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March 2006

Editors:
Michael Bissell, MD, PhD, MPH
Ronald Domen, MD

bullet Performance of different natriuretic peptide assays

Of all investigated neurohormones and natriuretic peptides, Btype natriuretic peptide and N-terminal proBNP are the best markers for ruling out left ventricular dysfunction. Some studies have also proposed N-terminal pro-A-type natriuretic peptide (NT-proANP) as a useful marker for diagnosing left ventricular dysfunction (LVD). A consistent finding among all reports is the excellent negative predictive value of B-type natriuretic peptide (BNP). Furthermore, BNP has a good negative likelihood ratio for diagnosing LVD compared with standard clinical indices, such as clinical history, electrocardiogram, and chest x-ray. These clinical results led to the development of numerous commercial assays to determine different natriuretic peptide hormones. However, different epitopes and fragments of the same analyte are detected by different assays, and cross-reactivities of antibodies with prohormone fragments may vary. Because natriuretic peptide assays are not standardized, one must use caution when interpreting clinical study results when using different assays. The authors conducted a study to investigate which of the natriuretic peptides—BNP, N-terminal proBNP (NT-proBNP), or NT-proANP—performs best in diagnosing mild forms of LVD. They also investigated the impact of using different assays on the diagnostic performance of these natriuretic peptides. The authors measured BNP (Triage BNP), NT-proBNP (Biomedica), and NT-proANP (Biomedica) in 130 consecutive patients (age range, 28 to 38 years) who had clinically suspected mild LVD. In patients with sufficient sample volume, the authors measured BNP and NT-proBNP using additional assays (Shionoria and Roche, respectively). The authors found that BNP and NT-proBNP were the best markers for identifying patients with mild systolic LVD, with mean (95 percent confidence interval) areas under the curves (AUC) of 0.78 (0.63–0.89) and 0.75 (0.58–0.87), respectively. However, the diagnostic performance of NT-proANP [AUC, 0.64 (0.48–0.77)] was significantly worse than that of BNP (P=0.014). Both BNP assays (Triage and Shionoria) and both NT-pro- BNP assays (Biomedica and Roche) performed equally well for diagnosing systolic LVD, despite the poor agreement between NT-pro- BNP assays. In patients with isolated diastolic LVD, the diagnostic performance of the Triage BNP [AUC, 0.70 (0.56–0.81)] was significantly better (P=0.006) than that of Biomedica NT-proBNP [0.49 (0.34–0.65)]. Furthermore, the performance of the Biomedica NT-proBNP assay was significantly worse (P=0.03) than that of the Roche NT-proBNP assay for diagnosing isolated diastolic LVD. The authors concluded that the performance of BNP for the diagnosis of systolic or diastolic LVD is not affected by the assay used, whereas the performance of NT-proBNP for the diagnosis of isolated diastolic LVD is assay dependent.

Hammerer-Lercher A, Ludwig W, Falkensammer G, et al. Natriuretic peptides as markers of mild forms of left ventricular dysfunction: effects of assays on diagnostic performance of markers. Clin Chem. 2004;50:1174–1183.

Reprints: Angelika Hammerer-Lercher, Dept. of Medical Chemistry and Biochemistry, Division of Clinical Biochemistry, Innsbruck Medical University, Fritz- Pregl Strasse 3, A-6020, Innsbruck, Austria; Angelika.Lercher@uibk.ac.at

bullet Levels of C-reactive protein following myocardial infarction

Inflammation, manifested by elevated serum levels of C-reactive protein measured by high-sensitivity CRP assay, is associated with an increased risk of cardiovascular events. Little is known, however, about whether elevated serum CRP levels reflect an increased tendency for plaque rupture or only a high atherosclerotic burden. It is well recognized that myocardial damage promotes the synthesis of CRP, and the level of CRP has been reported to be associated with poor prognosis after acute myocardial infarction (AMI). However, CRP is primarily synthesized and secreted rapidly in the liver six hours after an acute inflammatory stimulus. Thus, serum levels of CRP within six hours after the onset of AMI are suggested to offer valuable information about cell biology activity on ruptured plaque without being influenced by the effects of myocardial necrosis after AMI. The authors conducted a study in which they enrolled patients with AMI who were undergoing primary percutaneous coronary intervention within six hours of the onset of symptoms in order to evaluate whether serum high-sensitivity CRP (hs-CRP) levels are elevated prior to cardiomyocyte damage following AMI. CRP was prospectively measured by hs-CRP in 157 consecutive patients (106 patients within six hours and 51 patients at six hours or more but less than 12 hours after the onset of AMI), with ST-segment elevation AMI undergoing primary percutaneous coronary intervention. Serum levels of hs-CRP were also measured in 30 patients with stable angina who were undergoing elective percutaneous coronary intervention and in 30 healthy control subjects. The serum level of hs-CRP was significantly higher in patients with onset of AMI at less than six hours than in patients with angina pectoris (2.7±2.3 mg/L versus 1.4±0.7 mg/L, P<0.0001 [mean ± SD]) and in healthy subjects (2.7±2.3 mg/L versus 1.0±0.6 mg/L, P<0.0001). There were no significant differences in serum levels of hs-CRP in patients with onset of AMI at three hours or less than in those patients with onset of AMI after three hours but before six hours (2.7±2.5 mg/L versus 2.7±2.2 mg/L, P=0.87). However, the serum level of hs-CRP was significantly higher in patients with onset of AMI at six hours or more than in patients with onset before six hours (14.1±16.5 mg/L versus 2.7±2.3 mg/L, P<0.0001). The authors concluded that serum levels of hs-CRP were significantly higher in patients with onset of AMI before six hours than in healthy subjects and patients with angina pectoris undergoing percutaneous coronary intervention. The inflammatory process has been proven to be one of the mechanisms causing plaque rupture. Elevated serum hs-CRP levels in patients with AMI at less than six hours may portend vulnerable plaque rupture.

Yip HK, Wu CJ, Chang HW, et al. Levels and values of serum high-sensitivity C-reactive protein within 6 hours after the onset of acute myocardial infarction. Chest. 2004;126:1417–1422.

Reprints: Dr. Morgan Fu, Division of Cardiology, Dept. of Internal Medicine, Chang Gung Memorial Hospital, Kaohsiung, 123, Ta Pei Rd., Niao Sung Hsiang, Kaohsiung Hsien, 83301, Taiwan, ROC; tang@adm.cgmh.org.tw

bullet Acquired activated protein C resistance

Anti-phospholipid antibodies are a heterogeneous group of autoantibodies that includes anti-cardiolipin/ beta-2 glycoprotein I (anti- CL/β2-GPI) antibodies, anti-phosphatidylserine/ prothrombin (anti- PS/PT) antibodies, and lupus anticoagulant. These antibodies are frequently found in the plasma of patients with systemic lupus erythematosus (SLE) and are reported to be associated with clinical events such as arterial or venous thrombosis, or both, thrombocytopenia, and obstetric complications. Thromboembolic events are reported to occur in approximately 30 percent of SLE patients with antiphospholipid (aPL) antibodies. Venous thromboembolic events, such as deep vein thrombosis and pulmonary embolism, are common manifestations in SLE patients. Although the association between aPL antibodies and venous thromboembolism in patients with SLE has been established, the precise mechanism responsible for venous thromboembolism in these patients remains unclear. Several clinical studies have established that the prevalence of venous thromboembolism is associated with congenital or acquired abnormalities of the protein C pathway. The authors of this study examined the concentration of anti-CL/β2-GPI antibody concentrations, anti-PS/PT antibody concentrations, and lupus anticoagulant activity in 87 patients with SLE—21 with venous thromboembolism and 66 without thrombosis. Both anti-CL/β2-GPI and anti-PS/PT antibodies strongly correlated with lupus anticoagulant activity. Multivariate logistic analysis confirmed that anti-CL/β2-GPI and anti-PS/PT antibodies were significant independent risk factors for venous thromboembolism (odds ratios, 4.98 and 7.54, respectively; 95 percent confidence intervals, 1.51 to 16.4 and 2.30 to 24.7, respectively). The authors, therefore, studied the in vitro effects of IgG fractions containing anti-CL/β2- GPI or anti-PS/PT antibodies on the anticoagulant activity of activated protein C (APC) and found that purified IgG containing anti- CL/β2-GPI or anti-PS/PT antibodies significantly hampered the anticoagulant activity of APC. They also studied the ability of IgG fractions to impede the anticoagulant activity of APC before and after complete removal of anti-CL/β2-GPI or anti-PS/PT antibodies by adsorption. The authors concluded that anti-CL/β2- GPI and anti-PS/PT antibodies independently cause APC resistance, which may contribute to the risk of venous thromboembolism in patients with SLE.

Nojima J, Kuratsune H, Suehisa E, et al. Acquired activated protein C resistance associated with IgG antibodies against β2-glycoprotein I and prothrombin as strong risk factor for venous thromboembolism. Clin Chem. 2005;51:545–552.

Reprints: Junzo Nojima, Laboratory for Clinical Investigation, Osaka University Hospital, 2-15 Yamadaoka, Suita, Osaka 565-0871, Japan; nojima@hp-lab.med.osaka-u.ac.jp

bullet Visual assessment of red cell unit hemolysis

Red blood cell units are visually inspected for hemolysis before they are released from blood collection facilities and again before transfusion. Although thresholds defining excessive hemolysis exist in Europe, and in the United States for product licensure (0.8 percent and one percent, respectively), there are no U.S. standards applying to blood for transfusion. Laboratories use free plasma or serum hemoglobin (Hb) methods to assess hemolysis in patients. Spectrophotometric methods, such as the tetramethylbenzidine (TMB) chemical method, have been the traditional gold standard for measuring free Hb. However, these tests are not well suited to a blood manufacturing setting because they are complex and labor intensive, require equipment not routinely available in a blood center, have standardization problems, and sometimes require use of carcinogenic chemicals. In the absence of standards defining excessive hemolysis in blood components, and with the absence of reliable, convenient tests to detect and quantify hemolysis, blood bankers have relied primarily on subjective visual inspection to determine a unit’s suitability for transfusion. However, user-friendly methods to quantitate hemolysis in blood components are now available. They provide a means to develop objective standards for hemolysis in blood components for transfusion. The authors conducted a study in which they collected packed RBCs (10 CPDA-1, 10 Adsol). Half of each unit was leukoreduced. Plasma Hb was measured and compared in segments and units using the HemoCue plasma/ low Hb photometer system, a TMB chemical method, and a free Hb visual comparator. The authors found that visual assessment tended to overestimate hemolysis. Chemical methods were comparable (r2=0.894; HemoCue=0.043 + [0.770] × TMB; n=400; range, 0.01–0.05 g/dL), although the mean plasma Hb (g/dL) for the HemoCue method was higher than that of the TMB method (0.12 versus 0.10 g/dL, respectively; P<0.001). No units would have been discarded based on a hemolysis level of at least 0.6 g/dL (approximately one percent) if measured by a chemical method. However, 50 percent of CPDA-1 and 10 percent of Adsol units would have been discarded if only visual criteria were used. Leukoreduction did not increase plasma Hb levels. Discrepancies in plasma Hb levels were noted between units and their corresponding segments. The authors concluded that visual assessment of hemolysis can result in unnecessary waste of blood components. Hemo- Cue offers an alternative, objective method to assess plasma Hb in the setting of blood collection and processing facilities for routine quality control and process validation. Furthermore, it may aid in developing objective criteria for excessive hemolysis in blood components.

Janatpour KA, Paglieroni TG, Crocker VL, et al. Visual assessment of hemolysis in red blood cell units and segments can be deceptive. Transfusion. 2004;44:984–989.

Reprints: Dr. Kim Janatpour, University of California Davis Medical Center, Dept. of Pathology and Laboratory Medicine, 4400 V Street, Sacramento, CA 95817; kim.janatpour@ucdmc.ucdavis.edu

bullet POCT hemoglobin for field use in the Third World

The Malawi government’s Ministry of Health and Population has developed an essential health package, which aims to provide better access to quality services for common conditions that disproportionately affect the poor. To complement the essential health package, the ministry is developing a model package of essential laboratory services to improve the cost-effectiveness of district hospital laboratories. The government is piloting the model in select typical district hospitals in Malawi before releasing it nationally. Hemoglobin measurement is a key component of this service, so it is necessary to identify a method that is practical, accurate, and economically viable for use at the district level. In subSaharan Africa, hemoglobin estimations are used in district hospitals for individual patient management, to guide transfusion practice, and in the management of antiretroviral therapy. Surveys of hemoglobin concentrations are also used as tools to provide public health data, such as nutritional status, and to monitor malaria interventions. Despite the wide range of manual methods available for measuring hemoglobin in developing countries, no single technique has emerged as the most appropriate for this setting. To support its essential health package, the Ministry of Health and Population needed to identify a method for measuring hemoglobin that was simple, accurate, fast, and inexpensive and that required minimal training and supervision and could be used in district hospital laboratories. Therefore, the authors assessed the effectiveness, including technical, economic, and managerial aspects, of different manual techniques for measuring hemoglobin in routine practice in a typical district hospital in Malawi against criteria predetermined by local health professionals. To the authors’ knowledge, this is the first time that such a detailed effectiveness study, involving all the processes required to determine the suitability of a test, has been undertaken in a district hospital in a developing country. The project received ethical approval from the Liverpool School of Tropical Medicine, in the United Kingdom, and Malawi’s Ministry of Health and Population. Local health professionals determined criteria on accuracy, clinical usefulness, user friendliness, speed, training time, and economic costs and used them to compare six different manual hemoglobin methods. These methods were introduced sequentially in a district hospital in Malawi alongside the reference method. The authors determined that HemoCue was the optimal method based on most of the outcome measures, but it was also the most expensive ($0.75 per test in U.S. dollars). The DHT meter and Jenway colorimeter were the second choices because they were less expensive ($0.20 to $0.35 per test), but they were not as accurate or user-friendly as the HemoCue.

Medina Lara A, Mundy C, Kandulu J, et al. Evaluation and costs of different haemoglobin methods for use in district hospitals in Malawi. J Clin Pathol. 2005;58:56–60.

Reprints: Dr. A. Medina Lara, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, United Kingdom; amedina@liverpool.ac.uk

bullet Direct identification of gram-positive cocci in blood cultures

A rapid and accurate method for directly identifying pathogens from positive blood culture broth, combined with automated, continuously monitored blood culture systems, may greatly reduce the overall time from specimen collection to final identification. However, relatively few methods for directly identifying bacteria from blood culture broth have been described. Standard assays comprising the immunological, tube coagulase, and stable endonuclease methods routinely used for identifying Staphylococcus aureus after subculture have been applied directly to positive blood culture bottles, but variable sensitivities and specificities have been reported. The use of PCR to directly identify Streptococcus pneumoniae from blood has also been described, but overall, its use for this purpose has been rather limited owing to the amplification inhibitors in blood. The extreme sensitivity of the method may also be a problem. However, rapid identification of S. aureus from blood culture using the LightCycler system has already been reported, and the use of real-time fluorescence PCR for rapid identification of bacteria from blood culture may increase in the future. Fluorescent in situ hybridization, as well as the hybridization protection assay, have also proved to be reasonable methods for directly identifying microorganisms from blood. The commercially available DNA probe kit AccuProbe (Gen-Probe, San Diego), which uses hybridization protection assay technology, can directly identify the most common gram-positive bacteria in blood cultures. The authors reported on their six-year routine clinical laboratory experience using AccuProbe for direct identification of S. aureus, S. pneumoniae, enterococci, and group Aand B streptococci from positive blood culture bottles. With the enterococcal and group Aand B streptococcal probes, the diagnostic performance of the test was excellent at a cutoff value of 50,000 relative light units (RLU), as recommended by the manufacturer. However, with the S. aureus probe, the specificity was very high (99.8 percent), but the sensitivity was low (72.4 percent). To improve the clinical usability of the direct AccuProbe identification, optimal cutoff values for the individual AccuProbe tests were defined using receiver-operating characteristic analysis. Consequently, cutoff values for S. aureus and S. pneumoniae tests were adjusted to 30,000 RLU for enterococci and 55,000 RLU for group Aand B streptococci. With these adjustments, the performance of the AccuProbe tests, especially that for S. aureus, was significantly improved.

Lindholm L, Sarkkinen H. Direct identification of gram-positive cocci from routine blood cultures by using AccuProbe tests. J Clin Microbiol. 2004;42:5609–5613.

Reprints: Laura Lindholm, Päijät-Häme Central Hospital, Dept. of Clinical Microbiology, Keskussairaalankatu 7, FIN-15850, Lahti, Finland; laura.lindholm@phks.fi


Dr. Cibull is professor of pathology and laboratory medicine and direct of surgical pathology, University of Kentucky Medical Center, Lexington. Dr. Lele is assistant professor of pathology and laboratory medicine, University of Kentucky Medical Center. Dr. Kesler is hematopathology fellow, University of Texas Southwestern Medical Center at Dallas.