Showing papers in "Clinical Chemistry in 2000"

Evaluation of Nine Automated High-Sensitivity C-Reactive Protein Methods: Implications for Clinical and Epidemiological Applications. Part 2

Abstract: Background: C-Reactive protein (CRP) can provide prognostic information about risk of future coronary events in apparently healthy subjects. This application requires higher sensitivity assays than have traditionally been available in the clinical laboratory. Methods: Nine high-sensitivity CRP (hs-CRP) methods from Dade Behring, Daiichi, Denka Seiken, Diagnostic Products Corporation, Iatron, Kamiya, Olympus, Roche, and Wako were evaluated for limit of detection, linearity, precision, prozone effect, and comparability with samples from 388 apparently healthy individuals. Results: All methods had limits of detection that were lower than the manufacturers’ claimed limit of quantification except for the Kamiya, Roche, and Wako methods. All methods were linear at 0.3–10 mg/L. The Diagnostic Products Corporation, Kamiya, Olympus, and Wako methods had imprecision (CVs) >10% at 0.15 mg/L. The Iatron, Olympus, and Wako methods demonstrated prozone effects at hs-CRP concentrations of 12, 206, and 117 mg/L, respectively. hs-CRP concentrations demarcating each quartile in a healthy population were method-dependent. Ninety-two to 95% of subjects were classified into the same quartile of hs-CRP established by the Dade Behring method by the Denka Seiken, Diagnostic Products Corporation, Iatron, and Wako methods. In contrast, 68–77% of subjects were classified into the same quartile by the Daiichi, Kamiya, Olympus, and Roche methods. No subject varied by more than one quartile by any method. Conclusions: Four of the nine examined hs-CRP methods classified apparently healthy subjects into quartiles of hs-CRP similar to the classifications assigned by the comparison method. Additional standardization efforts are required because an individual patient’s results will be interpreted using population-based cutpoints.

Diagnosis and Monitoring of Hepatic Injury. I. Performance Characteristics of Laboratory Tests

Abstract: Purpose: To review information on performance characteristics for tests that are commonly used to identify acute and chronic hepatic injury. Data Sources and Study Selection: A MEDLINE search was performed for key words related to hepatic tests, including quality specifications, aminotransferases, alkaline phosphatase, γ-glutamyltransferase, bilirubin, albumin, ammonia, and viral markers. Abstracts were reviewed, and articles discussing performance of laboratory tests were selected for review. Additional articles were selected from the references. Guideline Preparation and Review: Drafts of the guidelines were posted on the Internet, presented at the AACC Annual Meeting in 1999, and reviewed by experts. Areas requiring further amplification or literature review were identified for further analysis. Specific recommendations were made based on analysis of published data and evaluated for strength of evidence and clinical impact. The drafts were also reviewed by the Practice Guidelines Committee of the American Association for the Study of Liver Diseases and approved by the committee and the Association’s Council. Recommendations: Although many specific recommendations are made in the guidelines, some summary recommendations are discussed here. Alanine aminotransferase is the most important test for recognition of acute and chronic hepatic injury. Performance goals should aim for total error of <10% at the upper reference limit to meet clinical needs in monitoring patients with chronic hepatic injury. Laboratories should have age-adjusted reference limits for enzymes in children, and gender-adjusted reference limits for aminotransferases, γ-glutamyltransferase, and total bilirubin in adults. The international normalized ratio should not be the sole method for reporting results of prothrombin time in liver disease; additional research is needed to determine the reporting mechanism that best correlates with functional impairment. Harmonization is needed for alanine aminotransferase activity, and improved standardization for hepatitis C viral RNA measurements.

Flow Cytometry: Principles and Clinical Applications in Hematology

Abstract: The use of flow cytometry in the clinical laboratory has grown substantially in the past decade. This is attributable in part to the development of smaller, user-friendly, less-expensive instruments and a continuous increase in the number of clinical applications. Flow cytometry measures multiple characteristics of individual particles flowing in single file in a stream of fluid. Light scattering at different angles can distinguish differences in size and internal complexity, whereas light emitted from fluorescently labeled antibodies can identify a wide array of cell surface and cytoplasmic antigens. This approach makes flow cytometry a powerful tool for detailed analysis of complex populations in a short period of time. This report reviews the general principles in flow cytometry and selected applications of flow cytometry in the clinical hematology laboratory.

Plasma DNA as a Prognostic Marker in Trauma Patients

Abstract: Background: Recently, much interest has developed in the potential use of plasma DNA as a diagnostic and monitoring tool. We hypothesized that plasma DNA is increased in patients with trauma and may be prognostic in such patients. Methods: We studied 84 patients who had sustained an acute blunt traumatic injury. We measured plasma DNA by a real-time quantitative PCR assay for the β-globin gene. Blood samples were collected at a median time of 60 min following injury. Blood samples were also obtained from 27 control subjects. Results: The median plasma DNA concentrations in the control, minor/moderate trauma (Injury Severity Score <16; n = 47), and major trauma (Injury Severity Score ≥16; n = 37) groups were 3154 kilogenome-equivalents/L, 13 818 kilogenome-equivalents/L, and 181 303 kilogenome-equivalents/L, respectively. Plasma DNA concentrations in patients with adverse outcomes, including acute lung injury, acute respiratory distress syndrome, and death, had 11.6- to 12-fold higher plasma DNA concentrations than those who did not develop these complications. At a cutoff of 232 719 kilogenome-equivalents/L, the sensitivities of plasma DNA analysis for the prediction of acute lung injury, acute respiratory distress syndrome, and death were 100% (95% confidence interval, 100–100%), 100% (95% confidence interval, 100–100%), and 78% (95% confidence interval, 40–97%), respectively. The respective specificities were 81% (95% confidence interval, 71–89%), 80% (95% confidence interval, 70–88%), and 82% (95% confidence interval, 71–90%). Conclusions: Plasma DNA is increased after trauma and may be a potentially valuable prognostic marker for these patients.

Genetic Analysis of DNA Excreted in Urine: A New Approach for Detecting Specific Genomic DNA Sequences from Cells Dying in an Organism

Abstract: Background: Cell-free DNA from dying cells recently has been discovered in human blood plasma. In experiments performed on animals and humans, we examined whether this cell-free DNA can cross the kidney barrier and be used as a diagnostic tool. Methods: Mice received subcutaneous injections of either human Raji cells or purified 32P-labeled DNA. DNA was isolated from urine and analyzed by measurement of radioactivity, agarose gel electrophoresis, and PCR. In humans, the permeability of the kidney barrier to polymeric DNA was assessed by detection in urine of sequences that were different from an organism bulk nuclear DNA. Results: In the experiments on laboratory animals, we found that ∼0.06% of injected DNA was excreted into urine within 3 days in a polymeric form and that human-specific Alu sequences that passed through the kidneys could be amplified by PCR. In humans, male-specific sequences could be detected in the urine of females who had been transfused with male blood as well as in DNA isolated from urine of women pregnant with male fetuses. K- ras mutations were detected in the urine of patients with colon adenocarcinomas and pancreatic carcinomas. Conclusions: The data suggest that the kidney barrier in rodents and humans is permeable to DNA molecules large enough to be analyzed by standard genetic methodologies.

Diagnosis and Monitoring of Hepatic Injury. II. Recommendations for Use of Laboratory Tests in Screening, Diagnosis, and Monitoring

Abstract: Purpose: To review information on the use of laboratory tests in screening, diagnosis, and monitoring of acute and chronic hepatic injury. Data Sources and Study Selection: A MEDLINE search was performed for key words related to hepatic diseases, including acute hepatitis, chronic hepatitis, alcoholic hepatitis, cirrhosis, hepatocellular carcinoma, and etiologic causes. Abstracts were reviewed, and articles discussing use of laboratory tests selected for review. Additional articles were selected from the references. Guideline Preparation and Review: Drafts of the guidelines were posted on the Internet, presented at the AACC Annual Meeting in 1999, and reviewed by experts. Areas requiring further amplification or literature review were identified for further analysis. Specific recommendations were made based on analysis of published data and evaluated for strength of evidence and clinical impact. Recommendations: Although many specific recommendations are made in the guidelines, only some summary recommendations are listed here. In acute hepatic injury, prothrombin time and, to a lesser extent, total bilirubin are the best indicators of severity of disease. Although ALT is useful for detecting acute and chronic hepatic injury, it is not related to severity of acute hepatic injury and only weakly related to severity of chronic hepatic injury. Specific tests of viral markers should be the initial differential tests in both acute and chronic hepatic injury; when positive, they are also useful for monitoring recovery from hepatitis B and C.

Cobalamin and Folate Evaluation: Measurement of Methylmalonic Acid and Homocysteine vs Vitamin B12 and Folate

Abstract: Vitamin B(12) and folate are two vitamins that have interdependent roles in nucleic acid synthesis. Deficiencies of either vitamin can cause megaloblastic anemia; however, inappropriate treatment of B(12) deficiency with folate can cause irreversible nerve degeneration. Inadequate folate nutrition during early pregnancy can cause neural tube defects in the developing fetus. In addition, folate and vitamin B(12) deficiency and the compensatory increase in homocysteine are a significant risk factor for cardiovascular disease. Laboratory support for the diagnosis and management of these multiple clinical entities is controversial and somewhat problematic. Automated ligand binding measurements of vitamin B(12) and folate are easiest to perform and widely used. Unfortunately, these tests are not the most sensitive indicators of disease. Measurement of red cell folate is less dependent on dietary fluctuations, but these measurements may not be reliable. Homocysteine and methylmalonic acid are better metabolic indicators of deficiencies at the tissue level. There are no "gold standards" for the diagnosis of these disorders, and controversy exists regarding the best diagnostic approach. Healthcare strategies that consider the impact of laboratory tests on the overall costs and quality of care should consider the advantages of including methylmalonic acid and homocysteine in the early evaluation of patients with suspected deficiencies of vitamin B(12) and folate.

Laboratory Investigation of Hemoglobinopathies and Thalassemias: Review and Update

Abstract: Structural hemoglobin (Hb) variants typically are based on a point mutation in a globin gene that produce a single amino acid substitution in a globin chain. Although most are of limited clinical significance, a few important subtypes have been identified with some frequency. Homozygous Hb C and Hb S (sickle cell disease) produce significant clinical manifestations, whereas Hb E and Hb D homozygotes may be mildly symptomatic. Although heterozygotes for these variants are typically asymptomatic, diagnosis may be important for genetic counseling. Thalassemia, in contrast, results from quantitative reductions in globin chain synthesis. Those with diminished β-globin chains are termed β-thalassemias, whereas those with decreased α-chain production are called α-thalassemias. Severity of clinical manifestations in these disorders relates to the amount of globin chain produced and the stability of residual chains present in excess. The thalassemia minor syndromes are characterized clinically by mild anemia with persistent microcytosis. Thalassemia intermedia (i.e., Hb H disease) is typified by a moderate, variably compensated hemolytic anemia that may present with clinical symptoms during a period of physiologic stress such as infection, pregnancy, or surgery. The thalassemia major syndromes produce severe, life-threatening anemia. α-Thalassemia major usually is incompatible with extrauterine life; β-thalassemia major presents in infancy and requires life-long transfusion therapy and/or bone marrow transplantation for successful control of the disease. Double heterozygosity for certain structural variants and/or thalassemia syndromes may also lead to severe clinical disease. Several guidelines have been published that outline the required steps for hemoglobinopathy and thalassemia investigation. The availability of HPLC has streamlined many of these requirements, allowing an efficient stepwise diagnostic strategy for these complex disorders.

Cardiac Troponins I and T Are Biological Markers of Left Ventricular Dysfunction in Septic Shock

Abstract: Background: Cardiac depression in severe sepsis and septic shock is characterized by left ventricular (LV) failure. To date, it is unclear whether clinically unrecognized myocardial cell injury accompanies, causes, or results from this decreased cardiac performance. We therefore studied the relationship between cardiac troponin I (cTnI) and T (cTnT) and LV dysfunction in early septic shock. Methods: Forty-six patients were consecutively enrolled, fluid-resuscitated, and treated with catecholamines. Cardiac markers were measured at study entry and after 24 and 48 h. LV function was assessed by two-dimensional transesophageal echocardiography. Results: Increased plasma concentrations of cTnI (≥0.4 μg/L) and cTnT (≥0.1 μg/L) were found in 50% and 36%, respectively, of the patients at one or more time points. cTnI and cTnT were significantly correlated ( r = 0.847; P <0.0001). Compared with cTnI-negative patients, cTnI-positive subjects were older, presented higher Acute Physiology and Chronic Health Evaluation II scores at diagnosis, and tended to have a worse survival rate and a more frequent history of arterial hypertension or previous myocardial infarction. In contrast, the two groups did not differ in type of infection or pathogen, or in dose and type of catecholamine administered. Continuous electrocardiographic monitoring in all patients and autopsy in 12 nonsurvivors did not disclose the occurrence of acute ischemia during the first 48 h of observation. LV dysfunction was strongly associated with cTnI positivity (78% vs 9% in cTnI-negative patients; P <0.001). In multiple regression analysis, both cTnI and cTnT were exclusively associated with LV dysfunction ( P <0.0001). Conclusions: These findings suggest that in septic shock, clinically unrecognized myocardial cell injury is a marker of LV dysfunction. The latter condition tends to occur more often in severely ill older patients with underlying cardiovascular disease. Further studies are needed to determine the extent to which myocardial damage is a cause or a consequence of LV dysfunction.

Proteasome Inhibition Measurements: Clinical Application

Abstract: Background: PS-341, a selective inhibitor of the proteasome, currently is under evaluation as an anticancer agent in multiple phase I clinical trials. In animal-model studies, PS-341 was rapidly removed from the vascular compartment and distributed widely, quickly approaching the limits of detection. An accurate pharmacodynamic assay has been developed as an alternative or complement to pharmacokinetic measurements. Methods: Fluorogenic kinetic assays for both the chymotryptic and tryptic activities of the proteasome have been optimized for both whole blood and blood cells. Using the ratio of these activities and the catalytic mechanism of the proteasome, we developed a novel method of calculating percentage of inhibition, using two structurally unrelated inhibitors (PS-341 and lactacystin). Results: This ratio method was demonstrated to be sensitive (detection limit of 13% inhibition with 10 μg of cell lysate), specific to the proteasome (PS-341 provides >98% inhibition), accurate (112% analyte recovery), and precise (0% ± 5% inhibition at 0 nmol/L PS-341 and 74.5% ± 1.7% inhibition at 200 nmol/L PS-341). Using these assays, we found that both erythrocytes and leukocytes contain proteasome at 3 μmol/L. Pharmacodynamic results for PS-341 obtained from the whole-blood ratio method were comparable to those using leukocytes determined by another method. Conclusions: The described assay provides a reliable method for studying the pharmacodynamics of proteasome inhibitors and is now in use in concurrent phase I clinical trials with PS-341.

Presence of Fetal RNA in Maternal Plasma

Abstract: The discovery of fetal DNA in maternal plasma (1) has opened up a new horizon on prenatal molecular diagnosis. Many groups have since shown that fetal genetic traits, such as RhD status and inherited genetic diseases, can be determined from fetal DNA in maternal plasma (2)(3)(4)(5). However, it is not known whether fetal RNA is also present in maternal plasma. Here, using a two-step reverse transcription (RT)-PCR assay, we demonstrate the presence of fetal-derived, male-specific mRNA in plasma of pregnant women carrying male fetuses. Pregnant women attending the Prenatal Diagnosis Unit at the Department of Obstetrics and Gynecology, Prince of Wales Hospital, Hong Kong were recruited with informed consent. The study was approved by the Clinical Research Ethics Committee. Women early and late in their pregnancies (n = 21 and 37, respectively) were recruited in this study. The mean gestational ages of the subjects in early and late pregnancies were 16 weeks (range, 11–19 weeks) and 33 weeks (range, 26–40 weeks), respectively. All early-pregnancy samples were obtained before any invasive procedure. On the other hand, late-pregnancy samples were collected either from women who had invasive procedures in early pregnancy (n = 21) or from women who did not have any prenatal invasive procedure (n = 16). All plasma samples were harvested within 30 min from EDTA-blood samples as described previously (1). Total RNA from plasma samples was isolated with the Trizol LS Reagent (Life Technologies) as instructed …

Use of a Reference Material Proposed by the International Federation of Clinical Chemistry and Laboratory Medicine to Evaluate Analytical Methods for the Determination of Plasma Lipoprotein(a)

Abstract: Background: As part of the NIH/National Heart, Lung and Blood Institute Contract for the Standardization of Lipoprotein(a) [Lp(a)] Measurements, a study was performed in collaboration with the IFCC Working Group for the Standardization of Lp(a) Assays. The aims of the study, performed with the participation of 16 manufacturers and 6 research laboratories, were to evaluate the IFCC proposed reference material (PRM) for its ability to transfer an accuracy-based value to the immunoassay calibrators and to assess concordance in results among different methods. Methods: Two different purified Lp(a) preparations with protein mass concentrations determined by amino acid analysis were used to calibrate the reference method. A Lp(a) value of 107 nmol/L was assigned to PRM. After uniformity of calibration was demonstrated in the 22 evaluated systems, Lp(a) was measured on 30 fresh-frozen sera covering a wide range of Lp(a) values and apolipoprotein(a) [apo(a)] sizes. Results: The among-laboratory CVs for these samples (6–31%) were, in general, higher than those obtained for PRM (2.8%) and the quality-control samples (14%, 12%, and 9%, respectively), reflecting the broad range of apo(a) sizes in the 30 samples and the sensitivity of most methods to apo(a) size heterogeneity. Thus, although all of the assays were uniformly calibrated through the use of PRM, no uniformity in results was achieved for the isoform-sensitive methods. Conclusions: Linear regression analyses indicated that to various degrees, apo(a) size heterogeneity affects the outcome of the immunochemical methods used to measure Lp(a). We have also shown that the inaccuracy of Lp(a) values determined by methods sensitive to apo(a) size significantly affects the assessment of individual risk status for coronary artery disease.

Identification of Lignans as Major Components in the Phenolic Fraction of Olive Oil

Abstract: Background: Because olive oil is an important component of the Mediterranean diet, it is necessary to establish unequivocal identification of the major potential antioxidant phenolic compounds it contains. Methods: The major phenolic antioxidants in extra virgin olive oil were isolated and purified. Structural analysis was conducted using several spectroscopic techniques, including mass spectrometry and nuclear magnetic resonance (NMR). In particular, detailed 1H and 13C NMR data are presented, and several assignment errors in the literature are corrected. Results: The data show for the first time that the lignans (+)-1-acetoxypinoresinol and (+)-pinoresinol are major components of the phenolic fraction of olive oils. These lignans, which are potent antioxidants, are absent in seed oils and virtually absent in refined virgin oils but are present at concentrations of up to 100 mg/kg (mean ± SE, 41.53 ± 3.93 mg/kg; range, 0.65–99.97 mg/kg) in extra virgin oils. As with the simple phenols and secoiridoids, there is considerable interoil variation in lignan concentrations. Foods containing high amounts of lignan precursors have been found to be protective against breast, colon, and prostate cancer. Conclusion: Lignans, as natural components of the diet, may be important modulators of cancer chemopreventive activity.

Immunoradiometric Assay of Circulating C-Reactive Protein: Age-related Values in the Adult General Population

Abstract: Background: Increased values of C-reactive protein (CRP), the classical acute phase protein, within the range below 5 mg/L, previously considered to be within the reference interval, are strongly associated with increased risk of atherothrombotic events, and are clinically significant in osteoarthritis and neonatal infection. Methods: A robust new polyclonal-monoclonal solid- phase IRMA for CRP was developed, with a range of 0.05–10.0 mg/L. Results: Plasma CRP values in general adult populations from Augsburg, Germany (2291 males and 2203 females; ages, 25–74 years) and Glasgow, Scotland (604 males and 650 females; ages, 25–64 years) were very similar. The median CRP approximately doubled with age, from ∼1 mg/L in the youngest decade to ∼2 mg/L in the oldest, and tended to be higher in females. Conclusion: This extensive data set, the largest such study of CRP, provides valuable reference information for future clinical and epidemiological investigations.

Simple Microplate Method for Determination of Urinary Iodine

Abstract: Background: Urinary iodine is a good biochemical marker for control of iodine deficiency disorders. Our aim was to develop and validate a simple, rapid, and quantitative method based on the Sandell–Kolthoff reaction, incorporating both the reaction and the digestion process into a microplate format. Methods: Using a specially designed sealing cassette to prevent loss of vapor and cross-contamination among wells, ammonium persulfate digestion was performed in a microplate in an oven at 110 °C for 60 min. After the digestion mixture was transferred to a transparent microplate and the Sandell–Kolthoff reaction was performed at 25 °C for 30 min, urinary iodine was measured by a microplate reader at 405 nm. Results: The mean recovery of iodine added to urine was 98% (range, 89–109%). The theoretical detection limit, defined as 2 SD from the zero calibrator, was 0.11 μmol/L (14 μg/L iodine). The mean intra- and interassay CVs for samples with iodine concentrations of 0.30–3.15 μmol/L were ≤10%. The new method agreed well with the conventional chloric acid digestion method (n = 70; r = 0.991; y = 0.944 x + 0.04; S y|x = 0.10) and with the inductively coupled plasma mass spectrometry method (n = 61; r = 0.979; y = 0.962 x + 0.03; S y|x = 0.20). The agreement was confirmed by difference plots. The distributions of iodine concentrations for samples from endemic areas of iodine deficiency diseases showed similar patterns among the above three methods. Conclusions: Our new method, incorporating the whole process into a microplate format, is readily applicable and allows rapid monitoring of urinary iodine.

Quantitative Analysis of the Bidirectional Fetomaternal Transfer of Nucleated Cells and Plasma DNA

Abstract: Background: Recently, much interest has been generated on the fetomaternal transfer of nucleated cells and plasma DNA. However, there has been no systematic quantitative comparison of these two directions and two modalities of trafficking within the same study population. Methods: The fetus-to-mother transfer of nucleated cells and plasma DNA in pregnant women carrying male babies was studied using a real-time quantitative PCR assay for the SRY gene. For mother-to-fetus transfer, real-time quantitative PCR assays for the insertion/deletion polymorphisms involving the glutathione S -transferase M1 and angiotensin-converting enzyme genes were used. Results: Of the 50 informative mother-baby pairs, maternal DNA was detected in the cellular fraction of umbilical cord blood in 24% of cases (12 of 50), at a median fractional concentration of 2.6 × 10−4 (interquartile range, 1.7 × 10−4 to 3.6 × 10−4). In the plasma fraction of cord blood, maternal DNA was detected in 30% (15 of 50) of cases at a median fractional concentration of 3 × 10−3 (interquartile range, 1 × 10−3 to 1.6 × 10−2). For the other direction of trafficking, fetus-to-mother transfer of nucleated cells was detected in 26% of cases (13 of 50) at a median fractional concentration of 3.2 × 10−4 (interquartile range, 0.6 × 10−4 to 7.6 × 10−4). In the plasma fraction, fetal DNA was detected in 100% of maternal plasma (50 of 50) at a median fractional concentration of 3 × 10−2 (interquartile range, 1.4 × 10−2 to 5.3 × 10−2). Conclusions: This study indicated that significantly more fetal DNA is present in the plasma of pregnant women compared with DNA from the cellular fraction of maternal blood. In addition, maternal DNA was demonstrated in both the cellular and plasma fractions of cord blood after delivery. This study has therefore determined the fundamental quantitative values for the bidirectional fetomaternal cellular and plasma DNA traffic.

Protein Glycosylation and Diseases: Blood and Urinary Oligosaccharides as Markers for Diagnosis and Therapeutic Monitoring

Abstract: Background: N- and O-oligosaccharide variants on glycoproteins (glycoforms) can lead to alterations in protein activity or function that may manifest themselves as overt disease. Approach: This review summarizes those diseases that are known to be the result of an inherited or acquired glycoprotein oligosaccharide structural alteration and that are diagnosed in blood or urine by chemical characterization of that oligosaccharide alteration. Content: The biochemical synthesis steps and catabolic pathways important in determining glycoprotein function are outlined with emphasis on alterations that lead to modified function. Clinical and biochemical aspects of the diagnosis are described for inherited diseases such as I-cell disease, congenital disorders of glycosylation, leukocyte adhesion deficiency type II, hereditary erythroblastic multinuclearity with a positive acidified serum test, and Wiskott-Aldrich syndrome. We also review the laboratory use of measurements of glycoforms related to acquired diseases such as alcoholism and cancer. Conclusions: Identification of glycoprotein glycoforms is becoming an increasingly important laboratory contribution to the diagnosis and management of human diseases as more diseases are found to result from glycan structural alterations.

Prenatal Diagnosis of Myotonic Dystrophy Using Fetal DNA Obtained from Maternal Plasma

Abstract: Myotonic dystrophy (DM; MIM 160900) is an autosomal dominant disorder associated with expansion of an unstable CTG trinucleotide repeat in the 3′ untranslated region of the DM kinase gene (DMPK) on chromosome 19q13 (1). Patients are heterozygous for expanded alleles in the range of 50–4000 repeats (1). The molecular diagnosis of DM routinely is performed by analyzing the CTG number on genomic DNA extracted from various biological sources, including trophoblast cells sampled at 10–11 weeks of amenorrhea during the first trimester of pregnancy (2)(3). We evaluated the possibility of using maternal plasma for prenatal diagnosis of DM, by monitoring the pregnancy of an unaffected woman whose husband was affected by DM (70 CTG repeats). All participants gave oral and written informed consent. A blood sample (∼10 mL) was collected at 10 weeks of gestation before chorionic villus sampling (CVS) and was centrifuged at 3000 g for 10 min. Plasma was carefully removed from EDTA-containing tube and centrifuged again at 3000 …

General Deming Regression for Estimating Systematic Bias and Its Confidence Interval in Method-Comparison Studies

Abstract: Background: Various forms of least-squares regression analyses are used to estimate average systematic error (bias) and its confidence interval in method-comparison studies. When assumptions that underlie a particular regression method are inappropriate for the data, errors in estimated statistics result. In this report, I present an improved method for regression analysis that is free from the usual simplifying assumptions and is generally applicable to linearly related method-comparison data. Methods: Theoretical equations based on the Deming approach, further developed by physicists and extended herein, were applied to method-comparison data analysis. Monte Carlo simulations were used to demonstrate the validity of the new procedure and to compare its performance to ordinary linear regression (OLR) and simple Deming regression (SDR) procedures. Results: Simulation studies included three types of data commonly encountered in method-comparison studies: ( a ) constant within-method SDs for both methods, ( b ) constant within-method CVs for both methods, and ( c ) neither SDs nor CVs constant for both methods. For all cases examined, OLR produced unreliable confidence intervals of the estimated bias. However, OLR point estimates of systematic bias were reliable when the correlation coefficient was >0.975. SDR produced reliable estimates of systematic bias for all cases studied, but the confidence intervals of systematic bias were unreliable when SDs of methods varied as a function of analyte concentration. Conclusion: Only iteratively reweighted general Deming regression produced statistically unbiased estimates of systematic bias and reliable confidence intervals of bias for all cases.

Riboflavin as a Determinant of Plasma Total Homocysteine: Effect Modification by the Methylenetetrahydrofolate Reductase C677T Polymorphism

Abstract: Background: Plasma total homocysteine (tHcy) is a risk factor for cardiovascular disease. tHcy concentrations are partly determined by folate, cobalamin, and vitamin B 6 status, and methylenetetrahydrofolate reductase (MTHFR) and other flavoenzymes are important for the biotransformation of these vitamins. This motivates the investigation of the possible relationship between riboflavin status and tHcy. Methods: The study had a cross-sectional design and included 423 healthy blood donors, ages 19–69 years. We determined plasma tHcy, serum folate, serum cobalamin, serum creatinine, and MTHFR C677T genotype. In addition, we measured riboflavin and its two coenzyme forms, flavin mononucleotide and flavin adenine dinucleotide, in EDTA plasma by capillary electrophoresis and laser-induced fluorescence detection. Results: Riboflavin determined tHcy independently in a multiple linear regression model with adjustment for sex, age, folate, cobalamin, creatinine, and MTHFR genotype ( P = 0.008). tHcy was 1.4 μmol/L higher in the lowest compared with the highest riboflavin quartile. The riboflavin-tHcy relationship was modified by genotype ( P = 0.004) and was essentially confined to subjects with the C677T transition of the MTHFR gene. Conclusions: Plasma riboflavin is an independent determinant of plasma tHcy. Studies on deficient populations are needed to evaluate the utility of riboflavin supplementation in hyperhomocysteinemia.

Association of Serum Bilirubin Concentration with Risk of Coronary Artery Disease

Abstract: Background: Lipid oxidation and formation of oxygen radicals are important elements of arterial plaque formation and atherosclerosis, and are involved in the pathophysiology of coronary artery disease (CAD). Because bilirubin has antioxidant properties, it has been suggested that it may have a protective role in the atherosclerotic process. Approach: This review examines in vitro and in vivo studies indicating that bilirubin inhibits lipid oxidation and oxygen radical formation. Experimental and epidemiological evidence is presented that suggests that bilirubin may serve as a physiological antioxidant providing protection against atherosclerosis and CAD. Special attention is focused on studies that noted an inverse relationship between plasma bilirubin concentration and cardiovascular morbidity. Content: Serum bilirubin concentrations in the upper portion of the reference interval reportedly reduce atherogenic risk and provide protection against CAD. In contrast, serum bilirubin concentrations in the lower portion of the reference interval may be associated with increased risk of ischemic heart disease. Summary: Taken together, the evidence presented in this review supports the concept that bilirubin, via its antioxidant potential, has antiatherogenic properties and that an inverse relationship exists between circulating bilirubin concentrations and risk of CAD.

Development of Conventional and Real-Time PCR Assays for the Rapid Detection of Group B Streptococci

Abstract: Background: Group B streptococci (GBS), or Streptococcus agalactiae , are the leading bacterial cause of meningitis and bacterial sepsis in newborns. Currently available rapid methods to detect GBS from clinical specimens are unsuitable for replacement of culture methods, mainly because of their lack of sensitivity. Methods: We have developed a PCR-based assay for the rapid detection of GBS. The cfb gene encoding the Christie-Atkins-Munch-Petersen (CAMP) factor was selected as the genetic target for the assay. The PCR primers were initially tested by a conventional PCR method followed by gel electrophoresis. The assay was then adapted for use with the LightCyclerTM. For this purpose, two fluorogenic adjacent hybridization probes complementary to the GBS-specific amplicon were designed and tested. In addition, a rapid sample-processing protocol was evaluated by colony-forming unit counting and PCR. A total of 15 vaginal samples were tested by both standard culture method and the two PCR assays. Results: The conventional PCR assay was specific because it amplified only GBS DNA among 125 bacterial and fungal species tested, and was able to detect all 162 GBS isolates from various geographical areas. This PCR assay allowed detection of as few as one genome copy of GBS. The real-time PCR assay was comparable to conventional PCR assay in terms of sensitivity and specificity, but it was more rapid, requiring only ∼30 min for amplification and computer-based data analysis. The presence of vaginal specimens had no detrimental effect on the sensitivity of the PCR with the sample preparation protocol used. All four GBS-positive samples identified by the standard culture method were detected by the two PCR assays. Conclusion: These assays provide promising tools for the rapid detection and identification of GBS.

Bioconjugates of intelligent polymers and recognition proteins for use in diagnostics and affinity separations.

Abstract: Polymers that respond to small changes in environmental stimuli with large, sometimes discontinuous changes in their physical state or properties are often called “intelligent” or “smart” polymers. We have conjugated these polymers to different recognition proteins, including antibodies, protein A, streptavidin, and enzymes. These bioconjugates have been prepared by random polymer conjugation to lysine amino groups on the protein surface, and also by site-specific conjugation of the polymer to specific amino acid sites, such as cysteine sulfhydryl groups, that are genetically engineered into the known amino acid sequence of the protein. We have conjugated several different smart polymers to streptavidin, including temperature-, pH-, and light-sensitive polymers. The preparation of these conjugates and their many fascinating applications are reviewed here.

Hemolyzed Specimens: A Reason for Rejection or a Clinical Challenge?

Abstract: Clinical laboratories must improve the preanalytical phase, a phase highly susceptible to mistakes (1). In some reports, hemolyzed specimens, the most common reason for rejection, account for ∼60% of rejected specimens, fivefold more than the second most common cause (2). Cellular contents can falsely increase values for some plasma constituents, such as potassium, lactate dehydrogenase, and aspartate aminotransferase (3). Moreover, hemolysis produces spectrophotometric interference with other laboratory methods. In vitro hemolysis depends mainly on the way in which the blood samples are drawn and treated, and it may in particular depend on the blood being forced through too fine a needle (4) or through the large-bore needle of a syringe into a tube; it may also be caused by shaking the tube too vigorously and/or centrifuging blood specimens before clotting is complete. In vivo hemolysis, on the other hand, may have at least 50 causes. We evaluated the causes of hemolysis in samples received by our STAT section in the …

Plasma Nitric Oxide Concentrations and Nitric Oxide Synthase Gene Polymorphisms in Coronary Artery Disease

Abstract: Background: Plasma NOx (nitrate and nitrite) is a stable end product of the vasodilator NO. Several polymorphisms in the endothelial constitutive NO synthase (ecNOS) gene have been reported, including the 4a/4b VNTR polymorphism in intron 4, the E298D mutation in exon 7, and the G10-T polymorphism in intron 23. The aims of this study were to examine plasma NOx in patients with coronary artery disease (CAD) and to assess the association between plasma NOx concentrations and the three ecNOS gene polymorphisms. Methods: Plasma NOx was measured in samples from 128 healthy controls and from 110 CAD patients at least 2 months after myocardial infarction. Three genetic polymorphisms that are known or have been suggested to be associated with plasma NOx concentration were also analyzed by PCR-restriction fragment length polymorphism. Results: Median plasma NOx was significantly higher ( P <0.001) in CAD patients (95.9 μmol/L) than in controls (73.8 μmol/L). Furthermore, the median plasma NOx was significantly higher ( P <0.001) in hypertensive CAD patients (116.0 μmol/L) than in controls and normotensive CAD patients (86.0 μmol/L). The G-allele frequency of the G10-T polymorphism in intron 23 was significantly higher in CAD patients than in controls. Other polymorphisms showed no differences in allelic frequencies among the control and CAD groups. In controls, individuals with the E298D mutation in exon 7 (136.1 μmol/L) showed significantly higher ( P = 0.001) median plasma NOx than those without this mutation (64.5 μmol/L). Conclusions: Plasma NOx was higher in hypertensive CAD patients than in normotensive CAD patients and controls. The E298D polymorphism of the ecNOS gene was associated with increased plasma NOx. Further study is needed to understand the gene expression and enzyme activity of ecNOS and their association with genotypes.

Measurement of Plasma and Intracellular S–Adenosylmethionine and S–Adenosylhomocysteine Utilizing Coulometric Electrochemical Detection: Alterations with Plasma Homocysteine and Pyridoxal 5'–Phosphate. Concentrations

Abstract: Background: The relative changes in plasma and intracellular concentrations of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) may be important predictors of cellular methylation potential and metabolic alterations associated with specific genetic polymorphisms and/or nutritional deficiencies. Because these metabolites are present in nanomolar concentrations in plasma, methods of detection generally require time-consuming precolumn processing or metabolite derivatization. Methods: We used HPLC with coulometric electrochemical detection for the simultaneous measurement of SAM and SAH in 200 mL of plasma, 10 6 lymphocytes, or 10 mg of tissue. Filtered trichloroacetic acid extracts were injected directly into the HPLC system without additional processing and were eluted isocratically. Results: The limits of detection were 200 fmol/L for SAM and 40 fmol/L SAH. In plasma extracts, the interassay CV was 3.4 ‐5.5% and the intraassay CV was

Correlation of Antemortem Serum Creatine Kinase, Creatine Kinase-MB, Troponin I, and Troponin T with Cardiac Pathology

Abstract: Background: Spurious increases in serum troponins, especially troponin T, have been reported in patients with and without acute myocardial syndromes. Methods: We studied 78 autopsied patients without clinical myocardial infarction (MI) and correlated histologic cardiac findings with antemortem serum creatine kinase (CK), its MB isoenzyme (CK-MB), cardiac troponin I (cTnI), and cardiac troponin T (cTnT). Results: There was no significant myocardial pathology in 15 patients. Cardiac pathologies were in five groups: scarring from previous MI or patchy ventricular fibrosis (n = 9), recent MI (n = 27), healing MI (n = 7), degenerative myocyte changes consistent with congestive heart failure (CHF; n = 12), and other cardiac pathologies (n = 8). The median concentrations in the five groups were not significantly different for either CK or CK-MB. Compared with the no-pathology group, only the MI group was significantly different for cTnI, and the MI and other pathology groups were significantly different for cTnT. For patients with MI, 22%, 19%, 48%, and 65% had increased CK, CK-MB, cTnI, and cTnT, respectively; for CHF and other cardiac pathologies combined, the percentages were 28%, 17%, 22%, and 50%. For patients with increased cTnI, 72% and 28% had MI and other myocardial pathologies, respectively; patients with increased cTnT had 64% and 36%, respectively. Patients without myocardial pathology had no increases in CK-MB, cTnI, or cTnT. Conclusions: All patients with increased serum CK-MB, cTnI, and cTnT had significant cardiac histologic changes. The second-generation cTnT assay appears to be a more sensitive indicator of MI and other myocardial pathologies than the cTnI assay used in this study.

Cardiac Troponin T and I in End-Stage Renal Failure

Abstract: Background: In patients suffering from end-stage renal failure, cardiac troponin T (cTnT) and I (cTnI) may be increased in serum without other signs of acute myocardial damage. Whether these increases are specific to myocardial injury or nonspecific is not completely clear. Methods: We investigated time courses of cTnT and cTnI over 1 year and the clinical outcome over 2 years in 59 patients with end-stage renal failure undergoing chronic hemodialysis. At the start of the study, we divided the patients into two groups, group 1, without history of cardiac failure, and group 2, with history of cardiac failure, and looked for differences between the groups in later adverse outcome. cTnT was measured using the Enzymun® troponin T assay on an ES 700 analyzer (Roche). cTnI was measured on a Stratus® II analyzer (Dade Behring). Creatinine and blood urea nitrogen were measured on a Vitros® 950 IRC (Ortho). Results: Dialysis acutely increased cTnT ( P <0.01) and decreased cTnI ( P <0.001) regardless of the dialysis membrane used. Although statistically not significant, cTnT but not cTnI was increased more frequently in group 2 than in group 1, in some cases over the whole study period. Five patients (8.5%) died of cardiac complications within 2 years; all of them had mostly increased cTnT and, in one or more samples, increased cTnI. Conclusions: Dialysis alters measured cTnT and cTnI concentrations in serum. In patients suffering from end-stage renal failure, sporadic or persistently increased cTnT and cTnI appear to predict cardiac complications. Because of the effects of the dialysis procedure on troponin values, we recommend that blood be collected before dialysis.

Detection of Multiple Allergen-specific IgEs on Microarrays by Immunoassay with Rolling Circle Amplification

Abstract: First described in 1967, the radio allergo sorbent test (RAST) has been the standard technique for measuring allergen-specific IgE antibodies in serum (1). An updated version of the RAST test, termed CAP (Pharmacia), has been introduced (2). In clinical practice, CAP results must be interpreted with care. The diagnostic performance of CAP varies in an allergen-specific manner, and CAP scores do not always correlate with clinical severity (3)(4). CAP sensitivity, specificity, and positive predictive values agree well with skin prick tests (SPTs) for house dust mites and grasses, but poorly with tests for cat dander and peanuts (5). Microarray technology potentially offers advantages in diagnostic applications such as allergy testing because the amount of reagent required, and thus the cost per assay, is greatly reduced (6). This approach has been difficult to reduce to practice, however, because the extremely small volumes (∼0.5–5 nL) of sample used to create spots on these microarrays require extremely sensitive methods of analyte detection (7). We have used rolling circle amplification (RCA) (8) for the detection of antibody bound to antigen (9). In this “immunoRCA”, the 5′ end of a RCA primer is attached to an antibody; thus, in the presence of circular DNA, DNA polymerase, and nucleotides, the rolling circle reaction produces a concatamer of circular DNA sequence copies that remain attached to the antibody. The amplified DNA can be detected by hybridization of complementary oligonucleotide probes. ImmunoRCA, therefore, represents a novel approach for signal amplification of antibody-antigen recognition events on microarrays. ImmunoRCA can detect IgE in a format using high-density microarrays of anti-human IgE printed on glass slides by a pin-tool type microarraying robot (9). Here, we describe the production of microarrays of multiple allergens and demonstrate the utility of these microarrays in combination with immunoRCA to simultaneously detect …

Evaluation of DNA Fragment Sizing and Quantification by the Agilent 2100 Bioanalyzer

Abstract: Capillary electrophoresis (CE) achieves efficient separation of molecular species by the application of high voltages to samples in solution (1). Commercial CE units, available for slightly more than a decade, have found numerous applications (2)(3)(4)(5)(6), but are expensive (∼$60 000) and require substantial user training and experience. Recent advances have allowed CE to be performed on microchip devices (7)(8)(9)(10)(11). We evaluated the Agilent 2100 Bioanalyzer (Agilent Technologies), which represents a new generation of CE instruments that use this technology. The Bioanalyzer is relatively inexpensive (∼$18 000) and is simple to operate, requiring only routine pipetting and basic computer skills. Typically, 12 nucleic acid samples can be sized and quantified on a disposable chip within 30 min. Chips are fabricated from glass and comprise an interconnected network of fluid reservoirs and microchannels, which must be filled with a gel-dye mixture. Each chip contains 16 wells: 3 for loading the gel-dye mixture, 1 for a molecular size ladder, and 12 for experimental samples. The movement of nucleic acids through the microchannels is controlled by a series of electrodes, each of which is independently connected to a common power supply. The Bioanalyzer displays data as both migration-time plots and as computer-generated virtual gels. Traditional CE operating variables [temperature, voltage, capillary material, and pH, ionic strength, and viscosity of buffer (12)] cannot be modified. The instruments costs ∼$18 000, and chips cost ∼$12–18 per chip ($1–1.50 per sample). The gel-dye mixture consists of a linear polymer and a fluorescent, intercalating dye. The marker mixture consists of a buffer along with lower and upper molecular size markers, which the Bioanalyzer uses as references when sizing DNA fragments. The upper marker is also used as a reference for calculating the …