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Technical Briefs |
1 Institute of Clinical Chemistry, University Hospital Zurich, Raemistrasse 100, CH-8091 Zurich, Switzerland2 Department of Laboratory Medicine, Kantonsspital Basel, University Hospital, Petersgraben 4, Basel, Switzerland
aauthor for correspondence: fax 41-1-255-4590, e-mail hmr{at}ikc.unizh.ch)
Clinical specimens from patients carrying highly infectious agents such as corona, Lassa, Ebola, or Marburg viruses may present a biohazard to laboratory workers. Although specialized medical microbiology laboratories amplify and analyze such viruses under the required biosafety measures, few general clinical laboratories have the equipment to perform their analyses without putting their personnel at risk. The recent spread of severe acute respiratory syndrome (SARS) has heightened concern about personnel safety.
Although the WHO (1) and the CDC(2) recommend that blood samples from patients in whom SARS is suspected should be analyzed under biosafety level II conditions, most high-throughput analyzers in the clinical chemistry laboratory use open tubes and therefore do not meet the biosafety level II standards. In such cases, the CDC recommends administrative measures and/or additional personal protective equipment to reduce risk. Some manufacturers announced that their analyzers should not be used for samples from patients in whom SARS is suspected, citing possible aerosol production during analysis. Hence, laboratory directors and biosafety officers are in the dilemma of how to offer urgent diagnostic and surveillance tests to clinicians and at the same time protect their coworkers from potential biohazards.
Gamma irradiation and heat inactivation procedures have been investigated for the mentioned lipid-enveloped viruses (3)(4). Gamma irradiation seems not to affect some tests, but the intensity necessary to inactivate viruses is usually not available in hospitals, and the procedure decreases activities of enzymes and results of coagulation tests (3)(5). Heat inactivation can be done simply in a biological safety cabinet with a waterbath at 60 °C, treating samples for 30 min to inactivate corona viruses or for 60 min to inactivate Lassa, Ebola, or Marburg viruses (3). Only limited and nonquantitative data are available on the effects of these procedures on tests for common, clinically important analytes (3)(4)(6). We examined the effect of these two heat inactivation procedures as well as the effect of a virus-envelope-destroying detergent on tests used in intensive and emergency care.
Residual routine blood samples were selected to cover a wide range of concentrations (Table 1
). Blood samples anticoagulated with either lithium heparinate or citrate (Vacutainer; Becton Dickinson) were centrifuged at 2500g for 10 min, and plasmas were transferred to plastic vials. The sealed plastic vials were totally immersed in a 60 °C waterbath for either 30 or 60 min, and the plasmas were analyzed together with a nontreated aliquot on a Roche-Hitachi Clinical Chemistry and Immunoassay Analyzer (Modular) with commercial tests from Roche Diagnostics GmbH and on a CA7000 Blood Coagulation Analyzer with commercial tests from Dade-Behring.
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After either heat inactivation procedure, measured concentrations ranged between 90% and 110% of pretreatment concentrations for electrolytes, creatinine, urea, uric acid, bilirubin, glucose, lactate, total protein, albumin, C-reactive protein, troponin T, the N-terminal fragment of pro-B-type natriuretic peptide, and the ß-chain of human chorionic gonadotropin (Table 1
). For thyroid-stimulating hormone, aspartate aminotransferase, and pancreatic amylase, measured concentrations were 
90% of values in untreated samples after the 30-min inactivation procedure and decreased to 70–80% after the 1-h incubation. In contrast, creatine kinase, myoglobin, alanine aminotransferase, 
-glutamyl transferase, lactate dehydrogenase, alkaline phosphatase, and the blood coagulation indicators were virtually inactivated, and free thyroxine was increased 2.4-fold after the shorter incubation procedure.
We searched for another inactivation method with minimal interference in these tests. Because Triton X-100 treatment of plasma samples inactivates other lipid-enveloped viruses, such as HIV and Berne viruses (7)(8), we investigated it. The inactivation procedure consisted of mixing (on a Vortex-type mixer) 1 mL of plasma and 10 µL of a solution containing 100 mL/L Triton X-100 and incubating the mixture for 60 min at room temperature. Measured concentrations ranged from 91% to 107% of untreated values for electrolytes, metabolites, enzymes, proteins, and hormones (Table 1
), offering a virus inactivation procedure for heat-labile analytes such as creatine kinase, myoglobin, alanine aminotransferase, -glutamyl transferase, lactate dehydrogenase, alkaline phosphatase, and free thyroxine. More importantly, even the results for blood coagulation indicators were little affected by the Triton X-100 inactivation procedure, although there was a 20% increase in the International Normalized Ratio and a 10% increase in the activated partial thromboplastin time.
To investigate whether Triton X-100 inactivation interferes with assays from other suppliers, we measured electrolytes, metabolites, and proteins on a RXL Dimension Clinical Chemistry Analyzer from Dade-Behring; hormones, B-type natriuretic peptide, and troponin I on an AxSYM Immunoassay Analyzer from Abbott; and the coagulation tests on a STA-R Blood Coagulation Analyzer from Stago. Results ranged between 96% and 109% of untreated values for all analytes, including the blood coagulation indicators, except the free thyroxine test on the AxSYM, which showed an increase to 122%. From these data it is tempting to speculate that the Triton X-100 inactivation treatment could be useful for most commercial assays on high-throughput analyzers. However, it may well be that certain assays are more influenced by the presence of 1 mL/L Triton X-100 than others.
These data suggest that it is possible to protect laboratory workers while offering essential analyses to the intensive or emergency care unit in case of a suspected viral infection with corona, Lassa, Ebola, or Marburg viruses. However, there are two limitations to the use of these inactivation procedures. The first limitation is that all of these procedures require that the transfer of plasma into the second tube and the inactivation procedure be performed by trained personnel in a biosafety level 2 safety cabinet under rigorous safety practices. The second limitation is that although there is evidence for an inactivation of these viruses by heat, there is evidence for effectiveness of Triton X-100 treatment only for the lipid-enveloped HIV and Berne viruses (7)(8) and no published investigation of inactivation of the lipid-enveloped corona, Lassa, Ebola, or Marburg viruses. Nevertheless, the presented data can serve as a resource to estimate the effects on an analyte when one of these inactivation procedures is used as a safety measure to protect workers from lipid-enveloped viruses.
Acknowledgments
We thank Ruth Böswald, Isabelle Peereboorn, Sabine Pfister, and the staff of the Institute of Clinical Chemistry of the University Hospital Zurich for their efforts to analyze the serum samples before and after the inactivation procedures.
References
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