Vol. 13, No. 4JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1981, p. 742-7490095-1 137/81/040742-08$02.00/0

Evaluation of Five Gentamicin Assay Procedures For ClinicalMicrobiology Laboratories

SALLY T. SELEPAK, FRANK G. WITEBSKY,* E. ARTHUR ROBERTSON,AND JAMES D. MAcLOWRY

Clinical Pathology Department, National Institutes of Health, Bethesda, Maryland 20205

Received 10 October 1980/Accepted 13 January 1981

Five gentamicin assay procedures (a bioassay, an enzyme immunoassay, a latexagglutination inhibition test, a fluorescence immunoassay, and a radioimmunoas-say) were evaluated to determine which was optimal for our laboratory. Theevaluation was based on recovery and precision studies and results of analyses ofpatient samples, as well as technical assay performance factors. The latex agglu-tination inhibition test appears useful for laboratories performing only occasionalassays for gentamicin; however, the fact that some rheumatoid factor-positivesera, as well as some other sera for unknown reasons, may give falsely low valuesis a potential drawback to this procedure. Because of its accuracy, precision, rapidturn-around time, and relative simplicity of performance, we selected the enzymeimmunoassay procedure for routine use for gentamicin assays in our laboratory.

Gentamicin continues to be a widely usedantimicrobial agent for the treatment of a vari-ety of serious infections. It is important to beable to determine quickly and accurately theconcentration of gentamicin in serum, and oc-casionally in other both fluids, because of its lowtherapeutic index. A variety of techniques isavailable for gentamicin measurement. It wasthe purpose of this study to evaluate five differ-ent techniques, a bioassay, an enzyme immu-noassay (EMIT), a latex agglutination inhibition(card) test, a fluorescence immunoassay (FIA),and a radioimmunoassay (RIA), to determinewhich was optimal for our laboratory.

MATERIALS AND METHODS

Recovery studies. Gentamicin sulfate (ScheringCorp., Kenilworth, N.J.) concentrated solution wasprepared by drying powdered gentamicin sulfate for 3h at 60°C under partial vacuum and then weighingand dissolving the gentamicin in 0.1 M potassiumphosphate buffer (pH 8.0) to a final concentration of1,000 Mg/ml. This solution was then diluted in 18separate human sera to give final concentrations of 0,1, 2, 4, 8, 12, and 16 dg/ml. Four categories of sera wereselected from patients not receiving gentamicin: nor-mal to visual inspection (nine patients); lipemic (threepatients); icteric (three patients); hemolyzed (threepatients). Portions (250 ,l) of each serum at eachconcentration were stored at -70°C until just beforeassay. These studies were not performed using thebioassay or RIA methods. For statistical handling ofthe data, when an assay gave a result of "less than"some lower limit on a sample to which no gentamicinhad been added, a concentration of 0 Mg/ml was as-signed to that sample for that determination. If any

gentamicin had been added and a result less than somelower limit was obtained, a concentration of 0.5 kg/mlwas assigned to that sample for that determination. Ifa result of "greater than" some upper limit was ob-tained on one or both of duplicate determinations,neither of the values was used in the statistical analysisof the data.

Precision studies. Concentrated gentamicin sul-fate solution (described above) was added to pooledhuman serum from Clinical Center patients to givefinal concentrations of 3, 6, and 12 ig of gentamicinper ml. Portions of 250 fl and 5 ml of serum at eachconcentration were stored at -70°C until just beforeassay. To determine intrarun precision, 30 assays ateach concentration were performed on the same day.Precision studies were not performed by the bioassayprocedure.

Patient samples. A total of 110 serum sampleswere selected from Clinical Center patients who werebeing treated with gentamicin and other antimicrobialagents. Samples were selected after assay by our rou-tine bioassay procedure to include a range of genta-micin concentrations from < 1 to 10.5 fig/ml. Aftercompletion of the bioassay, the residual serum waskept frozen at -20°C in screw-capped glass vials forup to 7 months before being thawed and divided into250-dl portions, which were refrozen and stored at-70°C. These were thawed just before assay by one ofthe non-bioassay procedures. For statistical handlingof the data, when an assay gave a result less than somelower limit (generally 1 tig/ml), a gentamicin concen-tration of 0.5 lig/ml was assigned to that specimen. Ifa result greater than some upper limit was obtained,that value was not used in the statistical analysis ofthe data.

Bioassay procedure. Bioassay results were ob-tained by our routine procedure, performed as previ-ously described (2), except that the gentamicin sulfatewas dissolved in 0.1 M potassium phosphate buffer

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EVALUATION OF FIVE GENTAMICIN ASSAYS 743

(pH 8.0), standards were prepared on a weekly basisrather than daily, and specimens and standards werestored in glass, rather than plastic, vials. The highestconcentration measurable in our routine procedure is16 ,ug/ml.EMIT. EMIT (Syva, Palo Alto, Calif.) was per-

formed according to the directions of the reagentsmanufacturer (Syva), except that calibrator curvepoints were all established by duplicate rather thansingle determinations. (The manufacturer recom-mends that only the 0 calibrator be run in duplicate.)The principle of this procedure involves competitivebinding between gentamicin in a sample and a knownamount of enzyme-labeled gentamicin for an anti-gen-tamicin antibody. The enzyme employed is glucose 6-phosphate dehydrogenase derived from the bacteriumLeuconostoc mesenteroides. Binding of the enzyme-labeled gentamicin decreases the activity of the en-zyme. The enzymatic activity remaining results inconversion of the coenzyme nicotinamide adenine di-nucleotide to reduced nicotinamide adenine dinucleo-tide; the resulting absorbance change is measuredspectrophotometrically. The instrumentation usedwas a model 1500 automatic pipetter-diluter (CAVROScientific Instruments, Los Altos, Calif.), a GilfordStasar III spectrophotometer (Gilford InstrumentLaboratories, Inc., Columbia, Md.) and a Syva CP-1000 timer-printer (manufactured for Syva by Ox-bridge, Inc., Mountain View, Calif.). Values for theabsorbance changes of the calibrator standards wereplotted on special graph paper provided with each kitof reagents; gentamicin concentrations for the sampleswere determined from the resulting curves. The high-est concentration measurable by this procedure is 16ig/ml.Card test. The latex agglutination inhibition test

(Macro-Vue Card Test, Hynson, Westcott and Dun-ning, Division of Becton Dickinson, Baltimore, Md.)was done as specified in the directions of the manu-facturer, except that all determinations were done induplicate and the results were averaged. The principleof this procedure involves the inhibition of agglutina-tion of gentamicin-sensitized latex particles which areadded to a mixture of sample and a known amount ofanti-gentamicin antibody. Quantitation of gentamicinconcentration is achieved by multiplying the recipro-cal of the highest dilution of sample showing aggluti-nation inhibition by the lowest concentration of gen-tamicin standard also showing agglutination inhibi-tion. The highest concentration measurable by thisprocedure varies depending upon the endpoint of thestandard; it ranges from 12.8 to 19.2 ,g/ml.

FIA. FIA (Ames Division, Miles Laboratories, Inc.,Elkhart, Ind.) was done as specified in the directionsof the reagents manufacturer, except that up to 39samples were run per curve; this was specified as apermissible procedure by the manufacturer. The prin-ciple of this procedure involves competitive bindingbetween gentamicin in a sample and a known amountof sisomicin labeled with a fluorogenic substrate (aderivative of umbelliferyl-,/-D-galactoside) for an anti-gentamicin-sisomicin antibody. Binding of the labeledsisomicin to antibody prevents hydrolysis of the fluo-rogenic substrate. /3-Galactosidase hydrolyzes avail-

able fluorogenic substrate to produce a fluorescentproduct, the amount of which is measured fluoromet-rically. The fluorometer used was the Aminco Fluoro-Colorimeter (American Instrument Co., Savage, Md.).Fluorescence units of the standards were plottedagainst gentamicin concentration on graph paper pro-vided by Ames; the gentamicin concentrations of thesamples were determined from the resulting curves.The highest concentration measurable by this proce-dure is 12 fig/ml.

RIA. RIAs were performed by Herner Analytics,Inc. (Rockville, Md.) using the Monitor Science ra-dioimmunoassay kit (Monitor Science Corp., NewportBeach, Calif.) as specified in the directions of thereagents manufacturer. The highest concentrationmeasurable by this procedure is 16 tig/ml.

RESULTSTable 1 shows the results of the recovery

studies on normal, hemolyzed, icteric, and li-pemic sera. The results from only seven normalsera for the FIA are shown in the concentrationrange of O to 8 lig/ml because the sera from theother two patients were used up in runs fromwhich no results could be obtained because ofunsatisfactory standard curves. Table 2 showsthe results of linear regression analysis of thedata presented in Table 1 except that the anal-ysis has been done only for the concentrationrange of O to 8 fig/ml. The 12- and 16-ftg/mlconcentrations were not used in this analysisbecause a full set of data was not available fromall the procedures at these concentrations. Anideal method for measuring gentamicin wouldgive a slope (m) of 1.0 and a y-intercept (b) ofzero for each of the different types of sera. Inaddition, the correlation coefficient (ofmeasuredconcentrations compared with concentrations ofgentamicin actually added) would be 1.00, andthe standard error of the estimate (interpretableas a standard deviation [5]) would be 0.00. Allthree procedures evaluated by recovery studiesappeared to perform satisfactorily with normaland hemolyzed sera. With icteric sera the FIAtended to overestimate the amount of gentami-cin present. With lipemic sera the EMIT proce-dure tended to underestimate the amount ofgentamicin present. This can be seen moreclearly from the results in Table 1 than from thelinear regression analysis in Table 2, which doesnot include the results obtained at 12 and 16 jug/ml. The FIA appeared to overestimate slightlythe amount of gentamicin present in lipemicsera, but the number of measurements wassmall.Table 3 shows the results of the studies on

intra-run precision.Table 4 shows the correlation coefficients for

the linear regression lines obtained by plotting

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TABLE 1. Recovery studiesRecovery by method

Gentamicin FIA EMIT Card testSerum concn added

(,ugrnl No.of o.f No. ofMean + SD' No. o Mean + SD Nolueof Mean ± SD values

Visually O 0.0 0.0 7 0.0 ± 0.0 9 0.0 ± 0.0 9Cnormal 1 1.1 0.5 7 1.2 ± 0.2 9 0.9 ± 0.3 9C

2 1.9+0.4 7 2.1±0.3 9 1.5±0.8 9C4 4.2±0.6 7 3.9±0.2 9 3.4± 1.2 9C8 8.3+0.8 7 7.8± 1.4 9 8.0±0.9 912 11.0 4d 10.7 8e 10.9 ± 1.6 916 -f 14.1 35 13.8 4

Hemolyzed O 0.0 ± 0.0 3 0.0 ± 0.0 3 0.0 ± 0.0 31 1.4±0.3 3 1.4±0.1 3 0.7±0.4 32 2.1±0.4 3 2.3±0.1 3 2.2±0.6 34 4.4±0.5 3 4.3±0.3 3 3.6±0.0 38 8.8±0.4 3 8.6±0.8 3 7.9± 1.5 312 - 11.5 1' 13.2± 1.2 316 J J 14.4 2

Icteric 0 0.6 ± 1.0 3 0.0 ± 0.0 3 0.0 ± 0.0 31 1.7±0.6 3 1.1 ±0.0 3 1.0±0.5 32 3.1 0.2 3 2.0±0.0 3 1.7±0.1 34 5.2 0.2 3 4.1 ± 0.2 3 3.5 ± 0.2 38 10.1±0.3 3 7.6±0.0 3 7.6±0.7 312 11.8±0.5 3 12.8± 1.4 316 - 16.0 li 14.4 2'

Lipemic 0 0.4 ± 0.7 3 0.0 ± 0.0 3 0.0 ± 0.0 31 1.7±0.6 3 1.0±0.4 3 0.9±0.4 32 2.9±0.8 3 1.8±0.3 3 2.0±0.3 34 4.9±0.8 3 3.6±0.2 3 3.7±0.5 38 9.0±1.5 3 7.1±0.8 3 7.4±0.3 312 11.2 lm 8.8 ± 0.6 3 12.0 ± 0.0 316 l 10.8 ± 1.6 3 14.4 2

a Mean measured gentamicin concentration (micrograms per milliliter) and standard deviation (SD).b Number of values used for determining each mean and standard deviation.c See text for discussion of problems with two sera.d By FIA three samples at this concentration were measured as >12,g/ml.e By EMIT one sample at this concentration was measured as >16 jug/ml.f By FIA all samples at this concentration were measured as >12 ,ug/ml.g By EMIT six samples at this concentration were measured as >16 jg/ml.h By card test five samples at this concentration were measured as greater than the highest concentration

measurable by at least one of the two cards used.By EMIT two samples at this concentration were measured as >16 ,ug/ml.By EMIT all three samples at this concentration were measured as >16 ,tg/ml.

k By card test one sample at this concentration was measured as greater than the highest concentrationmeasurable by one of the two cards used.'By card test one sample at this concentration was measured as greater than the highest concentration

measurable by both of the cards used.m By FIA two samples at this concentration were measured as >12 jg/ml.

gentamicin concentration measured by eachmethod against that measured by each othermethod for ail the patient samples tested. Thebest correlation was obtained between theEMIT and the RIA.

Figures 1, 2, 3, and 4 show the results ofplotting the data for patient samples as obtained

by RIA, bioassay, FIA, and card test, respec-tively, against the results obtained by EMIT.The data are displayed in this fashion becausewe ultimately selected the EMIT for routine usein our laboratory. Note in Fig. 4 that there werefour patient samples for which the card test gavea value of less than 1, whereas the EMIT gave

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EVALUATION OF FIVE GENTAMICIN ASSAYS 745

TABLE 2. Linear regression analysis of data from Table IParameters of equation

Serum Method rh SEE'y m x b

Visually FIA FIA 1.0 Concn added +0.0 0.99 0.50normal EMIT EMIT 1.0 Concn added +0.1 0.97 0.65

Card Card 1.0 Concn added -0.3 0.96 0.81

Hemolyzed FIA FIA 1.1 Concn added +0.1 0.99 0.36EMIT EMIT 1.0 Concn added +0.2 0.99 0.37Card Card 1.0 Concn added -0.1 0.97 0.70

Icteric FIA FIA 1.2 Conen added +0.6 0.99 0.51EMIT EMIT 0.9 Concn added +0.1 1.00 0.12Card Card 0.9 Concn added -0.1 0.99 0.38

Lipemic FIA FIA 1.1 Concn added +0.6 0.97 0.83EMIT EMIT 0.9 Concn added +0.1 0.99 0.38Card Card 0.9 Concn added +0.0 0.99 0.31

a Equation of regression line in the form y = mx + b.b r, Correlation coefficient.SEE, Standard error of the estimate.

TABLE 3. Intra-run precisionRecovery by method

Gentamicin FIA EMIT Cardconcn added()4/mMean ± SD<a No. of val- Mean ± SD No. of val- Mean ± SD No. of val--eaSI)a uesh - ues ues3 3.1 0.3 30 3.3±0.1 30 3.0±0.6 306 7.7+0.6 30 6.4±0.1 30 5.8±0.7 3012 - 11.1 ± 0.6 30 10.8 ± 1.8 28

a Mean measured gentamicin concentration and standard deviation (SD) (micrograms per milliliter).b Number of values used for determining each mean and standard deviation.C No mean or standard deviation was calculated for FIA at this sample concentration because 20 of the 30

samples were measured as >12 ,tg/ml.d By card test two samples at this concentration were measured as greater than the highest concentration

measurable by one of the two cards used for each sample.

TABLE 4. Correlation coefficients for resultsobtained on patient samples

Method Bioassay EMIT RIA FIA Card

Bioassay 1.00EMIT 0.88 1.00RIA 0.86 0.95 1.00FIA 0.86 0.90 0.89 1.00Card 0.82 0.85 0.84 0.85 1.00

values for these samples of 2.5 to 8.1 ,ug/ml. Forthese samples the results of the other assayprocedures (except for the bioassay on one sam-ple) were in agreement with the EMIT results.After this discrepancy was noted, these sampleswere checked for the presence of rheumatoidfactor. Rheumatoid factor was present in two ofthe samples, but not in the sample showing thegreatest discrepancy. We retained the two rheu-matoid factor-positive sera in the data base for

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FIG. 1. Linear regression line for values obtainedby RIA versus EMIT for 108 clinical specimens (12data points superimposed on others). See Table 5 forregression statistics.

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746 SELEPAK ET AL. J. CLIN. MICROBIOL.

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FIG. 2. Linear regression line for values obtainedby bioassay versus EMIT for 99 clinical specimens(eight data points superimposed on others). See Table5 for regression statistics.

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the card test because we felt that only veryrarely would a clinical laboratory performinggentamicin assays know that a particular samplewas positive for rheumatoid factor.Table 5 presents the results of the linear

regression analyses of the data in Fig. 1 to 4. Thelast column in this table shows the 95% confi-dence interval for a sample determined to be 6.0kg/ml by the EMIT procedure, when assayed bythe other procedures (1). The numbers may beapproximated by substituting the number 6 forthe EMIT value (x value) in the equations ofthe form y = mx + b and adding the quantitytwo times the standard error of the estimate.Table 6 shows the reagent cost of measuring

a gentamicin serum concentration, either as asingle sample or as a single sample analyzed ina batch of six samples, for the EMIT, FIA, andcard test.

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FIG. 4. Linear regression line for values obtainedby card test versus EMIT for 109 clinical specimens(16 data points superimposed on others). See Table 5for regression statistics.

TABLE 5. Linear regression analysis of data in Fig.1, 2, 3, and 4

Parameters of equation" 95% confi-dence in-

Method rh SEE' terval fory m x b predicted

value"RIA RIA 1.1 EMIT -0.1 0.95 0.82 4.8-8.1

valueBioassay Bioassay 0.9 EMIT +0.3 0.88 1.08 3.8-8.1

valueFIA FIA 1.1 EMIT +0.3 0.90 1.12 4.9-9.4

valueCard Card 1.0 EMIT -0.1 0.85 1.39 3.3-8.9

value

" Equation of regression line in the form y = mx + b.h r, Correlation coefficient.'SEE, Standard error of the estimate." For a sample with a gentamicin concentration of 6 jug/ml

as determined by EMIT.

TABLE 6. Cost of measuring a gentamicin serumconcentration'

Cost of procedure"

Method Single sampleSingle sample in batch of six

samples

EMIT $20.46 $6.51FIA $9.00 $2.75Card test $4.00 $4.00

a Includes only cost of kits. Does not include cost ofmaterials (e.g., test tubes) not provided with kits, anddoes not include labor cost.

h Cost based on manufacturers' recommendationsfor number of replicates of standards, controls, andpatient samples.

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EVALUATION OF FIVE GENTAMICIN ASSAYS 747

TABLE 7. Times for measuring gentamicin serumconcentrations of a single sample and of a batch of

30 samplesTime (min) for procedure'

Single sample Batch of 30 samplesMethod

Elapsed Tech- Elasped Technol-time nologist time ogisttime tirne

EMIT 14 14 170b 170bFIA 45 30 160 160Card test 12 4 225 220

a Time required to complete indicated procedureplus a standard curve when applicable.

b This time can be reduced to 2 h when the CP5000(Clinical Processor) is used.

Table 7 shows times required for the deter-mination of gentamicin serum concentrationseither as a single specimen or as a batch of 30specimens.Technical considerations. (i) Bioassay.

Advantages of the bioassay include the lack ofneed for sophisticated and costly instrumenta-tion, relative simplicity of performance, andbroad applicability to virtually any antimicrobialagent. The techniques for setting up a bioassayare familiar to most microbiologists. Disadvan-tages of the bioassay are the time required fromspecimen receipt to result availability (a mini-mum of 5 h) and interference by other antibioticswhich either cannot be inactivated or for whichresistant assay organisms are not available.

(ii) EMIT. Advantages of the EMIT assay areextremely rapid turn-around time (about 2 minper specimen after a standard curve has beenestablished), relative ease of performance, andestablishment of a standard curve (usable for atleast 24 h) independently of specimen assays.This last feature means that "stat" specimenscan easily be handled, and also means that,should there be any problems with the establish-ment of a standard curve, there has been no lossof time, reagents, or samples from specimenprocessing. We also found the quality controlfeatures of the EMIT to be especially useful.Specifically, precise intervals are given withinwhich certain calibrator points should fall, andprecise limits for acceptability of the results ofthe gentamicin control are stated. Only 100 pl ofserum is required for a duplicate determination.According to the package insert there is noknown interference with the assay by antibioticsother than sisomicin or netilmicin. A disadvan-tage of the procedure is that occasionally therewas some problem establishing an acceptablestandard curve; this difficulty was usually re-lated to the 16-,ug/ml calibrator and generally

required only re-assaying this calibrator one tothree times more to establish a satisfactoryvalue. It was our impression that, after most ofthe reagents in a given bottle (designated "A" or"B" by the manufacturer) had been used up, itwas occasionally difficult to establish a standardcurve with these small volumes. This problemmay have been due to difficulties with accurateaspiration when only small volumes of reagentremained in the bottles; we have since foundthat the problem disappears when such smailvolumes (with material of the same lot number)are pooled.

(iii) Card test. Advantages of this procedureinclude its lack of dependence on complex equip-ment, lack of need for a standard curve, andrapidity. According to information received fromthe manufacturer, sisomicin does cross-reactwith the assay; it is not known whether netil-micin also cross-reacts with the assay. Accordingto the package insert there are no other knowncross-reacting antibiotics. Disadvantages wefound were as follows. Since there is no "batch-ing" with this technique, the time involved forrunning large numbers of specimens becomesconsiderable. Also, as noted by the manufac-turer, an elevated rheumatoid factor may causea falsely low assay result. We found this to be aproblem up to a gentamicin concentration of 4,ug/mI in one patient sample, in which the levelwas <1.0 ,g/mI by latex agglutination inhibitionbut at least 4.0 ,ug/ml by all the other procedures.In addition, as noted above, two other patientsamples gave inexplicably low results by thisprocedure. In the recovery studies, in two serathat were visually normal, an unusual type ofagglutination occurred in the lower dilutions ofall gentamicin concentrations. This agglutina-tion was presumed to be due to an interferingsubstance, since it disappeared at higher serumdilutions. The presence of this phenomenonmeant that in these two sera only the higherconcentrations of gentamicin could be accu-rately determined. For these two sera, gentami-cin concentrations up to and including 4,g/mlwere all read as less than the lowest concentra-tion measurable by each of the two cards usedat each concentration. Therefore, for the sam-ples to which 0, 1, 2, and 4 ,ug of gentamicin hadbeen added per ml, values of 0, 0.5, 0.5, and 0.5,ug/ml, respectively, were assigned. Finally, thereis sometimes difficulty in determining the pre-cise agglutination inhibition endpoint; this diffi-culty decreases but does not disappear with fa-miliarity with the procedure. The clarity of theendpoints was also noted to vary somewhat be-tween lot numbers.

(iv) FIA. We were not able to discern any

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748 SELEPAK ET AL.

advantages to this procedure as compared withthe others evaluated. According to the packageinsert there is no known interference with theassay by antibiotics other than sisomicin or ne-tilmicin. A principal disadvantage of this proce-dure is that the standards on which the standardcurve for a given run is based are incorporatedin the same run as the samples. If the standardcurve is not satisfactory, the entire run, includingboth standards and samples, must be repeatedfrom the step in which standards and sampleshad been initially diluted in buffer. During thecourse of the evaluation we used two differentfluorometers; with the first instrument, slightlyover one-third of the runs had to be repeated.Performance was considerably better with thesecond instrument. Another disadvantage is thatmanual mixing of dilutions is required, increas-ing the risk of spillage. In addition, quality con-trol is not well defined for this procedure. Forexample, acceptable ranges of fluorescent unitsfor the standards are not specified. Also, dupli-cate readings are supposed to be done, but nostatement is made about maximum allowabledifferences, if any, between duplicate readings.Finally, since the standard curve is constructedfrom point to point, rather than using a "best-fit" line through ail the points, controls shouldprobably be run at a concentration in the inter-val between each two consecutive standards,rather than using only the one control, "a com-mercial control," recommended by the manufac-turer.

DISCUSSIONWe have previously evaluated a number of

different RIA procedures (2). Although many ofthese are accurate and precise, they require theuse of expensive and sophisticated equipmentand are not cost-effective for low-volume oper-ation, as a standard curve must be developed foreach run.The particular FIA procedure we evaluated

suffers from a number of technical problems, asdetailed above. We attempted to evaluate theFIA procedure of BioRad (BioRad Laboratories,Richmond, Calif.) but had to abandon that partof the study because of numerous technicalproblems with the reagents and instrumenta-tion.The card test appears useful for laboratories

performing only occasional assays for gentami-cin; however, the fact that some rheumatoidfactor-positive sera, as weil as some other serafor unknown reasons, will give falsely low valuesis a potential source of difficulty with this pro-cedure. After this study had been completed, wewere informed that others have found that the

use of serum which has been repeatedly frozenand thawed may produce results with the cardtest that correlate poorly with the results of bothEMIT and RIA procedures, whereas the use offresh serum or serum which has been frozen onlyonce produces results that correlate better withthese procedures (D. Bernstein, of Hynson,Westcott and Dunning, personal communica-tion). It should be noted that the patient sampleswe used in this study were all frozen twice;hence, it is possible that a better correlation ofthe card test results with both EMIT and RIAresults might be obtained with fresh serum orserum frozen and thawed only once.The bioassay procedure appears less accurate

than either RIA or EMIT and also has an exces-sively long turn-around time.The EMIT procedure has previously been

compared with a bioassay (3) and an RIA (4)and was found to perform well in those studies.Because of its accuracy, precision, rapid turn-around time, and relative simplicity of perform-ance as determined in our study, we selected theEMIT procedure for routine use for gentamicinassays in our laboratory. Since the conclusion ofthis study, Syva has developed a computer pro-gram for use with the assay; this program elim-inates the need for manual plotting of a standardcurve and makes performance of the assay pro-cedure still more simple. We should note thatour laboratory has established the policy of di-luting any specimen and reassaying the dilutionif the initial determination is -10 ig/ml.Only small numbers of severely hemolyzed,

icteric, and lipemic sera were tested, and all ofthese were sera to which gentamicin had beenadded in the laboratory. No patient sera thatwere severely hemolyzed, icteric, or lipemic weretested. Therefore, despite the fact that we de-tected no problem with assaying hemolyzed sera,and no problem assaying icteric sera with theEMIT procedure, we are not prepared to statethat one can accurately assay severely hemo-lyzed or icteric sera by either the FIA or EMITprocedure. We do believe, however, that ourdata demonstrate that lipemic sera cannot beadequately assayed by the EMIT procedure.With all of the procedures, it is imperative thatthe manufacturer's instructions be followed pre-cisely.There is considerable variation among the

methods both in the cost of reagents alone andin technologist time, as noted in Tables 6 and 7.Actual reagent costs per test may differ greatlyfrom these theoretical costs if standard curvesor entire runs have to be repeated, as notedabove. As with reagent costs per test, labor costsper test vary significantly with batch size for any

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EVALUATION OF FIVE GENTAMICIN ASSAYS 749

procedure for which the number of standardsand controls required is largely independent ofthe number of clinical specimens in a given run.

It is important to note also that we have notattempted to analyze the effect of costs of non-reagent materials, such as test tubes, nor theeffect of instrument costs on the total cost pertest.

ACKNOWLEDGMENTS

We thank M. V. Ratnaparkhi and Mark H. Zweig for helpwith study design and data analysis, James E. Byrkit and RitaG. Minker for help with data handling, and Virginia M. Hol-comb for technical assistance.

LITERATURE CITED1. Afifi, A. A., and S. P. Azen. 1972. Statistical analysis: a

computer oriented approach, p. 96. Academic Press,New York.

2. Lantz, C. H., D. J. Lawrie, F. G. Witebsky, and J. D.MacLowry. 1980. Evaluation of serum gentamicin as-

say procedures for a clinical microbiology laboratory. J.Clin. Microbiol. 12:583-589.

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