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April 2002

A novel detection platform for nucleic acids on microarrays
DNA microchips work by having DNA-capture probes immobilized on a solid support in a miniature array. These capture probes are deposited individually at defined locations that are close together, which allows one to test for the presence of many distinct nucleic acid targets in a single hybridization reaction. These nucleic acid targets are allowed to hybridize to the capture probes. After hybridization, the target is detected through a reporter molecule. This reporter molecule is frequently a fluorescent species that can be incorporated into the target before hybridization or in a separate signaling probe that forms a ternary complex with the capture probe and target nucleic acid. DNA chip systems that require an optical reader often use a high density of capture probe sites. This type of setup is most useful for gene and drug discovery efforts, for which the high cost of instrumentation is not a problem. Other systems will be required for the more economically sensitive environment of the molecular diagnostics laboratory. The authors have developed a novel platform to electronically detect nucleic acids on microarrays. It involves use of a gold electrode in a printed circuit board. The electrode is coated with a self-assembled monolayer (SAM) containing DNA capture probes. Unlabeled nucleic acid targets are immobilized on the surface of the SAM through sequence-specific hybridization with the DNA capture probe. A separate signaling probe that contains ferrocene-modified nucleotides and is complementary to the target in the region adjoining the capture-binding site is held close to the SAM. This configuration allows electron transfer between the immobilized ferrocenes and the gold, and it insulates the electrode from soluble redox species, including unbound signaling probes. The authors demonstrated sequence-specific detection of amplicons after simple dilution of the reaction product into hybridization buffer. They also showed the capability of single nucleotide polymorphism discrimination. The C282Y single nucleotide polymorphism associated with hereditary hemochromatosis was used to confirm the genotype of six patients' DNA. The authors concluded that the versatility of this detection platform made it suitable for multiple applications in diagnostics, pharmacogenetics, and infectious disease testing.

Umek RM, Lin SW, Vielmetter J, et al. Electronic detection of nucleic acids: a versatile platform for molecular diagnostics. J Mol Diag. 2001;3:74-84.

Reprints: Daniel H. Farkas, Clinical Micro Sensors, Division of Motorola Inc., 757 S. Raymond, Pasadena, CA 91105; dan.farkas@motorola.com

Serum vs. plasma levels of genomic DNA
Evidence indicates that cell-free genomic DNA increases in concentration in a variety of pathologic conditions, including systemic lupus erythematosus, pulmonary embolism, malignancies, and maternal chimerisms. The mechanisms by which DNA is released into the serum and plasma are not fully understood. It is known that circulating levels of free DNA in cancer patients generally correlate with tumor size, persistence of pain, and other clinical parameters. It has been suggested that measurements of circulating DNA be used as a prognostic tool in cancer patients and to monitor the effects of cancer therapy. Chimerism has also been studied with regard to the survival of donor white blood cells in transfusion recipients to more fully understand the relationship of donor/recipient white blood cell interactions to such complications as transfusion-associated graft versus host disease and alloimmunization. For all of these reasons, the authors thought it worthwhile to assess the baseline serum and plasma levels of cell-free genomic DNA using a protocol from PCR. DNA was extracted from normal donor serum that was fresh or stored at 4ºC for one to six days or from plasma samples using reagents from an HIV assay kit. After the samples were incubated and washed, the purified DNA was amplified with HLA DQ alpha primers (GH26 and 27) or human Y chromosome primers (SA and SD) to quantitate the concentration of genomic DNA. The authors found that the serum levels of cell-free DNA were about 20-fold higher than the concentrations in the fresh plasma. The serum concentrations increased daily to a level of more than 100 times baseline after clotted tubes were stored at 4°C for four to five days. Only a small increase was noted in the cell-free DNA in stored ACD whole blood samples. It was noted that male white blood cells spiked into fresh, non-anticoagulated female blood were lysed during clotting with male DNA liberated into the serum samples. The authors concluded that most of the cell-free DNA encountered in serum samples is generated during clotting in the original collection tube. The plasma concentration of cell-free genomic DNA is likely the same as that in the circulation. They further concluded that, while serum samples are not likely to be useful in monitoring the concentration of cell-free DNA in patient circulation, serum collected from sample tubes containing clots—that is, without anticoagulant present—three to five days after the date of phlebotomy, potentially could be useful as a specimen for determining DNA in the screening of post-transfusion microchimerism.

Lee TH, Montalvo L, Chrebtow V, Busch MP. Quantitation of genomic DNA in plasma and serum samples: higher concentrations of genomic DNA found in serum than in plasma. Transfusion. 2001;41:271-282.

Reprints: Dr. Michael P. Busch, Blood Centers of the Pacific, 270 Masonic Ave., San Francisco, CA 94118; mpbusch@its.ucsf.edu

Relationship between androgen levels and obesity in males
Total testosterone blood concentrations tend to be inversely correlated with body weight in males. Obese males, therefore, have lower mean testosterone concentrations than normal weight, healthy males. Obese patients also tend to have decreased levels of sex hormone-binding globulin (SHBG) synthesis and decreased pituitary gonadotropin secretion and pulse amplitude. In general, levels of free testosterone (FT) tend to be less affected than total testosterone (TT) concentrations. SHBG increases with weight loss in obese males, and hyperinsulinemia, frequently found in these men, has an inhibitory effect on SHBG synthesis. The authors proposed studying the effect of weight loss on serum TT, FT, luteinizing hormone (LH), and insulin concentrations to provide in vivo evidence to support the pathogenic explanations for the decreased androgen levels in these men. The authors studied a cohort of 37 unselected obese men who ranged in age from 20 to 68 years. This group was subsequently divided into two groups: the first group—14 individuals—were a mean age of 49±15 years and had a body mass index of 35 kg/m2 or less. The second group—23 individuals—were a mean age of 39±8.5 years and had a body mass index of 35.1 kg/m2 or greater. Both groups were studied before and after six months of a low-energy diet (1,200 kcal/day), during which every patient received a therapeutic prescription for dexfenfluramine (15 mg b.i.d.). Twenty normal-weight men were also studied as a control group. The authors measured TT and FT, serum LH, and insulin by radioimmunoassay methods. The levels were obtained before and after weight loss. The moderately obese men (group one) showed significantly decreased levels of TT (390±120 ng/dL) as well as FT (16.0±4.8 pg/mL), compared with normal controls. The FT serum levels correlated significantly and negatively with body mass index, while TT concentrations were less significant in this correlation. Concentrations of luteinizing hormone were normal, and insulin levels were elevated in all patients (46.3±30.1 µU/mL). Significant increases in TT, FT, and LH levels and a significant decrease in insulin occurred after weight loss (P<0.01). In the more obese men (group two), TT, FT, and LH were decreased and significantly lower than in the moderately obese group and the controls. As expected, TT, FT, and LH levels increased significantly after weight loss, while insulin concentrations decreased. The authors concluded that FT levels are dependent on degree of obesity. They proposed that a functional decrease of LH pulse and amplitude and serum LH levels, as well as the possible negative action of excess of circulating leptin on the steroidogenesis pathways, may be related to decreased androgen levels in massively obese men.

Lima N, Cavaliere H, Knobel M, et al. Decreased androgen levels in massively obese men may be associated with impaired function of the gonadostat. Int J Obesity. 2000;24:1433-1437.

Reprints: G. Medeiros-Neto, Hospital das Clinicas, Av. Dr. Eneas C. Agular, 255-8A-bl.3, 05403-900 Sao Paulo, SP, Brazil, medneto@uol.com.br

Functional C3a receptors on activated human T lymphocytes
The C3a molecule is one of the anaphylatoxins of the C system, a family of factors comprising C3a, C4a, and C5a. A wide spectrum of effects has been measured for C3a, including the release of histamine and intracellular calcium ions, chemotaxis in human mast cells, and basophils. C3a is also a potent stimulus for eosinophils, which respond with immobilization of calcium ion, superoxide anion production, degranulation, chemotaxis, and adhesion to postcapillary venules. And C3a induces the production of reactive oxygen species and the release of IL-8 and lysosomal enzymes from neutrophils. It also affects human monocytes and macrophages, in which it induces the release of PGE2 and calcium ions. Furthermore, it is a potent suppressor of antigen-specific and polyclonal antibody responses via the induction of nonspecific T lymphocytes. The receptor for C3a, C3aR, which belongs to the family of G protein-coupled receptors, recently was cloned. The receptor is widely expressed in different tissues, including lymphoid organs, suggesting that C3a may play a central role in inflammatory processes. The authors conducted a study to demonstrate that T cell clones from patients with atopic dermatitis expressed the functional C3a receptor. They used receptor-specific monoclonal antibodies. The authors detected C3a receptor expression in CD4+ and CD8+ blood- or skin-derived clones of T lymphocytes. These cells were taken from birch pollen-sensitized patients with atopic dermatitis. No significant difference in the expression of C3a receptors was noted between CD4+ and CD8+ cells. C3a led to a transient calcium flux in the T cells expressing the C3a receptor, whereas the C3a-negative T cells were unreactive. The C3a receptor was expressed in circulating T cells from patients suffering from severe inflammatory skin diseases, but no -expression could be found in unstimulated T lymphocytes from patients with mild inflammatory diseases or healthy individuals. The authors identified type 1 interferons as up-regulators of C3a receptor expression in vitro in freshly isolated or cloned T lymphocytes. C3a receptor-positive T cells were also found at the sites of injection in patients with multiple sclerosis who were treated with interferon beta. The authors concluded that their observations provide direct evidence for the expression of C3a receptor on activated human T lymphocytes.

Werfel T, Kirchhoff K, Wittmann M, et al. Activated human T lymphocytes express a functional C3a receptor. J Immunol. 2000; 165: 6599-6605.

Reprints: Dr. T. Werfel, Dept. of Dermatology, Mediziniscfhe Hochschule Hannover, Ricklingerstrasse 5, 30449 Hannover, Germany; Thomas_Werfel@t-online.de

TNF-a and soluble TNF-a receptors as serum markers in obesity
Tumor necrosis factor-a is a cytokine that is primarily produced by macrophages in response to endotoxemia, inflammation, and cancer. Human fat cells are a significant source of endogenous TNF-a production. It is uncertain whether adipose tissue is a source of circulating TNF-a. TNF-a is a mediator of insulin resistance; binding of insulin to its receptor initiates a phosphorylation cascade, which starts with autophosphorylation of the insulin receptor. This is followed by insulin--receptor activation of the receptor tyro-sine kinase and subsequent phos-phor-yla-tion of the insulin-receptor substrate 1. Obesity, of course, is linked to and predisposes one to insulin resistance. Animal studies have indicated that TNF-a expression in adipose tissue is significantly elevated in obesity. The authors studied whether weight-reduction treatment modulates serum concentration of TNF-a and two soluble TNF-a receptors in obese subjects. They examined 27 obese subjects, aged 48±12 years, who, on average, had a body mass index of 36±6 kg/m2. These individuals were studied before and after three months of weight-reduction treatment, which consisted of physical exercise and a 1,000-calorie per day diet. The authors measured for each subject serum concentration of TNF-a, the soluble receptors sTNF-R1 and sTNF-R2, plasma glucose, insulin, total cholesterol, high-density lipoprotein cholesterol, and triglycerides. The subjects showed a mean weight loss during the three months of 9.3±3.3 kg. The serum concentration of TNF-a decreased after weight loss, while both receptors increased significantly. The authors concluded that the observed decrease in the serum concentration of TNF-a and the increase in the TNF receptors after weight loss may represent a counter-regulatory effect that prevents additional weight loss.

Zahorska-Markiewicz B, Janowska J, Olszanecka-Glinianowicz M, et al. Serum concentrations of TNF-a and soluble TNF-a receptors in obesity. Int J Obes. 2000;24: 1392-1395.

Reprints: B. Zahorska-Markiewicz, Dept. of Pathophysiology, Medykow 18, 40-752 Katowice, Poland

Free-to-total PSA ratio in patients with increased PSA
Measurement of the ratio between total PSA and unbound or free PSA has been shown to be more accurate than PSA alone in detecting prostate cancer and to be most useful in patients with total PSA values of 4 to 10 ng/mL. The biologic basis for this phenomenon is unknown. Results of studies to determine the usefulness of free PSA and the prediction of pathology on radical prostatectomy have generally demonstrated a correlation between free-to-total PSA ratio and prostate cancer specimen pathology. These results, however, are controversial and have prompted the authors to evaluate the role of free-to-total PSA ratio in the prediction of prostate cancer specimen pathology. The authors incorporated tumor volume and prostate pathology into the end points of the study. They prospectively analyzed 54 consecutive patients with prostate cancer who had undergone radical prostatectomy and in whom frozen serum was available to assess the free-to-total PSA ratio. Whole mount sections were taken for pathological review, and total tumor volume was determined by planimetry. Comparisons of the free-to-total PSA ratio and pathological parameters were analyzed using the Pearson correlation coefficient. The mean total and free-to-total PS ratios were 5.81 and 14.2 ng/mL, respectively, among the 54 patients. The free-to-total PSA ratio directly correlated with prostate volume (P=0.037) and inversely correlated with Gleason score (P=0.012) and extracapsular disease (P=0.0074). A significant relationship was also noted between the free-to-total PSA ratio and pathological stage pT2a/b in 39 cases versus pT3a/b in 15 (P=0.005). Overall, no correlation was found between free-to-total PSA ratio and tumor volume, but, among 37 patients with an increased PSA, which was defined as more than 4 ng/mL, a significant inverse relationship was identified between free-to-total PSA ratio and tumor volume (P=0.01) This subset showed only a weak correlation with prostate volume. The authors concluded that the free-to-total PSA ratio may be predictive of tumor biology among those patients with a total PSA of more than 4 ng/mL.

Grossklaus DJ, Shappell SB, Gautam S, et al. Ratio of free-to-total prostate specific antigen correlates with tumor volume in patients with increased prostate specific antigen. J Urol. 2001;165:455-458.

Reprints not available.

Comparison of instruments for measuring pleural fluid pH
Of the common pleural fluid measurements for assessing parapneumonic effusions, pleural fluid pH has been shown to be the most consistent diagnostic predictor of the need for pleural space drainage. Many authors cite a pleural fluid pH of less than 7.2 as an indication of the need for chest tube drainage. The most accurate instrument for measuring pleural fluid is a blood gas analyzer. Several studies have demonstrated that other methods of measuring pleural fluid pH, including the pH meter and indicator strip, are far less accurate. Many hospital laboratories, however, are reluctant to measure pleural pH with a blood gas analyzer because of concerns about obstructing the machine with coagulated proteins. The authors surveyed hospital laboratories nationwide regarding methods used to measure pleural fluid pH and to compare differences between university, community, and pediatric institutions. The authors also prospectively compared measurement of pleural fluid pH between the traditional blood gas analyzer and a new method for analyzing pleural fluid pH—the handheld i-Stat portable clinical analyzer. Pleural fluid was collected prospectively from 19 pediatric and adult patients, and pleural effusions and pleural fluid pH were measured simultaneously with a traditional tabletop blood gas analyzer and the handheld i-Stat unit. The mean pH for the traditional blood gas analyzer was 7.358±0.189, and the mean pH for the handheld unit was 7.382± 0.203. The absolute difference between the two machines was 0.024 units, and the two methods were correlated (r=0.993, P<0.1). A total of 220 hospital laboratories were contacted by phone for the survey and 166 responded. PH meters were used in 35 percent of the hospital laboratories, blood gas analyzers in 32 percent of the labs, and litmus paper in 31 percent. Two percent of the laboratories did not perform the test. University hospitals were more likely to use the blood gas analyzer than community hospitals or children's hospitals. The authors concluded that most U.S. hosptal laboratories do not measure pleural fluid pH using a blood gas analyzer and instead use alternative methods that previously have been shown to be inaccurate. Pleural fluid pH obtained by handheld units shows a high degree of correlation to that obtained by the traditional tabletop blood gas analyzer. Handheld units, therefore, offer a satisfactory alternative to blood gas analyzers.

Kohn GL, Hardie WD. Measuring pleural fluid pH. Chest. 2000;118:1626-1629.

Reprints: Dr. William D. Hardie, Children's Hospital Medical Center, Division of Pulmonary Medicine, OSB-5 Burnet Ave., Cincinnati, OH 45229; bill.hardie@chmcc.org

Glucose diurnal variation
Diagnosis of diabetes mellitus under current American Diabetes Association recommendations calls for measuring fasting plasma glucose. Diagnostic criteria generally are based on studies of plasma glucose measured in the morning, after an overnight fast of at least eight hours. Plasma glucose levels generally have been observed to be higher in the morning. Many patients, however, are seen in the afternoon, often in an uncertain state of fasting. It is, therefore, unclear whether the current diagnostic criteria for diabetes mellitus can be applied to patients who are examined in the afternoon. The author investigated this using the Third National Health and Nutrition Examination Survey (NHANES III). Participants in the NHANES III were interviewed for sociodemographic information, medical history, and life style characteristics and then randomly assigned a morning or afternoon examination that included anthropometric measurements and phlebotomy. The subjects assigned to morning examination were asked to not eat or drink anything other than water after 8:30 PM the day before the exam. Those examined in the early afternoon were asked to eat breakfast but to not eat or drink anything other than water after 7:30 AM the day of the exam. The survey included 18,825 subjects who were 20 years or older. The authors excluded those who had a prior diagnosis of diabetes or who were pregnant (n=1,827), who did not have plasma glucose measured (n=2,288), or who attended an examination session other than that to which they were assigned (n=617), as well as morning subjects who fasted fewer than eight hours and afternoon subjects who fasted fewer than four hours (n=1,211). For the 6,483 subjects who were examined in the morning, the median fasting time was 13.5 hours; 82 percent fasted 12 through 16 hours. Of the 6,399 subjects who were examined in the afternoon, median fasting time was seven hours; 76 percent fasted four through 10 hours. The morning and afternoon groups did not differ in age, body mass index, waist-to-hip ratio, physical activity index, glycosylated hemoglobin level, and other factors. The morning group had higher mean fasting plasma glucose levels than the afternoon group. These levels were 97.4 mg/dL in the morning group versus 9.4 mg/dL in the afternoon group (P<.001). The prevalence of fasting plasma hyperglycemia in the participants examined in the afternoon was therefore half that of the participants examined in the morning. The diagnostic fasting plasma glucose value for patients examined in the afternoon that resulted in the same prevalence of diabetes found in the participants examined in the morning was 114 mg/dL or greater. The authors concluded that if current diabetes diagnostic criteria are applied to patients seen in the afternoon, approximately half of all cases of undiagnosed diabetes will be missed.

Troisi RJ, Cowie CC, Harris MI. Diurnal variation in fasting plasma glucose: Implications for diagnosis of diabetes in patients examined in the afternoon. JAMA. 2000;284: 3157-3159.

Reprints: Dr. Maureen I. Harris, National Institute of Diabetes and Digestive and Kidney Disease, Room 695, 6707 Democracy Blvd., MSC-5460, Bethesda, MD 20892; mh63q@nih.gov