bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of Capillary Zone Electrophoresis for Differentiating Excessive from Moderate Alcohol Consumption
Authors: Legros Franz J , Nuyens Vincent , Baudoux Michel , Zouaoui Boudjeltia Karim , Ruelle Jean-Luc , Colicis Jacques , Cantraine Francis , Henry Jean-Pol
Published: February 2004

Background: The poorly sialylated transferrin isoforms in serum were analyzed by capillary zone electrophoresis (CZE) to differentiate moderate from heavy alcohol consumption.

Methods: We enrolled 614 volunteers, classified after interviews, self-reported drinking habits, and AUDIT scores as alcohol abusers (consuming >50 g/day ethanol for the previous 3 months or longer; n = 413) or moderate drinkers (<30 g/day ethanol; n = 201). Serum transferrin isoforms were separated at 28 kV and monitored at 214 nm on a P/ACE 5500 CZE with use of fused-silica capillaries and the related CEofix CDT reagent set. Immunosubtraction by anti-human transferrin and electrophoretic migration times identified the isoforms. Previous markers of alcohol abuse and an assay combining anion-exchange minicolumn chromatography with immunoturbidimetry (%CDT) were included in the study. Sensitivities and specificities were compared by ROC analysis.

Results: The asialylated isoform was missing in 95% of moderate drinkers but present in 92% of alcohol misusers. Disialotransferrin had a specificity and sensitivity of 0.75 at a cutoff of 0.7% of total transferrin, whereas the sum (asialo- + disialotransferrin) at a threshold of 1.2% had a sensitivity of 0.73 and a specificity of 0.92. Trisialotransferrin values did not distinguish between the two populations. Sensitivities and specificities of %CDT averaged 0.77 and 0.74, respectively, at a 2.6% cutoff; 0.67 and 0.83 at 2.8%; and 0.63 and 0.90 at 3%. CDT data were more sensitive and specific for males. Conventional biomarkers appeared less discriminating.

Conclusions: Asialotransferrin detected by CZE in sera of alcohol abusers offers the highest discrimination between excessive and moderate drinking.
Validation of the FibroTest Biochemical Markers Score in Assessing Liver Fibrosis in Hepatitis C Patients
Authors: Rossi Enrico , Adams Leon , Prins Alexander , Bulsara Max , de Boer Bastiaan , Garas George , MacQuillan Gerry , Speers David , Jeffrey Gary
Published: February 2004

Background: Determining the stage of fibrosis by liver biopsy is important in managing patients with hepatitis C virus infection. We investigated the predictive value of the proprietary FibroTest score to accurately identify significant fibrosis in Australian hepatitis C patients.

Methods: Serum obtained from 125 confirmed hepatitis C patients before antiviral therapy was analyzed for haptoglobin, α2-macroglobulin, apolipoprotein A1, bilirubin, and γ-glutamyltransferase activity, and the FibroTest score was computed. Liver fibrosis pathology was staged according to a defined system on a scale of F0 to F4. We used predictive values and a ROC curve to assess the accuracy of FibroTest scores.

Results: The prevalence of significant fibrosis defined by liver biopsy was 0.38. The most useful single test for predicting significant fibrosis was serum α2-macroglobulin (cutoff value, 2.52 g/L; sensitivity, 75%; specificity, 67%). The negative predictive value of a FibroTest score <0.1 was 85%, and the positive predictive value of a score >0.6 was 78%. Although 33 of the 125 patients had FibroTest scores <0.1 and were therefore deemed unlikely to have fibrosis, 6 (18%) had significant fibrosis. Conversely, of the 24 patients with scores >0.6 who were likely to have significant fibrosis, 5 (21%) had mild fibrosis. Of the 125 patients in the cohort, 57 (46%) could have avoided liver biopsy, but discrepant results were recorded in 11 of those 57 (19%).

Conclusion: The FibroTest score could not accurately predict the presence or absence of significant liver fibrosis.
Certification of Standard Reference Material 970, Ascorbic Acid in Serum, and Analysis of Associated Interlaboratory Bias in the Measurement Process
Authors: Margolis Sam A , Vangel Mark , Duewer David L
Published: February 2004

Background: The accurate and reproducible measurement of ascorbic acid is essential in delineating the role of ascorbic acid as a diagnostic tool for human disease and for the comparison of data acquired by different laboratories. A stabilized pair of standards of ascorbic acid in human serum, which is compatible with most analytical methods, have been prepared.

Methods: The certification was based on the gravimetric addition of ascorbic acid to metaphosphoric acid-stabilized, ascorbic acid-depleted serum and NIST liquid chromatography–electrochemical measurements. The NIST results were analyzed statistically for homogeneity, and the expanded uncertainty of each SRM was calculated using all of the NIST data. An interlaboratory comparison exercise was also performed.

Results: These materials, Standard Reference Material (SRM) 970 Ascorbic Acid in Serum, Level I and Level II, are homogeneous and are certified to contain (10.07 ± 0.21) and (30.57 ± 0.28) mmol ascorbic acid/L of solution (expanded uncertainty), respectively. In the interlaboratory comparison (n = 17), the relative SDs for the two materials were 22% and 19%.

Conclusions: Two lots of serum, each containing different amounts of ascorbic acid stabilized in metaphosphoric acid, have been prepared and characterized. Many laboratories provide inaccurate results.
Toxicity of Free p-Cresol: A Prospective and Cross-Sectional Analysis
Authors: De Smet Rita , Van Kaer Jacqueline , Van Vlem Bruno , De Cubber Antoine , Brunet Philippe , Lameire Norbert , Vanholder Raymond
Published: February 2004

Background: Uremic syndrome is the consequence of the retention of solutes usually cleared by the healthy kidneys. p-Cresol can be considered a prototypic protein-bound uremic toxin. It is conceivable, analogous with drugs, that the non-protein-bound fraction of p-cresol exerts toxicity. This aspect had never been evaluated, nor have the factors influencing the free fraction of p-cresol.

Methods: In a transsectional study we evaluated the relationship between prehemodialysis free p-cresol and the ratio of free to total p-cresol (F:T) to clinical and biological factors in 44 chronic renal failure patients. The evolution of free p-cresol was assessed prospectively in 12 patients showing a change in serum albumin of at least 5 g/L over time. Hospitalization days attributable to infection and the free p-cresol concentrations were noted over a 1-year period. The impact of free p-cresol in vitro on leukocyte functional capacity was evaluated by chemiluminescence.

Results: We observed a correlation between total and free p-cresol (r = 0.84; P <0.001). In the multivariate analyses, free p-cresol and F:T showed a negative correlation with albumin. A shift from normal serum albumin to hypoalbumininemia in 12 patients led to an increase in free p-cresol from 5.9 ± 3.2 to 8.2 ± 4.5 μmol/L (P <0.05; 0.64 ± 0.35 to 0.89 ± 0.49 mg/L). Free p-cresol (P <0.05) was higher in the patients hospitalized for infectious disease. In vitro, free p-cresol was higher in a 25 g/L than in a 50 g/L albumin solution (P <0.05). Leukocyte chemiluminescence production was more inhibited in the low albumin (high free p-cresol) solution (28% ± 6% vs 21% ± 8%; P <0.05).

Conclusions: Hypoalbuminemia and total p-cresol increase the free fraction of p-cresol. Patients hospitalized for infections have higher free p-cresol. In vitro, high free p-cresol has a negative impact on leukocyte chemiluminescence production. These data demonstrate the toxicity of free p-cresol.
Low-Positive Anti-Hepatitis C Virus Enzyme Immunoassay Results: An Important Predictor of Low Likelihood of Hepatitis C Infection
Authors: Dufour D Robert , Talastas Mageli , Fernandez Maria D A , Harris Barbara , Strader Doris B , Seeff Leonard B
Published: February 2004

Background: Tests for hepatitis C antibodies (anti-HCV enzyme immunoassays) are usually described as positive or negative. Several studies, mainly in blood donors, have found that specimens with low signal/cutoff (S/C) ratios are commonly negative when tested with a recombinant immunoblot assay (RIBA) or for HCV RNA.

Methods: We retrospectively reviewed 17 418 consecutive anti-HCV results from a screening program for high-risk veterans; 2986 (17.1%) samples were anti-HCV-positive, and 490 (16.4%) had S/C ratios ≤3.7 (low positive). Additional tests were performed in 1814 anti-HCV-positive individuals.

Results: RIBA was performed in 263 patients with low-positive anti-HCV; results were negative in 86%, indeterminate in 12%, and positive in 2%. Only 16 of 140 individuals (11%) with low-positive anti-HCV values were HCV RNA-positive, whereas HCV RNA was positive in 90% of 1435 individuals with high-positive anti-HCV values (P <0.0001). Compared with those with high-positive anti-HCV, individuals with low-positive anti-HCV values were older (P <0.0001) and were less likely to have risk factors for HCV (P <0.0001 for most), multiple increased alanine aminotransferase (ALT) activity values (30% vs 81%; P <0.0001), or positive anti-hepatitis B core antigen (19% vs 59%; P <0.0002). Among 634 individuals with high anti-HCV titers and multiple increased ALT activity values, 95% were HCV RNA-positive.

Conclusions: The S/C ratio is important even in high-risk individuals; laboratories should report the S/C ratio along with anti-HCV EIA results and perform supplemental RIBA testing in those with low-positive values to avoid reporting false-positive results.
Cerebrospinal Fluid Lactate and Pyruvate Concentrations and Their Ratio in Children: Age-related Reference Intervals
Authors: Benoist Jean-François , Alberti Corinne , Leclercq Sandrine , Rigal Odile , Jean-Louis Rosalie , Ogier de Baulny Hélene , Porquet Dominique , Biou Daniel
Published: February 2004

Background: Lactate (L) and pyruvate (P) concentrations in cerebrospinal fluid (CSF) and the L/P ratio have diagnostic value in numerous primary and acquired disorders affecting the central nervous system, but age-related reference values are not available for children.

Methods: We analyzed CSF and blood lactate and pyruvate concentrations and their ratio in a 4-year retrospective survey of a children’s hospital laboratory database. Reference intervals (10th–90th percentiles) were established from data on 197 hospitalized children. A recent regression modeling method was used to normalize and smooth values against age. The model equation of best fit was calculated for each variable.

Results: Slight age-related variations were shown by the model, with an increase in lactate, a decrease in pyruvate, and a resulting increase in the L/P ratio with increasing age. However, the SD did not vary with age. We defined the upper limit of the reference intervals as the 90th percentiles, which from birth to 186 months of age varied continuously from 1.78 to 1.88 mmol/L (6%), 148 to 139 μmol/L (6%), and 16.9 to 19.2 (14%) for lactate, pyruvate, and the L/P ratio, respectively. At a threshold of 2 (in Z-score units), the sensitivity for a subgroup of inborn errors of metabolism (respiratory chain disorders) was 73%, 42%, and 31% for lactate, pyruvate, and the L/P ratio, respectively.

Conclusions: In children, CSF lactate and pyruvate concentrations and their ratio appear to vary slightly with age. Average 90th percentile values of 1.8 mmol/L, 147 μmol/L, and 17, respectively, could be used in infants up to 24 months of age. In older children, age-adjusted reference intervals should be used, especially when values are close to the 90th percentile.
Comparison of Two CYP2D6 Genotyping Methods and Assessment of Genotype-Phenotype Relationships
Authors: Chou Wen-Hwei , Yan Feng-Xiang , Robbins-Weilert Doris K , Ryder Thomas B , Liu Wei Wei , Perbost Clotilde , Fairchild Maureen , de Leon Jose , Koch Walter H , Wedlund Peter J
Published: February 2004

Background: There have been no published reports comparing the CYP450 GeneChip® microarray assay with more standard methods of genetic testing.

Methods: We collected 20-mL blood samples from 236 volunteers for DNA isolation and testing before each individual ingested 60 mg of dextromethorphan, and collected their urine. CYP2D6 alleles *3 to *7, *9, *17, and *41, and multiple CYP2D6 gene copies were tested by allele-specific PCR (AS-PCR), whereas alleles *2 to *4 and *6 to *11 were tested by the Affymetrix CYP450 GeneChip assay. Five of the CYP2D6 alleles (*3, *4, *6, *7, and *9) were tested by both AS-PCR and the CYP450 GeneChip assay in an independent and blinded fashion in 232 of the 236 healthy volunteers. The combined CYP2D6 genotype from both methods was used to divide the population into four subgroups, poor metabolizers (PMs), intermediate metabolizers (IMs), extensive metabolizers (EMs), and ultrarapid metabolizers (UMs), based on their relative function and ability to express the CYP2D6 gene. The urinary elimination of dextromethorphan was assessed in each of these CYP2D6 subgroups.

Results: The CYP2D6*3, *4, *6, *7, and *9 alleles showed a high degree of concordance between the CYP450 GeneChip and AS-PCR methods (>99% concordance). The mean (SD) of the log[dextromethorphan metabolic ratio (MR)] in the four CYP2D6 subgroups was PM = 0.49 (0.38); IM = −1.24 (0.53); EM = −2.35 (0.61); and UM = −2.43 (0.38).

Conclusions: Oligonucleotide microarray technology is an efficient and reliable way to test for CYP2D6 gene variation based on five alleles compared by separate methods. The methodology is influenced by the quality and amount of DNA present. The log(dextromethorphan MR) is a highly variable index that appears to reflect the crude nature of the dextromethorphan MR as an indicator of CYP2D6 in vivo enzyme activity.
Prognostic Use of Circulating Plasma Nucleic Acid Concentrations in Patients with Acute Stroke
Authors: Rainer Timothy H , Wong Lawrence K S , Lam Wynnie , Yuen Eddie , Lam Nicole Y L , Metreweli Constantine , Lo Y M Dennis
Published: February 2004

Background: At present there is no simple, accurate blood test that may be used to determine the severity of stroke or to predict mortality and morbidity in stroke patients presenting to emergency departments.

Methods: Patients with stroke-like symptoms who presented to an emergency department of a university hospital in Hong Kong were recruited for the study. DNA extracted from patients’ plasma was analyzed for the β-globin gene with a fluorescent-based PCR test. The primary outcome measures were in-hospital and 6-month mortality and morbidity using the post-stroke modified Rankin Score.

Results: Among the 88 consecutive patients recruited to the study, 70 (80%) had ischemic stroke, 11 (13%) had intracerebral hemorrhage, and 7 (8%) had transient ischemic attacks. Median plasma DNA concentrations taken within 3 h of symptom onset were higher in patients who died compared with those who survived at discharge (6205 vs 1334 kilogenome-equivalents/L; P = 0.03). Among patients with NIH Stroke Scale scores >8, median plasma DNA concentrations were higher in patients who died compared with those who survived to 6 months (2273 vs 968 kilogenome-equivalents/L; P = 0.002). Plasma DNA concentrations correlated with the volume of cerebral hematoma (r = 0.66; P = 0.03). Plasma DNA concentrations >1400 kilogenome-equivalents/L had a sensitivity of 100% and a specificity of 74.4% for predicting hospital mortality after stroke, and the area under the ROC curve was 0.89 (95% confidence interval, 0.80–0.94). The adjusted odds ratio for plasma DNA concentrations predicting 6-month mortality was 1.6 (1.1–2.4; P = 0.03) and for predicting 6-month post-Rankin Score >2 was 1.8 (1.0–3.3; P = 0.05).

Conclusion: Plasma DNA concentrations correlate with stroke severity and may be used to predict mortality and morbidity in the emergency room.
The D-Dimer Test for Deep Venous Thrombosis: Gold Standards and Bias in Negative Predictive Value
Authors: Philbrick John T , Heim Steven
Published: February 2004

Background: Because venous ultrasound (US) fails to fully image the calf veins, there is the potential for US gold standard studies to classify patients with calf deep venous thrombosis (DVT) in the nondiseased category, causing bias in test index calculations. A false increase in negative predictive value (NPV) is especially likely because calf DVT false-negative tests will be counted in the numerator along with the true-negative tests in NPV calculations. We verified the presence and magnitude of this bias for the d-dimer test.

Methods: We abstracted data on overall (calf and thigh) and thigh-only test sensitivity, specificity, and NPV from the six English language studies published between March 1995 and October 2001 that compared d-dimer to a gold standard (GS) capable of imaging both thigh and calf veins and that also stratified results by thigh and calf location. Thigh specificity and NPV were calculated classifying calf DVT patients as free of disease.

Results: The six studies included 81–214 participants and provided 26 comparisons of 16 different d-dimer assays to the GS. Thigh sensitivity was higher than overall sensitivity in 22 of 26 comparisons (range, −0.3 to 8.6); thigh specificity was lower than overall specificity in all comparisons (range, −0.7 to −7.8); and thigh NPV was higher than overall NPV in 22 of 26 comparisons and unchanged in 4 comparisons (range, 0.0–9.2). NPV was >95% in 20 of the thigh results but >95% in only 8 of the overall results.

Conclusions: Different GS can produce clinically significant differences in test indices. Care must be taken in interpreting DVT studies that evaluate d-dimer as a rule-out test and that use US as a GS, because missed calf DVT can falsely increase the NPV.
Cleaved Protein S (PS), Total PS, Free PS, and Activated Protein C Cofactor Activity as Risk Factors for Venous Thromboembolism
Authors: Borgel Delphine , Reny Jean-Luc , Fischelis David , Gandrille Sophie , Emmerich Joseph , Fiessinger Jean-Noël , Aiach Martine
Published: February 2004

Background: Although hereditary protein S (PS) deficiency is clearly associated with venous thromboembolism (VTE), the importance of low PS concentrations as a risk factor for VTE in other patients is still a matter of debate. To clarify this issue, we designed a case-control study to evaluate the role of different molecular forms of plasma PS.

Methods: We quantified plasma cleaved, total, and free PS and activated protein C (APC) cofactor activity in 87 VTE patients and 174 controls matched for age, sex, and hormonal treatment. Free PS was measured by ELISA or by enzyme-linked ligand sorbent assay (ELSA). Cleaved and total PS were measured by ELISA.

Results: In controls, the mean (SD) concentration of circulating cleaved PS was 39 (14) nmol/L, corresponding to 10% (3.5%) of total PS. Concentrations of cleaved PS and total PS were not significantly different in patients with VTE compared with controls. However, in our population, low free PS measured by ELISA or ELSA, as well as APC cofactor activity values were significantly associated with VTE with odds ratios (95% confidence intervals) of 2.9 (1.3–6.3), 2.5 (1.1–5.6), and 2.9 (1.3–6.4), respectively, in multivariate analyses.

Conclusion: Phenotypic low PS detected by APC cofactor activity assay or by an assay specific for free PS should be considered a risk factor for VTE.
Evaluation of Human Serum Albumin Cobalt Binding Assay for the Assessment of Myocardial Ischemia and Myocardial Infarction
Authors: Bhagavan Nadhipuram V , Lai Ernest M , Rios Patricia A , Yang Jinsheng , Ortega-Lopez Anna M , Shinoda Hiroko , Honda Stacey A A , Rios Carlos N , Sugiyama Cheryl E , Ha Chung-Eun
Published: February 2004

Background: Clinical diagnoses were correlated with results of a Co(II)–albumin binding assay in 167 patients treated at an emergency department of a health maintenance organization.

Methods: Patients were evaluated as being nonischemic or potentially ischemic through standard coronary disease indicators [creatine kinase (CK), CK-MB, cardiac troponin I, and electrocardiographic findings] and were tested by a Co(II)–albumin binding assay. Samples were tested anonymously, and the study was double-blinded. The sensitivity and specificity of this assay for the detection of ischemia were evaluated by ROC curve analysis. Known Co(II) binding sites on albumin were analyzed by N-terminal amino acid sequencing.

Results: The mean absorbance units (ABSU) ± 2 SD for non-myocardial ischemic and myocardial ischemic individuals measured at 470 nm were 0.43 ± 0.10 and 0.63 ± 0.25, respectively (P <0.0001). The area under the ROC curve was 0.95 [95% confidence interval (CI), 0.92–0.99], and at a cutoff value of 0.50 ABSU, sensitivity and specificity were 88% (78–94%) and 94% (86–98%), respectively, suggesting a high distinction between the two groups. When we compared non-acute myocardial infarction (AMI) and AMI ischemic individuals, the area under the ROC curve was 0.66 (95% CI, 0.53–0.79) and was considered a poor discriminator between these two groups. N-Terminal amino acid sequencing data for purified albumin showed normal amino acid residues for six of seven high-ABSU (≥0.70) individuals and one nonischemic individual tested. However, only one individual with a high ABSU (0.80) had two missing amino acid residues (DA) from the N-terminal region. Clinical diagnosis for this patient did not reveal an ischemic event.

Conclusions: The Co(II)–albumin binding test may serve as a useful diagnostic tool in emergency facilities for the assessment of myocardial ischemia. High and low ABSU were associated with myocardial ischemic individuals and non-myocardial ischemic individuals, respectively. However, the Co(II)–albumin binding was a poor discriminator between ischemic individuals with and without MI.
Catecholamine-Synthesizing Enzymes in Carcinoid Tumors and Pheochromocytomas
Authors: Meijer Wim G , Copray Sjef C V M , Hollema Harry , Kema Ido P , Zwart Nynke , Mantingh-Otter Ietje , Links Thera P , Willemse Pax H B , de Vries Elisabeth G E
Published: February 2004

Background: Serotonin is the principal endocrine product of carcinoid tumors, but simultaneously increased production of catecholamines has been described in these tumors. As it is not clear whether these tumors contain specific enzymes for catecholamine synthesis, we aimed to detect catecholamine-synthesizing enzymes [tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT)] in midgut carcinoid tumors and pheochromocytoma and to correlate enzyme expression to serotonin production as well as catecholamines and metabolites excreted in urine.

Methods: Paraffin-embedded tumor specimens from 21 midgut carcinoid patients and 20 pheochromocytoma patients (10 sporadic and 10 MEN type IIa-related tumors) were stained for TH, DBH, and PNMT, using a three-step biotin-avidin-peroxidase method.

Results: TH was demonstrated in 9 (43%) of 21 carcinoids and in all (100%) of 20 pheochromocytomas, DBH in 8 (38%) carcinoids and in 15 (75%) pheochromocytomas, and PNMT in 7 (33%) carcinoids and in 13 (65%) pheochromocytomas. Increased urinary excretion of catecholamines and metabolites was observed in 10 (48%) carcinoid patients and in all pheochromocytoma patients. No clinically relevant association between enzyme expression and urinary excretion of catecholamines and metabolites was found.

Conclusions: Catecholamine-synthesizing enzymes are present in many carcinoid tumors. This finding possibly indicates the existence of a catecholamine-synthesizing pathway in carcinoids similar to that found in pheochromocytoma.
New ELISA System for Myeloid-related Protein Complex (MRP8/14) and Its Clinical Significance as a Sensitive Marker for Inflammatory Responses Associated with Transplant Rejection
Authors: Ikemoto Masaki , Tanaka Tomoko , Takai Yuri , Murayama Hiroshi , Tanaka Koichi , Fujita Masatoshi
Published: February 2004

Background: C-reactive protein (CRP), a useful marker for inflammatory diseases, is not always sensitive to inflammatory reaction in the liver or other tissues. The aim of this study was to develop a sensitive and specific method for detecting inflammatory responses associated with transplant rejection.

Methods: We developed a new, highly sensitive ELISA system for the measurement of serum human myeloid-related protein complex (MRP8/14), using monoclonal antibodies against MRP8/14, and applied it to specimens obtained from patients undergoing small intestine or liver transplantation.

Results: This assay could detect MRP8/14 concentrations as low as 2 μg/L. Within-run CVs were 3.7–6.1% and between-day CVs were 5.6–8.7% for MRP8/14 concentrations of 117-3300 μg/L. Mean recovery was 104% (range, 80–128%). We observed a marked increase in serum MRP8/14 postoperatively in most recipients of transplants, followed by an increase in CRP 1–7 days after the increase in the complex. The increase in serum MRP8/14 occurred simultaneously with permeation of lymphocytes into the transplanted tissues as a result of rejection of the graft tissues.

Conclusions: Accurate measurement of serum MRP8/14 provides a useful clinical diagnostic method tool for detecting inflammation associated with rejection of transplanted tissues.
Standardization of Two Immunoassays for Human Glandular Kallikrein 2
Authors: Haese Alexander , Vaisanen Ville , Finlay Judith A , Pettersson Kim , Rittenhouse Harry G , Partin Alan W , Bruzek Debra J , Sokoll Lori J , Lilja Hans , Chan Daniel W
Published: February 2004

Background: Measurement of human kallikrein 2 (hK2) has improved early detection and staging of prostate cancer. However, reported concentrations of hK2 among currently used assays have not been standardized in any way. We compared two hK2 assays and five different recombinant hK2 variants (rhK2) and suggest a common calibrator as an important step and putative reference substance in hK2 assay standardization.

Methods: We measured 146 sera by two hK2 assays, using assay-specific calibrators to assess the difference between the two assays. Serial dilutions of five rhK2 preparations were measured repeatedly, with one preparation assigned as calibrator and the others as unknowns to define which variant provided the closest match between the two assays. This rhK2 variant was used to recalibrate both assays. We measured hK2 concentrations in the same 146 patients to evaluate the change in the difference.

Results: Use of assay-specific calibrators for comparison of the two assays yielded a Deming regression equation of: y = 0.789 (95% confidence interval, 0.674–0.922)x + 0.014 (0.004–0.025) μg/L; R2 = 0.667. Analysis of five rhK2 variants revealed that the enterokinase (ek)-rhK2 form provided the best match between both assays. Using the ek-rhK2 as a common calibrator, we observed a change in the slope of the regression curve to: y = 1.106 (0.872–1.340)x + 0.006 (−0.002 to 0.016) μg/L; R2 = 0.648, suggesting an increase in the mean estimate of agreement between the two assays.

Conclusion: Calibration with a common calibrator substantially increased agreement between the assays. The ek-rhK2 variant provided the best performance of all tested rhK2 variants and should undergo mass spectrometry and amino acid analysis for exact mass determination and value assignment to evaluate its potential as a reference material for immunoassays for hK2.
Quantitative Urine Particle Analysis: Integrative Approach for the Optimal Combination of Automation with UF-100 and Microscopic Review with KOVA Cell Chamber
Authors: Ottiger Cornelia , Huber Andreas R
Published: February 2004

Background: Automated systems have enabled the counting of particles in urine to be standardized. Their superiority over traditional sediment analysis has been well documented, but they have not gained wide acceptance. The reasons for this are that sediment analysis has been performed and interpreted for decades. Additionally, pathologic casts and other unknown particles still must be confirmed under the microscope. Furthermore, comparison between the methods has revealed outliers and thus decreased confidence in automation.

Methods: We used the standardized KOVA cell chamber system to count particles and compared the results with UF-100 flow cytometry as an alternative to traditional sediment analysis.

Results: We compared 252 randomly selected urine samples and obtained a review rate of 33%. Microscopic verification was necessary because of the presence of casts, yeast, sperm, dysmorphic erythrocytes, and some misclassified erythrocytes or leukocytes that were detected by incongruent dipstick results and abnormal scattergrams. We obtained correlation coefficients of 0.966 for erythrocytes and 0.935 for leukocytes. Criteria for an algorithm to identify samples that needed microscopic review were derived from comparisons between the number of particles from UF-100, dipstick results, cell chamber counting, and sediment analysis.

Conclusions: Automated cell counting combined with microscopic counting with a standardized cell chamber system is useful. An objective algorithm for review criteria can be developed via systematic comparison of UF-100 flow cytometry and microscopy. Only urine samples that meet these criteria need to be confirmed microscopically.
Identification of Rat Targets of Anti-Soluble Liver Antigen Autoantibodies by Serologic Proteome Analysis
Authors: Ballot Eric , Bruneel Arnaud , Labas Valérie , Johanet Catherine
Published: February 2004

Background: Anti-soluble liver antigen (SLA) autoantibodies are specific for autoimmune hepatitis type 1 and are the only immunologic marker found in 15–20% of hepatitis cases previously considered cryptogenic. Anti-SLA antibodies react with the 100 000g supernatant from rat liver homogenate, but the molecular targets remain controversial.

Methods: We characterized anti-SLA targets by one- and two-dimensional immunoblotting analysis. The recognized proteins were identified by peptide mass fingerprint analysis after matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.

Results: Three proteins of 35 kDa and pI 6.0, 50 kDa and pI between 6.0 and 6.5, and 58 kDa and pI between 6.5 and 7.0 were stained more intensely by anti-SLA positive-sera than by control sera. After in-gel tryptic digestion, MALDI-TOF analysis of the generated peptides enabled the clear identification of N-hydroxyarylamine sulfotransferase, isoforms of α-enolase, and isoforms of catalase.

Conclusions: Possible antigens for anti-SLA antibodies include a sulfotransferase, α-enolase(s), and catalase(s). Two-dimensional electrophoresis combined with mass spectrometry offers a versatile tool to identify molecular targets of autoantibodies and thus to improve diagnostic tools and the understanding of the immune process.
Distribution of Fasting Plasma Insulin, Free Fatty Acids, and Glucose Concentrations and of Homeostasis Model Assessment of Insulin Resistance in a Representative Sample of Quebec Children and Adolescents
Authors: Allard Pierre , Delvin Edgard E , Paradis Gilles , Hanley James A , O’Loughlin Jennifer , Lavallée Claudette , Levy Emile , Lambert Marie
Published: February 2004

Background: Plasma fasting insulin and the homeostasis model assessment of insulin resistance (HOMA-IR) are markers of IR, which, at least in part, mediates the relation of obesity to increased cardiovascular risk. Increased free fatty acids (FFAs) may be involved in the pathogenesis of IR. Our objectives were to describe the distributions of fasting plasma insulin, glucose, and FFAs and HOMA-IR in youth and to assess the relationship between FFAs and markers of IR.

Methods: Fasting plasma insulin, glucose, and FFAs were measured in a representative sample of Quebec youth comprising 2244 individuals 9, 13, and 16 years of age.

Results: In all age and sex groups, glucose exhibited remarkably tight distributions (median CV, 7.1%) in contrast to insulin, HOMA-IR, and FFAs (median CVs, 52%, 54% and 45%, respectively). For every percentile examined, 9-year-olds had lower insulin concentrations and HOMA-IR values than 13- and 16-year-olds. We observed strong correlations between insulin concentrations and HOMA-IR values, as well as close similarity in their rankings of individuals. The mean concentrations of glucose were higher in our population than in other Caucasian pediatric populations. No positive correlations were detected between FFAs and markers of IR.

Conclusions: We report some of the first data on the distributions of fasting plasma insulin, HOMA-IR, and FFAs from a representative sample of youth. HOMA-IR does not appear more informative than fasting insulin as a marker of IR. Our findings on higher mean glucose concentrations in this population require confirmation in other representative samples of youth to assess whether the North American distribution of glucose concentrations is shifting positively.
False-Positive Acetaminophen Results in a Hyperbilirubinemic Patient
Authors: Bertholf Roger L , Johannsen Laura M , Bazooband Alireza , Mansouri Vafa
Published: February 2004

Background: Acetaminophen was falsely detected in the plasma of a severely jaundiced patient, and a methodologic interference from bilirubin was suspected.

Methods: Acetaminophen was measured by an enzymatic method (GDS Diagnostics). The putative bilirubin interference was investigated in 12 hyperbilirubinemic specimens and in bilirubin linearity calibrators. The analytical method was modified to correct for background absorbance at a second wavelength. Hyperbilirubinemic specimens were fortified with acetaminophen to assess the effect of the interference on acetaminophen measurements.

Results: Acetaminophen was detected in 12 specimens from hyperbilirubinemic patients without a history of recent acetaminophen exposure. Dilution of hyperbilirubinemic specimens produced a nonproportional decrease in apparent acetaminophen concentrations, and no acetaminophen was detected when bilirubin was <50 mg/L. Background correction at a second wavelength failed to compensate for the interference. Although erroneous acetaminophen concentrations were detected in all specimens with high bilirubin, acetaminophen measurements in fortified specimens were accurate.

Conclusion: The data are consistent with bilirubin interference in the enzymatic and/or chromogenic reactions involved in the acetaminophen method.
Quantitative Analysis of Circulating Mitochondrial DNA in Plasma
Authors: Chiu Rossa W K , Chan Lisa Y S , Lam Nicole Y L , Tsui Nancy B Y , Ng Enders K O , Rainer Timothy H , Lo Y M Dennis
Published: February 2004

Background: Recent studies have demonstrated the existence of circulating mitochondrial DNA in plasma and serum, but the concentrations and physical characteristics of circulating mitochondrial DNA are unknown. The aim of this study was to develop an assay to quantify mitochondrial DNA in the plasma of healthy individuals.

Methods: We adopted a real-time quantitative PCR approach and evaluated the specificity of the assay for detecting mitochondrial DNA with a cell line (ρ0) devoid of mitochondria. The concentrations and physical characteristics of circulating mitochondrial DNA were investigated by experiments conducted in three modules. In module 1, we evaluated the concentrations of mitochondrial DNA in plasma aliquots derived from four blood-processing protocols. In module 2, we investigated the existence of both particle-associated and free forms of mitochondrial DNA in plasma by subjecting plasma to filtration and ultracentrifugation. In module 3, we used filters with different pore sizes to investigate the size characteristics of the particle-associated fraction of circulating mitochondrial DNA.

Results: The mitochondrial DNA-specific, real-time quantitative PCR had a dynamic range of five orders of magnitude and a sensitivity that enabled detection of one copy of mitochondrial DNA in plasma. In module 1, we found significant differences in the amounts of circulating mitochondrial DNA among plasma aliquots processed by different methods. Data from module 2 revealed that a significant fraction of mitochondrial DNA in plasma was filterable or pelletable by ultracentrifugation. Module 3 demonstrated that filters with different pore sizes removed mitochondrial DNA from plasma to different degrees.

Conclusions: Both particle-associated and free mitochondrial DNA are present in plasma, and their respective concentrations are affected by the process used to harvest plasma from whole blood. These results may have implications in the design of future studies on circulating mitochondrial DNA measured in different disease conditions.
The Concentration of Circulating Corticotropin-releasing Hormone mRNA in Maternal Plasma Is Increased in Preeclampsia
Authors: Ng Enders K O , Leung Tse N , Tsui Nancy B Y , Lau Tze K , Panesar Nirmal S , Chiu Rossa W K , Lo Y M Dennis
Published: February 2004

Background: Increased fetal DNA in maternal plasma/serum has been reported in pregnancies complicated by preeclampsia. We hypothesize that fetal RNA may also be increased in maternal plasma in preeclampsia.

Methods: We developed a real-time quantitative reverse transcription-PCR assay to measure the concentration of the mRNA of the corticotropin-releasing hormone (CRH) locus. Peripheral blood samples were obtained from healthy pregnant women both before and 2 h after delivery. Peripheral blood samples were also obtained from women suffering from preeclampsia and controls matched for gestational age. Plasma was harvested from these samples, and RNA was extracted. Plasma RNA was subjected to analysis by the reverse transcription-PCR assay.

Results: CRH mRNA was detected in the plasma of 10 healthy pregnant women in the third trimester. CRH mRNA was found to be cleared very rapidly after cesarean section, with no detectable signal by 2 h postpartum. Plasma CRH mRNA concentrations were 1070 and 102 copies/mL, respectively, in 12 preeclamptic women and 10 healthy pregnant women matched for gestational age (Mann–Whitney test, P <0.001).

Conclusion: Plasma CRH mRNA represents a new molecular marker for preeclampsia. Maternal plasma RNA is gender- and polymorphism-independent and may allow noninvasive gene-expression profiling of an unborn fetus.
Purification of Human Kallikrein 6 from Biological Fluids and Identification of its Complex with α1-Antichymotrypsin
Authors: Hutchinson Shirley , Luo Liu-Ying , Yousef George M , Soosaipillai Antoninus , Diamandis Eleftherios P
Published: February 2004

Background: Human kallikrein 6 (hK6) is significantly increased in serum in many patients with ovarian cancer and may have a role in amyloid precursor processing and Alzheimer disease. The forms of hK6 in biological fluids are poorly characterized.

Methods: hK6 protein was immunoaffinity-purified and positively identified by Western blotting, N-terminal sequencing, and mass spectrometry. hK6 in cerebrospinal fluid (CSF), milk, ascites, and serum was size-fractionated by chromatography and then measured by a highly sensitive and specific immunoassay. Hybrid assays were performed to detect the possible interactions between hK6 and proteinase inhibitors in CSF, milk, ascites fluid, and serum.

Results: N-Terminal sequencing identified hK6 in the proform in both CSF and milk. hK6 exists in two forms in milk and ascites fluid: a free form with a molecular mass of ∼25 kDa and a higher molecular mass form. Hybrid sandwich assays (capture antibody for hK6 and detection antibody for inhibitors), utilizing a panel of known serine protease inhibitors, indicated that α1-antichymotrypsin forms a complex with hK6 in milk and ascites fluid. Only the free form of hK6 was detected in CSF and serum.

Conclusions: hK6 exists mainly as a proenzyme in milk and CSF. A fraction of this enzyme is partially complexed with α1-antichymotrypsin in milk and ascites fluid of ovarian cancer patients.
Comprehensive Proteomic Profiling Identifies Serum Proteomic Signatures for Detection of Hepatocellular Carcinoma and Its Subtypes
Authors: Poon Terence C W , Yip Tai-Tung , Chan Anthony T C , Yip Christine , Yip Victor , Mok Tony S K , Lee Conrad C Y , Leung Thomas W T , Ho Stephen K W , Johnson Philip J
Published: February 2004

Background: Detection of hepatocellular carcinoma (HCC) in patients with chronic liver disease (CLD) is difficult. We investigated the use of comprehensive proteomic profiling of sera to differentiate HCC from CLD.

Methods: Proteomes in sera from 20 CLD patients with α-fetoprotein (AFP) <500 μg/L (control group) and 38 HCC patients (disease group) were profiled by anion-exchange fractionation (first dimension), two types (IMAC3 copper and WCX2) of ProteinChip® Arrays (second dimension), and time-of-flight mass spectrometry (third dimension). Bioinformatic tests were used to identify tumor-specific proteomic features and to estimate the values of the tumor-specific proteomic features in the diagnosis of HCC. Cross-validation was performed, and we also validated the models with pooled sera from the control and disease groups, serum from a CLD patient with AFP >500 μg/L, and postoperative sera from two HCC patients.

Results: Among 2384 common serum proteomic features, 250 were significantly different between the HCC and CLD cases. Two-way hierarchical clustering differentiated HCC and CLD cases. Most HCC cases with advanced disease were clustered together and formed two subgroups that contained significantly more cases with lymph node invasion or distant metastasis. For differentiation of HCC and CLD by an artificial network (ANN), the area under the ROC curve was 0.91 (95% confidence interval, 0.82–1.01; P <0.0005) for all cases and 0.954 (95% confidence interval, 0.881–1.027; P <0.0005) for cases with nondiagnostic serum AFP (<500 μg/L). At a specificity of 90%, the sensitivity was 92%. Both cluster analysis and ANN correctly classified the pooled serum samples, the CLD serum sample with increased AFP, and the HCC patient in complete remission.

Conclusion: Tumor-specific proteomic signatures may be useful for detection and classification of hepatocellular cancers.
Scanning for Mutations of the Ryanodine Receptor (RYR1) Gene by Denaturing HPLC: Detection of Three Novel Malignant Hyperthermia Alleles
Authors: Tammaro Angela , Bracco Adele , Cozzolino Santolo , Esposito Maria , Di Martino Antonietta , Savoia Gennaro , Zeuli Laura , Piluso Giulio , Aurino Stefania , Nigro Vincenzo
Published: February 2004

Background: Malignant hyperthermia (MH) is a fatal autosomal dominant pharmacogenetic disorder characterized by skeletal muscle hypertonicity that causes a sudden increase in body temperature after exposure to common anesthetic agents. The disease is genetically heterogeneous, with mutations in the gene encoding the skeletal muscle ryanodine receptor (RYR1) at 19q13.1 accounting for up to 80% of the cases. To date, at least 42 RYR1 mutations have been described that cause MH and/or central core disease. Because the RYR1 gene is huge, containing 106 exons, molecular tests have focused on the regions that are more frequently mutated. Thus the causative defect has been identified in only a fraction of families as linked to chromosome 19q, whereas in others it remains undetected.

Methods: We used denaturing HPLC (DHPLC) to analyze the RYR1 gene. We set up conditions to scan the 27 exons to identify both known and unknown mutations in critical regions of the protein. For each exon, we analyzed members from 52 families with positive in vitro contracture test results, but without preliminary selection by linkage analysis.

Results: We identified seven different mutations in 11 MH families. Among them, three were novel MH alleles: Arg44Cys, Arg533Cys, and Val2117Leu.

Conclusion: Because of its sensitivity and speed, DHPLC could be the method of choice for the detection of unknown mutations in the RYR1 gene.
Rapid Screening of Multiple β-Globin Gene Mutations by Real-Time PCR on the LightCycler: Application to Carrier Screening and Prenatal Diagnosis of Thalassemia Syndromes
Authors: Vrettou Christina , Traeger-Synodinos Joanne , Tzetis Maria , Malamis George , Kanavakis Emmanuel
Published: February 2004

Background: Hemoglobinopathies are priority genetic diseases for prevention programs. Rapid genotype characterization is fundamental in the diagnostic laboratory, especially when offering prenatal diagnosis for carrier couples.

Methods: As a model, we designed a protocol based on the LightCycler™ technology to screen for a spectrum of β-globin gene mutations in the Greek population. Design was facilitated by dual fluorochrome detection and close proximity of many mutations. Three probe sets were capable of screening 95% of β-globin gene mutations in the Greek population, including IVSII-745C→G, HbS, Cd5-CT, Cd6-A, Cd8-AA, IVSI-1G→A, IVSI-5G→A, IVSI-6T→C, IVSI-110G→A, and Cd39 C→T.

Results: The protocol, standardized by analysis of 100 β-thalassemia heterozygotes with known mutations, was 100% reliable in distinguishing wild-type from mutant alleles. Subsequent screening of 100 Greek β-thalassemia heterozygotes with unknown mutations found 96 of 100 samples heterozygous for 1 of the 10 mutations, although melting curves were indistinguishable for mutations ΗbS/Cd6 and IVSI-5/IVSI-1, indicating a need of alternative methods for definitive diagnosis. One sample demonstrating a unique melting curve was characterized by sequencing as Cd8/9+G. Three samples carried mutations outside the gene region covered by the probes. The protocol was 100% accurate in 25 prenatal diagnosis samples, with 14 different genotype combinations diagnosed. The protocol was also flexible, detecting five β-globin gene mutations from other population groups (IVSI-1G→T, IVSI-5G→C, IVSI-116T→G, Cd37 TGG→TGA, and Cd41/42 −TCTT).

Conclusions: The described LightCycler system protocol can rapidly screen for many β-globin gene mutations. It is appropriate for use in many populations for directing definitive mutation diagnosis and is suited for rapid prenatal diagnosis with low cost per assay.
Rapid Detection of β-Globin Gene Mutations and Polymorphisms by Temporal Temperature Gradient Gel Electrophoresis
Authors: Shaji Ramachandran V , Edison Eunice Sindhuvi , Poonkuzhali Balasubramanian , Srivastava Alok , Chandy Mammen
Published: February 2004

Background: Inherited hemoglobin disorders represent the most common Mendelian disease worldwide. Prevention programs based on molecular diagnosis of heterozygous carriers and/or patients require the use of reliable mutation scanning methods in at-risk populations.

Methods: We developed a rapid and highly specific mutation-screening test based on temporal temperature gradient gel electrophoresis (TTGE). We analyzed 889 β-thalassemia genes from homozygous β-thalassemia patients and unrelated individuals with heterozygous β-thalassemia. Previously reported common mutations were screened by reverse dot blots using allele-specific probes. The rare mutations were analyzed by TTGE.

Results: We found common mutations in 753 β-thalassemia genes. TTGE analysis in the rest of the genes showed the presence of mutations in different regions of the β-globin gene in 134 of them, and these mutations were characterized by DNA sequencing. In the two genes in which mutations were not identified, large deletions spanning β-globin gene were suspected.

Conclusions: Compared with other approaches for comprehensive mutation screening, the reported method is rapid, highly sensitive, cost-effective, and suitable for high-throughput screening of a large number of samples.