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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
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