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: 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 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 to perform verification and identification of interfering compounds.
RESULTS: We observed that the cases involving false-positive results involved a metabolite of zolpidem that might have been mistaken for lysergic acid diethylamide, a benzoylecgonine that might have been mistaken for atropine or 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%, 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: 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: 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: 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: 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: 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: 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 artifacts.
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: This report presents updated National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines summarizing quality requirements for the use of tumor markers.
METHODS: One subcommittee developed guidelines for analytical quality relevant to serum and tissue-based tumor markers in current clinical practice. Two other subcommittees formulated recommendations particularly relevant to the developing technologies of microarrays and mass spectrometry.
RESULTS: Prerequisites for optimal use of tumor markers in routine practice include formulation of the correct clinical questions to ensure selection of the appropriate test, adherence to good clinical and laboratory practices (e.g., minimization of the risk of incorrect patient and/or specimen identification, tube type, or timing), use of internationally standardized and well-characterized methods, careful adherence to manufacturer instructions, and proactive and timely reactions to information derived from both internal QC and proficiency-testing specimens. Highly desirable procedures include those designed to minimize the risk of the reporting of erroneous results attributable to interferences such as heterophilic antibodies or hook effects, to facilitate the provision of informative clinical reports (e.g., cumulative and/or graphical reports, appropriately derived reference intervals, and interpretative comments), and when possible to integrate these reports with other patient information through electronic health records. Also mandatory is extensive validation encompassing all stages of analysis before introduction of new technologies such as microarrays and mass spectrometry. Provision of high-quality tumor marker services is facilitated by dialogue involving researchers, diagnostic companies, clinical and laboratory users, and regulatory agencies.
CONCLUSIONS: Implementation of these recommendations, adapted to local practice, should encourage optimization of the clinical use of tumor markers.
]]>BACKGROUND: The utility of insulin-like growth factor (IGF) axis and collagen markers for a growth hormone (GH) doping test in sport depends on their stability and reproducibility. We sought to determine short-term within-subject variability of these markers in a large cohort of healthy individuals.
METHODS: We measured IGF-1, IGF binding protein 3 (IGFBP-3), acid labile subunit (ALS), and the collagen markers N-terminal propeptide of type I procollagen (PINP), C-terminal telopeptide of type I collagen (ICTP), and N-terminal propeptide of type III procollagen (PIIINP) in serum samples obtained on multiple occasions (median 3 per participant) over a 2- to 3-week period from 1103 elite athletes (699 men, 404 women) ages 22.2 (5.2) years [mean (SD)]. We estimated between-subject and within-subject variances by mixed–effects ANOVA.
RESULTS: Within-subject variance accounted for 32% to 36% and 4% to 13% of the total variance in IGF markers and collagen markers, respectively. The within-subject CV ranged from 11% to 21% for the IGF axis markers and from 13% to 15% for the collagen markers. The index of individuality for the IGF axis markers was 0.66–0.76, and for the collagen markers, 0.26–0.45. For each marker, individuals with initial extreme measured values tended to regress toward the population mean in subsequent repeated measurements. We developed a Bayesian model to estimate the long-term probable value for each marker.
CONCLUSIONS: These results indicate that in healthy individuals the within-subject variability was greater for IGF-1 than for the collagen markers, and that where a single measurement is available, it is possible to estimate the long-term probable value of each of the markers by applying the Bayesian approach. Such an application can increase the reliability and decrease the cost of detecting GH doping.
]]>BACKGROUND: The recent interest of clinical laboratories in developing serum testosterone assays based on isotope dilution liquid chromatography/tandem mass spectrometry (ID-LC/tandem MS) stems from the lack of accuracy of direct immunoassays. In this study, we assessed the accuracy and state of standardization (traceability) of 4 published ID-LC/tandem MS procedures in a method comparison with an ID-gas chromatography (GC)/MS reference measurement procedure listed in the database of the Joint Committee for Traceability in Laboratory Medicine.
METHODS: The study used 58 specimens from different patient categories. Each specimen was measured in triplicate (ID-LC/tandem MS) and quadruplicate (ID-GC/MS) in independent runs.
RESULTS: The testosterone concentrations by ID-GC/MS were 0.2–4.4 nmol/L (women), 0.2–2.0 nmol/L (hypogonadal man), and 10.1–31.3 nmol/L (normogonadal men). For ID-GC/MS, the CV was nearly constant, with a median of 1.0%; for ID-LC/tandem MS, it was concentration-dependent, with a median of up to 8%. Weighted Deming regression gave mean slopes, intercepts, and correlation coefficients of 0.90–1.11, -0.055–0.013 nmol/L, and 0.993–0.997, respectively. The % difference plot showed between 7% and 26% of the results outside a total error limit of 14%, with median deviations from ID-GC/MS between -9.6 and 0.4%.
CONCLUSIONS: This study demonstrated fairly good accuracy and standardization of the tested ID-LC/tandem MS procedures. Performance differences between procedures were evident in some instances, due to improper calibration and between-run calibration control. This emphasizes the need for thorough validation, including traceability, of new ID-LC/tandem MS procedures.
]]>BACKGROUND: Adequate vitamin D status is important for bone growth and mineralization and has been implicated in the regulation of autoimmunity, metabolic function, and cancer prevention. There are no reports of population-based studies on the vitamin D status of Canadian youth, a population with mandatory fortification of foods.
METHODS: We measured plasma 25-hydroxyvitamin D [25(OH)D], the best indicator of vitamin D status, in a school-based cross-sectional sample of representative French Canadian youth (n = 1753) ages 9, 13, and 16 years living in Québec (latitude: (45°–48°N). Blood samples were collected from January to May 1999. We defined 25(OH)D deficiency as ≤27.5 nmol/L, hypovitaminosis as ≤37.5 nmol/L, and optimal as >75.0 nmol/L.
RESULTS: More than 93% of youth in each age and sex group had suboptimal 25(OH)D concentrations. The prevalence of 25(OH)D deficiency increased with age in both sexes (P < 0.0001). It was 2%, 3%, and 13% in 9-, 13-, and 16-year-old boys and 2%, 8%, and 10% in 9-, 13-, and 16-year-old girls. Girls with higher body mass index and girls from households with lower income had lower 25(OH)D concentrations. These effects were not observed in boys.
CONCLUSIONS: Inadequate vitamin D status is a potentially serious public health problem among children and adolescents in Québec. Youth living at high latitudes in countries with and without mandatory fortification of vitamin D are likely at heightened risk of 25(OH)D deficiency. These results call for renewed efforts to ensure adequate vitamin D intake among growing children and adolescents.
]]>BACKGROUND: Glycohemoglobin (GHB), reported as hemoglobin (Hb) A1c, is a marker of long-term glycemic control in patients with diabetes and is directly related to risk for diabetic complications. HbE and HbD are the second and fourth most common Hb variants worldwide. We investigated the accuracy of HbA1c measurement in the presence of HbE and/or HbD traits.
METHODS: We evaluated 23 HbA1c methods; 9 were immunoassay methods, 10 were ion-exchange HPLC methods, and 4 were capillary electrophoresis, affinity chromatography, or enzymatic methods. An overall test of coincidence of 2 least-squares linear regression lines was performed to determine whether the presence of HbE or HbD traits caused a statistically significant difference from HbAA results relative to the boronate affinity HPLC comparative method. Deming regression analysis was performed to determine whether the presence of these traits produced a clinically significant effect on HbA1c results with the use of ±10% relative bias at 6% and 9% HbA1c as evaluation limits.
RESULTS: Statistically significant differences were found in more than half of the methods tested. Only 22% and 13% showed clinically significant interference for HbE and HbD traits, respectively.
CONCLUSIONS: Some current HbA1c methods show clinically significant interferences with samples containing HbE or HbD traits. To avoid reporting of inaccurate results, ion-exchange chromatograms must be carefully examined to identify possible interference from these Hb variants. For some methods, manufacturers' instructions do not provide adequate information for making correct decisions about reporting results.
]]>BACKGROUND: A serum ceruloplasmin concentration below 0.20 g/L is conventionally considered as one of the major diagnostic criteria for Wilson disease. This decision threshold has not been fully validated for its diagnostic characteristics, however. In this study, we evaluated various decision thresholds of serum ceruloplasmin concentration in the diagnosis of Wilson disease based on genotype-verified Wilson disease patients, carriers, and normal individuals.
METHODS: Serum ceruloplasmin concentration was measured by a nephelometric method in 57 Wilson disease patients and 71 family members (49 heterozygotes and 22 wild-type homozygotes), a validation group of 25 subjects clinically suspected of Wilson disease, and 690 normal individuals. We performed ROC analysis using Analyze-it software and confirmed the genotypes by direct DNA sequencing of ATP7B.
RESULTS: Serum ceruloplasmin concentrations <0.20, 0.14, and 0.10 g/L showed positive predictive values of 48.3%, 100%, and 100%, respectively, and negative predictive values of 98.7%, 97.1%, and 91.9%. In the validation group, a serum ceruloplasmin threshold of 0.14 g/L rendered 100% sensitivity and specificity. Forty of 690 healthy subjects had serum ceruloplasmin concentrations <0.20 g/L; however, these 40 individuals had normal genotypes by DNA sequencing, and none of the 40 had ceruloplasmin concentrations <0.14 g/L.
CONCLUSIONS: The diagnostic accuracy for Wilson disease using a serum ceruloplasmin concentration of 0.14 g/L as the local decision threshold was better than that using a threshold of 0.20 g/L. We suggest that laboratories providing ceruloplasmin assays determine decision thresholds based on local populations.
]]>BACKGROUND: Hypertriglyceridemia is a cardiovascular risk factor. Apolipoprotein C-III (apoC-III) is an important determinant of the catabolic rate of triglyceride-rich lipoproteins. The aim of this study was to investigate the prognostic value of plasma apoC-III concentrations for cardiovascular mortality.
METHODS: We performed this prospective study in 2244 subjects ages 49–77 years who participated in the Hoorn Study. During a mean follow-up of 15 years, 504 individuals died: 231 of cardiovascular disease, 180 of cancer, and 93 of other causes. Cardiovascular disease risk factors and plasma apoC-III concentrations were measured at baseline.
RESULTS: The age- and sex-adjusted plasma apoC-III concentration was prospectively associated with cardiovascular mortality (P < 0.001). After adjustment for traditional risk factors, including fasting triglycerides, the hazard ratio (95% CI) for cardiovascular death between the highest and the lowest quartile of apoC-III was 1.85 (1.02–3.38). High concentrations of apoC-III did not appear to be associated with noncardiovascular mortality.
CONCLUSIONS: In this general population cohort, a high apoC-III concentration in plasma, independently of fasting triglycerides and other traditional risk factors, predicts cardiovascular mortality.
]]>BACKGROUND: Smith-Lemli-Opitz syndrome (SLOS), a severe disorder of cholesterol synthesis, is classically diagnosed prenatally by GC-MS analysis of sterols in amniotic fluid. Considering the current trend toward tandem mass spectrometry (MS/MS) methodologies, we developed prototype LC-MS/MS methods for accurate diagnosis of the disorder.
METHODS: 3β-Hydroxysterols in amniotic fluid are oxidized with cholesterol oxidase to their corresponding 3-ketones, which are then derivatized with Girard P (GP) hydrazine in a "one-pot" reaction. The resulting GP-hydrazones give an improved response in electrospray (ES)-MS/MS owing to the presence of a charged quaternary nitrogen and are analyzed by reversed-phase LC-ES-MS/MS. Both capillary and conventional LC-MS/MS formats are suitable, and the method is also applicable to paper-absorbed blood spots.
RESULTS: In a double-blind analysis of 18 amniotic fluid samples comprising 6 SLOS and 12 controls, the ratio of 7 + 8-dehydrocholesterol (7 + 8-DHC) to cholesterol was <0.02 [range 0.00–0.02, mean (SD) 0.01 (0.007)] in all control samples (intraassay variation 5.91%) and >0.20 [0.20–1.13, 0.79 (0.35)] in SLOS (intraassay variation 4.56%), corresponding to a difference in ratios between the 2 groups of at least a factor of 10. The limit of quantification was equivalent to that of 2 nL amniotic fluid injected on-column.
CONCLUSIONS: We describe a proof-of-concept for the prenatal diagnosis of SLOS. Further developments will be necessary to automate sample handling and reduce chromatographic time for the methodology to be used in pre- and postnatal diagnosis.
]]>BACKGROUND: The American Diabetes Association (ADA)/European Association for the Study of Diabetes (EASD)/International Diabetes Federation (IDF)/IFCC Consensus Statement on the worldwide standardization of HbA1c states that "HbA1c results are to be reported world-wide in IFCC units and derived NGSP units, using the IFCC-NGSP master equation."
METHODS: We describe statistical methods to evaluate and monitor the relationships as expressed in master equations (MEs) between the IFCC Reference Measurement procedure (IFCC-RM) and designated comparison methods (DCMs) [US National Glycohemoglobin Standardization Program (NGSP), Japanese Diabetes Society/Japanese Society for Clinical Chemistry (JDS/JSCC), and Mono-S in Sweden]. We applied these statistics, including uncertainty calculations, to 12 studies in which networks of reference laboratories participated, operating the IFCC-RM and DCMs.
RESULTS: For NGSP and Mono-S, slope, intercept, and derived percentage HbA1c at the therapeutic target show compliance with the respective MEs in all 12 studies. For JDS/JSCC, a slight deviation is seen in slope and derived percentage HbA1c in 2 of the 12 studies. Using the MEs, the uncertainty in an assigned value increases from 0.42 mmol/mol HbA1c (IFCC-RM) to 0.47 (NGSP), 0.49 (JDS/JSCC), and 0.51 (Mono-S).
CONCLUSIONS: We describe sound statistical methods for the investigation of relations between networks of reference laboratories. Application of these statistical methods to the relationship between the IFCC-RM and DCMs in the US, Japan, and Sweden shows that they are suitable for the purpose, and the results support the applicability of the ADA/EASD/IDF/IFCC Consensus Statement on HbA1c measurement.
]]>BACKGROUND: The emergence of type 2 diabetes in young populations has mirrored a rising prevalence of obesity and insulin resistance during childhood and adolescence. At the same time, the role of adipokines as links between obesity and insulin resistance is becoming more appreciated. We sought to establish age- and sex-specific distributions of metabolic correlates of insulin resistance in healthy prepubertal children.
METHODS: We collected fasting blood samples from a contemporary cohort of 307 British children at ages 5, 6, 7, and 8 years and measured insulin, glucose, triglycerides, total and HDL cholesterol, urate, glycohemoglobin, sex hormone–binding globulin (SHBG), leptin, and adiponectin. We used homeostasis model assessment (HOMA 2) to estimate insulin sensitivity (HOMA-%S) and β-cell function (HOMA-%B). Anthropometric measures included body mass index.
RESULTS: Body mass index increased from age 5 to 8 years (P < 0.001). HOMA-%B decreased (P < 0.001) and HOMA-%S increased (P < 0.05), but glucose also increased (P < 0.001) whereas glycohemoglobin decreased (P < 0.001). Consistent with the rise in insulin sensitivity, HDL cholesterol increased (P < 0.001) and triglycerides decreased (NS), whereas adiponectin decreased (P = 0.02). The patterns were similar in boys and girls, although girls were less insulin sensitive throughout. Accordingly, triglycerides tended to be higher in the girls, and HDL cholesterol and SHBG lower.
CONCLUSIONS: The metabolic disturbances associated with insulin resistance appear to be more advanced in girls. Markers of metabolic health improve in both sexes from 5 to 8 years, despite rising adiposity.
]]>BACKGROUND: Increased N-terminal pro-B-type natriuretic peptide (NT-proBNP) concentrations are associated with increased cardiovascular mortality in chronic hemodialysis patients. Previous studies focused on prevalent dialysis patients and examined single measurements of NT-proBNP in time.
METHODS: We measured NT-proBNP concentrations in 2990 incident hemodialysis patients to examine the risk of 90-day and 1-year mortality associated with baseline NT-proBNP concentrations. In addition, we calculated the change in concentrations after 3 months in a subset of 585 patients to examine the association between longitudinal changes in NT-proBNP and subsequent mortality.
RESULTS: Increasing quartiles of NT-proBNP were associated with a monotonic increase in 90-day [quartile 1, referent; from quartile 2 to quartile 4, hazard ratio (HR) 1.7–6.3, P < 0.001] and 1-year (quartile 1, referent; from quartile 2 to quartile 4, HR 1.7–4.9, P < 0.001) all-cause mortality. After multivariable adjustment, these associations remained robust. When examined using a multivariable fractional polynomial, increased NT-proBNP concentrations were associated with increased 90-day (HR per unit increase in log NT-proBNP 1.5, 95% CI 1.3–1.7) and 1-year (HR per unit increase in log NT-proBNP 1.4, 95% CI 1.3–1.5) all-cause mortality. In addition, patients with the greatest increase in NT-proBNP after 3 months of dialysis had a 2.4-fold higher risk of mortality than those with the greatest decrease in NT-proBNP.
CONCLUSIONS: NT-proBNP concentrations are independently associated with mortality in incident hemodialysis patients. Furthermore, the observation that longitudinal changes in NT-proBNP concentrations were associated with subsequent mortality suggests that monitoring serial NT-proBNP concentrations may represent a novel tool for assessing adequacy and guiding therapy in patients initiating hemodialysis.
]]>BACKGROUND: The availability of commutable calibrator materials may ease considerably the task of harmonizing assay results and ensuring their traceability to reference procedures. We sought to verify the commutability of potential calibrator materials and evaluate their effectiveness in harmonizing LDH results by 2 measurement methods.
METHODS: We measured LDH in 109 serum samples and 31 materials, including frozen serum pools (with either normal or abnormal isoenzyme patterns), commercial stabilized materials, and the ERM-AD453/IFCC reference material. We assayed LDH activity with the IFCC reference procedure and with 2 commercial methods, 1 using the lactate-to-pyruvate (LP) reaction, and the other the pyruvate-to-lactate (PL) reaction. We selected a commutable material, with LDH value assigned by the reference procedure, as a calibrator for recalculating the results for patient sera by both LP and PL, thereby making them traceable to the IFCC reference procedure.
RESULTS: Original values for patient sera (n = 109) by the 2 commercial methods showed a mean (SD) PL/LP ratio of 1.97 (0.03); this ratio changed to 1.06 (0.02) after recalculation of results. Linear regression of PL vs LP recalibrated values gave y = 1.108x – 9.7. At the clinically important concentration of 250 U/L (upper reference limit), the systematic difference between methods was 6.8%, which met our proposed quality specifications for inaccuracy and total error.
CONCLUSIONS: By properly selecting the calibrator, the results of serum LDH measurement by 2 different methods may be harmonized and made traceable to the selected highest (reference) metrological level.
]]>BACKGROUND: The area under the receiver operating characteristics curve (AUC) is widely used to measure the diagnostic accuracy of noninvasive fibrosis indices. However, use of the AUC assumes a binary gold standard, whereas fibrosis staging is based on an ordinal scale and also depends on the distribution of fibrosis stages in the study sample. We explored other fibrosis staging accuracy measures designed for ordinal gold standards, the C-statistic and the Obuchowski measure.
METHODS: We performed a simulation study to assess the bias in estimating the accuracy measures when the distribution of fibrosis stages in the study sample do not fit the reference distribution in the population to which the indices are applied. We also estimated the type I error of the tests comparing these measures in 2 samples with different distributions of fibrosis stages. We illustrated the practical use of these measures by reanalyzing real data.
RESULTS: Compared with the AUC or the C-statistic, the Obuchowski measure showed limited bias when the distribution of fibrosis stages in the study sample differed from the reference distribution. The type I error was strongly inflated with the AUC or the C-statistic but was preserved in the Obuchowski measure. When we compared noninvasive indices on real data, AUC analysis led to discordant results depending on how the fibrosis stages were grouped together. One single conclusion was drawn from the analysis based on the Obuchowski measure.
CONCLUSIONS: We recommend using the Obuchowski measure for assessing the diagnostic accuracy of noninvasive indices of fibrosis.
]]>BACKGROUND: Individuals with severe deficiency in serum 1-antitrypsin (AAT) concentrations are at high risk for developing chronic obstructive pulmonary disease (COPD), whereas those carrying the PI*MZ genotype are at slightly increased risk. Testing appropriate subgroups of the population for AAT deficiency (AATD) is therefore an important aspect of COPD prevention and timely treatment. We decided to perform an exhaustive investigation of SERPINA1 gene variants in individuals from the general population with a moderately reduced serum AAT concentration, because such information is currently unavailable.
METHODS: We determined the Z and S alleles of 1399 individuals enrolled in the Swiss Cohort Study on Air Pollution and Lung Diseases in Adults (SAPALDIA) with serum AAT concentrations <113 mg/dL and submitted 423 of these samples for complete exon 2->5 sequencing.
RESULTS: We found that 900 of 1399 samples (64%), carried the normal PI*MM genotype, whereas 499 samples (36%) carried at least 1 SERPINA1 deficiency variant. In the subpopulations in which AAT concentrations ranged from 103 to 113 and from 93 to 103 mg/dL, individuals with the PI*MM genotype represented the majority (86.5% and 53.8%, respectively). The PI*MS genotype was predominant (54.9%) in the AAT range of 83 to 93 mg/dL, whereas PI*MZ represented 76.4% in the AAT range of 73 to 83 mg/dL.
CONCLUSIONS: This analysis provided a detailed molecular definition of intermediate AATD, which would be helpful in the diagnostic setting.
]]>BACKGROUND: Cellular markers help identify different components of a pathological process and may contribute to the diagnosis, prognostic assessment, and management of patients with suspected syndromes. Flow cytometry can be used to accurately assess markers of platelet and leukocyte activation and cellular aggregation in whole blood. To use cell markers as predictors of disease requires that they be measured reliably and show modest within-individual, day-to-day variation.
METHODS: We used whole blood flow cytometry to analyze monocyte and platelet markers in the Atherosclerosis Risk in Communities (ARIC) Carotid MRI study. We estimated laboratory variability using 20 split samples, process variation using replicate blood tubes taken from 112 subjects, and biologic plus process variation using replicate blood samples taken 4–8 weeks apart from 55 people.
RESULTS: For most analytes, the laboratory CV was <10% (mean 3.6%, range 0%–14.5%) and reliability was excellent (75% of analytes had R > 0.90). Reliability coefficients based on repeat-visit data indicated substantial to high repeatability (R > 0.60) for CD14, Toll-like receptor (TLR)-2, CD162, CD61, CD41, CD62P, CD154, and platelet–leukocyte aggregates. In contrast, TLR-4, CD45, myeloperoxidase (MPO), and cyclooxygenase (COX)-2 had slight to moderate repeat visit reliability.
CONCLUSIONS: The high repeatability results for selected platelet and monocyte markers indicate that they can be reliably measured in multicenter studies with delayed sample processing, provided that rigorous standardization of sample collection, shipping, and flow cytometry procedures is applied.
]]>BACKGROUND: Current methods for measuring the concentrations of lipoprotein particles and their distributions in particle subpopulations are not standardized. We describe here and validate a new gas-phase differential electrophoretic macromolecular mobility-based method (ion mobility, or IM) for direct quantification of lipoprotein particles, from small, dense HDL to large, buoyant, very-low-density lipoprotein (VLDL).
METHODS: After an ultracentrifugation step to remove albumin, we determined the size and concentrations of lipoprotein particles in serum samples using IM. Scan time is 2 min and covers a particle range of 17.2–540.0 Å. After scanning, data are pooled by totaling the particle number across a predetermined size range that corresponds to particular lipoprotein subclasses. IM results were correlated with those of standard methods for cholesterol and apolipoprotein analysis.
RESULTS: Intra- and interassay coefficients of variation for LDL particle size were <1.0%. The intra- and interassay variation for LDL and HDL particle subfraction measurements was <20%. IM-measured non-HDL correlated well with apolipoprotein B (r = 0.92).
CONCLUSIONS: The IM method provides accurate, reproducible, direct determination of size and concentration for a broad range of lipoprotein particles. Use of this methodology in studies of patients with cardiovascular disease and other pathologic states will permit testing of its clinical utility for risk assessment and management of these conditions.
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