bluuebunny/crossref_metadata_2025 huggingface dataset:
External Quality Assurance Program for PCR Amplification of Genomic DNA: An Italian Experience
Authors: Raggi Claudia Casini , Pinzani Pamela , Paradiso Angelo , Pazzagli Mario , Orlando Claudio
Published: February 2004

Background: External quality assurance (EQA) programs for diagnostic tests based on nucleic acid amplification have not been widely implemented in clinical laboratories and remain limited to few tests. Development of specific EQA programs based on application-based proficiency testing for any diagnostic molecular target is challenging. Development of EQA trials based on methodologic proficiency testing and directed to the evaluation of analytical aspects common to the majority of PCR-based tests may be valuable.

Methods: We developed an EQA program for evaluation of DNA extraction and amplification and analysis of products after PCR. Participants received a package containing primers and reference materials to evaluate three specific controls for, respectively, DNA extraction (quality and quantity), PCR performance (specificity and efficiency), and interpretation of results after electrophoresis. Each participant was asked to return to the organizers a form with their numerical results and an aliquot of all amplified samples for joint evaluation.

Results: Results varied in all phases of the experimental procedure: preamplification, amplification, and post-PCR interpretation. To give a general estimation on the quality of performances for each laboratory, we designed a score scheme in which the results of any specific action were evaluated on the basis of the distribution around the median consensus values. The maximum possible score was 84. On the basis of total score obtained by each laboratory, we created a qualitative ranking list that provided the final interpretation of results as excellent (>63 points; n = 4 laboratories), good (53–63 points; n = 13), sufficient (42–52 points; n = 15), poor (31–41 points; n = 3), and not acceptable (<31 points; n = 4).

Conclusions: This survey demonstrates the importance of EQA trials based on methodologic proficiency testing directed to evaluation of analytical aspects common to the majority of PCR-based tests.
Indicators of Erythrocyte Damage after Microwave Warming of Packed Red Blood Cells
Authors: Hirsch Jan , Menzebach Axel , Welters Ingeborg Dorothea , Dietrich Gerald Volker , Katz Norbert , Hempelmann Gunter
Published: February 2004

Background: Localized overheating of packed red blood cells (PRBCs) after microwave warming with consequent damage to erythrocytes has been reported. We therefore compared possible cellular markers of erythrocyte damage, as measured by flow cytometry, with laboratory indicators of hemolysis to evaluate the effects of microwave warming on PRBCs.

Methods: PRBC samples were warmed to room temperature or to 37, 42, 47, 52, or 57 °C in a water bath. Flow cytometry was performed after fluorescein labeling using antibodies to spectrin, Ca2+-ATPase, and Na+-K+-ATPase. The forward-to-sideward scatter (FSC/SSC) ratio and antibody binding were evaluated. Plasma free hemoglobin (FHb) and α-hydroxybutyrate dehydrogenase (HBDH) were measured immediately after heating and after 48 h. In addition, all measurements were made before and after the heating of PRBCs to 35 °C by a microwave blood warmer.

Results: Analysis of 15 000 erythrocytes showed a decrease in the FSC/SSC ratio and antibody binding above 47 °C [at 37 °C, median (SD) of 94.2 (7.4) with 0.07 (0.05)% fluorescein-positive; at 52 °C, median (SD) of 177.0 (19.0) with 18.5 (6.4)% positively gated; P <0.001]. FHb [room temperature, 0.3 (0.2) g/L] was increased 2-fold at 37 and 42 °C, 4-fold at 47 °C, and 25-fold at 52 °C. HBDH increased in parallel. Hemolysis markers showed an additional twofold increase 48 h after heating to 42 and 47 °C. Microwave heating to 35 °C did not produce significant changes of any marker.

Conclusions: All markers of cellular damage were altered after heating to >47 °C, and a substantial part of hemolysis was delayed. The methodology can be used for future testing of other blood warming devices.
High-Resolution Genotyping by Amplicon Melting Analysis Using LCGreen
Authors: Wittwer Carl T , Reed Gudrun H , Gundry Cameron N , Vandersteen Joshua G , Pryor Robert J
Published: February 2004

Background: High-resolution amplicon melting analysis was recently introduced as a closed-tube method for genotyping and mutation scanning (Gundry et al. Clin Chem 2003;49:396–406). The technique required a fluorescently labeled primer and was limited to the detection of mutations residing in the melting domain of the labeled primer. Our aim was to develop a closed-tube system for genotyping and mutation scanning that did not require labeled oligonucleotides.

Methods: We studied polymorphisms in the hydroxytryptamine receptor 2A (HTR2A) gene (T102C), β-globin (hemoglobins S and C) gene, and cystic fibrosis (F508del, F508C, I507del) gene. PCR was performed in the presence of the double-stranded DNA dye LCGreen, and high-resolution amplicon melting curves were obtained. After fluorescence normalization, temperature adjustment, and/or difference analysis, sequence alterations were distinguished by curve shape and/or position. Heterozygous DNA was identified by the low-temperature melting of heteroduplexes not observed with other dyes commonly used in real-time PCR.

Results: The six common β-globin genotypes (AA, AS, AC, SS, CC, and SC) were all distinguished in a 110-bp amplicon. The HTR2A single-nucleotide polymorphism was genotyped in a 544-bp fragment that split into two melting domains. Because melting curve acquisition required only 1–2 min, amplification and analysis were achieved in 10–20 min with rapid cycling conditions.

Conclusions: High-resolution melting analysis of PCR products amplified in the presence of LCGreen can identify both heterozygous and homozygous sequence variants. The technique requires only the usual unlabeled primers and a generic double-stranded DNA dye added before PCR for amplicon genotyping, and is a promising method for mutation screening.
Diagnostic Accuracy of Fecal Calprotectin Assay in Distinguishing Organic Causes of Chronic Diarrhea from Irritable Bowel Syndrome: A Prospective Study in Adults and Children
Authors: Carroccio Antonio , Iacono Giuseppe , Cottone Mario , Di Prima Lidia , Cartabellotta Fabio , Cavataio Francesca , Scalici Calogero , Montalto Giuseppe , Di Fede Gaetana , Rini GiovamBattista , Notarbartolo Alberto , Averna Maurizio R
Published: February 2004

Background: Fecal calprotectin (FC) has been proposed as a marker of inflammatory bowel disease (IBD), but few studies have evaluated its usefulness in patients with chronic diarrhea of various causes. We evaluated the diagnostic accuracy of a FC assay in identifying “organic” causes of chronic diarrhea in consecutive adults and children.

Methods: We consecutively enrolled 70 adult patients (30 males, 40 females; median age, 35 years) and 50 children (20 males, 30 females; median age, 3.5 years) with chronic diarrhea of unknown origin. All patients underwent a complete work-up to identify the causes of chronic diarrhea. FC was measured by ELISA.

Results: In adult patients, FC showed 64% sensitivity and 80% specificity with 70% positive and 74% negative predictive values for organic causes. False-positive results (8 of 40 cases) were associated with the use of aspirin (3 cases) or nonsteroidal antiinflammatory drugs (1 case) and with the presence of concomitant liver cirrhosis (3 cases). False-negative results mainly included patients suffering from celiac disease (5 cases). Patients with IBD (9 cases) were identified with 100% sensitivity and 95% specificity. In pediatric patients, sensitivity was 70%, specificity was 93%, and positive and negative predictive values were 96% and 56%. False-negative results (11 of 35 cases) were associated mainly with celiac disease (6 cases) or intestinal giardiasis (2 cases).

Conclusions: FC assay is an accurate marker of IBD in both children and adult patients. In adults, false negatives occur (e.g., in celiac disease) and false-positive results are seen in cirrhosis or users of nonsteroidal antiinflammatory drugs. Diagnostic accuracy is higher in children.
Convenient and Effective Method for Removing Fibrinogen from Serum Specimens before Protein Electrophoresis
Authors: Qiu Ling L , Levinson Stanley S , Keeling Kristen L , Elin Ronald J
Published: February 2004

Background: Fibrinogen in serum specimens can be misinterpreted on protein electrophoresis as a monoclonal protein. We evaluated selective precipitation of fibrinogen with ethanol.

Methods: Pooled human plasma was mixed with absolute ethanol or saline (final concentrations of 40, 80, 100, 120, and 160 mL/L) and incubated at 4 °C overnight or placed in an ice bath for 15 min. After centrifugation, the supernatants and resuspended pellets were used for protein electrophoresis and quantitative measurements of protein and fibrinogen.

Results: The fibrinogen band was effectively eliminated from the electrophoretic pattern in the plasma samples treated with ethanol at 100 mL/L and incubated in an ice bath for 15 min without a significant change in immunoglobulin concentrations. The 100 mL/L ethanol did not noticeably change the electrophoretic pattern of monoclonal immunoglobulins. This approach allowed analysis of a sample collected from an arteriovenous shunt kept open with heparin.

Conclusions: Ethanol, 100 mL/L, can selectively precipitate fibrinogen without significantly interfering with the immunoglobulins. The precipitation process can be completed in 15 min at 0–4 °C and can avoid the need to obtain another blood sample.
Strategy for Analysis of Cardiac Troponins in Biological Samples with a Combination of Affinity Chromatography and Mass Spectrometry
Authors: Labugger Ralf , Simpson Jeremy A , Quick Michelle , Brown Heather A , Collier Christine E , Neverova Irina , Van Eyk Jennifer E
Published: February 2004

Background: Cardiac troponins are modified during ischemic injury and are found as a heterogeneous mixture in blood of patients with cardiovascular diseases. We present a strategy to isolate cardiac troponins from human biological material, by use of affinity chromatography, and to provide samples ready for direct analysis by mass spectrometry.

Methods: Cardiac troponins were isolated from human left ventricular tissue by affinity chromatography. Isolated troponins were either eluted and analyzed by Western blot or enzymatically digested while bound to affinity beads. The resulting peptide mixture was subjected to mass spectrometry for protein identification and characterization. The same method was used to analyze serum from patients with acute myocardial infarction (AMI).

Results: Affinity chromatography with antibodies specific for one cardiac troponin subunit facilitated the isolation of the entire cardiac troponin complex from myocardial tissue. The three different proteases used for enzymatic digestion increased the total protein amino acid sequence coverage by mass spectrometry for the three cardiac troponin subunits. Combined amino acid sequence coverages for cardiac troponin I, T, and C (cTnI, cTnT, cTnC) were 54%, 48%, and 40%, respectively. To simulate matrix effects on the affinity chromatography–mass spectrometry approach, we diluted tissue homogenate in cardiac troponin-free serum. Sequence coverages in this case were 44%, 41%, and 19%, respectively. Finally, affinity chromatography–mass spectrometry analysis of AMI serum revealed the presence of cardiac troponins in a wide variety of its free and/or complexed subunits, including the binary cTnI-cTnC and cTnI-cTnC-cTnT complexes.

Conclusions: Affinity chromatography–mass spectrometry allows the extraction and analysis of cardiac troponins from biological samples in their natural forms. We were, for the first time, able to directly confirm the presence of cardiac troponin complexes in human serum after AMI. This approach could assist in more personalized risk stratification as well as the search for reference materials for cardiac troponin diagnostics.
Determination of Non-α1-Antichymotrypsin-complexed Prostate-specific Antigen as an Indirect Measurement of Free Prostate-specific Antigen: Analytical Performance and Diagnostic Accuracy
Authors: Wesseling Sebastian , Stephan Carsten , Semjonow Axel , Lein Michael , Brux Brigitte , Sinha Pranav , Loening Stefan A , Jung Klaus
Published: February 2004

Background: A new assay measures prostate-specific antigen (PSA) not complexed to α1-antichymotrypsin (nACT-PSA) after removing PSA complexed to ACT by use of anti-ACT antibodies. We evaluated nACT-PSA and its ratio to total PSA (tPSA) as alternatives to free PSA (fPSA) and its ratio to tPSA in differentiating prostate cancer (PCa) and benign prostatic hyperplasia (BPH) in patients with tPSA of 2–20 μg/L.

Methods: PSA in serum of 183 untreated patients with PCa and 132 patients with BPH was measured retrospectively on the chemiluminescence immunoassay analyzer LIAISON® (Byk-Sangtec Diagnostica) with the LIAISON tPSA and LIAISON fPSA assays. The nACT-PSA fraction was determined with a prototype assay measuring the residual PSA after precipitation of ACT-PSA with an ACT-precipitating reagent.

Results:nACT-PSA was higher than fPSA in samples with fPSA concentrations <1 μg/L but lower in samples with >1 μg/L fPSA. The median ratios of fPSA/tPSA and of nACT-PSA/tPSA were significantly different between patients with BPH and PCa (19.4% vs 12.2% and 17.4% vs 13.0%, respectively). Within the tPSA ranges tested (2–20, 2–10, and 4–10 μg/L), areas under the ROC curves for the fPSA/tPSA ratios were significantly larger than those for nACT-PSA/tPSA. In the tPSA ranges <10 μg/L, the areas under the ROC curves for fPSA/tPSA were significantly larger than those for tPSA, whereas the areas for nACT-PSA/tPSA were not. At decision limits for 95% sensitivity and specificity, both ratios significantly increased specificity and sensitivity, respectively, compared with tPSA, but the fPSA/tPSA ratio showed higher values.

Conclusions: nACT-PSA and its ratio to tPSA provide lower diagnostic sensitivity and specificity than fPSA/tPSA. The fPSA/tPSA ratio represents the state-of-the-art method for differentiating between PCa and BPH.
Plasma Total Cysteine, Mortality, and Cardiovascular Disease Hospitalizations: The Hordaland Homocysteine Study
Authors: El-Khairy Lina , Vollset Stein E , Refsum Helga , Ueland Per M
Published: February 2004

Background: We have previously reported a positive association between tHcy and mortality and cardiovascular disease (CVD) hospitalizations in the Hordaland Homocysteine Study cohort. Using the same data set, we assessed the relationship between plasma total cysteine (tCys) and mortality from all causes and from cardiovascular and noncardiovascular conditions, and the association between tCys and the risk of hospitalizations from CVD.

Methods: We measured plasma tCys in blood samples from 12 595 men and women 40–42 years of age and from 4766 men and women 65–67 years of age, collected as part of the Hordaland Homocysteine Study in the year 1992–1993. Follow-up data on mortality were collected through 1999. Data on CVD hospitalizations were collected from hospital records up to May 31, 1998.

Results: After a follow-up time of 6.6–7.6 years, there were a total of 610 deaths, of which 243 were cardiovascular deaths and 367 were noncardiovascular deaths. There was no association between tCys and all-cause, cardiovascular, or noncardiovascular mortality. When we used tCys values <247.6 μmol/L (lowest quartile) as the reference category, the adjusted mortality ratio (MR) for all-cause mortality at tCys concentrations of 247.6–270.79, 270.8–295.79, and ≥295.8 μmol/L (highest quartile) were 1.0, 0.9, and 1.0, respectively. The adjusted MRs for cardiovascular mortality were 1.0, 1.1, and 1.1, respectively. There were no associations between tCys and 1275 CVD hospitalizations, except that tCys was significantly associated with hospitalizations from coronary artery bypass grafting.

Conclusion: Plasma tCys is not associated with mortality or CVD hospitalizations.
Detection of Recombinant Human Erythropoietin in Urine by Isoelectric Focusing
Authors: Breidbach Andreas , Catlin Don H , Green Gary A , Tregub Inna , Truong Henry , Gorzek Jeffrey
Published: February 2004

Background: Doping with erythropoietic proteins such as recombinant human erythropoietin (rHuEPO) and darbepoetin alfa is a serious issue in sport. There is little information on the time course of detection of rHuEPO in urine and on methods to evaluate electrophoresis-based data.

Methods: We used a recently described isoelectric focusing method for detecting rHuEPO and endogenous EPO in urine obtained from individuals treated with placebo or epoetin alfa. The latter was administered subcutaneously at 50 IU/kg on days 0, 2, 4, 7, 9, 11, 14, 16, and 18. Blood and urine samples were collected during the morning of study days −3, 0, 2, 4, 7, 9, 11, 14, 16, and 18 and on days 2, 3, 4, and 7 postadministration. We developed visual and numerical (two-band ratio) techniques to evaluate the electropherograms for the presence of rHuEPO.

Results: Compared with the placebo group, the epoetin alfa-treated group responded with increases in hematocrit, reticulocytes, macrocytes, serum EPO, and serum soluble transferrin receptor. The electropherograms showed that the pattern of bands arising from urinary rHuEPO is different from that of endogenous urinary EPO. Both the two-band ratio and the visual technique detected rHuEPO in all 14 epoetin alfa-treated individuals 3 days after the last dose. On the 7th day after the last dose, both techniques detected rHuEPO in approximately one-half of the participants. rHuEPO was not detected in the placebo-treated individuals.

Conclusions: The isoelectric focusing method detects rHuEPO in most urine samples collected 3 days after nine doses of epoetin alfa. The numerical two-band ratio was equivalent to a visual method for detecting rHuEPO in urine.
Time-resolved Fluorometric Assay for Detection of Autoantibodies to Glutamic Acid Decarboxylase (GAD65)
Authors: Ankelo Matti , Westerlund-Karlsson Annette , Ilonen Jorma , Knip Mikael , Savola Kaisa , Kankaanpää Pasi , Meriö Liisa , Siitari Harri , Hinkkanen Ari
Published: February 2004

Background: Type 1 diabetes mellitus results from destruction of the pancreatic insulin-producing beta cells by a chronic autoimmune process. Methods are needed for the detection of circulating autoantibodies to glutamic acid decarboxylase (GAD65), a major marker of this process.

Methods: Streptavidin-coated microtiter plates were incubated with biotinylated GAD65, and after incubation with serum samples from patients with type 1 diabetes mellitus and control individuals, europium-labeled GAD65 was added. After washing steps, the delayed fluorescence was measured in duplicate in a fluorometer. Samples collected from 100 patients with newly diagnosed type 1 diabetes mellitus and 100 healthy controls were measured by the new assay and by a radiobinding assay.

Results: The detection limit of the new assay was 1.49 WHO units/mL, the calibration curve was linear to 4 140 WHO units/mL, and no hook effect was observed up to 41 400 WHO units/mL. The intraassay CV was 2.1–6.3% over the calibration range. For patient serum samples, the intraassay, interassay, and total CVs were 5.4–7.0%, 9.8–13%, and 12–14%, respectively. Compared with conventional radioimmunologic methods, the analytical range was broader and the analysis time required to perform the measurements was shorter. At a cutoff with 99% specificity, the new assay and the radiobinding assay were positive in 71 and 67 patients, respectively.

Conclusions: The new assay provides a rapid and sensitive nonradioactive method applicable for large-scale screening for beta-cell autoimmunity. It has a broad linear analytical range, is easy to perform and automate, and has sensitivity and specificity comparable to those for the conventional radioisotope assay.
Monitoring Blood Glucose Changes in Cutaneous Tissue by Temperature-modulated Localized Reflectance Measurements
Authors: Yeh Shu-jen , Hanna Charles F , Khalil Omar S
Published: February 2004

Background: Most proposed noninvasive methods for glucose measurements do not consider the physiologic response of the body to changes in glucose concentration. Rather than consider the body as an inert matrix for the purpose of glucose measurement, we exploited the possibility that noninvasive measurements of glucose can be approached by investigating their effects on the skin’s thermo-optical response.

Methods: Glucose concentrations in humans were correlated with temperature-modulated localized reflectance signals at wavelengths between 590 and 935 nm, which do not correspond to any near-infrared glucose absorption wavelengths. Optical signal was collected while skin temperature was modulated between 22 and 38 °C over 2 h to generate a periodic set of cutaneous vasoconstricting and vasodilating events, as well as a periodic change in skin light scattering. The method was tested in a series of modified meal tolerance tests involving carbohydrate-rich meals and no-meal or high-protein/no-carbohydrate meals.

Results: The optical data correlated with glucose values. Changes in glucose concentrations resulting from a carbohydrate-rich meal were predicted with a model based on a carbohydrate-meal calibration run. For diabetic individuals, glucose concentrations were predicted with a standard error of prediction <1.5 mmol/L and a prediction correlation coefficient 0.73 in 80% of the cases. There were run-to-run differences in predicted glucose concentrations. Non-carbohydrate meals showed a high degree of scatter when predicted by a carbohydrate meal calibration model.

Conclusions: Blood glucose concentrations alter thermally modulated optical signals, presumably through physiologic and physical effects. Temperature changes drive cutaneous vascular and refractive index responses in a way that mimics the effect of changes in glucose concentration. Run-to-run differences are attributable to site-to-site structural differences.
Preanalytical Factors That Influence the Abbott TDx Fetal Lung Maturity II Assay
Authors: Grenache David G , Parvin Curtis A , Gronowski Ann M
Published: February 2004

Background: The TDx Fetal Lung Maturity II (FLM II) assay uses amniotic fluid to assess lung maturity of the unborn infant. We investigated common preanalytical factors that influence FLM results, including centrifugation, sample storage, and contamination by whole blood.

Methods: We tested 18 specimens after centrifugation and after resuspension by vortex-mixing. We also analyzed 23 specimens stored at −20 °C for up to 448 days and then thawed (duplicate measurements), 20 specimens stored at 4 °C, and 24 specimens stored at room temperature. In addition, we evaluated the effects of whole blood diluted into 19 different specimens.

Results: Centrifugation significantly decreased FLM II results from baseline (P <0.0001), and resuspension returned results to baseline values (P = 0.286). Storage at −20 °C produced highly variable results that demonstrated a nonsignificant negative trend associated with storage time. Specimens were stable for 24 h when stored at 4 °C and 16 h at room temperature. Blood contamination produced significantly positive differences in results only in specimens with baseline values ≤39 mg/g with a 5.8 mg/g increase in FLM II for every 0.1 × 1012/L increase in the erythrocyte count (slope = 58.4).

Conclusions: Resuspension of centrifuged specimens produces clinically valid FLM II results. Results from specimens stored at −20 °C can be highly variable and decrease over time. Results from specimens stored at 4 °C and at room temperature are stable for 24 and 16 h, respectively. Blood contamination up to 0.03 × 1012 erythrocytes/L is acceptable for FLM II analysis.
Hydroxytyrosol Disposition in Humans
Authors: Miro-Casas Elisabet , Covas Maria-Isabel , Farre Magi , Fito Montserrat , Ortuño Jordi , Weinbrenner Tanja , Roset Pere , de la Torre Rafael
Published: February 2004

Background: Animal and in vitro studies suggest that phenolic compounds in virgin olive oil are effective antioxidants. In animal and in vitro studies, hydroxytyrosol and its metabolites have been shown to be strong antioxidants. One of the prerequisites to assess their in vivo physiologic significance is to determine their presence in human plasma.

Methods: We developed an analytical method for both hydroxytyrosol and 3-O-methyl-hydroxytyrosol in plasma. The administered dose of phenolic compounds was estimated from methanolic extracts of virgin olive oil after subjecting them to different hydrolytic treatments. Plasma and urine samples were collected from 0 to 12 h before and after 25 mL of virgin olive oil intake, a dose close to that used as daily intake in Mediterranean countries. Samples were analyzed by capillary gas chromatography–mass spectrometry before and after being subjected to acidic and enzymatic hydrolytic treatments.

Results: Calibration curves were linear (r >0.99). Analytical recoveries were 42–60%. Limits of quantification were <1.5 mg/L. Plasma hydroxytyrosol and 3-O-methyl-hydroxytyrosol increased as a response to virgin olive oil administration, reaching maximum concentrations at 32 and 53 min, respectively (P <0.001 for quadratic trend). The estimated hydroxytyrosol elimination half-life was 2.43 h. Free forms of these phenolic compounds were not detected in plasma samples.

Conclusions: The proposed analytical method permits quantification of hydroxytyrosol and 3-O-methyl-hydroxytyrosol in plasma after real-life doses of virgin olive oil. From our results, ∼98% of hydroxytyrosol appears to be present in plasma and urine in conjugated forms, mainly glucuronoconjugates, suggesting extensive first-pass intestinal/hepatic metabolism of the ingested hydroxytyrosol.
Biological Threat Detection via Host Gene Expression Profiling
Authors: Lin Baochuan , Vahey Maryanne T , Thach Dzung , Stenger David A , Pancrazio Joseph J
Published: February 2004

With the increased threat posed by biological weapons, detection techniques for biothreat pathogens are critically needed to monitor and assess the severity of the illness once exposure has occurred. Current approaches for detecting biological threats are either time-consuming or highly specific but provide little information regarding pathogenicity. Genotyping of pathogens by PCR provides a fast and definitive means for identifying pathogens, but reliance on pathogen genotypic endpoints has several limitations. Current progress in DNA microarrays technology provides an alternative way to address the issues faced by traditional detection systems through host gene expression profiles of peripheral blood cells. We discuss the advantages and critical issues facing the use of host gene expression profiling for biological threat detection.
Linked Linear Amplification for Simultaneous Analysis of the Two Most Common Hemochromatosis Mutations
Authors: Killeen Anthony A , Breneman John W , Carillo Arlene R , Liu Jason , Hixson Craig S
Published: February 2004

Background: Two mutations in HFE, G845A (amino acid substitution C282Y) and C187G (H63D), are associated with hereditary hemochromatosis. We developed and validated a novel method, linked linear amplification (LLA), for detection of these two mutations.

Methods: Two segments of HFE were amplified by a multiplex LLA reaction that generated biotinylated LLA products. Aliquots of the multiplex LLA reaction were captured in microwells by hybridization to immobilized allele-specific oligonucleotides (ASOs). One wild-type and one mutant ASO represented the DNA sequence at each of the two mutation sites. Hybridization was detected by a streptavidin–horseradish peroxidase-based colorimetric method. Genotypes obtained by LLA and PCR-restriction fragment length polymorphism (PCR-RFLP) methods for 320 individuals were compared.

Results: The amplified samples included the following genotypes as determined by PCR-RFLP: wild-type 282 and 63 codons (n = 105), C282Y homozygous mutant (n = 54), C282Y heterozygous (n = 52), H63D homozygous mutant (n = 17), H63D heterozygous (n = 59), and compound H63D and C282Y heterozygous mutant (n = 33). There was complete concordance between the results obtained by LLA and those obtained by PCR-RFLP analysis. The presence of another HFE mutation, A193T (encoding S65C), did not interfere with genotyping at codon 63.

Conclusions: LLA provides a reliable method to detect the common mutations in HFE that cause hereditary hemochromatosis.
DNA Integrity as a Potential Marker for Stool-based Detection of Colorectal Cancer
Authors: Boynton Kevin A , Summerhayes Ian C , Ahlquist David A , Shuber Anthony P
Published: February 2004

Background: Molecular genetic analysis of DNA in patient stools has been proposed for screening of colorectal cancer (CRC). Because nonapoptotic cells shed from tumors may contain DNA that is less degraded than DNA fragments from healthy colonic mucosa, our aim was to show that DNA fragments isolated from stools of patients with CRC had higher integrity than DNA isolated from stools of patients with healthy colonic mucosa.

Methods: We purified DNA from the stools of a colonoscopy-negative control group and patients with CRC and examined the relationship between long DNA fragments and clinical status by determining stool DNA integrity, using oligonucleotide-based hybrid captures with specific target sequences in increasingly long PCR reactions (200 bp, 400 bp, 800 bp, 1.3 kb, 1.8 kb, 24 kb). DNA fragments obtained from CRC patients were compared with fragments obtained from colonoscopy-negative individuals for length and/or integrity.

Results: DNA fragments isolated from CRC patients were of higher molecular weight (>18 bands detected of a total of 24 possible bands) than fragments isolated from fecal DNA of the colonoscopy-negative control group.

Conclusions: The presence of long DNA fragments in stool is associated with CRC and may be related to disease-associated differences in the regulation of proliferation and apoptosis. An assay of fecal DNA integrity may be a useful biomarker for the detection of CRC.
Screening and Quantification of Multiple Chromosome Translocations in Human Leukemia
Authors: Shi Run Zhang , Morrissey Joseph M , Rowley Janet D
Published: February 2004

Background: Characterization of fusion gene transcripts in leukemia that result from chromosome translocations provides valuable information regarding appropriate treatment and prognosis. However, screening for multiple fusion gene transcripts is difficult with conventional PCR and state-of-the-art real-time PCR and high-density microarrays.

Methods: We developed a multiplex reverse transcription-PCR (RT-PCR) assay for screening and quantification of fusion gene transcripts in human leukemia cells. Chimeric primers were used that contained gene-specific and universal sequences. PCR amplification of fusion and control gene transcripts was achieved with use of an excess of universal primers to allow the ratio of abundance of fusion gene to endogenous or exogenous controls to be maintained throughout PCR. Multiplex RT-PCR products analyzed by an ABI 310 Genetic Analyzer were consistent with those of duplex RT-PCR (single analytical sample plus control). In addition, multiplex RT-PCR results were analyzed by an assay using an oligonucleotide microarray that contained probes for the splice-junction sequences of various fusion transcripts.

Results: The multiplex RT-PCR assay enabled screening of >10 different fusion gene transcripts in a single reaction. RT-PCR followed by analysis with the ABI Prism 310 Genetic Analyzer consistently detected 1 fusion-transcript-carrying leukemia cell in 100–10 000 cells. The assay covered a 1000-fold range. Preliminary results indicate that multiplex RT-PCR products can also be analyzed by hybridization-based microarray assay.

Conclusions: The multiplex RT-PCR analyzed by either ABI Prism 310 Genetic Analyzer or microarray provides a sensitive and specific assay for screening of multiple fusion transcripts in leukemia, with the latter an assay that is adaptable to a high-throughput system for clinical screening.
Molecular Beacon Reverse Transcription-PCR of Human Chorionic Gonadotropin-β-3, -5, and -8 mRNAs Has Prognostic Value in Breast Cancer
Authors: Span Paul N , Manders Peggy , Heuvel Joop J T M , Thomas Chris M G , Bosch Remko R , Beex Louk V A M , Sweep C G J (Fred)
Published: February 2004

Background: The β-subunit of human chorionic gonadotropin (hCG) is encoded by four genes, of which expression of the hCGβ-3, -5, and -8 genes could have prognostic value in breast cancer.

Methods: Applying a new, modified Molecular Beacon reverse transcription-PCR assay, we investigated the prognostic value of the hCGβ-3, -5, and -8 gene transcripts in 129 sporadic unilateral breast cancer samples from patients with a median follow-up of 62.3 months.

Results: Expression of hCGβ-3, -5, -8 was significantly (P = 0.020) associated with relapse-free survival (RFS). In multivariate survival analysis, hCGβ-3, -5, and -8 maintained prognostic value for RFS, with high expression predicting shorter RFS (P = 0.015; hazard ratio, 2.25; 95% confidence interval, 1.17–4.34). Only 1 of 24 (4%) node-negative patients with low hCGβ-3, -5, -8 expression relapsed, in contrast to 7 of 26 (27%) patients with high expression (P = 0.046).

Conclusions: Expression of hCGβ-3, -5, -8, which differ by only one nucleotide from other hCGβ genes, can be assessed by our modified Molecular Beacon assay in breast cancer tissues. Expression of hCGβ-3, -5, -8 has independent, prognostic value for RFS in breast cancer and may help identify node-negative patients with poor prognosis.
Plasminogen Activator Inhibitor 1 4G/5G Polymorphism and Coagulation Factor XIII Val34Leu Polymorphism: Impaired Fibrinolysis and Early Pregnancy Loss
Authors: Dossenbach-Glaninger Astrid , van Trotsenburg Michael , Dossenbach Martin , Oberkanins Christian , Moritz Anne , Krugluger Walter , Huber Johannes , Hopmeier Pierre
Published: February 2004

Background: A successful outcome of pregnancy depends on proper placental formation. In the very beginning of this process, trophoblast invasion and fibrin deposition into the wall of the decidual veins play an important part. Two polymorphisms, coagulation factor XIII (FXIII) Val34Leu and plasminogen activator inhibitor 1 (PAI-1) 4G/5G, interfere with fibrin cross-linking and regulation of fibrinolysis and may therefore contribute to early pregnancy loss.

Methods: We enrolled 49 unrelated Caucasian women with a history of two consecutive or three to six nonconsecutive early pregnancy losses and 48 unrelated parous healthy controls without a history of pregnancy loss and evaluated them for the following genetic variants: the factor V Leiden and prothrombin G20210A gene mutations, the methylenetetrahydrofolate reductase C677T and A1298C polymorphisms, and the PAI-1 4G/5G and FXIII Val34Leu polymorphisms.

Results: For the isolated occurrence of PAI-1 4G/5G or FXIII Val34Leu, we found no statistically significant difference between cases and controls. For homozygosity of either or compound carrier status of both mutations, the overall relative risk for early pregnancy loss was significantly increased (odds ratio = 2.4; 95% confidence interval, 1.1–5.5; P = 0.032). We observed no statistically relevant association of any of the other tested mutations with early pregnancy loss.

Conclusion: Homozygosity for PAI-1 4G or FXIII 34Leu polymorphisms as well as compound carrier status is associated with early pregnancy loss.
Rapid Detection of Aneuploidy (Trisomy 21) by Allele Quantification Combined with Melting Curves Analysis of Single-Nucleotide Polymorphism Loci
Authors: Pont-Kingdon Genevieve , Lyon Elaine
Published: February 2004

Background: Molecular approaches for the detection of chromosomal abnormalities will allow the development of rapid, cost-effective screening strategies. We present here a molecular alternative for the detection of aneuploidies and, more specifically, trisomy 21.

Methods: We used the quantitative value of melting curve analysis of heterozygous genetic loci to establish a relative allelic count. The two alleles of a given single-nucleotide polymorphism (SNP) were differentiated by thermodynamic stability with a fluorescently labeled hybridization probe and were quantified by relative areas of derivative melting curves detected after fluorescence resonance energy transfer. Heterozygous SNPs provided internal controls for the assay.

Results: We selected six SNPs, heterozygous in at least 30% of a random population, to form a panel of informative loci in the majority of a random population. After normalization to a heterozygous control, samples segregated into three categories; nontrisomic samples had mean allele ratios of 0.96–1.09, whereas trisomic samples had mean ratios of 1.84–2.09 or 0.46–0.61, depending on which allele was duplicated. Within-run mean CVs of ratios were 6.5–27%, and between-assay mean CVs were 13–24%.

Conclusions: The use of melting curve analysis of multiple SNPs is an alternative to the use of small tandem repeats for the detection of trisomies. Because of the high density of SNPs, the approach may be specifically useful for very fine mapping of the regions of chromosome 21 that are critical for Down syndrome; it is also applicable to aneuploidies other than trisomy 21 and to specimens that are not amenable to cytogenetic analysis.
Negative Interference in Cardiac Troponin I Immunoassays from a Frequently Occurring Serum and Plasma Component
Authors: Eriksson Susann , Junikka Miia , Laitinen Päivi , Majamaa-Voltti Kirsi , Alfthan Henrik , Pettersson Kim
Published: February 2004

Background: Cardiac troponin I (cTnI) is a sensitive marker of cardiac injury, but cTnI assays, like other immunoassays, are susceptible to interferences. We evaluated the presence of interfering substances by measuring the recovery of cTnI added to samples from volunteers and from patients with acute coronary syndromes (ACS).

Methods: We added a ternary complex of human cardiac troponin (30–500 μg/L) or cTnI from serum to samples from healthy volunteers and ACS patients. We measured cTnI with a two-site sandwich time-resolved immunofluorometric assay using two antibodies against epitopes in the central stable part of cTnI. We also analyzed 108 heparin-plasma samples from 16 ACS patients with this assay, with an assay based on four antibodies, and with two commercial cTnI assays, AxSYM and ACS:180.

Results: In samples from both healthy persons and ACS patients, recoveries for our assay were 1–167% (range). Recoveries were increased by addition of an antibody with an epitope in the N-terminal region of cTnI to the solid phase and an antibody with an epitope in the C-terminal region as a second detection antibody. In 2 of 16 patients with ACS, normal cTnI concentrations found when measured with the original assay demonstrated clinically abnormal (up to 10-fold higher) results with the additional N- and C-terminal antibodies in the early phase of infarction. Both commercial cTnI assays also demonstrated clinically misleading, falsely low cTnI concentrations.

Conclusions: Some yet unidentified, variable component, present in the blood from healthy volunteers and ACS patients, interferes with the binding of antibodies against epitopes in the central part of cTnI used in two commercial assays. Our approach to supplement the mid-fragment cTnI antibodies with antibodies in the N- and C-terminal parts of the molecule in an experimental assay represents a step in resolving this interferent.
Time-resolved Fluorescence Resonance Energy Transfer Assay for Point-of-Care Testing of Urinary Albumin
Authors: Qin Qiu-Ping , Peltola Olli , Pettersson Kim
Published: February 2004

Background: Microalbuminuria is an established early marker of diabetic nephropathy and an important cardiovascular risk factor in diabetes and hypertension. We aimed to develop a rapid point-of-care assay for the measurement of urine albumin.

Methods: The competitive homogeneous assay used an albumin-specific monoclonal antibody labeled with a stable fluorescent europium chelate as donor and an albumin labeled with cyanine 5 (Cy5) as acceptor. The assay was performed at room temperature in single microtitration wells that contained all the required dry-form reagents. The close proximity between the two labels in the immune complex allowed fluorescence resonance energy to be transferred from the pulse-excited europium chelate to the acceptor Cy5. The emission of long-lived energy transfer signal from the sensitized Cy5 was measured at 665 nm with time-resolved fluorometry that eliminated short-lived background.

Results: The assay procedure required 12 min for a 10-μL urine sample. The working range was from 10 to ∼320 mg/L, and the lower limit of detection was 5.5 mg/L. The within- and between-run CVs were 6.9–10% and 7.5–13%, respectively. Recovery was 103–122%. The assay correlated well (r2 = 0.98; n = 37) with a laboratory-based immunoassay, although mean (SD) results were 7 (29)% lower.

Conclusions: The speed and ease of performance of this assay recommend it for near-patient use. The assay is the first to combine a fluorescence resonance energy transfer-type rapid competitive assay with an all-in-one dry reagent.
Infrared Spectroscopic Identification of β-Thalassemia
Authors: Liu Kan-Zhi , Tsang Kam Sze , Li Chi Kong , Shaw R Anthony , Mantsch Henry H
Published: February 2004

Background: The aim of this study was to investigate the potential of infrared (IR) spectroscopy as a fast and reagent-free adjunct tool in the diagnosis and screening of β-thalassemia.

Methods: Blood was obtained from 56 patients with β-thalassemia major, 1 patient with hemoglobin H disease, and 35 age-matched controls. Hemolysates of blood samples were centrifuged to remove stroma. IR absorption spectra were recorded for duplicate films dried from 5 μL of hemolysate. Differentiation between the two groups of hemoglobin spectra was by two statistical methods: an unsupervised cluster analysis and a supervised linear discriminant analysis (LDA).

Results: The IR spectra revealed changes in the secondary structure of hemoglobin from β-thalassemia patients compared with that from controls, in particular, a decreased α-helix content, an increased content of parallel and antiparallel β-sheets, and changes in the tyrosine ring absorption band. The hemoglobin from β-thalassemia patients also showed an increase in the intensity of the IR bands from the cysteine −SH groups. The unsupervised cluster analysis, statistically separating spectra into different groups according to subtle IR spectral differences, allowed separation of control hemoglobin from β-thalassemia hemoglobin spectra, based mainly on differences in protein secondary structure. The supervised LDA method provided 100% classification accuracy for the training set and 98% accuracy for the validation set in partitioning control and β-thalassemia samples.

Conclusion: IR spectroscopy holds promise in the clinical diagnosis and screening of β-thalassemia.
Disease-related Metabolites in Culture Medium of Fibroblasts from Patients with d-2-Hydroxyglutaric Aciduria, l-2-Hydroxyglutaric Aciduria, and Combined d/l-2-Hydroxyglutaric Aciduria
Authors: Struys Eduard A , Verhoeven Nanda M , Roos Birthe , Jakobs Cornelis
Published: February 2004

Background: d-2-Hydroxyglutaric aciduria (D-2-HGA), l-2-hydroxyglutaric aciduria (L-2-HGA), and the combined d/l-2-hydroxyglutaric aciduria (D/L-2-HGA) are poorly understood organic acidurias. To investigate the usefulness of cultured human skin fibroblasts for both diagnostic and research purposes, we measured disease-related metabolites in the cell culture medium.

Methods: We measured d-2-hydroxyglutarate (D-2-HG), l-2-hydroxyglutarate (L-2-HG), succinate, 2-ketoglutarate, and citrate in fibroblast cell medium by stable-isotope-dilution gas chromatography–mass spectrometry and glutamine, glutamic acid, and lysine with an amino acid analyzer. We used six cell lines from patients with D-2-HGA, two from patients with L-2-HGA, three from patients with D/L-2-HGA, and seven control cell lines. Culture medium was analyzed after a 96-h incubation period.

Results: Culture media from cell lines from D-2-HGA patients contained D-2-HG at concentrations 5- to 30-fold higher than media from controls, whereas the concentration of L-2-HG in media was not increased. Media from L-2-HGA cell lines showed a fivefold increase in L-2-HG compared with controls. Media containing fibroblasts from D/L-2-HGA patients contained moderately increased amounts of both D-2-HG and L-2-HG. For all cell lines, succinate concentrations in the blank medium were higher than after 96 h of incubation with the exception of two of three D/L-2-HGA cell lines. Media of D-2-HGA cell lines had 2-ketoglutarate concentrations that were 40% of that for controls. Glutamic acid concentrations in media of these cell lines were 60% lower than in controls.

Conclusions: Cell culture media from fibroblasts from patients with D-2-HGA, L-2-HGA, or D/L-2-HGA contain increased amounts the corresponding 2-HGs, demonstrating the suitability of fibroblasts for both diagnosis of and research concerning 2-HGAs.
Urinary Estrone Conjugate and Pregnanediol 3-Glucuronide Enzyme Immunoassays for Population Research
Authors: O’Connor Kathleen A , Brindle Eleanor , Holman Darryl J , Klein Nancy A , Soules Michael R , Campbell Kenneth L , Kohen Fortüne , Munro Coralie J , Shofer Jane B , Lasley Bill L , Wood James W
Published: February 2004

Background: Monitoring of reproductive steroid hormones at the population level requires frequent measurements, hormones or metabolites that remain stable under less than ideal collection and storage conditions, a long-term supply of antibodies, and assays useful for a range of populations. We developed enzyme immunoassays for urinary pregnanediol 3-glucuronide (PDG) and estrone conjugates (E1Cs) that meet these criteria.

Methods: Enzyme immunoassays based on monoclonal antibodies were evaluated for specificity, detection limit, parallelism, recovery, and imprecision. Paired urine and serum specimens were analyzed throughout menstrual cycles of 30 US women. Assay application in different populations was examined with 23 US and 42 Bangladeshi specimens. Metabolite stability in urine was evaluated for 0–8 days at room temperature and for 0–10 freeze-thaw cycles.

Results: Recoveries were 108% for the PDG assay and 105% for the E1C assay. Serially diluted specimens exhibited parallelism with calibration curves in both assays. Inter- and intraassay CVs were <11%. Urinary and serum concentrations were highly correlated: r = 0.93 for E1C–estradiol; r = 0.98 for PDG–progesterone. All Bangladeshi and US specimens were above detection limits (PDG, 21 nmol/L; E1C, 0.27 nmol/L). Bangladeshi women had lower follicular phase PDG and lower luteal phase PDG and E1Cs than US women. Stability experiments showed a maximum decrease in concentration for each metabolite of <4% per day at room temperature and no significant decrease associated with number of freeze-thaw cycles.

Conclusions: These enzyme immunoassays can be used for the field conditions and population variation in hormone metabolite concentrations encountered in cross-cultural research.