bluuebunny/crossref_metadata_2025 huggingface dataset:
Iron Deficiency and Erythropoiesis: New Diagnostic Approaches
Authors: Brugnara Carlo
Published: February 2004

Iron deficiency anemia is one of the most common diseases worldwide. In the majority of cases, the presence of hypochromic microcytic anemia and biochemical evidence for depletion of body iron stores makes the diagnosis relatively straightforward. However, in several clinical conditions, classic biochemical indices such as serum iron, transferrin saturation, and ferritin may not be informative or may not change rapidly enough to reflect transient iron-deficient states (functional iron deficiency), such as the ones that develop during recombinant human erythropoietin (r-HuEPO) therapy. The identification and treatment of iron deficiency in settings such as r-HuEPO therapy, anemia of chronic disease, and iron deficiency of early childhood may be improved by the use of red cell and reticulocyte cellular indices, which reflect in almost real time the development of iron deficiency and the response to iron therapy. In the anemia of chronic disease, measurements of plasma cytokines and iron metabolism regulators such as hepcidin (when available) may be helpful in the characterization of the pathophysiologic basis of this condition. The ratio of serum transferrin receptor (sTfR) to serum ferritin (R/F ratio) has been shown to have excellent performance in estimating body iron stores, but it cannot be used widely because of the lack of standardization for sTfR assays. The combination of hematologic markers such as reticulocyte hemoglobin content, which decreases with iron deficiency, and R/F ratio may allow for a more precise classification of anemias.
Potential Clinical Utility of Serum HER-2/neu Oncoprotein Concentrations in Patients with Breast Cancer
Authors: Carney Walter P , Neumann Rainer , Lipton Allan , Leitzel Kim , Ali Suhail , Price Christopher P
Published: February 2004

Background: The HER-2/neu oncogene and its p185 receptor protein are indicators of a more aggressive form of breast cancer. HER-2/neu status guides Herceptin therapy, specifically directed to the extracellular domain (ECD) of the HER-2/neu oncoprotein. The HER-2/neu ECD is shed from cancer cells into the circulation and is measurable by immunoassay.

Methods: We performed a systematic review of the peer-reviewed literature on circulating ECD with respect to prevalence, prognosis, prediction of response to therapy, and monitoring of breast cancer.

Results: The prevalence of increased ECD in patients with primary breast cancer varied between 0% and 38% (mean, 18.5%), whereas in metastatic disease the range was from 23% to 80% (mean, 43%). Some women with HER-2/neu-negative tumors by tissue testing develop increased ECD concentrations in metastatic disease. Increased ECD has been correlated with indicators of poor prognosis, e.g., overall survival and disease-free survival. Increased ECD predicts a poor response to hormone therapy and some chemotherapy regimens but can predict improved response to combinations of Herceptin and chemotherapy. Many studies support the value of monitoring ECD during breast cancer progression because serial increases precede the appearance of metastases and longitudinal ECD changes parallel the clinical course of disease.

Conclusions: The monitoring of circulating HER-2/neu ECD provides a tool for assessing prognosis, for predicting response to therapy, and for earlier detection of disease progression and timely intervention with appropriate therapy.
Quality Control and Peak Finding for Proteomics Data Collected from Nipple Aspirate Fluid by Surface-Enhanced Laser Desorption and Ionization
Authors: Coombes Kevin R , Fritsche Herbert A , Clarke Charlotte , Chen Jeng-neng , Baggerly Keith A , Morris Jeffrey S , Xiao Lian-chun , Hung Mien-Chie , Kuerer Henry M
Published: February 2004

Background: Recently, researchers have been using mass spectroscopy to study cancer. For use of proteomics spectra in a clinical setting, stringent quality-control procedures will be needed.

Methods: We pooled samples of nipple aspirate fluid from healthy breasts and breasts with cancer to prepare a control sample. Aliquots of the control sample were used on two spots on each of three IMAC ProteinChip® arrays (Ciphergen Biosystems, Inc.) on 4 successive days to generate 24 SELDI spectra. In 36 subsequent experiments, the control sample was applied to two spots of each ProteinChip array, and the resulting spectra were analyzed to determine how closely they agreed with the original 24 spectra.

Results: We describe novel algorithms that (a) locate peaks in unprocessed proteomics spectra and (b) iteratively combine peak detection with baseline correction. These algorithms detected ∼200 peaks per spectrum, 68 of which are detected in all 24 original spectra. The peaks were highly correlated across samples. Moreover, we could explain 80% of the variance, using only six principal components. Using a criterion that rejects a chip if the Mahalanobis distance from both control spectra to the center of the six-dimensional principal component space exceeds the 95% confidence limit threshold, we rejected 5 of the 36 chips.

Conclusions: Mahalanobis distance in principal component space provides a method for assessing the reproducibility of proteomics spectra that is robust, effective, easily computed, and statistically sound.
Optimized Strategy for Rapid Cytochrome P450 2D6 Genotyping by Real-Time Long PCR
Authors: Müller Burkhardt , Zöpf Konstanze , Bachofer Julia , Steimer Werner
Published: February 2004

Background: Because of genetic polymorphisms, cytochrome P450 2D6 (Cyp2D6) activity in humans varies widely and alters the metabolism of commonly used drugs such as antidepressants, neuroleptics, and cardioactive agents. Severe adverse effects or resistance to therapy may result.

Methods: We performed long PCR on the LightCycler™ and used the product as a template for a previously validated multiplex PCR that examines the *3, *4, *6, *7, and *8 alleles of Cyp2D6. We used real-time PCR to identify the *5 null allele and duplication of Cyp2D6 with detection by either hybridization probes or SYBR Green®. The *2 −1584 C/G polymorphism and the *35 allele were identified by PCR with detection by hybridization probes. Products of all PCRs were visualized with gel electrophoresis using a 0.7–1.5% agarose gel and ethidium bromide. Samples containing the *35 allele were analyzed in parallel by digestion with NlaIII, MslI, and BstXI and SmaI. We analyzed samples from volunteers and patients (105 samples for deletion and duplication and 116 samples for preamplification). Of those samples, 59 were from depressive inpatients taking part in a trial not yet published.

Results: Identical genotyping results for both real-time and conventional PCR were obtained and verified by gel electrophoresis. Use of long-PCR methods on the LightCycler enabled comprehensive analysis of all relevant polymorphisms of the Cyp2D6 gene in 1 working day with a hands-on time of ∼3–4 h.

Conclusions: This is the first description of a successful long-PCR application on the LightCycler and the fastest technique for amplification and specific detection of a PCR product of comparable length. The method appears suitable for large clinical and epidemiologic studies.
Rapid Detection of Flt3 Mutations in Acute Myeloid Leukemia Patients by Denaturing HPLC
Authors: Bianchini Michele , Ottaviani Emanuela , Grafone Tiziana , Giannini Barbara , Soverini Simona , Terragna Carolina , Amabile Marilina , Piccaluga Pier Paolo , Malagola Michele , Rondoni Michela , Bosi Costanza , Baccarani Michele , Martinelli Giovanni
Published: February 2004

Background: fms-related tyrosine kinase 3 (Flt3) is the most commonly mutated gene in human acute myeloid leukemia (AML) and has been implicated in its pathogenesis. Because screening of Flt3 in AML patients by PCR followed by gel electrophoresis is time-consuming and fails to detect some very small internal tandem duplications (ITDs), we developed a method for screening of FLT3 receptor mutations with PCR plus denaturing HPLC (D-HPLC).

Methods: Total mRNAs extracted from 34 AML patients were first analyzed for the presence of juxtamembrane length mutations and tyrosine kinase domain point mutations by a conventional method involving PCR amplification, restriction enzyme digestion, and agarose gel electrophoresis (PCR-RED-AGE). Subsequently, the same patient panel was analyzed by D-HPLC, using specifically designed primers and optimized running temperatures for the length and point mutation analysis.

Results: Thirty-four patients were analyzed by PCR-RED-AGE; 9 were positive for known Flt3 mutations: 6 of 34 (18%) for ITDs in exon 14 and 3 of 34 (9%) for point mutations in exon 20. The same patient panel was analyzed by D-HPLC, and additional nucleotide changes were discovered; in total, 14 sequence variations were identified: 7 of 34 (21%) for ITDs in exon 14; 2 of 34 (6%) for point mutations in exon 20; 1 of 34 (3%) for a new point mutation in exon 16; and 4 of 34 (12%) for polymorphisms in exons 13 and 14. Direct sequencing analysis identified nucleotide alterations in each of the “D-HPLC positives” but in none of the “D-HPLC negatives”, yielding a specificity and sensitivity of 100% for D-HPLC-based screening.

Conclusions: This novel D-HPLC-based procedure, which is optimized for identification of new point mutations in the catalytic and regulatory domains of FLT3 receptor, could potentially be useful for studies involving precise genotype determination, which could be critical for selection of innovative AML therapies targeting the FLT3 protein.
Blood Loss from Laboratory Tests
Authors: Wisser Dirk , van Ackern Klaus , Knoll Ernst , Wisser Hermann , Bertsch Thomas
Published: February 2004

Background: Laboratory tests can be an important source of blood loss in hospitals, especially for newborns and patients in intensive care. The aim of this study was to quantify blood loss for laboratory diagnostic tests in a large number of patients in a teaching hospital.

Methods: We estimated blood loss by multiplying the number and volumes of sampling tubes collected from 2654 adult inpatients. We compared the number of tests per patient for all inpatients and intensive care unit patients during the first period and again in the same time period 1 year later when cumulative blood-loss volumes were being reported to physicians and educational information had been given to decrease blood loss from laboratory tests.

Results: For 95% of the patients, blood loss during hospitalization was <196 mL. The largest proportion of the blood samples was used for clinical chemical tests (median, 45%), followed by hematologic (median, 26%) and coagulation (median, 17%) tests. In the surgical and cardiovascular surgical intensive care units, however, blood gas analyses accounted for 19–34% (medians) of the use. For 5% of the patients, all undergoing intensive care, blood loss was >200 mL and for 0.7% was >600 mL during their hospital stay. Such high blood losses were observed in patients with long-term ventilation, coagulation disorders, and repeated surgery. The largest median blood loss was in patients undergoing cardiovascular surgery (median, 201 mL). The mean number of tests was 44 per inpatient before cumulative blood loss was being reported and 46 when it was being reported.

Conclusions: Blood loss from laboratory diagnostic testing is not likely to pose a problem for most hospitalized patients. Blood loss is greater in intensive care patients and after cardiovascular surgical procedures. Reporting of the cumulative individual blood loss did not decrease blood loss for laboratory testing.
Justus Liebig and Animal Chemistry
Authors: Rosenfeld Louis
Published: February 2004

Justus Liebig was one of the individuals making chemistry almost a German monopoly in the 19th century. At Giessen he established the first organic chemistry laboratory and offered a systematic course for training new chemists. His comprehensive survey of plant nutrition changed the nature of scientific agriculture. In a study of animal chemistry, Liebig treated physiologic processes as chemical reactions and inferred the transformations from the chemical properties of the elements and compounds in laboratory reactions. He constructed hypothetical chemical equations derived from the formulae of the participating compounds. Liebig generalized that all organic nitrogenous constituents of the body are derived from plant protein and demonstrated how the application of quantitative methods of organic chemistry can be applied to the investigation of the animal organism. Liebig’s theories were attractive, but his method of converting one substance to another by moving atoms around on paper was speculative because of the lack of knowledge as to how the elements were arranged. His dynamic personality helped win widespread acceptance by many, but others were antagonized by his wishful thinking and speculative excesses. Liebig’s views on catalysis and fermentation brought him into a controversy with Louis Pasteur. Liebig’s Animal Chemistry stimulated an interest in clinical chemistry because it introduced a quantitative method into physiological chemistry. However, the isolated pieces of test results on blood and urine were unconnected and did not fit anywhere. Physicians found that chemistry was not helpful at the bedside and they lost interest in its application.
Familial Frontotemporal Dementia: From Gene Discovery to Clinical Molecular Diagnostics
Authors: Van Deerlin Vivianna M , Gill Lisa H , Farmer Jennifer M , Trojanowski John Q , Lee Virginia M-Y
Published: February 2004

Genetic testing is important for diagnosis and prediction of many diseases. The development of a clinical genetic test can be rapid for common disorders, but for rare genetic disorders this process can take years, if it occurs at all. We review the path from gene discovery to development of a clinical genetic test, using frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) as an example of a complex, rare genetic condition. An Institutional Review Board-approved multidisciplinary research program was developed to identify patients with familial frontotemporal dementia. Genetic counseling is provided and DNA obtained to identify mutations associated with FTDP-17. In some cases it may be appropriate for individuals to be given the opportunity to learn information from the research study to prevent unnecessary diagnostic studies or the utilization of inappropriate therapies, and to make predictive testing possible. Mutations identified in a research laboratory must be confirmed in a clinical laboratory to be used clinically. To facilitate the development of clinical genetic testing for a rare disorder, it is useful for a research laboratory to partner with a clinical laboratory. Most clinical molecular assays are developed in research laboratories and must be properly validated. We conclude that the transition of genetic testing for rare diseases from the research laboratory to the clinical laboratory requires a validation process that maintains the quality-control elements necessary for genetic testing but is flexible enough to permit testing to be developed for the benefit of patients and families.
Early Biomarkers of Stroke
Authors: Reynolds Mark A , Kirchick Howard J , Dahlen Jeffrey R , Anderberg Joseph M , McPherson Paul H , Nakamura Kevin K , Laskowitz Daniel T , Valkirs Gunars E , Buechler Kenneth F
Published: February 2004

Background: The diagnosis and management of acute ischemic stroke are limited by the lack of rapid diagnostic assays for use in an emergency setting. Computed tomography (CT) scanning is used to diagnose hemorrhagic stroke but is relatively ineffective (<33% sensitive) in detecting ischemic stroke. The ability to correlate blood-borne protein biomarkers with stroke phenotypes would aid in the development of such rapid tests.

Methods: ELISAs for >50 protein biomarkers were developed for use on a high-throughput robotic workstation. These assays were used to screen plasma samples from 214 healthy donors and 223 patients diagnosed with stroke, including 82 patients diagnosed with acute ischemic stroke. Marker assay values were first compared by univariate analysis, and then the top markers were subjected to multivariate analysis to derive a marker panel algorithm for the prediction of stroke.

Results: The top markers from this analysis were S-100b (a marker of astrocytic activation), B-type neurotrophic growth factor, von Willebrand factor, matrix metalloproteinase-9, and monocyte chemotactic protein-1. In a panel algorithm in which three or more marker values above their respective cutoffs were scored as positive, these five markers provided a sensitivity of 92% at 93% specificity for ischemic stroke samples taken within 6 h from symptom onset.

Conclusion: A marker panel approach to the diagnosis of stroke may provide a useful adjunct to CT scanning in the emergency setting.
Heterogeneity Analysis of the Human Pituitary Proteome
Authors: Zhan Xianquan , Desiderio Dominic M
Published: February 2004

Background: A human proteome is relatively dynamic compared with its corresponding genome. Our aim was to study the heterogeneity of a human pituitary proteome as a function of gender, age, and race.

Methods: Pituitary control tissues (n = 8) were used to extract proteins; each control tissue was analyzed (n = 3–5) with two-dimensional gel electrophoresis (2DGE) and PDQuest software. We obtained 30 high-resolution 2DGE gels and conducted a comparative analysis as a function of gender, age, and race. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography-electrospray ionization-quadrupole-ion trap tandem mass spectrometry were used to characterize the protein in each differential spot.

Results: We detected ∼1000 protein spots in each 2DGE map, and 51 differential spots (7 differing with gender, 17 with age, 15 with race, and 12 with the coeffect of age and race). Among those 51, we characterized 28 proteins [5 differing with gender, 8 with age, 6 with race, 8 with the coeffect of age and race, and 1 (somatotropin chain 1) with all of these]. Somatotropin was related to gender, age, and race, and prolactin was higher in females than males. The differentially expressed proteins that were related to age were mainly those proteins associated with cell growth, proliferation, differentiation, apoptosis, and death; those proteins showed no difference with gender and race. Age and race affected some proteins associated with hormone regulation (e.g., follistatin, thyroid hormone receptor β-2, adenylate cyclase-inhibiting Gα protein).

Conclusions: A heterogeneity exists in the human pituitary proteome as a function of gender, age, and race. These findings will serve as a basis for our comparative proteomics studies of human pituitary adenomas.
Blood Test Detecting Autoantibodies to N-Methyl-d-aspartate Neuroreceptors for Evaluation of Patients with Transient Ischemic Attack and Stroke
Authors: Dambinova Svetlana A , Khounteev Guerman A , Izykenova Galina A , Zavolokov Igor G , Ilyukhina Anna Y , Skoromets Alexander A
Published: February 2004

Background: Stroke is a multisystemic disorder that includes mechanisms of thrombosis and neurotoxic coupling. Key metabolites of the molecular cascade following biochemical events appear simultaneously in brain tissue, the blood–brain barrier, and brain vessels, activating the immune system and generating autoantibodies (aAbs) to brain-specific antigens. We developed an ELISA blood test to measure aAbs to a subtype of N-methyl-d-aspartate (NMDA) receptors, which are the key markers of neurotoxicity underlying cerebral ischemia. We investigated the diagnostic accuracy of serum aAbs to NR2A/2B, a subtype of NMDA receptors, in assessing transient ischemic attack (TIA) and ischemic stroke (IS) and its ability to distinguish cerebral ischemia from intracerebral hemorrhage (ICH).

Methods: Autoantibodies to NR2A/2B were measured in 360 serum samples: 105 from TIA/stroke patients and 255 from controls, including patients with controlled hypertension/atherosclerosis and gender- and age-matched healthy individuals.

Results: Patients with TIA (n = 56) and acute IS (n = 31) had significantly higher NR2A/2B aAb concentrations than controls (P <0.0001). The test sensitivities for TIA and IS were 95% and 97%, respectively, and predictive values were 86% and 91% at a cutoff point of 2.0 μg/L. The area under the ROC curve was 0.99. Monitoring NR2A/2B aAbs within 72 h differentiated IS and ICH (P <0.001) and was confirmed by magnetic resonance imaging and computed tomography.

Conclusions: NR2A/2B aAbs are independent and sensitive serologic markers capable of detecting TIA with a high posttest probability and, in conjunction with neurologic observation and neuroimaging, ruling out ICH. The test may help assess risk of TIA in routine general practice and may potentially be useful in assisting diagnosis of acute IS in the emergency setting.
Detecting Subsecond Dopamine Release with Fast-Scan Cyclic Voltammetry in Vivo
Authors: Robinson Donita L , Venton B Jill , Heien Michael L A V , Wightman R Mark
Published: February 2004

Background: Dopamine is a potent neuromodulator in the brain, influencing a variety of motivated behaviors and involved in several neurologic diseases. Measurements of extracellular dopamine in the brains of experimental animals have traditionally focused on a tonic timescale (minutes to hours). However, dopamine concentrations are now known to fluctuate on a phasic timescale (subseconds to seconds).

Approach: Fast-scan cyclic voltammetry provides analytical chemical measurements of phasic dopamine signals in the rat brain.

Content: Procedural aspects of the technique are discussed, with regard to appropriate use and in comparison with other methods. Finally, examples of data collected using fast-scan cyclic voltammetry are summarized, including naturally occurring dopamine transients and signals arising from electrical stimulation of dopamine neurons.

Summary: Fast-scan cyclic voltammetry offers real-time measurements of changes in extracellular dopamine concentrations in vivo. With its subsecond time resolution, micrometer-dimension spatial resolution, and chemical selectivity, it is the most suitable technique currently available to measure transient concentration changes of dopamine.
Multiplex Minisequencing Screen for Common Southeast Asian and Indian β-Thalassemia Mutations
Authors: Wang Wen , Kham Shirley K Y , Yeo Gare-Hoon , Quah Thuan-Chong , Chong Samuel S
Published: February 2004

Background: β-Thalassemia is endemic to many regions in Southeast Asia and India, and <20 β-globin gene mutations account for ≥90% of β-thalassemia alleles in these places. We describe a multiplex minisequencing assay to detect these common mutations.

Methods: Gap-PCR was used to simultaneously amplify the β-globin gene from genomic DNA and to detect the Δ619bp deletion mutation. Multiplex minisequencing was then performed on the amplified β-globin fragment to detect an additional 15 common Southeast Asian and Indian β-thalassemia mutations. Site-specific primers of different lengths were subjected to multiple rounds of annealing and single-nucleotide extension in the presence of thermostable DNA polymerase and the four dideoxynucleotides, each labeled with a different fluorophore. Minisequencing products were separated and detected by capillary electrophoresis, followed by automated genotyping. The optimized assay was subjected to a double-blind validation analysis of 89 β-thalassemia and wild-type DNA samples of known genotype.

Results: Homozygous wild-type or mutant DNA samples produced electropherograms containing only a single colored peak for each mutation site, whereas samples heterozygous for a specific mutation displayed two different-colored peaks for that mutation site. Samples were automatically genotyped based on color and position of primer peaks in the electropherogram. In the double-blind validation analysis, all 89 DNA samples were genotyped correctly (100% assay specificity).

Conclusions: The described semiautomated multiplex minisequencing assay can detect the most common Southeast Asian and Indian β-thalassemia mutations, is amenable to high-throughput scale up, and may bring population-based screening of β-thalassemia in endemic regions a step closer to implementation.
HER-2/neu Gene Copy Number Quantified by Real-Time PCR: Comparison of Gene Amplification, Heterozygosity, and Immunohistochemical Status in Breast Cancer Tissue
Authors: Königshoff Melanie , Wilhelm Jochen , Bohle Rainer M , Pingoud Alfred , Hahn Meinhard
Published: February 2004

Background: Amplification of the oncogene HER-2/neu influences breast cancer pathogenesis, and therapy and prognosis may be affected by the degree of amplification. The extent of amplification or protein overexpression typically is analyzed by fluorescence in situ hybridization or immunohistochemistry (IHC), but quantitative PCR techniques have been described that may provide alternatives to these methods.

Methods: We developed a rapid-cycle, real-time PCR assay for quantification of HER-2/neu gene status. We compared results obtained with this assay with short tandem repeat findings by capillary electrophoresis (CE) and with protein overexpression assessments by IHC. Accuracy and linearity were tested on cell lines and with simulation experiments. We analyzed the amplification of HER-2/neu in 51 clinical tissue samples from patients with suspected breast cancer.

Results: The intra- and interrun CVs for HER-2/neu quantification by real-time PCR were 12% and 18%, and the CV for different simulated amplification and deletion experiments was <7%. The results for HER-2/neu gene status in cell lines matched the values reported in literature. We detected HER-2/neu amplification by real-time PCR in 11 samples, all from patients with invasive ductal carcinoma. Allelic imbalances were found by CE analyses in three samples and by protein overexpression in six samples; five of these were also detected by real-time PCR. Comparison of the quantification results with known prognostic indices yielded results similar to those reported in several other published studies.

Conclusions: The assay is suitable for accurate and precise quantification of HER-2/neu copy numbers in tumor tissue samples obtained in routine clinical practice.
Real-Time Reverse Transcription-PCR Expression Profiling of the Complete Human ATP-Binding Cassette Transporter Superfamily in Various Tissues
Authors: Langmann Thomas , Mauerer Richard , Zahn Alexandra , Moehle Christoph , Probst Mario , Stremmel Wolfgang , Schmitz Gerd
Published: February 2004

Background: ATP-binding cassette (ABC) transporters are involved in many physiologic processes, such as lipid transport, sterol homeostasis, immune mechanisms, and drug transport, and cause various human inherited diseases. Thus, the analysis of ABC transporter mRNA expression profiles for basic research, especially in the field of lipid metabolism, for clinical diagnosis, and for monitoring of drug effects is of great interest.

Methods: We have developed a rapid, accurate, and highly sensitive real-time reverse transcription-PCR (RT-PCR) method for detection and quantification of all 47 currently known members of the ABC transporter superfamily. Our expression analysis is based on relative quantification using a calibration curve method. With our assay, expression monitoring of a large number of RNA samples in a 384-well format with only 50 ng of total RNA is possible.

Results: In contrast to previous expression analyses of single ABC genes, our method allows the rapid and complete analysis of all ABC transporters in given RNA samples. We used our newly established expression panel to study the gene expression of all human ABC transporters in 20 different human tissues. As a result, we identified tissues with high transcriptional activity for ABC transporters. These organs are mainly involved in secretory function (adrenal gland), metabolic function (liver), barrier function (lung, trachea, small intestine), and tropic function (placenta, uterus).

Conclusions: Our RT-PCR assay allows rapid, high-throughput transcriptional profiling of the complete ABC transporter superfamily and thus provides a new enabling tool for research, clinical diagnosis of disease, and drug testing and development.
Minor Increases in Plasma Troponin I Predict Decreased Left Ventricular Ejection Fraction after High-Dose Chemotherapy
Authors: Sandri Maria Teresa , Cardinale Daniela , Zorzino Laura , Passerini Rita , Lentati Paola , Martinoni Alessandro , Martinelli Giovanni , Cipolla Carlo Maria
Published: February 2004

Background: Increased cardiac troponin I (cTnI) in patients treated with high-dose chemotherapy (HDCT) for aggressive malignancy has been proposed as an early marker of late HDCT-induced cardiac dysfunction. We investigated whether cTnI measured by the Stratus CS (Dade Behring) would allow detection of minimal cTnI increases in patients treated with HDCT.

Methods: Plasma cTnI concentrations were determined in 179 consecutive patients before HDCT, at the end of the treatment, and after 12, 24, 36, and 72 h. Cardiac function was explored by echocardiography, and left ventricular ejection fraction (LVEF) was recorded during follow-up. The greatest variation in LVEF from the baseline value was used as a measure of cardiac damage.

Results: In 99 healthy volunteers, the 99th percentile was at 0.07 μg/L. On the basis of ROC curve analysis (area under the curve, 0.89), a cutoff of 0.08 μg/L was chosen (sensitivity, 82%; specificity, 77%). cTnI ≥0.08 μg/L occurred in 57 patients (32%) with echocardiographic monitoring revealing a mean decrease in LVEF of 18%. In comparison, the group of cTnI-negative patients had a mean decrease in LVEF of 2.5% (P <0.001).

Conclusions: Plasma cTnI, as measured with the Stratus CS, can detect minor myocardial injury in patients treated with HDCT.
Differences in Nucleotide Hydrolysis Contribute to the Differences between Erythrocyte 6-Thioguanine Nucleotide Concentrations Determined by Two Widely Used Methods
Authors: Shipkova Maria , Armstrong Victor William , Wieland Eberhard , Oellerich Michael
Published: February 2004

Background: Measurement of 6-thioguanine nucleotide (6-TGN) concentrations in erythrocytes is widely accepted for use in optimization of thiopurine therapy. Various chromatographic methods have been developed for this purpose. In preliminary experiments we observed a considerable difference between 6-TGN concentrations determined with two widely used methods published by Lennard (Lennard L. J Chromatogr 1987;423:169–78) and by Dervieux and Boulieu (Dervieux T, Boulieu R. Clin Chem 1998;44:551–5). We therefore investigated methodologic differences between the two procedures with respect to hydrolysis of 6-TGNs to 6-thioguanine (6-TG) in more detail.

Methods: We analyzed 6-TGNs in erythrocyte preparations (n = 50) from patients on azathioprine therapy by both methods, using the original protocols. In one set of experiments, we replaced the 0.5 mol/L sulfuric acid in the Lennard method with the 1 mol/L perchloric acid used by Dervieux and Boulieu. In a second set of experiments, we investigated the effect of various dithiothreitol (DTT) concentrations on 6-TG recovery with both methods. In a third set of experiments, we determined the effect of hydrolysis time on both protocols.

Results: Direct comparison of both methods showed that 6-TGN concentrations were, on average, 2.6-fold higher in the Dervieux–Boulieu method over the concentration range tested, although the correlation (r = 0.99; P <0.001) was good. Replacement of sulfuric acid by perchloric acid reduced this difference to ∼1.4-fold (r = 0.99; P <0.001). Increasing the DTT concentration enhanced 6-TG recovery. The hydrolysis time used in the Lennard method (1 h) was not sufficient to achieve complete hydrolysis.

Conclusions: The difference between 6-TGN concentrations measured by the two methods is attributable, at least in part, to differences in the extent of nucleotide hydrolysis. For optimization of thiopurine therapy, method-dependent therapeutic ranges are necessary, which precludes comparison of results from clinical studies derived with these methods. Efforts must therefore be made to standardize the analytical procedures for the determination of 6-TGN.
Determination of Choline, Betaine, and Dimethylglycine in Plasma by a High-Throughput Method Based on Normal-Phase Chromatography–Tandem Mass Spectrometry
Authors: Holm Pål I , Ueland Per Magne , Kvalheim Gry , Lien Ernst A
Published: February 2004

Background: The quaternary ammonium compounds, choline and betaine, and dimethylglycine (DMG) reside along a metabolic pathway linked to the synthesis of neurotransmitters and membrane phospholipids and to homocysteine remethylation and, therefore, folate status. Lack of a convenient, high-throughput method for the determination of these compounds has prevented population-based studies of their possible associations with lifestyle, nutrition, and chronic diseases.

Methods: Serum or plasma samples were deproteinized by mixing with three volumes of acetonitrile that contained d9-choline and d9-betaine as internal standards. We used a normal-phase silica column for the separation of choline (retention time, 2.8 min), betaine (1.3 min), DMG (1.15 min), and internal standards, which were detected as positive ions by tandem mass spectroscopy in the multiple-reaction monitoring mode, using the molecular transitions m/z 104→60 (choline), m/z 113→69 (d9-choline), m/z 118→59 (betaine), m/z 127→68 (d9-betaine), and m/z 104→58 (DMG).

Results: For all three metabolites, the assay was linear in the range 0.4–400 μmol/L, and the lower limit of the detection (signal-to-noise ratio = 5) was ≤0.3 μmol/L. The within- and between-day imprecision (CVs) was 2.1–7.2% and 3.5–8.8%, respectively. The analytical recovery was 87–105%. The fasting plasma concentrations (median, 25th–75th percentiles) were 8.0 (7.0–9.3) μmol/L for choline, 31.7 (27.0–41.1) μmol/L for betaine, and 1.66 (1.30–2.02) μmol/L for DMG in 60 healthy blood donors. In individuals who had eaten a light breakfast, plasma concentrations of all three metabolites were significantly (25–30%) higher than in fasting individuals.

Conclusion: This is the first method for the combined measurement of choline, betaine, and DMG in human plasma or serum. The assay is characterized by simple sample preparation, no derivatization, high throughput, imprecision (CV) <10%, detection limits below the values seen in volunteers, and the high specificity provided by tandem mass spectroscopy.
Effect of Riboflavin Status on the Homocysteine-lowering Effect of Folate in Relation to the MTHFR (C677T) Genotype
Authors: Moat Stuart J , Ashfield-Watt Pauline A L , Powers Hilary J , Newcombe Robert G , McDowell Ian F W
Published: February 2004

Background: Riboflavin (vitamin B2) is the precursor for FAD, the cofactor for methylenetetrahydrofolate reductase (MTHFR). MTHFR catalyzes the formation of 5-methyltetrahydrofolate, which acts as a methyl donor for homocysteine remethylation. Individuals with the MTHFR 677C→T mutation have increased plasma total homocysteine (tHcy) concentrations, particularly in association with low folate status. It has been proposed that riboflavin may act together with folate to lower plasma tHcy, particularly in individuals with the thermolabile MTHFR T variant.

Methods: We measured B-vitamin status and plasma tHcy in 126 healthy individuals 20–63 years of age (42 CC, 42 CT, and 42 TT MTHFR genotypes) at baseline and after three interventions (4 months): placebo plus natural diet; daily 400 μg folic acid supplement plus natural diet; and increased dietary folate to 400 μg/day.

Results: At baseline and after nutritional intervention, lower riboflavin status was associated with increased plasma tHcy concentrations. Plasma tHcy was 2.6 μmol/L higher in the lowest plasma riboflavin quartile compared with the highest (P <0.02) and was 4.2 μmol/L higher in the highest erythrocyte glutathione reductase activation coefficient (EGRAC) quartile compared with the lowest (P <0.001). This effect was not restricted to those with the T allele. Folic acid given as a 400 μg/day supplement appeared to exacerbate a tendency toward riboflavin deficiency, as suggested by an increase in the proportion of individuals with EGRAC ≥1.4 from 52% to 65% after supplementation (P <0.05).

Conclusions: Folate and riboflavin interact to lower plasma tHcy, possibly by maximizing the catalytic activity of MTHFR. The effect may be unrelated to MTHFR genotype.
National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines: Recommendations for the Use of Laboratory Tests to Support Poisoned Patients Who Present to the Emergency Department
Authors: Wu Alan H B , McKay Charles , Broussard Larry A , Hoffman Robert S , Kwong Tai C , Moyer Thomas P , Otten Edward M , Welch Shirley L , Wax Paul
Published: February 2004

Background: Exposure to drugs and toxins is a major cause for patients’ visits to the emergency department (ED).

Methods: Recommendations for the use of clinical laboratory tests were prepared by an expert panel of analytical toxicologists and ED physicians specializing in clinical toxicology. These recommendations were posted on the world wide web and presented in open forum at several clinical chemistry and clinical toxicology meetings.

Results: A menu of important stat serum and urine toxicology tests was prepared for clinical laboratories who provide clinical toxicology services. For drugs-of-abuse intoxication, most ED physicians do not rely on results of urine drug testing for emergent management decisions. This is in part because immunoassays, although rapid, have limitations in sensitivity and specificity and chromatographic assays, which are more definitive, are more labor-intensive. Ethyl alcohol is widely tested in the ED, and breath testing is a convenient procedure. Determinations made within the ED, however, require oversight by the clinical laboratory. Testing for toxic alcohols is needed, but rapid commercial assays are not available. The laboratory must provide stat assays for acetaminophen, salicylates, co-oximetry, cholinesterase, iron, and some therapeutic drugs, such as lithium and digoxin. Exposure to other heavy metals requires laboratory support for specimen collection but not for emergent testing.

Conclusions:Improvements are needed for immunoassays, particularly for amphetamines, benzodiazepines, opioids, and tricyclic antidepressants. Assays for new drugs of abuse must also be developed to meet changing abuse patterns. As no clinical laboratory can provide services to meet all needs, the National Academy of Clinical Biochemistry Committee recommends establishment of regional centers for specialized toxicology testing.
Anomalous Structure of Urinary Glycosaminoglycans in Patients with Pseudoxanthoma Elasticum
Authors: Maccari Francesca , Gheduzzi Dealba , Volpi Nicola
Published: February 2004

Background: Pseudoxanthoma elasticum (PXE) is a hereditary connective tissue disease in which proteoglycans have altered properties. We investigated whether altered proteoglycan metabolism occurs in vivo and may be reflected in the urine of PXE individuals by analyzing the excreted polysaccharides.

Methods: We measured sulfated glycosaminoglycans in the urine of 10 PXE-affected patients, 12 healthy carriers, and 20 healthy controls by agarose gel electrophoresis. Chondroitin sulfate and heparan sulfate disaccharides were also quantified by treatment with specific lyases and separation of products by chromatography.

Results: Total polysaccharides were 34% lower in the urine of PXE-affected patients and 17% lower in healthy carriers than in the control group. Chondroitin sulfate was significantly (P <0.01) decreased, and heparan sulfate was significantly increased. The ratio of chondroitin sulfate to heparan sulfate was 2.7 for PXE-affected patients, 2.3 for healthy carriers, and 10.7 for controls. In PXE-affected individuals and carriers, chondroitin sulfate contained more 4-sulfated disaccharide, less 6-sulfated disaccharide, and decreased nonsulfated disaccharide. Heparan sulfate from PXE-affected individuals and healthy carriers produced significantly less N-sulfated disaccharide and more disaccharide sulfated at the C-6 position with no significant abnormality of the nonsulfated disaccharide percentage and sulfates:disaccharide ratio.

Conclusions: The urinary data support the concept that the inherited defect of the ABCC6/MRP6 transporter in PXE alters metabolism of key polysaccharides. Structural analysis of urinary sulfated polyanions may be useful in the diagnosis of PXE.
Amplicon Melting Analysis with Labeled Primers: A Closed-Tube Method for Differentiating Homozygotes and Heterozygotes
Authors: Gundry Cameron N , Vandersteen Joshua G , Reed Gudrun H , Pryor Robert J , Chen Jian , Wittwer Carl T
Published: February 2004

Background: Common methods for identification of DNA sequence variants use gel electrophoresis or column separation after PCR.

Methods: We developed a method for sequence variant analysis requiring only PCR and amplicon melting analysis. One of the PCR primers was fluorescently labeled. After PCR, the melting transition of the amplicon was monitored by high-resolution melting analysis. Different homozygotes were distinguished by amplicon melting temperature (Tm). Heterozygotes were identified by low-temperature melting of heteroduplexes, which broadened the overall melting transition. In both cases, melting analysis required ∼1 min and no sample processing was needed after PCR.

Results: Polymorphisms in the HTR2A (T102C), β-globin [hemoglobin (Hb) S, C, and E], and cystic fibrosis (F508del, F508C, I507del, I506V) genes were analyzed. Heteroduplexes produced by amplification of heterozygous DNA were best detected by rapid cooling (>2 °C/s) of denatured products, followed by rapid heating during melting analysis (0.2–0.4 °C/s). Heterozygotes were distinguished from homozygotes by a broader melting transition, and each heterozygote had a uniquely shaped fluorescent melting curve. All homozygotes tested were distinguished from each other, including Hb AA and Hb SS, which differed in Tm by <0.2 °C. The amplicons varied in length from 44 to 304 bp. In place of one labeled and one unlabeled primer, a generic fluorescent oligonucleotide could be used if a 5′ tail of identical sequence was added to one of the two unlabeled primers.

Conclusion: High-resolution melting analysis of PCR products amplified with labeled primers can identify both heterozygous and homozygous sequence variants.
Exploiting the Enzymatic Recognition of an Unnatural Base Pair to Develop a Universal Genetic Analysis System
Authors: Moser Michael J , Marshall David J , Grenier Jennifer K , Kieffer Collin D , Killeen Anthony A , Ptacin Jerod L , Richmond Craig S , Roesch Eric B , Scherrer Christopher W , Sherrill Christopher B , Van Hout Cris V , Zanton Sara J , Prudent James R
Published: February 2004

Background: With the invention of the DNA chip, genome-wide analysis is now a reality. Unfortunately, solid-phase detection systems such as the DNA chip suffer from a narrow range in quantification and sensitivity. Today the best methodology for sensitive, wide dynamic range quantification and genotyping of nucleic acids is real-time PCR. However, multiplexed real-time PCR technologies require complicated and costly design and manufacturing of separate detection probes for each new target.

Methods: We developed a novel real-time PCR technology that uses universal energy transfer probes constructed from An Expanded Genetic Information System (AEGIS) for both quantification and genotyping analyses.

Results: RNA quantification by reverse transcription-PCR was linear over four orders of magnitude for the simultaneous analysis of β-actin messenger RNA and 18S ribosomal RNA. A single trial validation study of 176 previously genotyped clinical specimens was performed by endpoint analysis for factor V Leiden and prothrombin 20210A mutation detection. There was concordance for 173 samples between the genotyping results from Invader® tests and the AEGIS universal energy transfer probe system for both factor V Leiden and prothrombin G20210A. Two prothrombin and one factor V sample gave indeterminate results (no calls).

Conclusion: The AEGIS universal probe system allows for rapid development of PCR assays for nucleic acid quantification and genotyping.
Application of Optical Trapping for Cells Grown on Plates: Optimization of PCR and Fidelity of DNA Sequencing of p53 Gene from a Single Cell
Authors: Gale James M , Romero Christopher P , Tafoya Gregory B , Conia Jérôme
Published: February 2004

Background: Optical trapping has traditionally been used to visually select and isolate nonadherent cells grown in suspension because cells grown in monolayers will rapidly reattach to surfaces if suspended in solution. We explored methods to slow cell reattachment that are also compatible with high-fidelity PCR.

Methods: Using HeLa cells grown on plates and suspended after trypsinization, we measured the efficiency of capture by retention and movement of the cell by the laser. Success for removing a captured cell by pipette was determined by PCR amplification of the 5S rRNA gene. After optimizing PCR amplification of a 2049-bp region of the p53 gene, we determined PCR fidelity by DNA sequencing.

Results: Addition of bovine serum albumin to suspended cells slowed reattachment from seconds to minutes and allowed efficient trapping. The success rate of removing a cell from the trap by pipette to a PCR tube was 91.5%. The 5S PCR assay also revealed that DNA and RNA that copurify with polymerases could give false-positive results. Sequence analysis of four clones derived from a single cell showed only three polymerase errors in 7200 bp of sequence read and revealed difficulties in reading the correct number in a run of 16 A:T. Comparison of the HeLa and wild-type human sequences revealed several previously unreported base differences and an (A:T)n length polymorphism in p53 introns.

Conclusions: These results represent the first use of optical trapping on adherent cells and demonstrate the high accuracy of DNA sequencing that can be achieved from a single cell.
PCR-based Calibration Curves for Studies of Quantitative Gene Expression in Human Monocytes: Development and Evaluation
Authors: Øvstebø Reidun , Haug Kari Bente Foss , Lande Knut , Kierulf Peter
Published: February 2004

Background: Quantitative reverse transcription-PCR (RT-PCR) used to detect small changes in specific mRNA concentrations is often associated with poor reproducibility. Thus, there is a need for stringent quality control in each step of the protocol.

Methods: Real-time PCR-based calibration curves for a target gene, tissue factor (TF), and a reference gene, β-actin, generated from PCR amplicons were evaluated by running cDNA controls. In addition, the reverse transcription step was evaluated by running mRNA controls. Amplification efficiencies of calibrators and targets were determined. Variances within and between runs were estimated, and power statistics were applied to determine the concentration differences that could reliably be detected.

Results: Within- and between-run variations (CVs) of cDNA controls (TF and β-actin), extrapolated from reproducible calibration curves (CVs of slopes, 4.3% and 2.7%, respectively) were 4–10% (within) and 15–38% (between) using both daily and “grand mean” calibration curves. CVs for the β-actin mRNA controls were 12% (within) and 19–28% (between). Estimates of each step’s contribution to the total variation were as follows: CVRT-PCR, 28%; CVPCR, 15%; CVRT, 23% (difference between CVRT-PCR and CVPCR). PCR efficiencies were as follows: β-actin calibrator/target, 1.96/1.95; TF calibrator/target, 1.95/1.93. Duplicate measurements could detect a twofold concentration difference (power, 0.8).

Conclusions: Daily PCR calibration curves generated from PCR amplicons were reproducible, allowing the use of a grand mean calibration curve. The reverse transcription step contributes the most to the total variation. By determining a system’s total variance, power analysis may be used to disclose differences that can be reliably detected at a specified power.