Showing papers in "Clinical Chemistry in 2005"

Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay (ELISA)

Abstract: This brief note addresses the historical background of the invention of the enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA). These assays were developed independently and simultaneously by the research group of Peter Perlmann and Eva Engvall at Stockholm University in Sweden and by the research group of Anton Schuurs and Bauke van Weemen in The Netherlands. Today, fully automated instruments in medical laboratories around the world use the immunoassay principle with an enzyme as the reporter label for routine measurements of innumerable analytes in patient samples. The impact of EIA/ELISA is reflected in the overwhelmingly large number of times it has appeared as a keyword in the literature since the 1970s. Clinicians and their patients, medical laboratories, in vitro diagnostics manufacturers, and worldwide healthcare systems owe much to these four inventors.

Hepascore: An Accurate Validated Predictor of Liver Fibrosis in Chronic Hepatitis C Infection

Abstract: Background: Staging hepatic fibrosis by liver biopsy guides prognosis and treatment of hepatitis C, but is invasive and expensive. We sought to create an algorithm of serum markers that accurately and reliably predict liver fibrosis stage among hepatitis C patients. Methods: Ten biochemical markers were measured at time of liver biopsy in 117 untreated hepatitis C patients (training set). Multivariate logistic regression and ROC curve analyses were used to create a predictive model for significant fibrosis (METAVIR F2, F3, and F4), advanced fibrosis (F3 and F4), and cirrhosis (F4). The model was validated in 104 patients from other institutions. Results: A model (Hepascore) of bilirubin, γ-glutamyltransferase, hyaluronic acid, α2-macroglobulin, age, and sex produced areas under the ROC curves (AUCs) of 0.85, 0.96, and 0.94 for significant fibrosis, advanced fibrosis, and cirrhosis, respectively. In the training set, a score ≥0.5 (range, 0.0–1.0) was 92% specific and 67% sensitive for significant fibrosis, a score <0.5 was 81% specific and 95% sensitive for advanced fibrosis, and a score <0.84 was 84% specific and 71% sensitive for cirrhosis. Among the validation set, the AUC for significant fibrosis, advanced fibrosis, and cirrhosis were 0.82, 0.90, and 0.89, respectively. A score ≥0.5 provided a specificity and sensitivity of 89% and 63% for significant fibrosis, whereas scores <0.5 had 74% specificity and 88% sensitivity for advanced fibrosis. Conclusions: A model of 4 serum markers plus age and sex provides clinically useful information regarding different fibrosis stages among hepatitis C patients.

Multiplex Human Papillomavirus Serology Based on In Situ-Purified Glutathione S-Transferase Fusion Proteins,

Abstract: Background: More than 100 different human papillomaviruses (HPVs) can cause proliferative diseases, many of which are malignant, such as cervical cancer. HPV serology is complex because infection and disease lead to distinct type-specific antibody responses. Using bead-based technology, we have developed an assay platform that allows the simultaneous detection of antibodies against up to 100 in situ affinity–purified recombinant HPV proteins. Methods: Twenty-seven HPV proteins were expressed as glutathione S -transferase fusion proteins and affinity-purified in one step by incubation of glutathione-displaying beads in bacterial lysate. Spectrally distinct bead sets, each carrying one particular antigen, were mixed, incubated with serum, and differentiated in a flow cytometer-like analyzer (xMAP; Luminex Corp). Antibodies bound to the antigens were detected via fluorescent secondary reagents. We studied 756 sera from 2 case-control studies of cervical cancer. Results: Glutathione S -transferase fusion proteins bound with high affinity to glutathione-displaying beads ( K d = 6.9 × 10−9 mol/L). The dynamic range of multiplex serology covered 1.5 orders of magnitude, and antibodies were detected at serum dilutions >1:1 000 000. Imprecision (median CV) was ≤5.4%, and assay reproducibility was high ( R 2 = 0.97). Results on clinical samples showed high concordance with ELISA (κ = 0.846), but multiplex serology exhibited increased detection of weak antibody responses. Antibodies to the E6 oncoproteins of the rare HPV types 52 and 58 were associated with cervical cancer ( P <0.001). Conclusion: Multiplex serology enables antibody analyses of large numbers of sera against up to 100 antigens in parallel and has the potential to replace ELISA technology.

Future Biomarkers for Detection of Ischemia and Risk Stratification in Acute Coronary Syndrome

Abstract: Background: Evaluation of patients who present to the hospital with a complaint of chest pain or other signs or symptoms suggestive of acute coronary syndrome (ACS) is time-consuming, expensive, and problematic. Recent investigations have indicated that increases in biomarkers upstream from biomarkers of necrosis (cardiac troponins I and T), such as inflammatory cytokines, cellular adhesion molecules, acute-phase reactants, plaque destabilization and rupture biomarkers, biomarkers of ischemia, and biomarkers of myocardial stretch may provide earlier assessment of overall patient risk and aid in identifying patients with higher risk of an adverse event. Approach and Content: The purpose of this review is to provide an overview of the pathophysiology and clinical and analytical characteristics of several biomarkers that may have potential clinical utility to identify ACS patients. These biomarkers (myeloperoxidase, metalloproteinase-9, soluble CD40 ligand, pregnancy-associated plasma protein A, choline, ischemia-modified albumin, unbound free fatty acids, glycogen phosphorylase isoenzyme BB, and placental growth factor) have demonstrated promise and need to be more thoroughly evaluated for commercial development for implementation into routine clinical and laboratory practice. Summary: Specifications that have been addressed for cardiac troponins and natriuretic peptides will need to be addressed with the same scrutiny for the biomarkers discussed in this review. They include validating analytical imprecision and detection limits, calibrator characterization, assay specificity and standardization, preanalytical issues, and appropriate reference interval studies. Crossing boundaries from research to clinical application will require replication in multiple settings and experimental evidence supporting a pathophysiologic role and, ideally, interventional trials demonstrating that monitoring single or multiple biomarkers improves outcomes.

Simple Cystatin C–Based Prediction Equations for Glomerular Filtration Rate Compared with the Modification of Diet in Renal Disease Prediction Equation for Adults and the Schwartz and the Counahan–Barratt Prediction Equations for Children

Abstract: Background: Serum creatinine is the most commonly used marker for estimation of glomerular filtration rate (GFR). To compensate for its drawbacks as a GFR marker, several prediction equations including several parameters are being used, with the Modification of Diet in Renal Disease (MDRD), Schwartz, and Counahan–Barratt equations being the ones most widely accepted for estimation of relative GFR in mL · min−1 · (1.73 m2)−1. The present study analyzes whether these GFR prediction equations for adults and children might be replaced by simple prediction equations based on plasma concentrations of cystatin C. Methods: Data from 536 patients (0.3–93 years), consecutively referred for determination of GFR by an invasive gold standard procedure, were used for the analysis. Calculations of bias (median percentage of error), correlation (adjusted R 2), and percentage of estimates within 30% and 50% of measured GFR were used in the comparisons. Results: A cystatin C–based prediction equation using only concentration in mg/L and a prepubertal factor: GFR [mL · min−1 · (1.73 m2)−1] = 84.69 × cystatin C (mg/L)−1.680 × 1.384 (if a child <14 years) assessed GFR equally well or better than the simplified MDRD, the Schwartz, and the Counahan–Barratt prediction equations for the adult (≥18 years) and juvenile groups of the investigated cohort. Age did not influence the cystatin C–based prediction equation for adults, whereas gender did, but with a factor close to unity (0.948 for females). Conclusion: A GFR prediction equation based solely on cystatin C (in mg/L) and a prepubertal factor might replace the simplified MDRD prediction equation for adults and the Schwartz and Counahan–Barratt prediction equations for children.

The Metabolic Syndrome: Requiescat in Pace

Abstract: Values for insulin-mediated glucose disposal vary continuously throughout a population of apparently healthy individuals, with at least a sixfold variation between the most insulin sensitive and most insulin resistant of these individuals. The more insulin resistant a person, the more insulin must be secreted to prevent decompensation of glucose tolerance. Insulin resistance is not a disease, but a description of a physiologic state, and approximately one third of an apparently healthy population is sufficiently insulin resistant to be at increased risk to develop a cluster of abnormalities and related clinical syndromes. The primary value of the concept of insulin resistance is that it provides a conceptual framework with which to place a substantial number of apparently unrelated biological events into a pathophysiological construct. In contrast, the metabolic syndrome was introduced as a diagnostic category to identify individuals that satisfy three of five relatively arbitrarily chosen criteria to initiate lifestyle changes with the goal of decreasing risk of cardiovascular disease. Consequently, the value of the notion of the metabolic syndrome must be considered not in pathophysiologic terms, but as a pragmatic approach to obtain a better clinical outcome. In this review, an effort is made to critically evaluate the concept of the metabolic syndrome, the criteria chosen to identify individuals with the syndrome, and the clinical utility of making, or not making, a diagnosis of the metabolic syndrome.

Simultaneous Measurement of β-Amyloid(1–42), Total Tau, and Phosphorylated Tau (Thr181) in Cerebrospinal Fluid by the xMAP Technology

Abstract: Background: To simultaneously study several biomarkers for Alzheimer disease (AD), we used the xMAP™ technology to develop and evaluate a multiparametric bead-based assay for quantification of β-amyloid(1–42) [Aβ(1–42)], total tau (T-TAU), and hyperphosphorylated tau [P-TAU(181P)] in cerebrospinal fluid (CSF). Methods: We compared the new multianalyte assay format with established ELISA techniques for the same proteins. We then performed a clinical study using CSF samples from patients with AD or mild cognitive impairment with progression to AD, healthy controls, and patients with other neurologic disorders. Results: The INNO-BIA AlzBio3 selectively and specifically measured Aβ(1–42), T-TAU, and P-TAU(181P) in the CSF. The new assay format had intra- and interassay CVs <10% for all analytes, even at low concentrations. The measurement range of the new assay was 3 to 4 logs compared with 1 to 2 logs for ELISAs. By plotting the mean of the values obtained in ELISA and the xMAP technology against the difference, we found that a correction factor could be used to convert xMAP results to ELISA values. The clinical study demonstrated that the new multiparametric assay could accurately distinguish patients with AD from patients with other neurologic disorders or control patients, with the diagnostic accuracy reaching recommended consensus criteria for specificity and sensitivity. Conclusion: The new multiparametric method may be able to replace the corresponding ELISA methods.

Routine isotope-dilution liquid chromatography-tandem mass spectrometry assay for simultaneous measurement of the 25-hydroxy metabolites of vitamins D2 and D3.

Abstract: Background: Measurement of 25-hydroxyvitamin D2 and D3 (25-OH D2 and D3) is essential for investigating vitamin D deficiency. Competitive binding techniques are unable to distinguish between the 2 metabolites and suffer from interference from other hydroxy metabolites of vitamin D. Methods: We used isotope-dilution liquid chromatography–tandem mass spectrometry (ID-LC-MS/MS) for routine determination of 25-OH D2 and D3 with a stable-isotope–labeled internal standard (IS). Serum samples (100 μL) were denatured with methanol–propanol containing IS, vortex-mixed, extracted into hexane, and dried under nitrogen. The reconstituted extract was chromatographed on a BDS C8 HPLC column, and the metabolites and IS were detected by electrospray ionization MS/MS in multiple-reaction monitoring mode. Results: 25-OH D2 and D3 and the IS nearly coeluted, whereas 1α-hydroxyvitamin D3 was separated; total run time was 8 min. The interassay CVs for 25-OH D2 were 9.5% and 8.4% at 52 and 76 nmol/L, respectively, and for 25-OH D3 were 5.1% and 5.6% at 55 and 87 nmol/L, respectively. The detection limit of the present method was <4 nmol/L for both metabolites. Method comparison with a commercial RIA measuring total 25-hydroxyvitamin D showed good correlation: y = 0.97 x − 2.7 nmol/L ( r = 0.91). The analytical system can assay 100 samples in 12.5 h. Conclusions: This simple robust interference-free LC-MS/MS assay is suitable for routine measurement of the 25-hydroxy metabolites of vitamins D2 and D3 in human serum. The assay has been in use for 9 months and has been used to assay more than 6000 routine samples.

Case–Control and Two-Gate Designs in Diagnostic Accuracy Studies

Abstract: Background: In some diagnostic accuracy studies, the test results of a series of patients with an established diagnosis are compared with those of a control group. Such case–control designs are intuitively appealing, but they have also been criticized for leading to inflated estimates of accuracy. Methods: We discuss similarities and differences between diagnostic and etiologic case–control studies, as well as the mechanisms that can lead to variation in estimates of diagnostic accuracy in studies with separate sampling schemes (“gates”) for diseased (cases) and nondiseased individuals (controls). Results: Diagnostic accuracy studies are cross-sectional and descriptive in nature. Etiologic case–control studies aim to quantify the effect of potential causal exposures on disease occurrence, which inherently involves a time window between exposure and disease occurrence. Researchers and readers should be aware of spectrum effects in diagnostic case–control studies as a result of the restricted sampling of cases and/or controls, which can lead to changes in estimates of diagnostic accuracy. These spectrum effects may be advantageous in the early investigation of a new diagnostic test, but for an overall evaluation of the clinical performance of a test, case–control studies should closely mimic cross-sectional diagnostic studies. Conclusions: As the accuracy of a test is likely to vary across subgroups of patients, researchers and clinicians might carefully consider the potential for spectrum effects in all designs and analyses, particularly in diagnostic accuracy studies with differential sampling schemes for diseased (cases) and nondiseased individuals (controls).

Measurement of Midregional Proadrenomedullin in Plasma with an Immunoluminometric Assay

Abstract: Background: Adrenomedullin (ADM) is a potent vasodilatory peptide, and circulating concentrations have been described for several disease states, including dysfunction of the cardiovascular system and sepsis. Reliable quantification has been hampered by the short half-life, the existence of a binding protein, and physical properties. Here we report the technical evaluation of an assay for midregional pro-ADM (MR-proADM) that does not have these problems. Methods: MR-proADM was measured in a sandwich immunoluminometric assay using 2 polyclonal antibodies to amino acids 45–92 of proADM. The reference interval was defined in EDTA plasma of 264 healthy individuals (117 male, 147 female), and increased MR-proADM concentrations were found in 95 patients with sepsis and 54 patients with cardiovascular disease. Results: The assay has an analytical detection limit of 0.08 nmol/L, and the interassay CV was 0.12 nmol/L. The assay was linear on dilution with undisturbed recovery of the analyte. EDTA-, heparin-, and citrate-plasma samples were stable (<20% loss of analyte) for at least 3 days at room temperature, 14 days at 4 °C, and 1 year at −20 °C. MR-proADM values followed a gaussian distribution in healthy individuals with a mean (SD) of 0.33 (0.07) nmol/L (range, 0.10–0.64 nmol/L), without significant difference between males or females. The correlation coefficient for MR-proADM vs age was 0.50 ( P <0.001). MR-proADM was significantly ( P <0.001) increased in patients with cardiovascular disease [median (range), 0.56 (0.08–3.9) nmol/L] and patients with sepsis [3.7 (0.72–25.4) nmol/L]. Conclusions: MR-proADM is stable in plasma of healthy individuals and patients. MR-proADM measurements may be useful for evaluating patients with sepsis, systemic inflammation, or heart failure.

Evaluation of Serum Protein Profiling by Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry for the Detection of Prostate Cancer: I. Assessment of Platform Reproducibility

Abstract: Background: Protein expression profiling for differences indicative of early cancer has promise for improving diagnostics. This report describes the first stage of a National Cancer Institute/Early Detection Research Network-sponsored multiinstitutional evaluation and validation of this approach for detection of prostate cancer. Methods: Two sequential experimental phases were conducted to establish interlaboratory calibration and standardization of the surface-enhanced laser desorption (SELDI) instrumental and assay platform output. We first established whether the output from multiple calibrated Protein Biosystem II SELDI-ionization time-of-flight mass spectrometry (TOF-MS) instruments demonstrated acceptable interlaboratory reproducibility. This was determined by measuring mass accuracy, resolution, signal-to-noise ratio, and normalized intensity of three m/z “peaks” present in a standard pooled serum sample. We next evaluated the ability of the calibrated and standardized instrumentation to accurately differentiate between selected cases of prostate cancer and control by use of an algorithm developed from data derived from a single site 2 years earlier. Results: When the described standard operating procedures were established at all laboratory sites, the across-laboratory measurements revealed a CV for mass accuracy of 0.1%, signal-to-noise ratio of ∼40%, and normalized intensity of 15–36% for the three pooled serum peaks. This was comparable to the intralaboratory measurements of the same peaks. The instrument systems were then challenged with sera from a selected group of 14 cases and 14 controls. The classification agreement between each site and the established decision algorithm were examined by use of both raw peak intensity boosting and ranked peak intensity boosting. All six sites achieved perfect blinded classification for all samples when boosted alignment of raw intensities was used. Four of six sites achieved perfect blinded classification with ranked intensities, with one site passing the criteria of 26 of 28 correct and one site failing with 19 of 28 correct. Conclusions: These results demonstrate that “between-laboratory” reproducibility of SELDI-TOF-MS serum profiling approaches that of “within-laboratory” reproducibility as determined by measuring discrete m/z peaks over time and across laboratories.

Use of Protein:Creatinine Ratio Measurements on Random Urine Samples for Prediction of Significant Proteinuria: A Systematic Review

Abstract: Background: Proteinuria is recognized as an independent risk factor for cardiovascular and renal disease and as a predictor of end organ damage. The reference test, a 24-h urine protein estimation, is known to be unreliable. A random urine protein:creatinine ratio has been shown to correlate with a 24-h estimation, but it is not clear whether it can be used to reliably predict the presence of significant proteinuria. Methods: We performed a systematic review of the literature on measurement of the protein:creatinine ratio on a random urine compared with the respective 24-h protein excretion. Likelihood ratios were used to determine the ability of a random urine protein:creatinine ratio to predict the presence or absence of proteinuria. Results: Data were extracted from 16 studies investigating proteinuria in several settings; patient groups studied were primarily those with preeclampsia or renal disease. Sensitivities and specificities for the tests ranged between 69% and 96% and 41% and 97%, respectively, whereas the positive and negative predictive values ranged between 46% and 95% and 45% and 98%, respectively. The positive likelihood ratios ranged between 1.8 and 16.5, and the negative likelihood ratios between 0.06 and 0.35. The cumulative negative likelihood ratio for 10 studies on proteinuria in preeclampsia was 0.14 (95% confidence interval, 0.09–0.24). Conclusion: The protein:creatinine ratio on a random urine specimen provides evidence to “rule out” the presence of significant proteinuria as defined by a 24-h urine excretion measurement.

Plasma concentrations of cystatin C in patients with coronary heart disease and risk for secondary cardiovascular events: more than simply a marker of glomerular filtration rate.

Abstract: Background: Renal impairment (RI) is associated with worse prognosis. Recently, cystatin C has been shown to represent a potentially superior marker of the glomerular filtration rate compared with creatinine clearance (CrCl). We evaluated the impact of cystatin C and other markers of RI on prognosis in a large cohort of patients with coronary heart disease (CHD). Methods: Cystatin C, creatinine (Cr), and CrCl were determined at baseline in a cohort of 1033 patients (30–70 years) with CHD. Patients were followed for a mean of 33.5 months, and a combined endpoint [fatal and nonfatal cardiovascular disease (CVD) events] was used as the outcome variable. Cystatin C was measured by immunonephelometry, and CrCl was calculated. Results: During follow-up, 71 patients (6.9%) experienced a secondary CVD event. Neither Cr ( P = 0.63) nor CrCl ( P = 0.10) were associated with incidence of CVD events, whereas cystatin C was clearly associated with risk of secondary CVD events ( P <0.0001). In multivariate analyses, patients in the top quintile of the cystatin C distribution at baseline had a statistically significantly increased risk of secondary CVD events even after adjustment for classic risk factors, severity of coronary disease, history of diabetes mellitus, treatment with angiotensin-converting enzyme inhibitors, and C-reactive protein (hazard ratio, 2.27; 95% confidence interval, 1.05–4.91) compared with patients in the bottom quintile. Conclusions: These data support the possibly important prognostic value of cystatin C among patients with known CHD and suggest that it may be a useful clinical marker providing complementary information to established risk determinants.

Simultaneous Measurement of 25 Inflammatory Markers and Neurotrophins in Neonatal Dried Blood Spots by Immunoassay with xMAP Technology

Abstract: Background: Inflammatory reactions and other events in early life may be part of the etiology of late-onset diseases, including cerebral palsy, autism, and type 1 diabetes. Most neonatal screening programs for congenital disorders are based on analysis of dried blood spot samples (DBSS), and stored residual DBSS constitute a valuable resource for research into the etiology of these diseases. The small amount of blood available, however, limits the number of analytes that can be determined by traditional immunoassay methodologies. Methods: We used new multiplexed sandwich immunoassays based on flowmetric Luminex® xMAP technology to measure inflammatory markers and neutrophins in DBSS. Results: The high-capacity 25-plex multianalyte method measured 23 inflammatory and trophic cytokines, triggering receptor expressed on myeloid cells-1 (TREM-1), and C-reactive protein in two 3.2-mm punches from DBSS. It also measured 26 cytokines and TREM-1 in serum. Standards Recovery in the 25-plex method were 90%–161% (mean, 105%). The low end of the working range for all 25 analytes covered concentrations found in DBSS from healthy newborns. Mean recovery of exogenous analytes added at physiologic concentrations in DBSS models was 174%, mean intra- and interassay CVs were 6.2% and 16%, respectively, and the mean correlation between added and measured analytes was r 2 = 0.91. In DBSS routinely collected on days 5–7 from 8 newborns with documented inflammatory reactions at birth, the method detected significantly changed concentrations of inflammatory cytokines. Measurements on DBSS stored at −24 °C for >20 years showed that most cytokines are detectable in equal concentrations over time. Conclusions: The method can reliably measure 25 inflammatory markers and growth factors in DBSS. It has a large potential for high-capacity analysis of DBSS in epidemiologic case–control studies and, with further refinements, in neonatal screening.

Influences of Blood Sample Processing on Low–Molecular-Weight Proteome Identified by Surface-Enhanced Laser Desorption/Ionization Mass Spectrometry

Abstract: Background: Profiling approaches in proteomics, such as surface-enhanced laser desorption/ionization (SELDI) mass spectrometry, are used in disease marker discovery. The aim of this study was to investigate the potential influence of selected preanalytical factors on the results obtained. Methods: Plasma samples anticoagulated with EDTA, citrate, or heparin, and serum samples from healthy volunteers were profiled by SELDI on CM10, immobilized metal affinity capture (IMAC) array with copper, and H50 chip surfaces. Using linear mixed-effects models, we examined the influence of elapsed time between venipuncture and sample separation (immediate to 24 h) and the type of serum tube used (Greiner Vacuette activator, gel serum separator, or plain tubes). We analyzed purified platelets, as well as platelet-poor and platelet-rich plasma samples treated with calcium and/or thrombin to determine the platelet contribution, directly or via the clotting process, to the profiles generated. We then used cluster analysis to identify samples with similar peak profiles. Results: Different plasma types and sera could be distinguished on the basis of cluster analyses of their spectral profiles. Elapsed time between venipuncture and separation of plasma and serum from blood samples altered the profiles obtained, particularly for serum samples and particularly on IMAC chips. The type of serum collection tube also affected the profiles because of differences in clotting time. In vitro manipulation of platelets revealed that specific peaks in IMAC profiles of serum appeared to be derived directly from platelets. Several other peaks, including some of those exhibiting time-dependent changes, arose during the clotting process. Conclusion: Preanalytical variables, such as sample handling, can markedly influence results.

Diagnostic Performance of Quantitative κ and λ Free Light Chain Assays in Clinical Practice

Abstract: Background: The quantitative assay for free light chains (FLCs) is a recently introduced commercial test reported to be sensitive and specific for detecting FLC diseases such as primary systemic amyloidosis (AL), light chain deposition disease (LCDD), nonsecretory multiple myeloma (NSMM), and light chain multiple myeloma. We evaluated its diagnostic performance in clinical practice. Methods: All FLC clinical test results generated in 2003 were abstracted from the Laboratory Information System. Diagnoses were obtained from the Dysproteinemia database and the patient medical history. Results: In 2003, we received samples for FLC assays from 1020 Mayo Clinic patients. The majority of these patients (88%) had bone marrow-derived monoclonal plasma cell disorders (PCDs). The 121 patients who did not have monoclonal gammopathy all had FLC κ/λ ratios within the range of values obtained for a reference population in our laboratory. Among the patients with monoclonal gammopathies were patients with multiple myeloma (330), AL (269), monoclonal gammopathy of undetermined significance (114), smoldering multiple myeloma (72), plasmacytoma (22), NSMM (20), macroglobulinemia (9), LCDD (7), and a variety of other PCDs. Among the 110 AL patients who had not been previously treated and who had a FLC assay performed within 120 days of diagnosis, the FLC κ/λ ratio was positive in 91% compared with 69% for serum immunofixation electrophoresis (IFE) and 83% for urine IFE. The combination of serum IFE and serum FLC assay detected an abnormal result in 99% (109 of 110) of patients with AL. Conclusion: The performance of the FLC assay in this analysis of clinical laboratory data is consistent with results from published retrospective validation studies.

Monitoring temporary immunodepression by flow cytometric measurement of monocytic HLA-DR expression: a multicenter standardized study.

Abstract: Background: Single-center trials have shown that monocytic HLA-DR is a good marker for monitoring the severity of temporary immunodepression after trauma, major surgery, or sepsis. A new test for measuring monocytic HLA-DR is now available. Methods: We evaluated a new test reagent set for monocytic HLA-DR expression (BD Quantibrite™ HLA-DR/Monocyte reagent; Becton Dickinson) in single-laboratory and interlaboratory experiments, assessing preanalytical handling, lyse-no-wash (LNW) vs lyse-wash (LW) values, reference values, and the effect of use of different flow cytometers and different instrument settings on test variance. Results: For preanalytical handling, EDTA anticoagulation, storage on ice as soon as possible, and staining within 4 h after blood collection gave results comparable to values obtained for samples analyzed immediately after collection (mean increase of ∼4% in monocytic HLA-DR). Comparison of LNW and LW revealed slightly higher results for LNW (∼18% higher for LNW compared with LW; r = 0.982). Comparison of different flow cytometers and instrument settings gave CVs <4%, demonstrating the independence of the test from these variables and suggesting that this method qualifies as a standardized test. CV values from the interlaboratory comparison ranged from 15% (blood sample unprocessed before transport) to 25% (stained and fixed before transport). Conclusions: For the BD Quantibrite HLA-DR/Monocyte test, preanalytical handling is standardized. Single-laboratory results demonstrated the independence of this test from flow cytometer and instrument settings. Interlaboratory results showed greater variance than single-laboratory values. This interlaboratory variance was partly attributable to the influence of transport and can be reduced by optimization of transport conditions.

Standardized Approach to Proteome Profiling of Human Serum Based on Magnetic Bead Separation and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Abstract: Background: Magnetic bead purification for the analysis of low-abundance proteins in body fluids facilitates the identification of potential new biomarkers by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The aims of our study were to establish a proteome fractionation technique and to validate a standardized blood sampling, processing, and storage procedure for proteomic pattern analysis. Methods: We used magnetic bead separation for proteome profiling of human blood by MALDI-TOF MS (mass range, 1000–10 000 Da) and studied the effects on the quality and reproducibility of the proteome analysis of anticoagulants, blood clotting, time and temperature of sample storage, and the number of freeze–thaw cycles of samples. Results: The proteome pattern of human serum was characterized by ∼350 signals in the mass range of 1000–10 000 Da. The proteome profile showed time-dependent dynamic changes before and after centrifugation of the blood samples. Serum mass patterns differed between native samples and samples frozen once. The best reproducibility of proteomic patterns was with a single thawing of frozen serum samples. Conclusion: Application of the standardized preanalytical blood sampling and storage procedure in combination with magnetic bead-based fractionation decreases variability of proteome patterns in human serum assessed by MALDI-TOF MS.

High-Resolution DNA Melting Analysis for Simultaneous Mutation Scanning and Genotyping in Solution

Abstract: Background: High-resolution DNA melting analysis with saturation dyes for either mutation scanning of PCR products or genotyping with unlabeled probes has been reported However, simultaneous PCR product scanning and probe genotyping in the same reaction has not been described Methods: Asymmetric PCR was performed in the presence of unlabeled oligonucleotide probes and a saturating fluorescent DNA dye High-resolution melting curves for samples in either capillaries (03 °C/s) or microtiter format (01 °C/s) were generated in the same containers used for amplification Melting curves of the factor V Leiden single-nucleotide polymorphism (SNP) and several mutations in exons 10 and 11 of the cystic fibrosis transconductance regulator gene were analyzed for both PCR product and probe melting transitions Results: Independent verification of genotype for simple SNPs was achieved by either PCR product or probe melting transitions Two unlabeled probes in one reaction could genotype many sequence variants with simultaneous scanning of the entire PCR product For example, analysis of both product and probe melting transitions genotyped ΔF508, ΔI507, Q493X, I506V, and F508C variants in exon 10 and G551D, G542X, and R553X variants in exon 11 Unbiased hierarchal clustering of the melting transitions identified the specific sequence variants Conclusions: When DNA melting is performed rapidly and observed at high resolution with saturating DNA dyes, it is possible to scan for mutations and genotype at the same time within a few minutes after amplification The method is no more complex than PCR and may reduce the need for resequencing

N-Terminal Pro-B-Type Natriuretic Peptide after High-Dose Chemotherapy: A Marker Predictive of Cardiac Dysfunction?

Abstract: Background: Chronic cardiac dysfunction may develop after administration of aggressive chemotherapy, sometimes leading to development of congestive heart failure (CHF). Recently, N-terminal pro-B-type natriuretic peptide (NT-proBNP) was implicated as a marker of CHF. In this study we evaluated the predictive role of NT-proBNP in patients treated with high-dose chemotherapy (HDC). Methods: NT-proBNP was measured after 62 chemotherapy treatments in 52 patients affected by aggressive malignancies. Blood samples were drawn before the start of HDC, at the end of HDC administration, and 12, 24, 36, and 72 h thereafter. In these patients, echocardiograms were performed regularly during a 1-year follow-up. Results: Seventeen patients (33%) had persistently increased NT-proBNP, 19 patients (36%) had only transient increases (concentrations went back to baseline at 72 h), and 16 (31%) had no increases [mean (SD) values at 72 h, 1163 (936) ng/L vs 185 (101) ng/L vs 39 (19) ng/L, respectively; P <0.0001]. Only patients with persistently increased NT-proBNP had a significant worsening of the left ventricular diastolic indexes from baseline to 12 months [ratio of peak early to peak late flow velocities from 1.42 (0.33) to 0.78 (0.11); P <0.0001; isovolumetric relaxation time from 90 (15) to 141 (26) ms; P <0.0001; E-wave deceleration time from 162 (17) to 224 (32) ms; P = 0.0004] and of the left ventricular ejection fraction [from 62.8 (3.4)% to 45.6 (11.5)%; P <0.0001]. Conclusions: Persistently increased NT-proBNP early after administration of HDC is strongly associated with development of cardiac dysfunction. This finding has important implications for identifying patients at risk of developing chemotherapy-induced cardiotoxicity.

Novel Isothermal, Linear Nucleic Acid Amplification Systems for Highly Multiplexed Applications

Abstract: Background: Global analysis of the genome, transcriptome, and proteome is facilitated by the recent development of tools for large-scale, highly parallel analysis. We describe a novel nucleic acid amplification system that generates products by several methods. 3′-Ribo-SPIA™ primes cDNA synthesis at the 3′ polyA tail, and whole transcript (WT)-Ribo-SPIA primes cDNA synthesis across the full length of the transcripts and thus provides whole-transcriptome amplification, independent of the 3′ polyA tail. Methods: We developed isothermal linear nucleic acid amplification systems, which use a single chimeric primer, for amplification of DNA (SPIA) and RNA (Ribo-SPIA). The latter allows mRNA amplification from as little as 1 ng of total RNA. Amplification efficiency was calculated based on the delta threshold cycle between nonamplified cDNA targets and amplified cDNA. The amounts and quality of total RNA and amplification products were determined after purification of the amplification products. GeneChip® array gene expression profiling and real-time PCR were used to test the accuracy and reproducibility of the method. Quantification of cDNA products (before and after amplification) at the 2 loci along the transcripts was used to assess product length (for evaluation of the 3′-initiated Ribo-SPIA) and equal representation throughout the length of the transcript (for evaluation of the whole transcript amplification system, WT-Ribo-SPIA™). Results: Ribo-SPIA–based global RNA amplification exhibited linearity over 6 orders of magnitude of transcript abundance and generated microgram amounts of amplified cDNA from as little as 1 ng of total RNA. Conclusions: The described methods enable comprehensive gene expression profiling and analysis from limiting biological samples. The WT-Ribo-SPIA procedure, which enables amplification of non–polyA-tailed RNA, is suitable for amplification and gene expression analysis of both eukaryotic and prokaryotic biological samples.

D-Dimer Concentrations in Normal Pregnancy: New Diagnostic Thresholds Are Needed

Abstract: Background: Pregnancy is known to increase the D-dimer concentration above the conventional normal threshold of 0.50 mg/L, leading to an increased false-positive D-dimer test when venous thromboembolism (VTE) is clinically suspected in a pregnant patient. Our aim was to determine the effect of normal pregnancy on the D-dimer concentration. Methods: Healthy women who were seeking to become pregnant and had no preexisting condition known to increase the D-dimer concentration were identified. Quantitative D-dimer measurements (MDA turbidimetric assay) and fibrinogen assays were performed before conception, at each trimester, and at 4 weeks postpartum. Patients were excluded for fetal loss or preeclampsia. Results: A total of 50 women were enrolled in the study, and blood samples were obtained at preconception and all trimesters from 23 women. The mean (SD) preconception D-dimer concentration was 0.43 (0.49) mg/L, and 79% of women had a D-dimer concentration <0.50 mg/L. D-Dimer increased with each trimester such that only 22% of women in the second trimester and none (of 23) in the third trimester (95% confidence interval, 0–14%) had a D-dimer concentration <0.50 mg/L. We found no correlation between either the D-dimer and fibrinogen concentrations or between the increases in D-dimer and fibrinogen with pregnancy. Conclusions: Normal pregnancy causes a progressive increase in circulating D-dimer. The D-dimer test has no use in ruling out VTE in the third trimester if a cutoff of 0.50 mg/L is used. A large management study is needed to establish new thresholds for the D-dimer to rule out VTE in each trimester.

Screening for Primary Aldosteronism in Essential Hypertension: Diagnostic Accuracy of the Ratio of Plasma Aldosterone Concentration to Plasma Renin Activity

Abstract: Background: The ratio of plasma aldosterone concentration to plasma renin activity (PRA) is considered the screening test of choice for primary aldosteronism. Uncertainty exists, however, regarding its diagnostic accuracy and the effects of antihypertensive drugs and dietary sodium balance on test characteristics. Methods: We measured PRA and aldosterone in 118 white adults [71 men and 47 women; mean (SD) age, 51 (7) years] with previously diagnosed essential hypertension. Measurements were made while individuals were on antihypertensive drug therapy, after a 2-week drug-free period, after 4 days of dietary sodium loading, and after acute furosemide diuresis. We measured 24-h urine aldosterone excretion and PRA on the 4th day of dietary sodium loading to establish the diagnosis of primary aldosteronism. ROC curves were constructed for ratios measured under each clinical condition, and likelihood ratios were determined for individuals on or off antihypertensive drug therapy. Results: Fifteen patients [13%; 95% confidence interval (CI), 7–20%] met the reference standard for primary aldosteronism. The mean (SD) areas under the ROC curves did not differ significantly across conditions of measurement [range, 0.80 (0.10) to 0.85 (0.04); P = 0.72]. When measured on and off antihypertensive drug therapy, the 95% CIs for the optimum cutpoint for the ratio overlapped. Point estimates of sensitivity on and off therapy were 73% (95% CI, 50–96%) and 87% (70–100%), respectively, and specificities were 74% (65–83%) and 75% (66–84%). Under either condition, increased ratios were associated with 2.4- to 13-fold increases of posttest odds above pretest odds. Conclusions: The aldosterone:PRA ratio provides only fair diagnostic accuracy in screening for primary aldosteronism, but concomitant antihypertensive drug therapy or acute variation in dietary sodium balance does not adversely affect test accuracy. Reporting of likelihood ratios associated with ranges of values of the aldosterone:PRA ratio, rather than use of a single “optimum” cutpoint, may enhance the usefulness of the test.

New Reference Intervals for Thyrotropin and Thyroid Hormones Based on National Academy of Clinical Biochemistry Criteria and Regular Ultrasonography of the Thyroid

Abstract: Background: The aim of our present study was to establish new reference intervals for thyrotropin (TSH) and thyroid hormones based on National Academy of Clinical Biochemistry (NACB) criteria and regular thyroid ultrasonography. We also assessed the effect of potentially confounding factors to modulate the limits of these intervals. Methods: We investigated 870 apparently healthy persons and excluded, step by step, those with a family history of thyroid disease, pathologic thyroid ultrasonography results, and increased anti-thyroid peroxidase or anti-thyroglobulin antibodies. Accordingly, only 453 of the 870 persons in the entire group were finally included as reference collective. We measured serum concentrations of TSH, total and free thyroxine (T4 and FT4), and total and free triiodothyronine (T3 and FT3) of the whole and the reference collective on the ELECSYS system assays (Roche Diagnostics) and calculated the 2.5th and 97.5th percentiles for comparison. Results: The calculated lower limit for TSH differed significantly between the reference intervals for healthy persons with an assessed normal thyroid gland vs the nonselected group of healthy blood donors. Age was the only independent factor and was significantly inversely associated with TSH ( P <0.0001). Use of oral contracep-tives was a significant predictor for variation in T4 concentrations ( P <0.001). Age and oral contraceptives were independently associated with T3 variations ( P <0.05). For FT4 vs FT3 variation, gender and (inversely) age ( P <0.01) were independent modulating factors. Conclusions: The selection of healthy persons according to NACB criteria combined with sonographic confirmation of a normal thyroid gland provide a valid basis for the reference interval for TSH. Factors indicating a preclinical disease state, such as family history, pathologic ultrasonography result, or increased anti-thyroid peroxidase and anti-thyroglobulin antibodies, can be associated with normal hormone concentrations. Additionally, patient age and gender as well as use of contraceptives should be considered in diagnostic evaluation of thyroid diseases.

Contribution of CYP3A5 to the in vitro hepatic clearance of tacrolimus.

Abstract: Background: Tacrolimus is metabolized predominantly to 13- O -demethyltacrolimus in the liver and intestine by cytochrome P450 3A (CYP3A). Patients with high concentrations of CYP3A5, a CYP3A isoenzyme polymorphically produced in these organs, require higher doses of tacrolimus, but the exact mechanism of this association is unknown. Methods: cDNA-expressed CYP3A enzymes and a bank of human liver microsomes with known CYP3A4 and CYP3A5 content were used to investigate the contribution of CYP3A5 to the metabolism of tacrolimus to 13- O -demethyltacrolimus as quantified by liquid chromatography–tandem mass spectrometry. Results: Demethylation of tacrolimus to 13- O -demethyltacrolimus was the predominant clearance reaction. Calculated K m and V max values for CYP3A4, CYP3A5, and CYP3A7 cDNA-expressed microsomes were 1.5 μmol/L and 0.72 pmol · (pmol P450)−1 · min−1, 1.4 μmol/L and 1.1 pmol · (pmol P450)−1 · min−1, and 6 μmol/L and 0.084 pmol · (pmol P450)−1 · min−1, respectively. Recombinant CYP3A5 metabolized tacrolimus with a catalytic efficiency (Vmax/ K m) that was 64% higher than that of CYP3A4. The contribution of CYP3A5 to 13-O-demethylation of tacrolimus in human liver microsomes varied from 1.5% to 40% (median, 18.8%). There was an inverse association between the contribution of CYP3A5 to 13-O-demethylation and the amount of 3A4 protein ( r = 0.90; P <0.0001). Mean 13-O-demethylation clearances in CYP3A5 high and low expressers, estimated by the parallel-tube liver model, were 8.6 and 3.57 mL · min−1 · (kg of body weight)−1, respectively ( P = 0.0088). Conclusions: CYP3A5 affects metabolism of tacrolimus, thus explaining the association between CYP3A5 genotype and tacrolimus dosage. The importance of CYP3A5 status for tacrolimus clearance is also dependent on the concomitant CYP3A4 activity.

The novel apolipoprotein A5 is present in human serum, is associated with VLDL, HDL, and chylomicrons, and circulates at very low concentrations compared with other apolipoproteins.

Abstract: Background: The recently discovered apolipoprotein A5 (ApoA5) is fast gaining attention as a key regulator of serum triglyceride concentrations. An ApoA5 mouse knock-out model produced an approximately fourfold increase in serum triglycerides, whereas a knock-in model with human ApoA5 produced 50–70% lower concentrations of mouse serum triglycerides. In addition, peroxisome proliferator-activated receptor-α agonists, which are used clinically to lower serum triglyceride concentrations, cause increased ApoA5 mRNA expression. Despite these compelling molecular biology data, relatively little is known about ApoA5 protein in human serum. Methods: To better understand circulating concentrations and lipoprotein particle distribution of ApoA5, we expressed the recombinant human ApoA5 protein and raised antibodies against both the NH2 and COOH termini. Results: Using the above reagents, we demonstrate for the first time that ApoA5 is present in human serum, although at much lower concentrations than other apolipoproteins such as ApoA1. Using a dual-antibody sandwich ELISA that we developed, we observed ApoA5 concentrations in human serum ranging from 24 to 406 μg/L compared with ∼1 g/L for ApoA1. We also examined the lipoprotein particle distribution of ApoA5 and found that ApoA5 was detectable in VLDL, HDL, and chylomicrons, but not LDL. Conclusions: These data demonstrate for the first time that ApoA5 is a secreted protein present in human serum and is associated with specific lipoprotein particles. In addition, our data indicate that the circulating concentration of human ApoA5 is very low compared with other apolipoproteins.

Early Enhanced Expression of Interferon-Inducible Protein-10 (CXCL-10) and Other Chemokines Predicts Adverse Outcome in Severe Acute Respiratory Syndrome

Abstract: Background: Exaggerated activation of cytokines/chemokines has been proposed as a factor in adverse outcome of severe acute respiratory syndrome (SARS). Previous studies on chemokines have included only small numbers of patients, and the utility of plasma chemokines as prognostic indicators is unclear. Methods: We studied 255 archival plasma samples collected during the first or second week after disease onset. The chemokines interferon-inducible protein-10 (IP-10), monokine induced by interferon-γ (MIG), interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and regulated upon activation normal T cell expressed and secreted (RANTES) were measured by cytometric bead array with a 4-color FACSCalibur flow cytometer. Reverse transcription and real-time quantitative PCR and immunohistochemical staining were performed to analyze the production of IP-10 in lung tissue at autopsy. Conditional logistic regression was used to identify independent predictors for adverse disease outcome. Results: Increases in IP-10, MIG, and IL-8 during the first week after onset of fever were associated with adverse outcome (intensive care unit admission or death) in the univariate analysis. During the second week, only MIG concentration was associated with prognosis. After adjusting for other risk factors, plasma IP-10 concentration at the first week remained as an independent prognostic factor, with an odds ratio for adverse outcome of 1.52 (95% confidence interval, 1.05–2.55) per fold increase in plasma IP-10 concentration above the median. During the second week, chemokines provided little independent prognostic information. IP-10 was increased in lung tissue from patients who died of SARS. Conclusions: Increased plasma IP-10 during the first week of SARS symptoms is an independent predictor of outcome. Chemokine activation may be an early event in SARS, and an exaggerated host response may produce complications.

Approved IFCC Recommendation on Reporting Results for Blood Glucose (Abbreviated)

Abstract: In current clinical practice, plasma and blood glucose are used interchangeably with a consequent risk of clinical misinterpretation. In human blood, glucose, like water, is distributed between erythrocytes and plasma. The molality of glucose (amount of glucose per unit of water mass) is the same throughout the sample, but the concentration is higher in plasma because the concentration of water and, therefore, glucose is higher in plasma than in erythrocytes. Different devices for the measurement of glucose may detect and report fundamentally different quantities. Different water concentrations in calibrators, plasma, and erythrocyte fluid can explain some of the differences. Results of glucose measurements depend on sample type and on whether methods require sample dilution or use biosensors in undiluted samples. If the results are mixed up or used indiscriminately, the differences may exceed the maximum allowable error of glucose determinations for diagnosing and monitoring diabetes mellitus, and complicate the treatment. The goal of the IFCC Scientific Division Working Group on Selective Electrodes and Point of Care Testing (IFCC-SD, WG-SEPOCT) is to reach a global consensus on reporting results. The document recommends reporting the concentration of glucose in plasma (with the unit mmol/L), irrespective of sample type or measurement technique. A constant factor of 1.11 is used to convert concentration in whole blood to the equivalent concentration in the pertinent plasma. The conversion will provide harmonized results, facilitating the classification and care of patients and leading to fewer therapeutic misjudgments.

Predictive Markers in Breast and Other Cancers: A Review

Abstract: Background: Unpredictable efficacy and toxicity are hallmarks of most anticancer therapies. Predictive markers are factors that are associated with response or resistance to a particular therapy. Methods: The English literature relating to predictive markers in oncology was reviewed. Particular attention was paid to metaanalyses, systematic reviews, prospective trials, and guidelines issued by expert panels. Results: The prototype predictive tests in oncology are the estrogen receptor (ER) and progesterone receptor (PR), which are used to select patients with breast cancer likely to respond to hormone therapy. A more recently introduced predictive marker is HER-2 for selecting patients with advanced breast cancer for treatment with the therapeutic antibody trastuzumab (Herceptin). In adjuvant breast cancer, overproduction of HER-2 may also indicate an enhanced sensitivity to high-dose anthracycline-based regimens. On the other hand, in both early and advanced breast cancer, high concentrations of HER-2 appear to correlate with a lower probability of response to hormone therapy. Although many different anticancer drugs appear to mediate tumor regression by inducing apoptosis, there is currently no consistent evidence that any of the molecules implicated in this process can be used as predictive markers. Conclusions: Currently, the only recommended predictive markers in oncology are ER and PR for selecting endocrine-sensitive breast cancers and HER-2 for identifying breast cancer patients with metastatic disease who may benefit from trastuzumab. For malignancies other than breast cancers, validated predictive markers do not exist at present.

Monitoring of Clopidogrel Action: Comparison of Methods

Abstract: Background: Clopidogrel is a potent drug for prevention of adverse effects during and after coronary intervention. Increasing experience indicates that a significant proportion of patients do not respond adequately to clopidogrel. Because failure of antiplatelet therapy can have severe consequences, there is need for a reliable assay to quantify the effectiveness of clopidogrel treatment. Methods: Of 24 healthy volunteers admitted to the study, 18 were treated for 1 week with clopidogrel (300-mg loading dose and 75-mg maintenance dose), and 6 with placebo. Platelet function was monitored by 2 assays, based on flow cytometry and enzyme immunoassay, that measure the phosphorylation status of vasodilator-stimulated phosphoprotein (VASP) and by aggregometry, flow cytometry of P-selectin, and the platelet function analyzer at baseline, on days 1–5, and on day 9 of treatment. Results: Aggregometry and VASP phosphorylation revealed a loss of platelet response to ADP within 12 h after clopidogrel intake. The phosphorylation status of VASP correlated with the inhibition of platelet aggregation. In contrast, neither P-selectin expression nor PFA-100 closure time was a clear indicator of clopidogrel effects on platelets. Conclusions: VASP phosphorylation assays are reliable for quantifying clopidogrel effects. Because the VASP assay directly measures the function of the clopidogrel target, the P2Y12 receptor, the assay is selective for clopidogrel effects rather than effects of other platelet inhibitors commonly in use. © 2005 American Association for Clinical Chemistry