Showing papers in "Clinical Chemistry in 2003"

Ion Suppression in Mass Spectrometry

Abstract: Background: Mass spectrometry (MS) is being introduced into a large number of clinical laboratories. It provides specificity because of its ability to monitor selected mass ions, sensitivity because of the enhanced signal-to-noise ratio, and speed because it can help avoid the need for intensive sample cleanup and long analysis times. However, MS is not without problems related to interference, especially through ion suppression effects. Ion suppression results from the presence of less volatile compounds that can change the efficiency of droplet formation or droplet evaporation, which in turn affects the amount of charged ion in the gas phase that ultimately reaches the detector. Content: This review discusses materials shown to cause ion suppression, including salts, ion-pairing agents, endogenous compounds, drugs, metabolites, and proteins. Experimental protocols for examining ion suppression, which should include, at a minimum, signal recovery studies using specimen extracts with added analyte, are also discussed, and a more comprehensive approach is presented that uses postcolumn infusion of the analyte to evaluate protracted ionization effects. Finally, this review presents options for minimizing or correcting ion suppression, which include enhanced specimen cleanup, chromatographic changes, reagent modifications, and effective internal standardization. Summary: Whenever mass spectrometric assays are developed, ion suppression studies should be performed using expected physiologic concentrations of the analyte under investigation.

The STARD Statement for Reporting Studies of Diagnostic Accuracy: Explanation and Elaboration

Abstract: The quality of reporting of studies of diagnostic accuracy is less than optimal. Complete and accurate reporting is necessary to enable readers to assess the potential for bias in the study and to evaluate the generalisability of the results. A group of scientists and editors has developed the STARD (Standards for Reporting of Diagnostic Accuracy) statement to improve the reporting the quality of reporting of studies of diagnostic accuracy. The statement consists of a checklist of 25 items and flow diagram that authors can use to ensure that all relevant information is present. This explanatory document aims to facilitate the use, understanding and dissemination of the checklist. The document contains a clarification of the meaning, rationale and optimal use of each item on the checklist, as well as a short summary of the available evidence on bias and applicability. The STARD statement, checklist, flowchart and this explanation and elaboration document should be useful resources to improve reporting of diagnostic accuracy studies. Complete and informative reporting can only lead to better decisions in healthcare.

High-Resolution Genotyping by Amplicon Melting Analysis Using LCGreen

Abstract: 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.

Angiogenesis assays: a critical overview.

Abstract: Background: Angiogenesis, the formation of new blood vessels, is an integral part of both normal developmental processes and numerous pathologies, ranging from tumor growth and metastasis to inflammation and ocular disease. Angiogenesis assays are used to test efficacy of both pro- and antiangiogenic agents. Methods: Most studies of angiogenesis inducers and inhibitors rely on various models, both in vitro and in vivo, as indicators of efficacy. In this report we describe the principal methods now in use: the in vivo Matrigel plug and corneal neovascularization assays, the in vivo/in vitro chick chorioallantoic membrane (CAM) assay, and the in vitro cellular (proliferation, migration, tube formation) and organotypic (aortic ring) assays. We include description of two new methods, the chick aortic arch and the Matrigel sponge assays. Conclusions: In vitro tests are valuable, can be carried out expeditiously, and lend themselves to quantification, but must be interpreted with extreme caution. In vitro tests are best viewed as providing initial information, subject to confirmation by in vivo assays. Multiple tests should be used to obtain maximum benefit from in vitro tests. In vivo tests are more difficult and time-consuming to perform, thereby limiting the number of tests that can run at any one time. Quantification is generally more difficult as well. However, in vivo assays are essential because of the complex nature of vascular responses to test reagents, responses that no in vitro model can fully achieve.

Towards Complete and Accurate Reporting of Studies of Diagnostic Accuracy: The STARD Initiative

Abstract: Background: To comprehend the results of diagnostic accuracy studies, readers must understand the design, conduct, analysis, and results of such studies. That goal can be achieved only through complete transparency from authors. Objective: To improve the accuracy and completeness of reporting of studies of diagnostic accuracy to allow readers to assess the potential for bias in the study and to evaluate its generalisability. Methods: The Standards for Reporting of Diagnostic Accuracy (STARD) steering committee searched the literature to identify publications on the appropriate conduct and reporting of diagnostic studies and extracted potential items into an extensive list. Researchers, editors, and members of professional organisations shortened this list during a two-day consensus meeting with the goal of developing a checklist and a generic flow diagram for studies of diagnostic accuracy. Results: The search for published guidelines on diagnostic research yielded 33 previously published checklists, from which we extracted a list of 75 potential items. The consensus meeting shortened the list to 25 items, using evidence on bias whenever available. A prototypical flow diagram provides information about the method of patient recruitment, the order of test execution and the numbers of patients undergoing the test under evaluation, the reference standard or both. Conclusions: Evaluation of research depends on complete and accurate reporting. If medical journals adopt the checklist and the flow diagram, the quality of reporting of studies of diagnostic accuracy should improve to the advantage of clinicians, researchers, reviewers, journals, and the public.

Testosterone measured by 10 immunoassays and by isotope-dilution gas chromatography-mass spectrometry in sera from 116 men, women, and children.

Abstract: Background: Commercially available testosterone immunoassays give divergent results, especially at the low concentrations seen in women. We compared immunoassays and a nonimmunochemical method that could quantify low testosterone concentrations. Methods: We measured serum testosterone in 50 men, 55 women, and 11 children with use of eight nonisotopic immunoassays, two isotopic immunoassays, and isotope-dilution gas chromatography–mass spectrometry (ID/GC-MS). Results: Compared with ID/GC-MS, 7 of the 10 immunoassays tested overestimated testosterone concentrations in samples from women; mean immunoassay results were 46% above those obtained by ID/GC-MS. The immunoassays underestimated testosterone concentrations in samples from men, giving mean results 12% below those obtained by ID/GC-MS. In women, at concentrations of 0.6–7.2 nmol/L, 3 of the 10 immunoassays gave positive mean differences >2.0 nmol/L (range, −0.7 to 3.3 nmol/L) compared with ID/GC-MS; in men at concentrations of 8.2–58 nmol/L, 3 of the 10 immunoassays tested gave mean differences >4.0 nmol/L (range, −4.8 to 2.6 nmol/L). Conclusion: None of the immunoassays tested was sufficiently reliable for the investigation of sera from children and women, in whom very low (0.17 nmol/L) and low (<1.7 nmol/L) testosterone concentrations are expected.

Use of Tandem Mass Spectrometry for Multianalyte Screening of Dried Blood Specimens from Newborns

Abstract: Background: Over the past decade laboratories that test for metabolic disorders have introduced tandem mass spectrometry (MS/MS), which is more sensitive, specific, reliable, and comprehensive than traditional assays, into their newborn-screening programs. MS/MS is rapidly replacing these one-analysis, one-metabolite, one-disease classic screening techniques with a one-analysis, many-metabolites, many-diseases approach that also facilitates the ability to add new disorders to existing newborn-screening panels. Methods: During the past few years experts have authored many valuable articles describing various approaches to newborn metabolic screening by MS/MS. We attempted to document key developments in the introduction and validation of MS/MS screening for metabolic disorders. Our approach used the perspective of the metabolite and which diseases may be present from its detection rather than a more traditional approach of describing a disease and noting which metabolites are increased when it is present. Content: This review cites important historical developments in the introduction and validation of MS/MS screening for metabolic disorders. It also offers a basic technical understanding of MS/MS as it is applied to multianalyte metabolic screening and explains why MS/MS is well suited for analysis of amino acids and acylcarnitines in dried filter-paper blood specimens. It also describes amino acids and acylcarnitines as they are detected and measured by MS/MS and their significance to the identification of specific amino acid, fatty acid, and organic acid disorders. Conclusions: Multianalyte technologies such as MS/MS are suitable for newborn screening and other mass screening programs because they improve the detection of many diseases in the current screening panel while enabling expansion to disorders that are now recognized as important and need to be identified in pediatric medicine.

Detecting subsecond dopamine release with fast-scan cyclic voltammetry in vivo.

Abstract: 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. © 2003 American Association for Clinical Chemistry

Iron Deficiency and Erythropoiesis: New Diagnostic Approaches

Abstract: 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.

LOINC, a Universal Standard for Identifying Laboratory Observations: A 5-Year Update

Abstract: The Logical Observation Identifier Names and Codes (LOINC) database provides a universal code system for reporting laboratory and other clinical observations. Its purpose is to identify observations in electronic messages such as Health Level Seven (HL7) observation messages, so that when hospitals, health maintenance organizations, pharmaceutical manufacturers, researchers, and public health departments receive such messages from multiple sources, they can automatically file the results in the right slots of their medical records, research, and/or public health systems. For each observation, the database includes a code (of which 25 000 are laboratory test observations), a long formal name, a "short" 30-character name, and synonyms. The database comes with a mapping program called Regenstrief LOINC Mapping Assistant (RELMA(TM)) to assist the mapping of local test codes to LOINC codes and to facilitate browsing of the LOINC results. Both LOINC and RELMA are available at no cost from http://www.regenstrief.org/loinc/. The LOINC medical database carries records for >30 000 different observations. LOINC codes are being used by large reference laboratories and federal agencies, e.g., the CDC and the Department of Veterans Affairs, and are part of the Health Insurance Portability and Accountability Act (HIPAA) attachment proposal. Internationally, they have been adopted in Switzerland, Hong Kong, Australia, and Canada, and by the German national standards organization, the Deutsches Instituts fur Normung. Laboratories should include LOINC codes in their outbound HL7 messages so that clinical and research clients can easily integrate these results into their clinical and research repositories. Laboratories should also encourage instrument vendors to deliver LOINC codes in their instrument outputs and demand LOINC codes in HL7 messages they get from reference laboratories to avoid the need to lump so many referral tests under the "send out lab" code.

γ-Glutamyltransferase Is a Predictor of Incident Diabetes and Hypertension: The Coronary Artery Risk Development in Young Adults (CARDIA) Study

Abstract: Background: γ-Glutamyltransferase (GGT), which maintains cellular concentrations of glutathione, may be a marker of oxidative stress, and GGT itself may produce oxidative stress. We performed a prospective study to examine whether serum GGT predicts diabetes and hypertension. Methods: Study participants were 4844 black and white men and women 18–30 years of age in 1985–1986; they were reexamined 2, 5, 7, 10, and 15 years later. Year 0 GGT cutpoints were 12, 17, 25, and 36 U/L (overall 25th, 50th, 75th, and 90th percentiles; the laboratory cutpoints for abnormal are 40 U/L in women and 50 U/L in men). We deleted 32 participants with prevalent diabetes and 140 participants with prevalent hypertension from the respective incidence analyses. Results: After adjustment for study center, race, sex, and age in proportional hazards regression, the hazard ratios across year 0 GGT categories were 1.0, 1.6, 1.7, 4.0 (95% confidence interval, 2.0–8.1), and 5.5 (2.7–11.1) for 15-year incident diabetes and 1.0, 1.2, 1.7 (1.2–2.2), 2.3 (1.7–3.2), and 2.3 (1.7–3.2) for hypertension. Additional adjustment for year 0 alcohol consumption, body mass index, cigarette smoking, and physical activity attenuated this relationship, but GGT remained a significant predictor. Conclusions: Serum GGT within a range regarded as physiologically normal is associated with incident diabetes and hypertension. Considering known functionality of GGT, these associations are consistent with a role for oxidative stress in risk for diabetes and hypertension.

Increased homocysteine and S-adenosylhomocysteine concentrations and DNA hypomethylation in vascular disease

Abstract: Background: The pathogenic mechanism of homocysteine’s effect on cardiovascular risk is poorly understood. Recent studies show that DNA hypomethylation induced by increases in S -adenosylhomocysteine (AdoHcy), an intermediate of Hcy metabolism and a potent inhibitor of methyltransferases, may be involved in homocysteine-related pathology. Methods: We measured fasting plasma total Hcy (tHcy), AdoHcy, and S -adenosylmethionine (AdoMet) and methylation in leukocytes in 17 patients with vascular disease and in 15 healthy, age- and sex-matched controls. Results: Patient with vascular disease had significantly higher plasma tHcy and AdoHcy concentrations and significantly lower plasma AdoMet/AdoHcy ratios and genomic DNA methylation. AdoMet concentrations were not significantly different between the two groups. More than 50% of the patients fell into the highest quartiles of plasma tHcy, AdoHcy, and [3H]dCTP incorporation/μg of DNA (meaning the lowest quartile of DNA methylation status) and into the lowest quartile of the AdoMet/AdoHcy ratios of the control group. Plasma tHcy was significantly correlated with plasma AdoHcy and AdoMet/AdoHcy ratios (n = 32; P < 0.001). DNA methylation status was significantly correlated with plasma tHcy and AdoHcy (n = 32; P < 0.01) but not with plasma AdoMet/AdoHcy ratios. Conclusion: Global DNA methylation may be altered in vascular disease, with a concomitant increase in plasma tHcy and AdoHcy.

Amplicon Melting Analysis with Labeled Primers: A Closed-Tube Method for Differentiating Homozygotes and Heterozygotes

Abstract: 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), -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.

Proteomic Patterns in Biological Fluids: Do They Represent the Future of Cancer Diagnostics?

Abstract: Writing on the future of cancer diagnostics, this author has predicted that multiparametric biomarker analysis, in combination with artificial neural networks and pattern recognition, will likely represent one of the most promising methodologies for diagnosing and monitoring cancer (1)(2). Over the last few years, we have witnessed publication of many reports dealing with proteomic patterns in biological fluids, and especially serum, by using the so-called “SELDI-TOF” technique (surface-enhanced laser desorption/ionization time-of-flight mass spectrometry), in combination with artificial intelligence (3)(4)(5)(6)(7). The reported sensitivities and specificities of this method for ovarian, prostate, and breast cancer diagnosis are clearly impressive, and they are superior to the sensitivities and specificities obtained with current serologic cancer biomarkers (8)(9)(10)(11)(12). In particular, these techniques appear to detect early as well as advanced disease with similar efficiency, making them candidate tools for cancer screening, an application that is not currently recommended, by utilizing the classical cancer biomarkers, e.g., CA125, carcinoembryonic antigen (CEA), and α-fetoprotein (AFP) (1). In addition to scientific journals, these reports have also been presented in international news media and have attracted public attention. Despite of some important shortcomings of these methodologies, criticism has been minimal (13)(14). It seems that the impressive bottom line (very high diagnostic sensitivity and specificity) overshadows potential problems. The recent publication of three reports, from two different research groups, on the use of this technology in the diagnosis of prostate cancer allows for comparison of the data and the methodology and for the presentation of some important questions that have not been adequately addressed. In the following paragraphs, I will focus on some critical questions and provide discussion that could form the basis for further investigations. I will concentrate only …

Plasma 99th Percentile Reference Limits for Cardiac Troponin and Creatine Kinase MB Mass for Use with European Society of Cardiology/American College of Cardiology Consensus Recommendations

Abstract: Background: The European Society of Cardiology/American College of Cardiology (ESC/ACC) consensus document for definition of myocardial infarction (MI) is predicated on increased cardiac troponin or creatine kinase (CK) MB mass above the 99th percentile reference limit. The purpose of this study was to determine the plasma (heparin) 99th percentile reference limits for the leading in vitro diagnostic cardiac troponin and CKMB mass assays. Methods: Blood (heparin plasma) was obtained from healthy adults (n = 696; age range, 18–84 years) stratified by gender and ethnicity. Cardiac troponin I (cTnI) and T (cTnT) and CKMB mass concentrations were measured by eight assays. Reference limits were determined by nonparametric statistical analysis. Results: Two cTnI assays demonstrated at least a 1.2- to 2.5-fold higher 99th percentile for males vs females, with the mean concentrations significantly higher for males ( P <0.05). Two cTnI assays also demonstrated a 1.1- to 2.8-fold higher 99th percentile for blacks vs Caucasians, with the mean concentrations significantly higher for blacks ( P = 0.05). There was a 13-fold variance between the lowest measured 99th percentile (0.06 μg/L) and the highest (0.8 μg/L). All CKMB assays demonstrated a 1.2- to 2.6-fold higher 99th percentile for males vs females, with mean concentrations significantly higher for males ( P <0.0001). Four CKMB assays also showed significantly higher (1.2- to 2.7-fold) mean concentrations for blacks ( P <0.02) vs Caucasians. Conclusions: The heparin-plasma 99th percentile reference limits for cardiac troponin and CKMB mass provide an evidence base in support of the ESC, ACC, and American Heart Association guidelines for detection of myocardial injury. Selective gender and ethnic differences were demonstrated. These data allow clinicians, trialists, and epidemiologists a common point for operational use.

Determination of Choline, Betaine, and Dimethylglycine in Plasma by a High-Throughput Method Based on Normal-Phase Chromatography–Tandem Mass Spectrometry

Abstract: 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.

Hydroxytyrosol Disposition in Humans

Abstract: 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.

Report of the National Heart, Lung, and Blood Institute Workshop on Lipoprotein(a) and Cardiovascular Disease: Recent Advances and Future Directions

Abstract: It has been estimated that ∼37% of the US population judged to be at high risk for developing coronary artery disease (CAD), based on the National Cholesterol Education Program guidelines, have increased plasma lipoprotein(a) [Lp(a)], whereas Lp(a) is increased in only 14% of those judged to be at low risk. Therefore, the importance of establishing a better understanding of the relative contribution of Lp(a) to the risk burden for CAD and other forms of vascular disease, as well as the underlying mechanisms, is clearly evident. However, the structural complexity and size heterogeneity of Lp(a) have hindered the development of immunoassays to accurately measure Lp(a) concentrations in plasma. The large intermethod variation in Lp(a) values has made it difficult to compare data from different clinical studies and to achieve a uniform interpretation of clinical data. A workshop was recently convened by the National Heart, Lung, and Blood Institute (NHLBI) to evaluate our current understanding of Lp(a) as a risk factor for atherosclerotic disorders; to determine how future studies could be designed to more clearly define the extent to which, and mechanisms by which, Lp(a) participates in these processes; and to present the results of the NHLBI-supported program for the evaluation and standardization of Lp(a) immunoassays. This report includes the most recent data presented by the workshop participants and the resulting practical and research recommendations.

Prognostic Use of Circulating Plasma Nucleic Acid Concentrations in Patients with Acute Stroke

Abstract: Background: At present there is no simple, accurate blood test that may be used to determine the severity of stroke or to predict mortality and morbidity in stroke patients presenting to emergency departments. Methods: Patients with stroke-like symptoms who presented to an emergency department of a university hospital in Hong Kong were recruited for the study. DNA extracted from patients’ plasma was analyzed for the β-globin gene with a fluorescent-based PCR test. The primary outcome measures were in-hospital and 6-month mortality and morbidity using the post-stroke modified Rankin Score. Results: Among the 88 consecutive patients recruited to the study, 70 (80%) had ischemic stroke, 11 (13%) had intracerebral hemorrhage, and 7 (8%) had transient ischemic attacks. Median plasma DNA concentrations taken within 3 h of symptom onset were higher in patients who died compared with those who survived at discharge (6205 vs 1334 kilogenome-equivalents/L; P = 0.03). Among patients with NIH Stroke Scale scores >8, median plasma DNA concentrations were higher in patients who died compared with those who survived to 6 months (2273 vs 968 kilogenome-equivalents/L; P = 0.002). Plasma DNA concentrations correlated with the volume of cerebral hematoma ( r = 0.66; P = 0.03). Plasma DNA concentrations >1400 kilogenome-equivalents/L had a sensitivity of 100% and a specificity of 74.4% for predicting hospital mortality after stroke, and the area under the ROC curve was 0.89 (95% confidence interval, 0.80–0.94). The adjusted odds ratio for plasma DNA concentrations predicting 6-month mortality was 1.6 (1.1–2.4; P = 0.03) and for predicting 6-month post-Rankin Score >2 was 1.8 (1.0–3.3; P = 0.05). Conclusion: Plasma DNA concentrations correlate with stroke severity and may be used to predict mortality and morbidity in the emergency room.

Fractional Esterification Rate of Cholesterol and Ratio of Triglycerides to HDL-Cholesterol Are Powerful Predictors of Positive Findings on Coronary Angiography

Abstract: Background: We examined the predictive value of various clinical and biochemical markers for angiographically defined coronary artery disease (aCAD). Specifically, we assessed the value of the ratio of plasma triglyceride (TGs) to HDL-cholesterol (HDL-C) and the fractional esterification rate of cholesterol in plasma depleted of apolipoprotein B (apoB)-containing lipoproteins (FERHDL), a functional marker of HDL and LDL particle size. Methods: Patients (788 men and 320 women) undergoing coronary angiography were classified into groups with positive [aCAD(+)] and negative [aCAD(−)] findings. Patient age, body mass index, waist circumference, blood pressure (BP), medications, drinking, smoking, exercise habits, and plasma total cholesterol (TC), LDL-cholesterol (LDL-C), HDL-unesterified cholesterol, HDL-C, TGs, FERHDL, apoB, log(TG/HDL-C), and TC/HDL-C were assessed. Lipids and apoproteins were measured by standard laboratory procedures; FERHDL was determined by a radioassay. Results: Members of the aCAD(+) group were older and had a higher incidence of smoking and diabetes than those in the aCAD(−) group. The aCAD(+) group also had higher TG, apoB, FERHDL, and log(TG/HDL-C) and lower HDL-C values. aCAD(+) women had greater waist circumference and higher plasma TC and TC/HDL-C. aCAD(+) men, but not women, had higher plasma LDL-C. In the multivariate logistic model, the significant predictors of the presence of aCAD(+) were FERHDL, age, smoking, and diabetes. If only laboratory tests were included in the multivariate logistic model, FERHDL appeared as the sole predictor of aCAD(+). Log(TG/HDL-C) was an independent predictor when FERHDL was omitted from multivariate analysis. Conclusions: FERHDL was the best laboratory predictor of the presence of coronary atherosclerotic lesions.

Real-Time Reverse Transcription-PCR Expression Profiling of the Complete Human ATP-Binding Cassette Transporter Superfamily in Various Tissues

Abstract: 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.

Detection of SARS coronavirus RNA in the cerebrospinal fluid of a patient with severe acute respiratory syndrome.

Abstract: Severe acute respiratory syndrome (SARS) is a recently emerged disease caused by a novel coronavirus, the SARS coronavirus (SARS-CoV) (1)(2). Although the respiratory manifestations of SARS are well recognized, the neurologic manifestations have been much less studied (1). Here we report a SARS patient with clinical and laboratory evidence of neurologic involvement. A 59-year-old woman with IgA nephropathy was admitted to the Prince of Wales Hospital in Hong Kong in early May 2003 because of swinging fever, chills, productive cough, and diarrhea. She was previously admitted in April with fungal peritonitis related to her peritoneal dialysis. Despite antifungal and antibiotic therapy, her respiratory function deteriorated. She became increasingly dyspneic and required supplemental oxygen. High-resolution computer tomography …

C-Reactive Protein, Interleukin-6, and Procalcitonin in the Immediate Postnatal Period: Influence of Illness Severity, Risk Status, Antenatal and Perinatal Complications, and Infection

Abstract: Background: Studies of the diagnostic accuracy of most laboratory tests for early-onset neonatal sepsis have yielded variable results. We investigated whether some of this variation might be attributable to differences in population baseline severity and risk status as well as to specific ante- and perinatal variables, independent of the presence of neonatal infection. Methods: The Score for Neonatal Acute Physiology (SNAP) was used to define illness severity, with SNAP Perinatal Extension (SNAP-PE) used to define the combined physiologic and perinatal mortality risk. A total of 134 ill newborns (19 with early-onset infection and 115 with no infection) were available for simultaneous analysis of the association of SNAP, SNAP-PE, and maternal and perinatal variables with C-reactive protein (CRP), interleukin-6 (IL-6), and procalcitonin (PCT) concentrations at birth and at 24 and 48 h of life. Results: Early-onset neonatal infection was associated with significant increases in CRP, IL-6, and PCT concentrations at all three time points, independent of illness severity. However, among babies without infection, higher SNAP and SNAP-PE scores were associated with higher IL-6 concentrations at birth. Certain maternal or perinatal variables altered IL-6 and PCT values in the infected as well as in the uninfected neonates. However, if different cutoff points were used at any of the three neonatal ages, PCT sensitivity and specificity were greater than those of CRP or IL-6. Conclusions: Illness severity and risk status are unlikely to interfere with the use of CRP and PCT for detection of early-onset neonatal sepsis. In contrast, the diagnostic value of IL-6 at birth may be altered by physiologic severity and risk indexes. The reliability of CRP, IL-6, and PCT for the diagnosis of early-onset neonatal infection requires specific cutoff values for each evaluation time point over the first 48 h of life.

Validation of the FibroTest Biochemical Markers Score in Assessing Liver Fibrosis in Hepatitis C Patients

Abstract: Background: Determining the stage of fibrosis by liver biopsy is important in managing patients with hepatitis C virus infection. We investigated the predictive value of the proprietary FibroTest score to accurately identify significant fibrosis in Australian hepatitis C patients. Methods: Serum obtained from 125 confirmed hepatitis C patients before antiviral therapy was analyzed for haptoglobin, α2-macroglobulin, apolipoprotein A1, bilirubin, and γ-glutamyltransferase activity, and the FibroTest score was computed. Liver fibrosis pathology was staged according to a defined system on a scale of F0 to F4. We used predictive values and a ROC curve to assess the accuracy of FibroTest scores. Results: The prevalence of significant fibrosis defined by liver biopsy was 0.38. The most useful single test for predicting significant fibrosis was serum α2-macroglobulin (cutoff value, 2.52 g/L; sensitivity, 75%; specificity, 67%). The negative predictive value of a FibroTest score 0.6 was 78%. Although 33 of the 125 patients had FibroTest scores 0.6 who were likely to have significant fibrosis, 5 (21%) had mild fibrosis. Of the 125 patients in the cohort, 57 (46%) could have avoided liver biopsy, but discrepant results were recorded in 11 of those 57 (19%). Conclusion: The FibroTest score could not accurately predict the presence or absence of significant liver fibrosis.

Evaluation of Human Serum Albumin Cobalt Binding Assay for the Assessment of Myocardial Ischemia and Myocardial Infarction

Abstract: Background: Clinical diagnoses were correlated with results of a Co(II)–albumin binding assay in 167 patients treated at an emergency department of a health maintenance organization. Methods: Patients were evaluated as being nonischemic or potentially ischemic through standard coronary disease indicators [creatine kinase (CK), CK-MB, cardiac troponin I, and electrocardiographic findings] and were tested by a Co(II)–albumin binding assay. Samples were tested anonymously, and the study was double-blinded. The sensitivity and specificity of this assay for the detection of ischemia were evaluated by ROC curve analysis. Known Co(II) binding sites on albumin were analyzed by N-terminal amino acid sequencing. Results: The mean absorbance units (ABSU) ± 2 SD for non-myocardial ischemic and myocardial ischemic individuals measured at 470 nm were 0.43 ± 0.10 and 0.63 ± 0.25, respectively ( P <0.0001). The area under the ROC curve was 0.95 [95% confidence interval (CI), 0.92–0.99], and at a cutoff value of 0.50 ABSU, sensitivity and specificity were 88% (78–94%) and 94% (86–98%), respectively, suggesting a high distinction between the two groups. When we compared non-acute myocardial infarction (AMI) and AMI ischemic individuals, the area under the ROC curve was 0.66 (95% CI, 0.53–0.79) and was considered a poor discriminator between these two groups. N-Terminal amino acid sequencing data for purified albumin showed normal amino acid residues for six of seven high-ABSU (≥0.70) individuals and one nonischemic individual tested. However, only one individual with a high ABSU (0.80) had two missing amino acid residues (DA) from the N-terminal region. Clinical diagnosis for this patient did not reveal an ischemic event. Conclusions: The Co(II)–albumin binding test may serve as a useful diagnostic tool in emergency facilities for the assessment of myocardial ischemia. High and low ABSU were associated with myocardial ischemic individuals and non-myocardial ischemic individuals, respectively. However, the Co(II)–albumin binding was a poor discriminator between ischemic individuals with and without MI.

Preanalytic and Analytic Sources of Variations in C-reactive Protein Measurement: Implications for Cardiovascular Disease Risk Assessment

Abstract: Background: C-reactive protein (CRP) is a widely recognized indicator of inflammation and is known to play an important role in atherogenesis. Recent prospective studies have demonstrated that increased CRP concentrations within the reference interval are a strong predictor of myocardial infarction, stroke, sudden cardiac death, and peripheral vascular disease in apparently healthy adults. On the basis of available evidence, the American Heart Association and the CDC have issued guidelines for the utility of CRP in the primary prevention of coronary heart disease and in patients with stable coronary disease or acute coronary syndromes. Nevertheless, there remains considerable work to optimize the utility of this marker for risk assessment. Issues: Most traditional CRP tests designed to monitor acute and chronic inflammation have inadequate sensitivity for risk stratification of coronary disease. Thus, manufacturers have had to develop tests with higher sensitivity. Because an individual’s CRP concentration will be interpreted according to fixed cut-points, issues related to the preanalytic and analytic components of CRP measurement must be considered and standardized where possible to avoid potential misclassification of cardiovascular risk. Conclusions: Efforts to define performance criteria for high-sensitivity CRP applications coupled with growing awareness of the physiologic aspects of CRP most likely will lead to refinements in standardization, improved performance in quality-assessment schemes, and enhanced risk prediction.

Methamphetamine and Amphetamine Pharmacokinetics in Oral Fluid and Plasma after Controlled Oral Methamphetamine Administration to Human Volunteers

Abstract: Background: Methamphetamine (METH) and amphetamine (AMP) concentrations in 200 plasma and 590 oral fluid specimens were used to evaluate METH pharmacokinetics and pharmacodynamics after oral administration of sustained-release METH. Methods: Eight participants received four oral 10-mg S -(+)-METH hydrochloride sustained-release tablets within 7 days. Three weeks later, five participants received four oral 20-mg doses. Blood samples were collected for up to 24 h and oral fluid for up to 72 h after drug administration. Results: After the first oral dose, initial plasma METH detection was within 0.25–2 h; c max was 14.5–33.8 μg/L (10 mg) and 26.2–44.3 μg/L (20 mg) within 2–12 h. In oral fluid, METH was detected as early as 0.08–2 h; c max was 24.7–312.2 μg/L (10 mg) and 75.3–321.7 μg/L (20 mg) and occurred at 2–12 h. The median oral fluid-plasma METH concentration ratio was 2.0 across 24 h and was highly variable. Neutral cotton swab collection yielded significantly higher METH and AMP concentrations than citric acid candy-stimulated expectoration. Mean (SD) areas under the curve for AMP were 21% ± 25% and 24% ± 11% of those observed for METH in plasma and oral fluid, respectively. After a single low or high dose, plasma METH was >2.5 μg/L for up to 24 h in 9 of 12 individuals (mean, 7.3 ± 5.5 μg/L at 24 h); in oral fluid the detection window was at least 24 h (mean, 18.8 ± 18.0 μg/L at 24 h). The plasma and oral fluid 24-h METH detection rates were 54% and 60%, respectively. After four administrations, METH was measurable for 36–72 h (mean, 58.3 ± 14.5 h). Conclusions: Perceived advantages of oral fluid for verifying METH exposure compared with urine include simpler specimen collection and reduced potential for adulteration, but urine offers higher analyte concentrations and a greater window of detection.

Comprehensive Proteomic Profiling Identifies Serum Proteomic Signatures for Detection of Hepatocellular Carcinoma and Its Subtypes

Abstract: Background: Detection of hepatocellular carcinoma (HCC) in patients with chronic liver disease (CLD) is difficult. We investigated the use of comprehensive proteomic profiling of sera to differentiate HCC from CLD. Methods: Proteomes in sera from 20 CLD patients with α-fetoprotein (AFP) 500 μg/L, and postoperative sera from two HCC patients. Results: Among 2384 common serum proteomic features, 250 were significantly different between the HCC and CLD cases. Two-way hierarchical clustering differentiated HCC and CLD cases. Most HCC cases with advanced disease were clustered together and formed two subgroups that contained significantly more cases with lymph node invasion or distant metastasis. For differentiation of HCC and CLD by an artificial network (ANN), the area under the ROC curve was 0.91 (95% confidence interval, 0.82–1.01; P <0.0005) for all cases and 0.954 (95% confidence interval, 0.881–1.027; P <0.0005) for cases with nondiagnostic serum AFP (<500 μg/L). At a specificity of 90%, the sensitivity was 92%. Both cluster analysis and ANN correctly classified the pooled serum samples, the CLD serum sample with increased AFP, and the HCC patient in complete remission. Conclusion: Tumor-specific proteomic signatures may be useful for detection and classification of hepatocellular cancers.

Early Biomarkers of Stroke

Abstract: 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.

Changes in Concentration of DNA in Serum and Plasma during Storage of Blood Samples

Abstract: Cell-free DNA in serum and plasma has been suggested to have diagnostic potential because associations between DNA concentrations and several disorders have been described (1). The concentration of cell-free DNA circulating in plasma and serum has been analyzed in several studies and used as an interchangeable index of the quantity of circulating DNA in blood (2)(3)(4). However, it is known that the DNA concentration in serum is ∼3- to 24-fold higher than in plasma (3)(4)(5)(6). Recently published articles in this journal showed that various preanalytical factors of blood sampling and processing can affect the DNA concentration in plasma (5)(7)(8), but these findings do not explain the difference between plasma and serum concentrations of DNA mentioned above. Comparative investigations of the preanalytical conditions influencing the DNA concentration in serum and plasma are lacking. In addition, reference intervals for the concentration of cell-free DNA in serum were established without considering these factors (9). Thus, to complement the data of Lui et al. (5), we analyzed the influence of time delay in blood processing for plasma and serum at room temperature and at 4 °C. Venous blood samples from 10 healthy volunteers (5 females and 5 males; mean age, 42 years) were simultaneously collected in …