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May 2006

Editor:
Michael Bissell, MD, PhD, MPH

An outbreak of listeriosis linked to delicatessen turkey
Measuring tacrolimus concentrations using MEIA versus LC-MS/MS
Thrombomodulin as a ­tumor marker
Quality assurance of ­molecular coagulation testing
Vascular endothelial growth factor levels and sleep disorders
Inductively coupled plasma mass spectrometry

bullet An outbreak of listeriosis linked to delicatessen turkey

Listeria monocytogenes was estimated to have caused 2,500 infections and 500 deaths in the United States in 1997. Despite a decreasing incidence of listeriosis over the past two decades, new tools, such as molecular subtyping, have increased the number of recognized outbreaks. Between 1980 and 2000, 15 foodborne outbreaks of listeriosis were published or reported to the Centers for Disease Control and Prevention; 10 of these outbreaks have occurred since 1998, and three of these involved multiple states. Centralization of the processing and production of food, with its subsequent widespread distribution, increases the possibility of diffuse, widespread outbreaks of foodborne disease, including listeriosis, when food is contaminated during the production process. Such outbreaks may not be recognized at the local level if there are only a few cases in any one jurisdiction; there is no routine integrated analysis of case-specific data among states. The authors reported on an outbreak that was detected using molecular subtyping tools, which allowed them to link cases occurring in many states. To determine the magnitude of the outbreak and identify risk factors for infection, the authors notified state health departments and conducted a case-control study. A case was defined as a patient or mother-infant pair infected with L. monocytogenes whose isolate yielded the outbreak pulsed-field gel electrophoresis (PFGE) pattern. Controls were patients infected with L. monocytogenes whose isolate yielded a different PFGE pattern. Patients were asked about food and drink consumed during the 30 days before the onset of illness. Between May and December 2000, 30 clinical isolates of L. monocytogenes with identical PFGE patterns were identified in 11 U.S. states. Cases of infection caused by these isolates were associated with four deaths and three miscarriages. A case-control study implicated sliced processed turkey from a delicatessen (Mantel-Haenszel odds ratio, 8.0; 95 percent confidence interval, 1.2–43.3). A trace-back investigation identified a single processing plant as the likely source of the outbreak, and the company voluntarily recalled 16 million pounds of processed meat. The same plant had been identified in a Listeria contamination event that had occurred more than a decade before. The authors concluded that prevention of persistent L. monocytogenes contamination in food processing plants presents a critical challenge to food safety professionals.

Olsen SJ, Patrick M, Hunter SB, et al. Multistate outbreak of Listeria monocytogenes infection linked to delicatessen turkey meat. Clin Infect Dis. 2005;40:962–967.

Reprints: Dr. Sonja J. Olsen, CDC Box 68, American Embassy, APO AP 96546; sco2@cdc.gov

bullet Measuring tacrolimus concentrations using MEIA versus LC-MS/MS

Tacrolimus is a potent calcineurin inhibitor that is increasingly used for primary immunosuppression after liver transplantation because of its perceived advantages over cyclosporine. Microparticle enzyme immunoassay (MEIA) has been the mainstay for therapeutic monitoring of tacrolimus (TRL) for the past decade. However, the MEIA has a relatively high limit of detection and shows particularly poor precision at lower TRL concentrations, with a lower limit of quantification (LLOQ) of 3.1 µg/L. Recent developments in liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology may address this problem, delivering assays for TRL with rapid turnaround times and improvements in accuracy, precision, and sensitivity in the lower therapeutic range. The authors of this study compared an established LC-MS/MS method with MEIA in a large number of blood samples from a diverse range of patients. Their study attempts to explain the differences between the two methods in relation to the hematocrit, which has been noted to adversely affect the extraction of TRL (for MEIA) from whole blood and markers of hepatic and renal function (thought to modulate TRL clearance). The authors collected 1,156 blood samples from 277 adult and 121 pediatric recipients of liver, renal, and bone marrow grafts or hepatocyte or pancreatic islet cell implants. They measured TRL in whole blood by MEIA and LC-MS/MS and collected hematologic and biochemical data when available. LC-MS/MS was significantly more precise (P<0.02) than the MEIA, with increased sensitivity. The MEIA had a median difference of 16.2 percent versus LC-MS/MS overall, and this was significantly affected by patient cohort (P<0.001). The difference was greater in adult and pediatric liver graft recipients while they were inpatients versus outpatients (31.8 percent and 14 percent versus 7.5 percent and 6.55 percent, respectively). The difference was also greater in bone marrow than kidney graft recipients (32.8 percent versus 15.8 percent, respectively). Multiple linear regression analysis showed significant inverse relationships of this difference with hematocrit (packed cell volume) and plasma albumin (P<0.001) in the total cohort and a positive relationship with plasma bilirubin in a subgroup of liver graft recipients. The authors concluded that patients with a low packed cell volume and plasma albumin are likely to show artificially high concentrations of TRL when measured by MEIA. The increased risk of underimmunosuppression must be considered should doses be reduced to lower these seemingly high TRL concentrations.

Brown NW, Gonde CE, Adams JE, et al. Low hematocrit and serum albumin concentrations underlie the overestimation of tacrolimus concentrations by microparticle enzyme immunoassay versus liquid chromatography-tandem mass spectrometry. Clin Chem. 2005;51:586–592.

Reprints: Nigel W. Brown, Immunosuppressive Drug Monitoring, Institute of Liver Studies, King’s College Hospital, Denmark Hill, London SE5 9RS, United Kingdom; nigel.brown@kingsch.nhs.uk

bullet Thrombomodulin as a tumor marker

Thrombomodulin was first described as an integral component of the hemostatic pathway in 1981. Constitutively expressed on the vascular and lymphatic endothelium, it interacts with thrombin to form a high-affinity complex that inhibits thrombin activity (fibrin formation) and accelerates protein C activation. Thrombomodulin also reduces fibrinolysis by activating thrombin-activatable fibrinolysis inhibitor in plasma. Therefore, induced thrombomodulin deficiency (genetic knockout in mice) results in a hypercoagulable state with risk of arterial thrombotic disease. Soluble molecules of thrombomodulin, released from endothelial cell surfaces, are found in plasma and urine, where higher levels indicate injury or enhanced turnover of the endothelium, or both. It is not surprising, therefore, that smokers display increased serum soluble thrombomodulin concentrations that correlate with activated protein C levels, number of cigarettes smoked (per day), duration of smoking (years), degree of endo­thelial damage, and risk of thrombosis. Serum thrombomodulin elevations, by neutrophil-­mediated proteoly­tic/oxidative cleavage or hypoxia-induced shedding from the endothelium, parallel the activity of inflammatory diseases—for example, vasculitides and ulcerative colitis; ischemia reperfusion injuries—that is, vascular, cardiac, and transplant surgery; and atherosclerotic complications—for example, in hypertension, smoking, and diabetes. The clinical use of exogenous soluble infusions is approaching, with a recent report that administration of human urinary thrombomodulin limited ischemic injury, reperfusion sequelae, and systemic coagulopathy in a canine model of hepatic surgery. Early literature focused on thrombomodulin (then termed fetomodulin) as a placental surface protein, where it was identified as a marker of fetal endodermal differentiation essential for survival. However, since it was recognized that embryonic lethality was independent of its anticoagulant activity, the role of thrombomodulin in tumor biology has drawn clinical interest. The authors presented the key points in this genesis with a focus on future directions in cancer care. They reviewed the electronic literature (1966–2004), with a specific focus on tumor biology. They found that thrombomodulin is expressed on the endothelium and tumor cells in several cancers. Loss of expression denotes a more malignant profile with poorer prognosis. Loss of expression is mediated by hypoxia, endotoxin, and various cytokines, while up-regulation can be achieved by pharmacological manipulation—for example, pentoxyfylline and statins. The authors concluded that thrombomodulin plays a key role in tumor biology and prognostics and provides a potential therapeutic target in impeding the spread of cancer.

Hanly AM, Hayanga A, Winter DC, et al. Thrombomodulin: tumour biology and prognostic implications. EJSO. 2005;31:217–220.

Reprints: A.M. Hanly, Dublin 9, Ireland; amhanly@indigo.ie

bullet Quality assurance of molecular coagulation testing

Factor V Leiden and prothrombin G20210A mutations are the most common polymorphisms among whites that thus far have been associated with an increased risk of venous thrombosis. Another polymorphism in the MTHFR (methylene-tetrahydrofolate reductase) gene, namely the C677T thermolabile variant, seems to be a weak risk factor for venous thrombosis when inherited in the homozygous state with low or borderline folate intake. The clinical value of detecting these mutations among individuals and families with a history of venous thrombosis remains controversial. Notwithstanding this, there has been a dramatic increase in the number of laboratories performing these molecular genetic assays and in the number of test requests. Molecular genetic analysis to detect common thrombophilic mutations can detect a mutation without relying on phenotypic parameters. Unlike phenotypic testing, genotypes are unequivocal, with no borderline values. Accordingly, referring physicians typically do not question the reliability of the results. Because results of genetic analyses might have important clinical and family implications, it is vital that laboratories undertake stringent internal quality assurance in relation to staff and laboratory practice. A small number of external quality control surveys of thrombophilia mutations have been reported. These have indicated that while concordance is relatively high, analytic and transcription errors occur. Therefore, molecular laboratories should continue to participate in external quality assurance programs. The Royal College of Pathologists of Australasia quality assurance program has conducted external QA testing of factor V Leiden G1691A, prothrombin G20210A, and MTHFR C677T gene mutations for the past five years, including 133 DNA samples in 10 multi-laboratory surveys. Of 3,799 responses, the overall success rate was 98.63 percent; the poorest individual sample result was 15 percent incorrect for a homozygous factor V Leiden sample. Success rates for identifying specific mutations were 98.13 percent for factor V Leiden, 98.84 percent for prothrombin G20210A, and 99.3 percent for the MTHFR C677T mutation. Among responding laboratories, 51 percent (20/39) made at least one error; three of 39 laboratories were responsible for 46 percent of all errors (24/52). Although encouraging, these data underscore the need for molecular diagnostic laboratories to continue participating in external quality assurance programs to ensure the provision of quality genetic testing services.

Hertzberg M, Neville S, Favaloro E, et al. External quality assurance of DNA testing for thrombophilia mutations. Am J Clin Pathol. 2005;123:189–193.

Reprints: Dr. Mark Hertzberg, Dept. of Haematology, Westmead Hospital, Westmead, NSW 2145, Australia

bullet Vascular endothelial growth factor levels and sleep disorders

Patients with sleep-disordered breathing are at increased risk for hypertension and cardiovascular disease. However, the risk for cardiovascular disease is the same for patients with mild and more severe sleep-disordered breathing (SDB). Therefore, it is unclear whether repetitive hypoxic episodes during the night or other mechanisms are responsible for the link between cardiovascular disease and SDB. Vascular endothelial growth factor (VEGF) is a cytokine with potent angiogenic properties, inducing proliferation of endothelial cells and modulating thrombogenicity. Increased VEGF expression occurs following hypoxia by enhanced transcription of the VEGF gene mediated by the hypoxia inducible factor-1 (HIF-1). Hypoxia may be involved in the development of cardiovascular disease via increased VEGF expression in human atherosclerotic lesions. Recent studies have reported elevated blood VEGF levels in patients with episodic hypoxia due to sleep apnea. In most of these reports, VEGF was measured in serum rather than plasma, which may not reflect VEGF synthesis by peripheral tissue. However, plasma VEGF may reflect more closely free, circulating VEGF. Therefore, the authors conducted a study to investigate plasma concentrations of VEGF in patients with a variable degree of nocturnal hypoxia due to SDB. VEGF levels were assessed by enzyme-linked immunosorbent assay in the nonactivated (VEGFb1) and thrombin-stimulated platelet-rich plasma (VEGFprp) of 45 patients with SDB: group one patients with obstructive sleep apnea and an apnea-hypopnea index (AHI) greater than 15 per hour, group two subjects with an AHI of less than five per hour, and group three patients on continuous positive airway pressure treatment for sleep apnea. Thirty-nine patients were included in the final analysis. Patients in group one had a higher percentage time of sleep with oxyhemoglobin saturation (SaO2) less than 90 percent and a significantly lower mean and minimum overnight oxygen saturation than subjects in group two and group three (P<0.05). Despite significant differences in overnight oxygenation, VEGFb1 and VEGFprp concentrations were not significantly different between the three study groups. However, plasma levels of VEGFb1 were significantly higher (P=0.02) in SDB patients with arterial hypertension (n=19; VEGFb1, 14.0±3.3 pg/mL) than in those without arterial hypertension (n=20; VEGFb1, 10.9±5.2 pg/mL). There were no relationships between VEGF levels and polysomnographic oxygenation parameters. In univariate analysis, the authors observed significant relationships for VEGFb1 with body mass index (C, 0.393; P<0.05) and serum fibrinogen (C, 0.399; P<0.05). The authors concluded that circulating plasma VEGF levels in patients with SDB may be unrelated to nighttime hypoxemia.

Valipour A, Litschauer B, Mittermayer F, et al. Circulating plasma levels of vascular endothelial growth factor in patients with sleep disordered breathing. Res Med. 2004;98:1180–1186.

Reprints: Arschang Valipour, Dept. of Respiratory and Critical Care Medicine, Otto-Wagner-Spital Sanatoriumsstr. 2, 1140 Wien, Austria; valipour@wienkav.at

bullet Inductively coupled plasma mass spectrometry

Potential preanalytic sources of variation in trace metal concentrations during specimen storage include solvent and analyte evaporation, analyte adsorption to the surface of the container, specimen heterogeneity resulting from precipitation or bacterial growth, and specimen contamination. In an attempt to combat potential causes of specimen variation, chemical additives often have been added to preserve trace element concentrations in urine specimens. A variety of methods are used to preserve urine specimens for trace element analysis, including refrigeration. Acidification traditionally has been used to try to prevent adsorption of trace metals to container surfaces. Using additives such as acids also may retard precipitation of urine components. However, using additives such as strong hydrochloric or nitric acid complicates the sample preparation process, and the additives themselves are a potential source of sample contamination. In a few cases, such as in measuring urinary arsenic, chromium, and nickel, the use of preservative additives has been demonstrated to have no benefit. Inductively coupled plasma mass spectrometry (ICP-MS) analysis of trace elements is a powerful and sensitive technique for simultaneous multielemental analysis in clinical samples. The objective of the authors’ study was to evaluate the effects of several commonly available chemical additives (or no additive) and storage temperatures on trace element analysis by ICP-MS for 16 elements. A 24-hour urine specimen was supplemented with concentrations of the elements. Aliquots containing one of four chemical additives were stored at three temperatures in sealed polypropylene containers. Elemental concentrations were determined by ICP-MS for the resulting samples after two, three, eight, and 65 days of storage. Initial element concentrations measured within eight hours of specimen preparation were consistent with expected concentrations, except for aluminum. For most elements, preservation and storage conditions yielded consistent measured concentrations throughout the experiment. Notable exceptions were for aluminum (general rise over time) and mercury (general decrease over time). Adding boric acid and potassium pyrosulfate resulted in sample contamination; elemental contamination was concentration dependent for both. Although the authors observed little microbial contamination during the experiment, they recommend that samples be refrigerated to curtail bacterial growth in nonsterile specimens. In light of these results, the authors concluded that refrigerated urine storage without the use of chemical additives is an effective preservation method for ICP-MS analysis of many trace elements.

Bornhorst JA, Hunt JW, Urry FM, et al. Comparison of sample preservation methods for clinical trace element analysis by inductively coupled plasma mass spectrometry. Am J Clin Pathol. 2005;123:578–583.

Reprints: Dr. Gwen A. McMillin, ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108


Dr. Bissell is Professor and Director of Clinical Services and Vice Chair, Department of Pathology, Ohio State University Medical Center, Columbus.