College of American Pathologists
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  Clinical Abstracts





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July 2001

CRP and fibrinogen levels in prognosis of ischemic stroke
Long-term studies of apparently healthy individuals have shown that increased fibrinogen levels are an important factor in future cardiovascular events. Fibrinogen is an acute-phase reactant protein that may indicate an inflammatory condition in ischemic stroke. Other signs of inflammation in ischemic stroke include increased levels of inflammatory cytokines and C-reactive protein. Recent studies have shown an association of increased CRP levels with a more adverse, short-term prognosis in patients with ischemic stroke. Prospective studies of patients with angina pectoris have shown a strong correlation between fibrinogen and CRP levels. The authors investigated and compared the one-year prognostic value of fibrinogen and CRP levels with respect to outcome in a well-defined population of patients with first-time ischemic stroke. They studied 128 patients admitted to a hospital in Italy with the diagnosis of ischemic stroke. They determined fibrinogen and CRP levels within 24 hours of the stroke and studied the followup of all patients with survival analysis by the Kaplan-Meier technique. Multiple logistic regression analysis was used to evaluate any association between risk factors and outcome. When all patients were stratified with respect to their tertiles of fibrinogen level, their probabilities of death or a new vascular event were 21.1 percent, 27.9 percent, and 51.7 percent, respectively, for the three tertiles of fibrinogen level. Similarly, their probabilities for a fatal or secondary vascular event outcome were 12.1 percent, 29.7 percent, and 54.8 percent, respectively, after stratification by the tertiles of CRP level. In multiple logistic regression analysis, higher CRP levels and stroke severity on the Canadian Neurological Stroke Scale were independently associated with death or a new vascular event. The authors concluded that increased levels of CRP are associated with the worst outcome in patients with ischemic stroke.

Di Napoli M, Papa F, Bocola V. Prognostic influence of increased CRP and fibrinogen levels in ischemic stroke. Stroke. 2001;32:133-138.

Reprints: Dr. Mario Di Napoli, Dept. of Neurology and Neurorehabilitation, Casa di Cura Villa Pini dAbruzzo, Via dei Frentani 228, 66100 Chieti, Italy

Use of botulinum toxin in bio-terrorism
Botulinum toxin potentially poses a major threat as a biological weapon because it is an extremely potent and lethal compound that is easily produced, transported, and misused. Subjects exposed to the compound require prolonged intensive care. Treatment of botulism requires prompt provision of botulinum antitoxin and, often, mechanical ventilation. An outbreak of botulism is a public health emergency that requires intervention to prevent additional cases and should be immediately reported to public health officials. Although botulinum toxin recently has been licensed in the United States as a pharmaceutical agent for treating various conditions, it is the most poisonous substance known. A single gram of crystalline toxin evenly dispersed and inhaled would kill more than 1 million people, though disseminating the compound in this manner would be extraordinarily difficult. The compound is an exquisitely precise blocker of acetylcholine released into the neuromuscular junction. The report of the Working Group on Civilian Biodefense offers consensus-based recommendations on what public health and medical professionals should do if botulinum toxin is used as a biological weapon against a civilian population. The working group is composed of 23 representatives from academic, government, and private institutions with expertise in public health, emergency management, and clinical medicine. The group systematically reviewed the world literature on botulinum toxin as a biological weapon. The first draft of the working group’s statement was issued in May 1999 and has undergone subsequent revision and input from a variety of sources worldwide. The final statement incorporates all relevant evidence obtained through this process. Conclusions of the statement are: “An aerosolized or foodborne botulinum toxin weapon would cause acute symmetric, descending flaccid paralysis with prominent bulbar palsies such as diplopia, dysarthria, dysphonia, and dysphagia that would typically present 12 to 72 hours after exposure. Effective rsponse to a deliberate release of botulism toxin will depend on timely clinical diagnosis, case reporting, and epidemiological investigation.” Those with signs of botulism require prompt treatment with antitoxin and supportive care, possibly assisted ventilation for weeks or months. Treatment with antitoxin should not be delayed for purposes of microbiological testing.

Arnon SS, Schechter R, Inglesby TV, et al. Botulinum toxin as a biological weapon: medical and public health management. JAMA. 2001;285:1059-1070.

Reprints: Dr. Stephen S. Arnon, Infant Botulism Treatment and Prevention Program, California Dept. of Health Services, 2151 Berkeley Way, Room 506, Berkeley, CA 94704

Homocysteine levels in type II diabetes
Total homocysteine level can be greatly increased by the rate of glomerular filtration. Glomerular hypofiltration is not uncommon in type II diabetes. Little is known about the relationship between total homocysteine levels and the degree of insulin resistance in type II diabetes. The authors, therefore, prospectively studied a cohort of type II diabetic patients and attempted to correlate total homocysteine levels to the degree of macroangiopathic and nephropathic changes. They used the homeostasis model assessment to analyze the relationship between total homocysteine levels and degree of insulin resistance. The authors measured fasting total homocysteine levels in 122 type II diabetic patients for whom chronic complications had been recorded. They also studied the relationship between plasma total homocysteine and the degree of insulin resistance as measured by homeostasis model assessment. Sixty-nine percent of the patients had normal values for total homocysteine, and 31 percent had elevated values. The group with elevated homocysteine had a higher prevalence of macroangiopathy and an especially high prevalence of coronary artery disease. Impaired renal function also was more prevalent in this group. The group with high homocysteine levels had lower levels of folic acid than the group with normal homocysteine levels, but other clinical and biological characteristics were comparable, and no differences between the groups were found for microalbuminuria, retinopathy, or neuropathy. Likewise, the degree of insulin resistance was similar in the two groups, as was assessment of beta cell function. When treatments were considered, no differences in total homocysteine levels were noted between subjects receiving metformin and those not receiving metformin; however, diabetics treated with fibrates showed higher homocysteine levels.

Buysschaert M, et al. Hyperhomocysteinemia in type 2 diabetes: relationship to macroangiopathy, nephropathy, and insulin resistance. Diabetes Care. 2000;23: 1816-1822.

Reprints: M. Buysschaert, Service d’Endocrinologie et Nutrition, Cliniques Universitaires St. Luc, Avenue Hippocrate 54, UCL 54.74, B-1200 Bruxelles, Belgium

Hypopituitarism and premature mortality
Patients with hypopituitarism have a variety of different endocrine deficiencies, all of which play an important role in pathophysiology. Patients undergo various treatments, including surgery and drug therapy, which might contribute to unfavorable outcomes. The authors prospectively examined total and specific-cause mortality in more than 1,000 patients with hypopituitarism and attempted to identify factors that contribute to excess mortality. They performed subgroup analysis to explore questions that exist about mortality in hypopituitarism and examined the use of radiotherapy in the context of the condition. They followed 514 men and 500 women in the United Kingdom who were diagnosed with hypopituitarism from January 1992 to January 2000. Nonfunctioning pituitary adenomas were found in 573 of these patients, and 18 had craniopharyngiomas and 93 had prolactinomas. The authors calculated standardized mortality ratios as the ratio of observed death to the number of deaths in an age-matched and sex-matched British population. They observed 181 deaths compared with the 96.7 that were expected (standardized mortality ratio, 1.87). Mortality was higher in women with an SMR of 2.29 than in men with an SMR of 1.57. It was also higher in younger patients—those with an underlying diagnosis of craniopharyngioma—and in 353 patients who had been treated with radiotherapy (SMR, 2.32). Excess mortality was attributed to cardiovascular disease (SMR, 1.82), respiratory disease (SMR, 2.66), and cerebrovascular disease (SMR, 2.44). The only effect of hormonal deficiency on mortality was gonadotropin deficiency, which was associated with excess mortality if untreated (SMR, 2.97). Age at diagnosis, gender, a diagnosis of craniopharyngioma, and untreated gonadotropin deficiency were found by multiple regression analysis to be significant independent risk factors for mortality.

Tomlinson JW, Holden N, Hills RK, et al. Association between premature mortality and hypopituitarism. Lancet. 2001;357:425-431.

Reprints: P. M. Stewart, Div. of Medical Sciences, Queen Elizabeth Hospital, University of Birmingham, Birmingham B15 2TH, United Kingdom

A proposed marker for renal function
Sequential measurements of serum creatinine concentration and creatinine clearance are inadequate as markers of glomerular filtration rate in patients with chronic renal failure. Creatinine concentration is influenced by muscle mass as well as the ingestion of high-protein meals. Furthermore, creatinine is a less than perfect marker for glomerular function since it is largely secreted in the tubules. The exogenous compound inulin is a more perfect measure of glomerular filtration but it is cumbersome, involving a timed infusion and measurements of radioactivity. The authors isolated a new compound from human urine that is apparently endogenously synthesized in most vertebrates. The compound, 2-(a-mannopyranosyl)-L-tryptophan (MPT), is a c-glycoside of the amino acid tryptophan. It apparently is stable in the body and is found in serum and cerebrospinal fluid, but its metabolism and physiologic role are not yet defined. It has been observed that serum MPT concentrations increase progressively as renal function declines in patients with chronic renal failure. The authors designed a study to determine whether serum concentrations of MPT could be used as a marker of renal function. They compared the clearances of MPT and inulin in normal rats and in rats with Cisplatin-induced acute renal failure. They also compared clearance of MPT and creatinine with inulin clearance in 25 patients with chronic renal disease. They further determined the serum concentrations of MPT and creatinine as a function of MPT clearance in 108 patients with chronic renal disease. MPT and inulin concentrations correlated very strongly in rats (r=0.97) and humans (r=0.87). Linear regression analyses indicated that MPT was a better indicator of inulin clearance in humans than was creatinine. Serum creatinine concentrations tended to be lower in patients with less muscle mass, whereas serum MPT concentrations were not affected by creatinine excretion. The authors proposed using a measurement of MPT clearance as a replacement for inulin clearance in the clinical setting and suggested that it is a better indicator of renal function than is creatinine concentration.

Takahira R, Yonemura K, Yonekawa O, et al. Tryptophan glycoconjugate as a novel marker of renal function. Am J Med. 2001;110:192-197.

Reprints: Dr. Katsuhiko Yonemura, Hemodialysis Unit, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu, 431-3192, Japan

Carbohydrate-deficient transferrin determined using CZE
Carbohydrate-deficient transferrin is considered the most clinically useful marker for monitoring chronic alcohol abuse. The most commonly occurring isoforms of transferrin occur as a result of variations in the carbohydrate content. Normal serum contains high concentrations of tetrasialotransferrin and low amounts of disialo-, trisialo-, and pentasialotransferrin. In alcoholics, serum typically shows increased fractions of di-, mono-, and asialylated transferrins. The half-life of the CDT marker is approximately 16 to 17 days. Chemical methods for CDT have used isoelectric focusing, anion- exchange chromatography, chromatofocusing, high-pressure liquid chromatography, and fast protein liquid chromatography. These methods have been too cumbersome for rapid clinical use. More recently, commercial methods for more rapid testing have used anion-exchange chromatography followed by radioimmunoassay, enzyme immunoassay, or turbidimetric immunoassay. Capillary isoelectric focusing was successfully applied to the measurement of CDT in 1989. The authors described a new capillary zone electrophoresis method for determining CDT. This method uses a Beckman P/ACE 5000 CZE unit. The method resolves separation of each of the different CDT fractions. The efficiency of separation is enormous, with the number of theoretical plates ranging from 200,000 to 300,000 for the various fractional separations. Separations are so good that carbohydrate-deficient glycoprotein syndrome could be detected, allowing false-positive results to be excluded. The method does not require sample pretreatment. It is a rapid method with good precision, which correlates well with anion-exchange chromatography. CDT values in 130 healthy nonalcoholics ranged from 1.84 percent (2.5th percentile) to 6.79 percent (97.5th percentile).

Wuyts B, Delanghe JR, Kasvosve I, et al. Determination of carbohydrate-deficient transferrin using capillary zone electrophoresis. Clin Chem. 2001:47;247-255.

Reprints: J. R. Delanghe, Dept. of Clinical Chemistry, Ghent University Hospital, De Pintelaan 185, B9000 Ghent, Belgium

Ruling out pulmonary embolism through use of bedside D-dimer assay and alveolar dead-space determination
An abnormal D-dimer measurement suggests an intravascular thrombus, and a measurement indicating an elevated alveolar dead-space is suggestive of pulmonary vascular obstruction. Alveolar dead-space volume occurs in parts of the lung that are ventilated but not perfused and that contain a very low partial pressure of carbon dioxide. This alveolar dead-space volume can be estimated by simultaneously measuring CO2 in exhaled breaths and the arterial partial pressure of CO2. Previous observations have suggested that finding a normal D-dimer and a normal alveolar dead-space volume could be used to exclude the diagnosis of pulmonary embolism in emergency room patients. The authors designed a prospective study involving 380 stable patients aged 18 years and older who had suspected acute pulmonary embolus. These patients were treated in the ERs of six U.S. urban teaching hospitals. Measurements of D-dimer and alveolar dead-space volume were obtained prior to standardized testing for pulmonary embolism. The standardized testing consisted of radionuclide lung scanning or contrast-enhanced computed tomography followed by a six-month followup, as well as venous ultrasonography and pulmonary angiography on a selective basis. All the imaging studies were interpreted by blinded observers. A positive screening test result was defined as an abnormal alveolar dead-space measurement or an abnormal D-dimer assay. Pulmonary embolism was diagnosed in 64 patients, of whom 20 had abnormal D-dimer results, three had abnormal alveolar dead-space determinations, and 40 had a combination of abnormal results in both assays. One of the 64 patients had normal results for both tests. Pulmonary embolism was not found in 316 patients. In this group, D-dimer and dead-space results were normal in 163. The sensitivity for diagnosis of pulmonary embolism was 98.4 percent, specificity was 51.6 percent, and posterior probability of pulmonary embolism with normal results on both tests was less than one percent.

Kline JA, et al. Diagnostic accuracy of a bedside D-dimer assay and alveolar dead-space measurement for rapid exclusion of pulmonary embolism. JAMA. 2001;285(6):761-768.

Reprints: Dr. Jeffrey A. Kline, Dept. of Emergency Medicine, Carolinas Medical Center, Charlotte, NC 28232-2816;

Measuring CMV infection in transplant recipients
Infection with cytomegalovirus is a frequent and sometimes life-threatening complication for those individuals in immunosuppressed states. Effective treatments are available, but they require use of a laboratory determination capable of accurately predicting the development of CMV disease. They must also allow active infection to be detected early. Pp65 antigenemia is a widespread method for quantifying CMV viral load, but the conventional procedures used are relatively time-consuming. Several groups have developed methods for the direct quantification of CMV antigenemia by flow cytometry. These methods appear to be feasible and rapid and provide objective results. The authors prospectively collected and tested 1,305 blood samples from 85 solid-organ transplant recipients and 25 stem-cell transplant recipients at risk for cytomegalovirus infection. They used a leukocytic qualitative PCR and a shell vial assay. A subset of 462 specimens also was tested for CMV antigenemia by flow cytometry direct quantification. A quantitative PCR was used to test 125, and 200 were tested for pp65 antigenemia using a slide method. In 73 percent of cases, flow cytometry was the first test to show positivity. Of the methods compared, flow cytometry showed the highest sensitivity and negative predictive value for the diagnosis of CMV disease in solid- organ transplant recipients. In the solid-organ transplant and stem-cell transplant recipients, 63.5 percent and 36 percent, respectively, acquired active CMV infection. CMV disease occurred in 53 percent and 24 percent, respectively.

Poirier-Toulemonde AS, Milpied N, Cantarovich D, et al. Clinical relevance of direct quantification of pp65 antigenemia using flow cytometry in solid organ and stem cell transplant recipients. J Clin Microbiol. 2000;38:3143-3149.

Reprints: Berthe-Marie Imbert-Marcille, Laboratoire de Virologie, Institut de Biologie du Centre Hospitalier Universitaire de Nantes, 9 quai Moncousu, 44093 Nantes Cedex 01, France;