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May 2003
Stability
of DNA after extended storage of clinical specimens
A recent advance in polymerase chain-reaction technology, known
as real-time PCR, consists of homogeneous assays that allow rapid
detection of the PCR product with a minimum of specimen handling
and provide quantitative measurements of viral nucleic acid in patient
specimens. PCR is often performed on specimens that have been stored
for an extended period. The quantitative stability of the specimens
is, therefore, of considerable importance. It is generally known,
and has been reported by several groups, that viral DNA stored for
extended periods generally remains positive by qualitative PCR.
However, whether standard storage conditions retain the quantitative
information on the viral DNA in patient specimens has not been investigated
fully. The authors, therefore, compared herpes simplex virus DNA
levels on specimens at baseline and then after 16 months of storage.
They found that levels of viral DNA remained stable whether the
DNA was stored as purified DNA or unextracted DNA in a whole specimen
for this period of time.
Jerome KR, Huang ML, Wald A, et al. Quantitative
stability of DNA after extended storage of clinical specimens as
determined by real-time PCR. J Clin Microbiol. 2002;40:2609-2611.
Reprints: Keith R. Jerome, Fred Hutchinson Cancer Research Center,
1100 Fairview Ave. North, D3-100, Seattle, WA 98109; kjerome@u.washington.edu.
Recombinant
factor VIIa and orthotopic liver transplantation
Cirrhosis causes severe changes in the hemostatic system, including
thrombocytopenia and platelet function defects, deficiencies of
clotting factors and inhibitors, dysfibrinogenemia, and deficiencies
of fibrinolytic proteins. These bleeding complications in cirrhosis
may be a consequence of a defective hemostatic system, but patients
may also bleed because of complications of portal hypertension,
such as esophageal varices. The bleeding diathesis of cirrhotic
patients becomes apparent during orthotopic liver transplantation,
which frequently is accompanied by excessive blood loss. Managing
bleeding in patients with cirrhosis involves administering vitamin
K, fresh frozen plasma, fibrinogen concentrate, and platelet concentrates.
A novel approach to treating these hemostatic defects in cirrhosis
involves administering recombinant factor VIIa (rFVIIa). Recent
reports show that rFVIIa reduced the need for transfusion of red
cells and plasma during orthotopic liver transplantation. It was
originally developed to treat hemophiliac patients with inhibitors
during bleeding episodes or surgery, in whom it was shown to be
safe and effective. It exerts its prohemostatic effect via enhancement
of the extrinsic coagulation pathway in a tissue factor-dependent
manner. One mechanism for the efficacy of rFVIIa might be down-regulation
of fibrinolytic system via enhanced activation of the thrombin-activatable
fibrinolysis inhibitor (TAFI). The authors showed previously that
rFVIIa serves as an antifibrinolytic agent in factor VIII-deficient
plasma by enhancing secondary thrombin generation required for TAFI
activation. In this study, the authors examined whether the efficacy
of rFVIIa in cirrhosis might be explained in part by this mechanism.
Adding therapeutic or supratherapeutic doses of rFVIIa to the plasma
of 12 patients with stable cirrhosis did not prolong clot lysis
time, though clotting times were significantly reduced. Clot lysis
assays of plasma samples taken during and after orthotopic liver
transplantation, which was performed with or without a single bolus
dose of rFVIIa, did not show any effect of the molecule on plasma
fibrinolytic potential. The authors concluded that the study shows
no evidence for an antifibrinolytic effect of rFVIIa in cirrhotic
patients and patients undergoing orthotopic liver transplantation.
Lisman T, Leebeek FWG, Meijer K, et al. Recombinant factor VIIa improves clot
formation but not fibrolytic potential in patients with cirrhosis and during
liver transplantation. Hepatology. 2002;35:616-621.
Reprints: Dr. Ton Lisman, Thrombosis and Haemostasis Laboratory, Dept. of Haematology
G.03.647, University Medical Center, P.O. Box 85500, 3508 GA, Utrecht, The Netherlands;
j.a.lisman@lab.azu.nl.
A
new technique for isolating fetal cells from maternal blood
The most promising candidate for the source of fetal cells from
maternal blood for purposes of noninvasive sampling for prenatal
genetic diagnosis seems to be fetal nucleated red blood cells (NRBCs).
These are morphologically distinguishable from maternal cells, bear
full complements of nuclear genes, have a limited lifespan, and
are abundant in first trimester maternal blood. A variety of approaches
have been used successfully to recover fetal NRBCs, such as density-gradient
centrifugation, fluorescence-activated cell sorting, magnetic-activated
cell sorting, and charged flow separation. These methods have been
used to separate fetal cells and are feasible for prenatal diagnosis
of aneuploidies such as trisomy 21 and genetic diseases such as
Duchenne muscular dystrophy, sickle cell anemia, and thalassemia.
None of these methods, however, have been shown to recover fetal
cells from maternal blood with sufficient reliability to allow routine
prenatal diagnosis. The authors developed a new method for isolating
fetal NRBCs using galactose-bearing conjugation. NRBCs, which highly
express galactose on their surface, were selectively attached to
a substrate coated with galactose-containing polymer using soybean
agglutinin, a galactose-specific lectin. The authors then used cord
blood samples to evaluate the enrichment efficacy of NRBCs by this
method. They obtained blood samples from 131 pregnant women between
six and 27 weeks’ gestation. After preliminary condensation
of the fetal cells using Ficoll gradient centrifugation, the NRBCs
were enriched using galactose-positive selection by adjusting the
soybean agglutinin concentration. The authors isolated one to several
hundred NRBCs in 2.3 mL of peripheral blood samples from 96 percent
of the pregnant women. The isolated NRBCs were analyzed with a fluorescence
in situ hybridization (FISH) probe for the Y chromosome in eight
cases carrying male fetuses. Y signals were detected in all eight
cases, and more than half of the NRBCs were of fetal origin. The
authors concluded that their new method using galactose-specific
lectin enriches fetal NRBCs, thus allowing noninvasive prenatal
diagnosis.
Kitagawa M, Sugiura K, Omi H, et al. New technique
using galactose-specific lectin for isolation of fetal cells from
maternal blood. Prenat Diagn. 2002;22:17-21.
Reprints: M. Kitagawa, Dept. of Obstetrics & Gynecology, National
Okura Hospital, 2-10-1, Okura, Setagaya-ku, Tokyo 157-8535, Japan;
kitagawa@sub-tky.hosp.go.jp.
An
assay for brown recluse spider venom
The brown recluse spider (Loxosceles reclusa), which is
indigenous to the United States, is associated with a particularly
disfiguring and dangerous type of bite. The typical bite begins
as an oval of pallor with peripheral erythema that may extend 5
to 10 cm or more in diameter. A centrally located necrotic ulcer
typically forms eight to 24 hours after envenomation. Definitive
diagnosis of this type of spider bite is a problem because patients
typically don’t appear with the spider in hand, and the pattern
of cutaneous necrosis is not specific for brown recluse envenomation.
A variety of treatable conditions may be mistaken for a brown recluse
bite, including dermal bacterial infections, Rocky Mountain spotted
fever, Lyme disease, pyoderma gangrenosum, and sporotrichosis. Delaying
the diagnosis of these treatable causes may result in significant
morbidity. The authors developed a sensitive and specific polyclonal
antibody-based Loxosceles species enzyme-linked immunosorbent
assay and characterized the specificity of the assay by evaluating
antigenic cross-reactivity with other North American spider venoms.
The authors assayed three venom amounts—16,000 ng, 2,000 ng,
and 40 ng—using North American arthropod venoms (14 spiders,
two scorpions, and one bee). At the lowest amount of venom tested
(40 ng), the ELISA detected only Loxosceles species positive
control. The authors concluded that the venom specificity of the
ELISA might allow clinical application of their assay in brown recluse
spider endemic regions of North America.
Gomez HF, Krywko DM, Stoecker WV. A new assay for
the detection of Loxosceles species (brown recluse) spider
venom. Ann Emerg Med. 2002;39:469-474.
Reprints: Dr. Hernan F. Gomez, Dept. of Emergency Medicine, University
of Michigan Medical Center, 1500 E. Medical Center Drive, TC B1382
Box 0305, Ann Arbor, MI 48109-0305; hfg@umich.edu.
A
meta-analysis of literature on D-dimer in the
diagnosis of pulmonary embolism
Evaluating patients presenting with symptoms and signs suspicious
of pulmonary embolism is often an expensive and complex undertaking.
This condition can present with a variety of symptoms and signs
that have been grouped into models that predict low, moderate, and
high pretest probabilities of disease. Although many emergency departments
use the ventilation-perfusion (V/Q) lung scan to identify V/Q mismatching,
this test is rarely definitive and usually requires additional testing.
Helical computed tomography (CT) scans, pulmonary angiography, and/or
imaging of the extremities—that is, Doppler ultrasonography,
impedance plethysmography, and venography—are alternatives.
The fibrin degradation product D-dimer is usually increased in the
presence of thromboembolic disease but may also be increased in
such common diseases as inflammatory arthritis, cancer, and infection.
Published studies of the use of D-dimer to rule out pulmonary embolus
vary in size and quality, so the accuracy and utility of the test
is still a matter of debate. The authors performed a meta-analysis
of the diagnostic literature to determine the sensitivity and specificity
of the enzyme-linked immunosorbent assay D-dimer test for the diagnostic
rule-out of pulmonary embolism in the adult emergency room population.
They performed Medline searches of the literature, consulted bibliographies
of previous reviews, consulted with experts in the field of pulmonary
embolism research, and limited their search to prospective investigations
involving predominantly outpatient populations suspected of pulmonary
embolus who used the ELISA D-dimer test. Two authors extracted data
independently and assessed study quality on the basis of the patient
spectrum and reference standard. The authors used Delphi conference
for consensus. The analysis consisted of constructing a summary
receiver operating characteristic curve and pooled estimates for
sensitivity and specificity using a random-effects model for meta-analysis.
The search yielded 52 publications and no unpublished studies. Eleven
of these studies met the authors’ inclusive criteria and provided
a total sample of 2,126 patients. There was significant heterogeneity
among the 11 studies. Subgroup analysis of the nine studies that
used traditional ELISA D-dimer methods yielded the most valid pooled
estimates. These had a sensitivity of 0.94 and a specificity of
0.45. Lower specificity occurred in the aged population. Prolonged
duration of symptoms decreased the sensitivity and specificity of
the test. The authors concluded that the ELISA D-dimer is highly
sensitive but nonspecific for detecting pulmonary embolus in the
clinical setting. The test, therefore, might help clinicians rule
out pulmonary embolism, especially in the face of low or low-to-moderate
pretest probabilities.
Brown MD, Rowe BH, Reeves MJ, et al. The accuracy
of the enzyme-liked immunosorbent assay D-dimer test in the diagnosis
of pulmonary embolism: a meta-analysis. Ann Emerg Med. 2002;40:133-144.
Reprint information not available.
Inhibin-A
as a marker for preeclampsia
The severe hypertensive complication of pregnancy known as preeclampsia
is one of the major causes of maternal mortality, particularly in
developing countries. It is associated with a fivefold increase
in perinatal mortality worldwide. Though the etiology of preeclampsia
is unknown, placental disorders are likely involved in the pathophysiologic
mechanism. An early and reliable placental marker could, therefore,
be extremely beneficial in screening for this condition. Inhibin-A
is a dimeric protein produced by the placental throphoblast during
pregnancy. Studies in the literature have reported that midtrimester
plasma levels of inhibin-A are significantly higher in pregnant
women who subsequently developed preeclampsia than in controls.
Other studies, however, suggest that inhibin-A may be a good predictor
of early-onset preeclampsia only because in later-onset preeclampsia
or gestational hypertension, no differences with normotensive pregnant
women were found. The authors evaluated maternal serum multiple
of median inhibin-A at midtrimester in women who subsequently developed
preeclampsia, gestational hypertension, and intrauterine growth
restriction (IUGR). They compared these results with those of controls.
This was done using a retrospective bank of stored serum originally
taken for Down syndrome screening over 15 to 18 weeks. There were
20 patients with gestational hypertension, 20 patients with preeclampsia,
10 patients with IUGR, and 40 controls. The authors failed to find
a statistically significant difference of inhibin-A multiple of
median values between the control group and the preeclamptic or
gestational hypertension groups. There was a statistically significant
elevation in the IUGR group compared with the control group. The
authors concluded that elevated maternal inhibin-A concentrations
in the second trimester are strongly associated with IUGR and not
with preeclampsia, as previously hypothesized.
D’Anna R, Baviera G, Corrado F, et al. Is
mid-trimester maternal serum inhibin-A a marker of preeclampsia
or intrauterine growth restriction? Acta Obstet Gynecol Scand.
2002;81:540-543.
Reprints: Rosario D’Anna, Via Setaioli, 15, 98121 Messina,
Italy; rosariodanna@tin.it.
Donor
hepatocytes and epithelial cells in recipients
of peripheral blood stem cells
Pluripotent bone marrow stem cells can differentiate into hematopoietic
or mesenchymal cell lineages. Studies in laboratory animals and
humans have indicated that bone marrow stem cells can develop into
hepatic oval cells, hepatocytes, cholangiocytes, skeletal-muscle
cells, astrocytes, and neurons. The authors studied biopsy specimens
of skin, liver, and gastrointestinal tract from recipients of peripheral
blood stem cells from HLA-matched, sex-mismatched siblings for the
presence of donor-derived epithelial cells and hepatocytes. They
sought to determine whether circulating stem cells have similar
potent potential. They examined biopsy specimens from liver, gastrointestinal
tract, and skin from 12 patients who had undergone transplantation
of hematopoietic stem cells from peripheral blood (11 patients)
or bone marrow (one patient). Six female patients received a transplant
from a male donor; five had received a sex-matched transplant and
one had received an autologous transplant. Hematopoietic stem cell
engraftment was verified by cytogenetic analysis or restriction-fragment–length
polymorphism analysis. Biopsies were studied for the presence of
donor-derived epithelial cells or hepatocytes with the use of fluorescence
in situ hybridization of interphase nuclei and immunohistochemical
staining for cytokeratin, CD45, and a hepatocyte-specific antigen.
All six recipients of the sex-mismatched transplants showed evidence
of complete hematopoietic donor chimerism. XY-positive epithelial
cells or hepatocytes counted for zero to seven percent of the cells
in histologic sections of the biopsy specimens. These cells were
detected in liver tissue as early as day 13 and in skin tissue as
late as day 354 post-transplant. The presence of donor cells in
the biopsy specimens did not seem to depend on the intensity of
the tissue damage induced by graft-versus-host disease. The authors
concluded that circulating stem cells can differentiate into mature
hepatocytes and epithelial cells of the skin and gastrointestinal
tract.
Korbling M, Katz RL, Khanna A, et al. Hepatocytes
and epithelial cells of donor origin in recipients of peripheral-blood
stem cells. N Engl J Med. 2002;346:738-746.
Reprints: Dr. Martin Korbling, University of Texas, M.D. Anderson
Cancer Center, Dept. of Blood and Marrow Transplantation, Box 423,
1515 Holcombe Blvd., Houston, TX 77030; mkorblin@mdanderson.org.
Studies
of oxidized lipoproteins, macrophages
Oxidized low-density lipoproteins (oxLDL) are generated in vitro
by auto-oxidation in the presence of transition metals or in vivo
via cell-mediated mechanisms. This chemical species has been shown
to be a powerful regulator of cell signaling in provoking various
responses. Among these are the expression of adhesion molecules
on endothelial cells, production of proinflammatory cytokines and
growth factors by vascular cells, or proliferation and migration
of vascular cells. OxLDL is also a potent chemoattractant for circulating
monocytes and a differentiating agent that promotes the transition
of macrophages to lipid-loaded foam cells. Receptor-mediated endocytosis
of oxLDL by several scavenger receptor molecules is implicated in
the process of atherogenesis. Atherosclerosis is considered a problem
of wound healing and chronic inflammation and can be viewed as a
response to injury, with lipoproteins or other risk factors as the
injurious agents, keeping in mind the idea that accumulation of
lipid-loaded foam cells in fatty streaks is a primary event in disease
progression. Activation of macrophages may elicit an oxidative burst
in response to agonists such as oxLDL. The authors questioned whether
an acute response to oxLDL might provoke an oxidative response in
macrophages, whereas a late answer may attenuate reactive oxygen
radical (ROS) production. Peroxisome proliferator-activated receptors
(PPARs) are a group of lipid-activated nuclear receptors that heterodimerize
with the 9-cis-retinoic acid receptor to form functional transcription
factors that regulate genes involved in lipid and glucose metabolism.
Exposure of monocytes and macrophages to oxLDL provokes activation
and expression of PPARg. The authors proposed an attenuated oxidative
response via activation of PPARg in macrophages after oxLDL preincubation
and that the oxLDL not only generates ROS on first contact but also
promotes PPARg activation, which in turn desensitizes macrophages—that
is, reduces ROS production. In contrast, preincubation of macrophages
with oxLDL for 16 hours showed an attenuated oxidative burst on
second contact with oxLDL. The authors then showed that a PPARg
agonist such as ciglitazone attenuated the reactivated oxygen species
formation. Major lipid peroxidation products of oxLDL, such as 9-
and 13-hydroxyoctadecadienoic acid, shared that performance. The
authors concluded that oxLDL not only elicits an oxidative burst
on first contact but also promotes desensitization of macrophages
via activation of PPARg. Desensitization of macrophages may have
important consequences for the behavior of macrophages and foam
cells in atherosclerotic lesions.
Fischer B, von Knethen A, Brune B. Dualism of oxidized lipoproteins in provoking
and attenuating the oxidative burst in macrophages: role of peroxisome proliferator-activated
receptor-γ.1 J Immunol. 2002;168:2828–2834.
Reprints: Dr. Bernhard Brune, Faculty of Biology, University of Kaiserslautern,
Erwin Schrodinger Strasse, 67663 Kaiserslautern, Germany; bruene@rhrk.uni-kl.de
Role
of procalcitonin levels in the prediction of sepsis
The number of patients presenting with sepsis or septic shock has
been steadily increasing. Emergency medicine departments should
expect to see a greater number of patients with this problem in
the future. In the emergency department, the focus will likely be
on differential diagnosis and investigating the clues that support
a diagnosis of sepsis. In this setting, prompt decisionmaking is
critical because early diagnosis not only decreases the mortality
rate but is necessary to perform further therapy simultaneously.
A number of biologic parameters have been described in the literature
that may be useful in the rapid diagnosis of sepsis, besides the
classic bacterial examinations. One of these is procalcitonin, which
was recently discovered to be a marker of bacterial infection. Several
animal and human studies of sepsis have shown a sustained increase
in the concentration of plasma procalcitonin that was ultimately
identified by a highly specific marker itself. The authors conducted
a prospective study to determine the accuracy of procalcitonin,
C-reactive protein (CRP), and white blood cell count (WBC) among
patients who have at least two criteria of systemic inflammatory
response syndrome (SIRS) and clinically suspected and nonsuspected
sepsis and who were admitted to the emergency department. Thirty-four
patients with signs of SIRS were enrolled in the study between January
1999 and September 2000. These patients were divided into two groups
according to nonsuspected sepsis and suspected sepsis clinically.
The admission procalcitonin level was significantly higher in the
suspected sepsis group, with a median value of 68.7 µg/L,
than in the unsuspected sepsis group, with a median value of 0.23
µg/L. The procalcitonin levels were compared with WBC and
CRP levels. The predictive accuracy for sepsis, expressed as the
area under the receiver operating characteristic curve, was .88
for procalcitonin, .44 for WBC, and .34 for CRP. The authors concluded
that procalcitonin probably can be used as a predictive marker in
bacterial infections in emergency departments.
Guven H, Altintop L, Baydin A, et al. Diagnostic
value of procalcitonin levels as an early indicator of sepsis. Am
J Emerg Med. 2002;20:202-206.
Reprints: Dr. Hakan Guven, Dept. of Emergency Medicine, Ondokuz
Mayis University School of Medicine, 55139 Samsun, Turkey.
Clinical
pathology abstracts editors
Michael Bissell, MD, PhD, MPH, professor and director of clinical
services and vice chair, Department of Pathology, Ohio State University
Medical Center, Columbus.
Ronald Domen, MD, professor of pathology, medicine, and humanities,
Penn State University College of Medicine, Hershey, Pa.
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