The accuracy of gentamicin, netilmicin, and tobramycin concentration determinations by enzyme multiplied immunoassay technique (EMIT; Syva Corp., Palo Alto ...
Vol. 20, No. 6
JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1984, p. 1080-1082 0095-1137/84/121080-03$02.00/0 Copyright ©) 1984, American Society for Microbiology
Analytical Accuracy of Determinations of Aminoglycoside Concentrations by Enzyme Multiplied Immunoassay, Fluorescence Polarization Immunoassay, and Radioimmunoassay in the Presence of Heparin MARY E. O'CONNELL, KAREN L. HEIM, CHARLES E. HALSTENSON, AND GARY R. MATZKE* Drug Evaluation Unit, Regional Kidney Disease Program, Hennepin County Medical Center, and College of Pharmacy, University of Minnesota, AMinneapolis, Minnesota 55415 Received 18 May 1984/Accepted 19 August 1984
The accuracy of gentamicin, netilmicin, and tobramycin concentration determinations by enzyme multiplied immunoassay technique (EMIT; Syva Corp., Palo Alto, Calif.), fluorescence polarization immunoassay (TDx; Abbott Diagnostics, Irving, Tex.), and radioimmunoassay were compared in the presence of 0 to 3,000 USP units of porcine heparin per ml. Gentamicin, netilmicin, and tobramycin concentrations determined by EMIT decreased by 10 and 50% in the presence of 75 and 1,000 USP units/ml, 2 and 5 USP units/mi, and 2 and 7.5 USP units/mi, respectively. Accuracy of the TDx and radioimmunoassay determinations, however, were not affected by the presence of heparin. Blood samples for the determinations of gentamicin, netilmicin, and tobramycin by EMIT should not be collected in evacuated heparinized tubes. Several assays have been developed for the determination of aminoglycoside concentrations (1, 6). Recently, the enzyme multiplied immunoassay technique, fluorescence immunoassay, and fluorescence polarization immunoassay have become available (2, 3). In general, the analytical accuracy of these techniques has been equivalent (2, 3, 6, 8). However, the accuracy has been questioned in certain clinical situations, e.g., in patients with hyperbilirubinemia (15), hypoalbuminemia (7), and end-stage renal disease (13). Controversy also exists as to whether serum can be used interchangeably with heparinized plasma for the analysis of aminoglycosides by these techniques. The determination of aminoglycoside concentrations in heparinized plasma has been evaluated for some bioassays (5, 10, 11, 16), radioimmunoasays (RIAs) (5, 12), and the enzyme multiplied immunoassay technique (4, 5, 9, 10, 14). Gentamicin concentrations determined by bioassay have been reported to be 11.4% lower when measured in the presence of as little as 2 USP units of heparin per ml (11). Aminoglycoside concentration determinations by RIA, however, have not been altered by the presence of heparin (5, 12). Krogstad et al. (5) observed a 10% decrease in tobramycin and gentamicin concentrations when determined by the enzyme multiplied immunoassay technique with more than 10 and 100 USP units of heparin per ml, respectively. Nilsson et al. (10) and Knight and Ukena (4), however, reported no inhibitor effect on the enzyme multiplied immunoassay technique for the determination of gentamicin concentrations at heparin concentrations of up to 1,000 USP
(Portions of this work were presented at the 5th Annual Meeting of the American College of Clinical Pharmacy, San Diego, Calif., 1984.) MATERIALS AND METHODS Stock solutions (-10 p.g/ml) were prepared in healthy human volunteer serum from gentamicin (561 jig/mg; Sigma Chemical Co., St. Louis, Mo.), tobramycin (942 pLg/mg; Sigma Chemical Co.), and netilmicin sulfate (670 ,ug/mg; Schering-Plough Corp., Kenilworth, N.J.). Porcine heparin stock solutions of 10, 100 (Invenex, Chagrin Falls, Ohio), 1,000 (Lypo Med Inc., Chicago, Ill.), 5,000, and 10,000 (The Upjohn Co., Kalamazoo, Mich.) USP units/ml were obtained or prepared by dilution of a commercially available solution with saline. The samples containing final gentamicin, netilmicin, and tobramycin concentrations of ca. 3 and 7 [Lg/ml and 0, 1, 2, 3, 5, 7.5, 10, 12.5, 15, 17.5, 20, 25, 50, 75, 100, 300, 500, 1,000, and 3,000 USP units of porcine heparin per ml were freshly prepared at 22°C by dilution of the aminoglycoside and heparin stock solutions. Each sample was promptly vortexed and separated into three portions. The portions were immediately assayed in duplicate. Gentamicin, netilmicin, and tobramycin concentrations were determined by the fluorescence polarization immunoassay (TDx; Abbott Diagnostics, Irving, Tex.) and enzyme multiplied immunoassay technique (EMIT; Syva Corp., Palo Alto, Calif.). Gentamicin and tobramycin concentrations
units/ml. This study characterizes the effect of various concentrations of heparin on the analytical accuracy of aminoglycoside concentration determinations by the enzyme multiplied immunoassay technique, fluorescence polarization im-
TABLE 1. The within-day coefficients of variation of EMIT, TDx, and RIA Aminoglycoside assays
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1.8 4.8
2.2 1.7 9.6
9.0 1.1 2.6
5.0 2.6 7.0
AMINOGLYCOSIDE ASSAY ACCURACY WITH HEPARIN PRESENT
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mycin concentrations were below quantifiable limits when heparin concentrations exceeded 10 USP units/ml at the low concentration and 50 USP units/ml at the high concentration. The apparent gentamicin concentration also decreased as the heparin concentration increased; however, gentamicin was still quantifiable (30 to 40% of initial value) at a heparin concentration of 3,000 USP units/ml. Heparin in concentrations of up to 3,000 USP units/ml had no appreciable effect on the accuracy of gentamicin, netilmicin, and tobramycin concentration determinations by TDx (Fig. 3). Similarly, the aminoglycoside concentration determinations by RIA were not significantly affected by the presence of heparin.
RESULTS The apparent aminoglycoside concentrations determined by EMIT were significantly reduced in the presence of heparin (Fig. 1 and 2). Although netilmicin and tobramycin concentration determinations were exquisitely sensitive to the presence of heparin (2 USP units/ml resulted in a 10% decrement in measured concentration), gentamicin determinations were less affected. A 10% decrement in the apparent high and low gentamicin concentrations was not observed until the heparin concentration exceeded 25 and 75 USP units/ml, respectively. The apparent netilmicin and tobra-
DISCUSSION The measurement by EMIT of gentamicin, netilmicin, and tobramycin concentrations was depressed in the presence of heparin. Gentamicin concentrations of 3 and 7 ,ug/ml were depressed to 90% of the control at heparin concentrations of 75 and 25 USP units/ml, respectively. Although Nilsson et al. (10) and Knight and Ukena (4) found no effect of heparin on the gentamicin assay, Krogstad et al. (5) and Walters and Roberts (14) reported similar degrees of depression in gentamicin concentrations (10 and 12.1%, respectively) with 100 USP units of heparin per ml.
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FIG. 3. Analytical accuracy of netilmicin serum concentration determinations (-3 and 7 jig/ml) by TDx (0) and RIA (A) in the presence of heparin. Similar results were observed for gentamicin and tobramycin.
The heparin concentration associated with a 10% depression in EMIT netilmicin and tobramycin concentrations was significantly lower (2 USP units/ml) than that observed with gentamicin. Krogstad et al. (5) and Nilsson et al. (10) also have noted significant depressions (ca. 10%) in tobramycin concentrations in the presence of 10 USP units of heparin per ml. The reason for the greater depression in tobramycin concentrations observed in this study is not readily apparent. Although we observed netilmicin concentrations to be depressed to a similar degree as tobramycin concentrations, Nilsson et al. (10) reported no effect on netilmicin concentrations in the presence of up to 1,000 USP units of heparin per ml. The use of a gentamicin EMIT kit by Nilsson et al. (10) to determine netilmicin concentrations may explain this discrepancy. Krogstad et al. (5) determined that the depression in gentamicin and tobramycin concentration determinations by EMIT was due to inhibition of the enzyme reaction rather than to an interference with the antigen antibody reaction. The fact that we did not find inhibition with the TDx or RIA supports this conclusion. The lack of alteration in gentamicin, netilmicin, and tobramycin determinations by TDx and RIA in the presence of heparin confirms the previous observations of Schmelter et al. (12) and the suggestion of Jolley et al. (3) that plasma samples can be utilized for the determination of aminoglycoside concentrations by these two assay techniques. This study demonstrates that the concentration of gentamicin, netilmicin, and tobramycin may be underestimated when assayed by EMIT in the presence of heparin concentrations of more than or equal to 25, 2, and 2 USP units/ml, respectively. Concentrations of heparin in these ranges can be inadvertently introduced into a blood sample, especially when blood is not properly collected from indwelling lines or catheters or when evacuated heparinized tubes are utilized.
Although the degree of depression in gentamicin concentrations may affect the accuracy of pharmacokinetic data determinations, it likely will have a minimal effect on clinical efficacy or toxicity. The greater reduction in netilmicin and tobramycin concentrations, however, suggests that the clinical efficacy or toxicity of these aminoglycosides may be affected by the utilization of EMIT-determined concentrations from heparin-containing samples. It is concluded that heparinized plasma cannot be used interchangeably with serum for the analysis of aminoglycoside concentrations by EMIT. ACKNOWLEDGMENTS We thank David Haughey and Catherine Ross for their technical support which allowed for the timely completion of this project. We also thank the Toxicology Laboratory of Metropolitan Medical Center for their assistance in this study and Kim Podany for her expert assistance in the preparation of this manuscript. This study was supported in part by a grant from Abbott Diagnostics Div., Abbott Laboratories, Irving, Tex. LITERATURE CITED 1. Barza, M., and M. Lauermann. 1978. Why monitor serum levels of gentamicin. Clin. Pharmacokinet. 3:202-215. 2. Hospes, W., R. J. Boskma, and J. R. Brouwers. 1982. Comparison of an HPLC method with a RIA, EMIT and FIA method for the assay of serum gentamicin with extensive statistical evaluation. Pharm. Weekbl. 4:32-37. 3. Jolley, M. E., S. D. Stroupe, C. J. Wang, H. N. Panas, C. L. Keegan, R. L. Schmidt, and K. S. Schwenzer. 1981. Fluorescence polarization immunoassay. I. Monitoring aminoglycoside antibiotics in serum and plasma. Clin. Chem. 27:1190-1197. 4. Knight, D., and T. Ukena. 1981. Heparin does not affect enzyme immunoassay of gentamicin. Clin. Chem. 27:640. 5. Krogstad, D. J., G. G. Granich, P. R. Murray, M. A. Pfallen, and R. Valdes. 1982. Heparin interferes with the radioenzymatic and homogenous enzyme immunoassay for aminoglycosides. Clin. Chem. 28:1517-1521. 6. Maitra, S. K., T. T. Yoshikawa, L. B. Guze, and M. C. Schotz. 1979. Determination of aminoglycoside antibiotics in biological fluids. A review. Clin. Chem. 25:1361-1367. 7. Manuel, C. H., E. Bouvet, and J. L. Wilchitz. 1978. Is the radioenzymatic assay of gentamicin in serum modified by the albumin concentration. Biomedicine (Paris) 29:152-153. 8. Matzke, G. R., C. Gwizdala, J. Wery, D. Ferry, and R. Starnes. 1982. Evaluation of three gentamicin serum assay techniques. Ther. Drug Monit. 4:195-200. 9. Matzke, G. R., K. Piveral, C. E. Halstenson, and P. A. Abraham. 1984. Heparin interferes with tobramycin serum concentration determinations by EMIT. Drug Intell. Clin. Pharm. 18:611-613. 10. Nilsson, L., R. Miller, and S. Ansen. 1981. Inhibition of amino11. 12. 13. 14.
15.
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glycoside activity by heparin. Antimicrob. Agents Chemother. 20:155-158. Regamey, C., D. Schaberg, and W. M. M. Kirby. 1972. Inhibitory effect of heparin on gentamicin concentrations in blood. Antimicrob. Agents Chemother. 1:329-332. Schmelter, R. F., J. W. Dirksen, J. T. Stalp, S. C. Augustine, and J. E. DuBe. 1981. Effect of heparin on radioimmunoassay of gentamicin. Am. J. Hosp. Pharm. 38:534-537. Stessman, J., J. Michel, and T. Sacks. 1977. Error in recovery rate of aminoglycosides from uraemic sera. Chemotherapy 12:286-287. Walters, M. I., and W. H. Roberts. 1984. Gentamicin/heparin interactions: effect on two immunoassays and on protein binding. Ther. Drug Monit. 6:199-202. Witebsky, F. G., and S. T. Selepak. 1983. Feasibility of gentamicin measurement in icteric sera by the Syva EMIT system. Antimicrob. Agents Chemother. 23:172-174. Yourassowsky, E., M. E. deBroe, and R. J. Wieme. 1972. Effect of heparin on gentamicin concentration in blood. Clin. Chim. Acta 42:189-191.
