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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.