were defined in which the combined use of novel imaging techniques and biomarkers would be most beneficial. This review addresses multiple cardiovascular conditions for which the useful aspects of imaging and biomarkers are likely to be positively synergistic, including acute and chronic ischemic heart disease, heart failure, myocarditis, hypertension, and atherosclerosis.
Conclusions: The synergistic use of imaging techniques and biomarkers will enhance the investigation of many key issues and questions and will be an important resource in the future.
Background: Precise genotyping of the intron 8 poly(TG) and poly(T) tracts of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is of clinical relevance in CFTR pathology. The (TG)m locus influences the penetrance of the (T)5 allele, which may be associated with male infertility by congenital bilateral absence of the vas deferens (CBAVD) or other CFTR-related disorders (CFTR-RD), in particular in the context of (TG)12 and (TG)13. Simple and accurate genotyping of both loci should thus be routinely offered in laboratories.
Methods: We designed a new single test method relying on multiplex allele-specific fluorescent PCR: (T)5-, (T)7-, and (T)9-specific primers, labeled with different fluorophores, in combination with a common primer. Each fluorescent PCR product was identified on a capillary sequencer by its fluorescence color, specific for (T)n, and size, indicative of the (TG) length. We first validated the assay in 2 different laboratories on 52 DNA samples with already known genotypes. We then evaluated the method prospectively, compared with sequencing, on 62 samples from healthy individuals and 108 samples from patients with CBAVD or other CFTR-RDs.
Results: We observed a 100% match in both validation steps. Results found in CBAVD and CFTR-RD patients are in keeping with data in the literature.
Conclusions: The assay proved to be simple, rapid, and accurate for single-test (TG)m(T)n genotyping and suited for analysis in clinical laboratories.
Background: Brain natriuretic peptide (BNP) is an unstable molecule that can rapidly lose immunologic activity in blood. Conventional sandwich BNP immunoassays use 2 antibodies specific to 2 different epitopes. Larger distances between epitopes are associated with a greater probability of proteolysis sites being located between the antibody-binding sites, and thus such assays have an increased susceptibility to underdetect BNP because of the increased likelihood of proteolytic degradation. The purpose of our study was to develop a sandwich immunoassay for the precise quantification of BNP and BNP precursor (proBNP) in human blood that is not susceptible to proteolysis.
Methods: Mice were immunized with an immune complex consisting of monoclonal antibody (MAb) 24C5 (specific for BNP peptide 11–22) and the entire BNP molecule. The MAb used in our assay (Ab-BNP2) recognizes the immune complex but neither free BNP nor MAb 24C5.
Results: We used MAbs 24C5 and Ab-BNP2 to develop a new type of sandwich BNP assay (the "single-epitope sandwich assay"), which requires only a short BNP fragment (fragment 11–22) for immunodetection. This assay recognizes both BNP and proBNP with the same efficiency and sensitivity and demonstrates both considerably less susceptibility to antigen degradation and greater stability of the measured antigen than conventional sandwich BNP immunoassays.
Conclusions: We have developed this sensitive single-epitope sandwich assay for detecting BNP, proBNP, and their fragments in human blood. This assay appears promising for use in clinical studies to assist in triage, management, and outcomes assessment in heart failure patients.
Background: The recent discovery and specific functions of d-amino acids in humans are bound to lead to the revelation of d-amino acid abnormalities in human disorders. Therefore, high-throughput analysis techniques are warranted to determine d-amino acids in biological fluids in a routine laboratory setting.
Methods: We developed 2 chromatographic techniques, a nonchiral derivatization with chiral (chirasil-l-val column) separation in a GC-MS system and a chiral derivatization with Marfey’s reagent and LC- MS analysis. We validated the techniques for d-serine, l-serine, and glycine determination in cerebrospinal fluid (CSF), evaluated several confounders, and determined age-dependent human concentration ranges.
Results: Quantification limits for d-serine, l-serine, and glycine in cerebrospinal fluid were 0.14, 0.44, and 0.14 µmol/L, respectively, for GC-MS and 0.20, 0.41, and 0.14 µmol/L for LC-MS. Within-run imprecision was <3% for both methods, and between-run imprecision was <13%. Comparison of both techniques with Deming regression yielded coefficients of 0.90 (d-serine), 0.92 (l-serine), and 0.96 (glycine). Sample collection, handling, and transport is uncomplicated—there is no rostrocaudal CSF gradient, no effect of storage at 4 °C for 1 week before storage at –80 °C, and no effect of up to 3 freeze/thaw cycles. Conversely, contamination with erythrocytes increased d-serine, l-serine, and glycine concentrations. CSF concentrations for 145 apparently healthy controls demonstrated markedly and specifically increased (5 to 9 times) d-serine concentrations during early central nervous system development.
Conclusions: These 2 clinically applicable analysis techniques will help to unravel pathophysiologic, diagnostic, and therapeutic issues for disorders associated with central nervous system abnormalities, NMDA-receptor dysfunction, and other pathology associated with d-amino acids.
Background: Coffee consumption has been associated with several risk factors for coronary heart disease, including increased cholesterol, increased blood pressure, and increased plasma total homocysteine (tHcy). tHcy is determined by several B-vitamins. However, reports about the association between coffee intake and B-vitamin status are few.
Methods: We measured plasma B-vitamins and tHcy in a cohort of 10 601 healthy, middle-aged Norwegian men and women. Information about lifestyle factors, including coffee consumption, smoking, alcohol use, height, and weight, was obtained by interview.
Results: Coffee consumption was dose-dependently associated with reduced plasma B-vitamin concentrations. Compared with coffee abstainers, individuals drinking ≥4 cups/day had 11.7% (P < 0.001), 14.1% (P < 0.001), and 5.5% (P = 0.01) lower plasma concentrations of folate, pyridoxal phosphate, and riboflavin, respectively, and the mean tHcy concentration was 6.8% (P < 0.001) higher. Quantile regression analysis showed essentially no difference in B-vitamin concentrations between coffee consumption categories at low vitamin concentrations but a progressive increase in the difference at higher concentrations. This pattern of differences (effect profile) was found independently of smoking status, alcohol intake, and sex. The decrease in folate explained approximately half of the increase in tHcy.
Conclusions: Coffee consumption was associated with reduced circulating B-vitamin concentrations. The observed effect profiles indicated that coffee consumption preferentially affected the upper, but not the lower, part of the B-vitamin concentration distributions. We hypothesize that coffee consumption may increase the loss of surplus B-vitamins by excretion in urine.
Background: The accurate and precise measurement of urinary albumin is critical, since even minor increases are diagnostically sensitive indicators of renal disease, cardiovascular events, and risk for death. To gain insights into potential measurement biases, we systematically compared urine albumin measurements performed by LC-MS, a clinically available immunoturbidimetric assay, and size-exclusion HPLC.
Methods: We obtained unused clinical urine samples from 150 patients who were stratified by degrees of albuminuria (<20 mg/L, 20–250 mg/L, >250 mg/L) as determined by the immunoturbidimetric assay used in our clinical laboratory (Roche Hitachi 912). Urine albumin was then remeasured via LC-MS and HPLC (AccuminTM) assays.
Results: The immunoturbidimetric assay, calibrated using manufacturer-supplied serum-derived calibrators (Diasorin), underestimated albumin compared with LC-MS. After calibration with purified HSA, this immunoturbidimetric assay correlated well with LC-MS. HPLC overestimated albumin compared with both LC-MS and immunoturbidimetry. The current LC-MS and HPLC assays both performed poorly at concentrations <20 mg/L.
Conclusions: Efforts are needed to establish gold-standard traceable calibrators for clinical assays. LC-MS is a specific method to quantify albumin in native urine when concentrations exceed 20 mg/L, and therefore could be employed for standardization among assays.
Background: Analysis of carnitine and acylcarnitines by tandem mass spectrometry (MS/MS) has limitations. First, preparation of butyl esters partially hydrolyzes acylcarnitines. Second, isobaric nonacylcarnitine compounds yield false-positive results in acylcarnitine tests. Third, acylcarnitine constitutional isomers cannot be distinguished.
Methods: Carnitine and acylcarnitines were isolated by ion-exchange solid-phase extraction, derivatized with pentafluorophenacyl trifluoromethanesulfonate, separated by HPLC, and detected with an ion trap mass spectrometer. Carnitine was quantified with d3-carnitine as the internal standard. Acylcarnitines were quantified with 42 synthesized calibrators. The internal standards used were d6-acetyl-, d3-propionyl-, undecanoyl-, undecanedioyl-, and heptadecanoylcarnitine.
Results: Example recoveries [mean (SD)] were 69.4% (3.9%) for total carnitine, 83.1% (5.9%) for free carnitine, 102.2% (9.8%) for acetylcarnitine, and 107.2% (8.9%) for palmitoylcarnitine. Example imprecision results [mean (SD)] within runs (n = 6) and between runs (n = 18) were, respectively: total carnitine, 58.0 (0.9) and 57.4 (1.7) µmol/L; free carnitine, 44.6 (1.5) and 44.3 (1.2) µmol/L; acetylcarnitine, 7.74 (0.51) and 7.85 (0.69) µmol/L; and palmitoylcarnitine, 0.12 (0.01) and 0.11 (0.02) µmol/L. Standard-addition slopes and linear regression coefficients were 1.00 and 0.9998, respectively, for total carnitine added to plasma, 0.99 and 0.9997 for free carnitine added to plasma, 1.04 and 0.9972 for octanoylcarnitine added to skeletal muscle, and 1.05 and 0.9913 for palmitoylcarnitine added to skeletal muscle. Reference intervals for plasma, urine, and skeletal muscle are provided.
Conclusions: This method for analysis of carnitine and acylcarnitines overcomes the observed limitations of MS/MS methods.
Background: We observed cases of false-positive results with the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Different LC-MS/MS techniques that use the selected reaction-monitoring mode, routinely employed for the analysis and quantification of drugs and toxic compounds in biological matrices, were involved in the false-positive and potentially false-positive results obtained. We sought to analyze the causes of and solutions to this problem.
Methods: We used a previously reported LC-MS/MS general unknown screening method, as well as manual spectral investigation in 1 case, to perform verification and identification of interfering compounds.
Results: We observed that false-positive results involved: a metabolite of zolpidem that might have been mistaken for lysergic acid diethylamide, benzoylecgonine mistaken for atropine, and clomipramine and 3 phenothiazines that share several common ion transitions.
Conclusions: To prevent problems such as those we experienced, we recommend the use of stable-isotope internal standards when possible, relative retention times, 2 transitions or more per compound when possible, and acceptable relative abundance ratios between transitions, with an experience-based tolerance of ±15% for transitions with a relative abundance >10% and with an extension to ±25% for transitions <10% when the concentration is at the limit of quantification. A powerful general unknown screening procedure can help to confirm suspected interferences. Our results indicate that the specificity of screening procedures is questionable for LC-MS/MS analyses performed in the selected reaction-monitoring mode and involving a large number of compounds with only 1 transition per compound.
Background: Biomarkers play a pivotal role in the diagnosis and treatment of patients with cardiovascular disease. Active investigation has brought forward an increasingly large number of novel candidate markers; however, few of these markers have yet to be incorporated into routine clinical use.
Content: This review discusses biomarkers currently used in the setting of acute coronary syndromes. In this context, we assess the contemporary unmet needs for novel biomarkers in acute ischemic heart disease and the related challenges faced in developing new biomarkers to the point of integration into clinical practice. In particular, we address the impact of the availability of increasingly sensitive biomarkers of myocardial necrosis on the potential roles for novel biomarkers of inflammation, thrombosis, and ischemia.
Summary: Although active investigation has produced a growing list of candidate novel biomarkers for the care of patients with cardiovascular disease, it has become increasingly challenging to find appreciable incremental clinical benefit for their addition to existing markers, in particular newer, more analytically sensitive cardiac troponin assays. A major challenge for researchers and clinicians will be to demonstrate whether candidate novel markers are useful in improving diagnosis and guiding clinical treatment.
Background: We developed and compared 2 different methods for quantifying uracil (U) and dihydrouracil (UH2) in BSA and human plasma. Special attention was paid to the selectivity/specificity and the absence of a matrix effect. The UH2/U ratio is intended as a biomarker to identify patients with deficiency in 5-fluorouracil metabolism.
Methods: We quantified U and UH2 with 2 liquid chromatography methods after solid-phase extraction, one with UV detection (LC-UV) and the other with mass spectrometric detection (LC-MS). We selected 2 internal standards to prevent the risk of interferences. Separation was achieved with a Waters Atlantis dC18 column (LC-MS) or a Waters SymmetryShield RP18 column connected with an Atlantis dC18 (LC-UV). Mass spectrometric data were acquired in single-ion monitoring mode.
Results: Assay imprecision in BSA solution was <15% (LC-UV) and <12% (LC-MS); in plasma, assay imprecision was <9.5% and <9.0%, respectively. Recoveries were 88.2%–110% (LC-UV) and 94.8%–107% (LC-MS). Extraction efficiencies were ≥89.0%. In BSA, the lower limits of quantification for U and UH2 were 2.5 µg/L and 6.25 µg/L, respectively, for the LC-UV method and 2.5 µg/L and 3.1 µg/L for LC-MS. The corresponding values in plasma were 11.6 µg/L and 21.5 µg/L, and 4.1 µg/L and 12.1 µg/L.
Conclusions: To estimate endogenous U and UH2 concentrations and their ratio, we recommend the use of a drug-free human plasma pool in which baseline U and UH2 concentrations have previously been measured with the standard-addition method. Our LC-MS method, which has the better test performance and is useful for measuring UH2/U ratios in cancer patients, is preferred when this equipment is available.
Background: Pseudohypoparathyroidism type Ib (PHPIb) is characterized by parathyroid hormone (PTH) resistance, which can lead to hypocalcemia, hyperphosphatemia, and increased serum PTH. The disorder is caused by mutations in regulatory regions of the GNAS gene (GNAS complex locus) that lead to interferences in the methylation status of alternative GNAS promoters, such as exon A/B, NESP55, and XL-s. PHPIb comprises disorders that show distinctive changes in methylation status but share the same clinical phenotype: (a) loss of methylation only at exon A/B of the GNAS gene and involving no other obvious epigenetic abnormalities [e.g., those caused by heterozygous microdeletions in the STX16 (syntaxin 16) region and found in many patients with autosomal dominant (AD) PHPIb]; (b) methylation abnormalities at several differentially methylated regions (DMRs), which are observed in most patients with sporadic PHPIb and some families with AD PHPIb.
Methods: To permit early and reliable diagnosis of suspected PHPIb, we designed methylation-sensitive restriction enzyme–based and bisulfite deamination–based PCR tests for exon A/B and NESP55 DMRs.
Results: Both PCR strategies permit proper methylation testing of GNAS and NESP55 DMRs and elucidate different disease subtypes. We have identified a novel microsatellite repeat polymorphism within GNAS exon A/B, and pedigree analyses have shown its presence to be conclusive evidence for familial disease.
Conclusions: We provide a simple diagnostic test for PHPIb, an imprinting disorder caused by different molecular changes within the GNAS complex locus. PHPIb, a complex and diagnostically challenging clinical phenotype, can be treated successfully by taking steps before the manifestation of symptoms to avoid clinical complications in affected patients or asymptomatic members of affected families who show positive results in genetic tests.
Background: Initial screening of potential biomarkers for monitoring dialysis was performed with saliva samples collected from patients with end-stage renal disease (ESRD). A more thorough analysis of the most promising markers identified in the initial screening was conducted with saliva samples acquired at hourly intervals throughout dialysis to monitor analyte concentrations as dialysis progressed. We observed that salivary nitrite (NO2–) and uric acid (UA) concentrations consistently decreased as dialysis proceeded.
Methods: Solution-based colorimetric-detection chemistries for NO2– and UA were converted to a test strip format to produce a simple method for semiquantitatively measuring NO2– and UA concentrations in the clinic or at the patient’s home. We assessed the test strips with saliva samples collected from both ESRD patients undergoing dialysis and healthy control volunteers to qualitatively monitor the effect of dialysis on salivary NO2– and UA. We used computer software to analyze digital images of the resulting test strip color intensities.
Results: Test strip measurements showed that mean salivary concentrations of NO2– and UA were decreased in ESRD patients by 86% and 39%, respectively, compared with 15% and 9% for time-matched controls. Comparison of test strip results with calibrated solution-based assays suggests that the test strips can semiquantitatively measure salivary concentrations of NO2– and UA.
Conclusions: The colorimetric test strips monitored changes in salivary NO2– and UA concentrations that occurred in ESRD patients during dialysis. The test strips may prove useful for noninvasively evaluating dialysis progress and may also be useful for monitoring renal disease status.
Background: Hypermethylation of the RASSF1A [Ras association (RalGDS/AF-6) domain family member 1A] gene is frequently observed in hepatocellular carcinoma (HCC). We evaluated the analysis of circulating hypermethylated RASSF1A for detecting HCC and assessing its prognosis.
Methods: In module 1, we studied 63 pairs of HCC patients and age- and sex-matched chronic hepatitis B virus (HBV) carriers, as well as 50 healthy volunteers. In module 2, we studied 22 HCC patients with cancer detected through a surveillance program. The concentrations of circulating hypermethylated RASSF1A sequences were measured by real-time PCR after digestion with a methylation-sensitive restriction enzyme.
Results: We detected hypermethylated RASSF1A sequences in the sera of 93% of HCC patients, 58% of HBV carriers, and 8% of the healthy volunteers. The median RASSF1A concentrations for the HCC patients and HBV carriers were 7.70 x 105 copies/L and 1.18 x 105 copies/L, respectively (P < 0.01). The detection of low concentrations in HBV carriers is consistent with previous findings that RASSF1A hypermethylation is an early event in HCC pathogenesis and can be found in premalignant liver tissues. Use of a marker cutoff value of 1 x 106 copies/L also identifies 50% of -fetoprotein-negative HCC cases. Patients with higher RASSF1A concentrations at diagnosis or 1 year after tumor resection showed poorer disease-free survival (P < 0.01). For the HBV carriers who underwent HCC surveillance and subsequently developed HCC, the circulating concentration of RASSF1A increased significantly from the time of enrollment to cancer diagnosis (P = 0.014).
Conclusions: Detection and quantification of circulating methylated RASSF1A sequences are useful for HCC screening, detection, and prognostication.
Background: The pyridinium cross-links pyridinoline (PYD) and deoxypyridinoline (DPD) are established markers of bone resorption. We evaluated the analytical and clinical performance of a commercially available PYD HPLC assay and established reference intervals in children and adults.
Methods: We used a commercially available reagent set (Chromsystems Instruments & Chemicals) to measure PYD and DPD in 319 healthy controls (156 premenopausal women, 80 healthy men, and 83 healthy children age 1 month to 14 years) and 397 patients with metabolic bone diseases (postmenopausal osteoporosis, n = 175; male osteoporosis, n = 176; hyperparathyroidism, n = 17; hyperthyroidism, n = 19; Paget disease, n = 10).
Results: The mean intraassay and interassay CVs were <6% and <8% for both PYD and DPD, respectively. The reference interval was constant for premenopausal women in the age group 20–49 years. In men, cross-link values peaked at 20–29 years and decreased thereafter. Women with postmenopausal osteoporosis had significantly higher PYD (51%) and DPD (58%) values compared to premenopausal women. Similar results were found in osteoporotic men. In children the highest values were found in the first weeks and months after birth, followed by a decrease of 50%–60% at age 11–14 years. In metabolic bone diseases cross-link concentrations were significantly increased. The DPD:PYD ratio (mean value approximately 0.2) was remarkably constant in all populations evaluated.
Conclusions: The automated HPLC assay is a precise and convenient method for PYD and DPD measurement. We established reference intervals for adult women and men and for children up to 14 years old. The cross-link concentrations we determined by use of this HPLC method confirm its clinical value in enabling identification of increased bone resorption in patients with metabolic bone diseases.
Background: Real-time quantitative reverse transcription PCR (RQ-PCR) assay for BCR-ABL is used to monitor treatment response in chronic myeloid leukemia (CML). BCR-ABL transcript levels decline over several years of imatinib treatment, and increasing numbers of patients have BCR-ABL transcripts at or below the limit of detection. More sensitive PCR methods are required to assess whether these patients have a long-term continuing decline in residual disease.
Methods: We used random pentadecamer (R15) primers for reverse transcription in RQ-PCR and compared the results with our established method that uses random hexamers. An increase in assay sensitivity would be detected as an increase in the number of BCR-ABL transcripts.
Results: BCR-ABL transcripts increased by 86% with R15 primers. We used R15 primers to retest 19 samples from selected CML patients who had no BCR-ABL transcripts recently detectable with hexamer primers and detected BCR-ABL transcripts in 68% of the samples. Use of R15 primers showed variable increases in the transcripts for control genes BCR (breakpoint cluster region), ABL1 (c-abl oncogene 1, receptor tyrosine kinase), and GUSB (glucuronidase, beta), depending on the gene examined. The reported BCR-ABL/control gene ratio was affected, and the estimated detection limit of the assay, which was based on increased control gene copy number, was different for each control gene.
Conclusions: This simple modification to the reverse transcription methodology improved the detection limit of the RQ-PCR assay for BCR-ABL transcripts. In the field of CML, these results have important implications for defining the detection limit of an assay when the BCR-ABL transcript is undetectable. Random pentadecamer primers may also be useful in other reverse transcription PCR assays for which the abundance of the target RNA is low.
Background: A reagentless sensor array for simultaneous multianalyte testing (SMAT) may enable accurate diagnosis and be applicable for point-of-care testing. We developed a disposable reagentless immunosensor array for simple immunoassay of panels of tumor markers.
Methods: We carried out SMAT with a direct capture format, in which colloidal gold nanoparticles with bound horseradish peroxidase (HRP)-labeled antibodies were immobilized on screen-printed carbon electrodes with biopolymer/sol-gel to trap their corresponding antigens from sample solution. Upon formation of immunocomplex, the direct electrochemical signal of the HRP decreased owing to increasing spatial blocking, and the analytes could be simultaneously determined by monitoring the signal changes.
Results: The proposed reagentless immunosensor array allowed simultaneous detection of carcinoma antigen 153, carcinoma antigen 125, carbohydrate antigen 199, and carcinoembryonic antigen in clinical serum samples in the ranges of 0.4–140 kU/L, 0.5–330 kU/L, 0.8–190 kU/L, and 0.1–44 µg/L, respectively, with detection limits of 0.2 kU/L, 0.5 kU/L, 0.3 kU/L, and 0.1 µg/L corresponding to the signals 3 SD above the mean of a zero standard. The interassay imprecision of the arrays was <9.5%, and they were stable for 35 days. The positivity detection rate of panels of tumor markers was >95.5% for 95 cases of cancer-positive sera.
Conclusions: The immunosensor array provides a SMAT with short analytical time, small sampling volume, no need for substrate, and, no between-electrode cross-talk. This method not only proved the capability of the array in point-of-care testing, but also allowed simultaneous testing of several tumor markers.
Background: Serum C-terminal cross-linked telopeptide of type I collagen (CTX-I), N-terminal propeptide of type I collagen (PINP), and osteocalcin (OC) are among the most sensitive bone turnover markers for evaluating osteoporosis. Each marker is currently measured individually by manual or automated immunoassays that are time consuming and require substantial sample volume. We evaluated the performance characteristics of a novel, fully automated, protein-array chip system that allows the simultaneous measurement of CTX-I, PINP, OC, and intact parathyroid hormone (PTH) in 20 µL of serum.
Methods: We measured CTX-I, PINP, OC, and PTH using multiplex and corresponding automated single assays in 157 healthy premenopausal women, 74 healthy men, and 56 postmenopausal osteoporotic women before and 6 months after treatment with oral ibandronate (150 mg/month).
Results: Within- and between-run CVs of the multiplex assay were similar to those of single measurement assays (<10% for all markers), whereas the limit of quantification was lower, except for OC. Multiplex values highly correlated (r > 0.93, P < 0.0001 for all markers) with the corresponding single assays, and measured concentrations were comparable. After 6 months of ibandronate, CTX-I, PINP, and OC decreased by a median of 48%, 63%, and 52%, respectively (P < 0.0001 for all 3 markers), magnitudes similar to those of the corresponding single assays.
Conclusions: The automated protein-array chip demonstrated similar analytical precision, improved analytical sensitivity, and comparable measured concentrations to those of single assays. The multiplex assay should be useful for assessing bone metabolism in large clinical studies, particularly when sample volume is limited.
Background: Risk assessments of patients should be based on objective variables, such as biological markers that can be measured routinely. The acute response to stress causes the release of catecholamines from the adrenal medulla accompanied by chromogranin A (CGA). To date, no study has evaluated the prognostic value of CGA in critically ill intensive care unit patients.
Methods: We conducted a prospective study of intensive care unit patients by measuring serum procalcitonin (PCT), C-reactive protein (CRP), and CGA at the time of admission. Univariate and multivariate analyses were performed to evaluate the ability of these biomarkers to predict mortality.
Results: In 120 consecutive patients, we found positive correlations between CGA and the following: CRP (r2 = 0.216; P = 0.02), PCT (r2 = 0.396; P < 0.001), Simplified Acute Physiologic Score II (SAPS II) (r2 = 0.438; P < 0.001), and the Logistic Organ Dysfunction System (LODS) score (r2 = 0.374; P < 0.001). Nonsurvivors had significantly higher CGA and PCT concentrations than survivors [median (interquartile range): 293.0 µg/L (163.5–699.5 µg/L) vs 86.0 µg/L (53.8–175.3 µg/L) for CGA, and 6.78 µg/L (2.39–22.92 µg/L) vs 0.54 µg/L (0.16–6.28 µg/L) for PCT; P < 0.001 for both comparisons]. In a multivariable linear regression analysis, creatinine (P < 0.001), age (P < 0.001), and SAPS II (P = 0.002) were the only significant independent variables predicting CGA concentration (r2 = 0.352). A multivariate Cox regression analysis identified 3 independent factors predicting death: log-normalized CGA concentration [hazard ratio (HR), 7.248; 95% confidence interval (CI), 3.004–17.487], SAPS II (HR, 1.046; 95% CI, 1.026–1.067), and cardiogenic shock (HR, 3.920; 95% CI, 1.731–8.880).
Conclusions: CGA is a strong and independent indicator of prognosis in critically ill nonsurgical patients.
BACKGROUND: microRNA (miRNA) expression profiles are being intensively investigated for their involvement in carcinogenesis. We evaluated the prognostic value of mature microRNA-21 (miR-21) and mature microRNA-205 (miR-205) overexpression in non–small cell lung cancer (NSCLC).
PATIENTS AND METHODS: We studied 48 pairs of NSCLC fresh frozen tissue specimens collected at time of surgery and before chemotherapy. Highly specific amplification and quantification of mature miR-21 and mature miR-205 was achieved using looped real time RT-PCR.
RESULTS: miRNA expression, determined by real time RT-PCR, was defined by Ct measurements. We detected overexpression of mature miR-21 in 25 (52.0%) of the 48 NSCLC paired specimens and overexpression of miR-205 in 31 (64.6%). Overexpression was assessed after comparison of miRNA expression in NSCLC tissues and in their corresponding noncancerous tissues with respect to U6 expression. During the follow-up period, 29 of 48 (60.4%) patients relapsed, and 23 of 48 died (47.9%). Mature miR-21 was upregulated in 16 of 29 (55.2%) patients who relapsed and 15 of 23 (65.2%) patients who died. Mature miR-205 was overexpressed in 19 of 29 patients who relapsed (65.5%) and 15 of 23 patients who died (65.2%). Mature miR-21 overexpression correlated with overall survival (OS) of the patients (P = 0.027), whereas overexpression of mature miR-205 did not.
CONCLUSIONS: Our results suggest that overexpression of mature miR-21 is an independent negative prognostic factor for OS in NSCLC patients.
BACKGROUND: Gene expression profiling has the potential to offer consistent, objective diagnostic test results once a standardized protocol has been established. We investigated the robustness, precision, and reproducibility of microarray technology.
METHODS: One hundred sixty individual patient samples representing 11 subtypes of acute and chronic leukemias, myelodysplastic syndromes, and nonleukemia as a control group were centrally collected and diagnosed as part of the daily routine in the Munich Leukemia Laboratory. The custom AmpliChip Leukemia research microarray was used for technical analyses of quadruplicate mononuclear cell lysates in 4 different laboratories in Germany (D), Austria (A), and Switzerland (CH) (the DACH study).
RESULTS: Total-RNA preparations were successfully performed in 637 (99.5%) of 640 cases. Mean differences between pairs of laboratories in the total-RNA yield from the same sample ranged from 0.02 µg to 1.03 µg. Further processing produced 622 successful in vitro transcription reactions (97.6%); the mean differences between laboratories in the cRNA yield from the same sample ranged from 0.40 µg to 6.18 µg. After hybridization to microarrays, a mean of 47.6%, 46.5%, 46.2%, and 46.4% of probe sets were detected as present for the 4 laboratories, with mean signal-intensity scaling factors of 3.1, 3.7, 4.0, and 4.2, respectively. In unsupervised hierarchical cluster and principal component analyses, replicates from the same patient always clustered closely together, with no indications of any association between gene expression profiles due to different operators or laboratories.
CONCLUSIONS: Microarray analysis can be performed with high interlaboratory reproducibility and with comparable quality and high technical precision across laboratories.
BACKGROUND: Trace element external quality assessment schemes monitor laboratory performance and provide a stimulus for improvement in accuracy. However, monitoring of participant performance varies according to the scheme and can lead to conflicting conclusions.
METHODS: Quality specifications based on biological intra- and interindividual variability were calculated and compared to those currently used by various trace element external quality assessment schemes for plasma or serum copper, zinc, and selenium concentrations. For this purpose, we evaluated results reported by participating laboratories in different schemes, at key concentrations, using z scores.
RESULTS: Minimal quality specifications developed from the biological intra- and interindividual variability were, for Cu, ±0.84 µmol/L or 12% of the assigned target concentration, whichever is greater; for Zn, ±1.20 µmol/L or 15% of the assigned target concentration, whichever is greater; and for Se, ±0.072 µmol/L or 12% of the assigned target concentration, whichever is greater. Reported performance of the participating laboratories depended on analyte, concentration, and the selected quality specification. In addition, the most commonly used methods for the determination of Cu, Zn, and Se may give different results.
CONCLUSIONS: The proposed minimal quality specifications based on biological variation are generally slightly less stringent than those currently in use, although they do not drastically change the performance evaluation in the different schemes. These specifications are a first step in the harmonization of practices among the schemes and remain to be evaluated.
BACKGROUND: MicroRNAs (miRNAs) are RNA molecules that are involved in the regulation of many cellular processes, including those related to human cancers. The aim of this study was to determine, as a proof of principle, whether specific candidate miRNAs could be detected in fine-needle aspirate (FNA) biopsies of pancreatic ductal adenocarcinoma (PDAC) and could accurately differentiate malignant from benign pancreatic tissues.
METHODS: We used TaqMan® assays to quantify miRNA levels in FNA samples collected in RNARetain (n = 16) and compared the results with a training set consisting of frozen microdissected pancreatic samples (n = 20).
RESULTS: Quantitative reverse-transcription PCR analysis confirmed that miRNA levels are affected in PDAC FNAs and correlate well with the changes observed in the training set of frozen pancreatic samples. Analysis of the amounts produced for a few specific miRNAs enabled identification of PDAC samples. The combination of miR-196a and miR-217 biomarkers further improved the ability to distinguish between healthy tissue, PDAC, and chronic pancreatitis in the training set (P = 8.2 x 10-10), as well as segregate PDAC FNA samples from other FNA samples (P = 1.1 x 10-5). Furthermore, we showed that miR-196a production is likely specific to PDAC cells and that its incidence paralleled the progression of PDAC.
CONCLUSIONS: To the best of our knowledge, this study is the first to evaluate the diagnostic potential of miRNAs in a clinical setting and has shown that miRNA analysis of pancreatic FNA biopsy samples can aid in the pathologic evaluation of suspicious cases and may provide a new strategy for improving the diagnosis of pancreatic diseases.
BACKGROUND: Reports of the use of multiplex enzyme assay screening for Pompe disease, Fabry disease, Gaucher disease, Niemann-Pick disease types A and B, and Krabbe disease have engendered interest in the use of this assay in newborn screening. We modified the assay for high-throughput use in screening laboratories.
METHODS: We optimized enzyme reaction conditions and procedures for the assay, including the concentrations of substrate (S) and internal standard (IS), assay cocktail composition, sample clean-up procedures, and mass spectrometer operation. The S and IS for each enzyme were premixed and bottled at an optimized molar ratio to simplify assay cocktail preparation. Using the new S:IS ratio, we validated the modified assay according to CLSI guidelines. Stability of the S, IS, and assay cocktails were investigated. Dried blood spots from 149 healthy adults, 100 newborns, and 60 patients with lysosomal storage disorder were tested using the modified assay.
RESULTS: In our study, the median enzyme activity measured in adults was generally increased 2–3 fold compared to the original method, results indicating higher precision. In the multiplex format, each of the 5 modified enzyme assays enabled unambiguous differentiation between samples from healthy individuals (adults and newborns) and the corresponding disease-specific samples.
CONCLUSIONS: The modified multiplex enzyme assay with premixed S and IS is appropriate for use in high-throughput screening laboratories.
BACKGROUND: Macroprolactin is an important source of immunoassay interference that commonly leads to misdiagnosis and mismanagement of hyperprolactinemic patients. We used the predominant immunoassay platforms for total prolactin and bioactive monomeric prolactin to assay serum samples treated with polyethylene glycol (PEG) and establish and validate reference intervals for macroprolactin.
METHODS: We used the Architect (Abbott), ADVIA Centaur and Immulite (Siemens Diagnostics), Access (Beckman Coulter), Elecsys (Roche Diagnostics), and AIA (Tosoh) analyzers with samples from healthy males (n = 53) and females (n = 93) to derive parametric reference intervals for total and post-PEG monomeric prolactin. Concentrations of immunoreactive prolactin isoforms in serum samples from healthy individuals were established by gel filtration chromatography (GFC). We then used samples from 22 individuals whose hyperprolactinemia was entirely attributable to macroprolactin and 32 patients with true hyperprolactinemia to compare patient classifications and prolactin concentrations measured by GFC with the newly derived post-PEG reference intervals.
RESULTS: Parametric reference intervals for post-PEG prolactin in male and female serum samples, respectively, were (in mIU/L): 61–196, 66–278 (Centaur); 63–245, 75–381 (Elecsys); 70–301, 92–469 (Access); 72–229, 79–347 (Architect); 73–247, 83–383 (AIA); and 78–263, 85–394 (Immulite). Concordance between GFC and immunoassay-specific post-PEG reference intervals was observed in 311 of 324 cases and for 31 of 32 patients with true hyperprolactinemia and 17 of 22 patients with macroprolactinemia. Results leading to misclassification occurred in a few analyzers for 5 macroprolactinemia patient samples with relatively minor increases in post-PEG prolactin (mean 61 mIU/L).
CONCLUSIONS: Our validated normative reference data for sera pretreated with PEG and analyzed on the most commonly used immunoassay platforms should facilitate the more widespread introduction of macroprolactin screening by clinical laboratories.
