Evaluation of four gentamicin and tobramycin assay procedures for clinical laboratories

Vol. 18, No. 4 JOURNALOFCLINICALMICROBIOLOGY, OCt. 1983,p.890-894

0095-1137/83/100890-05$02.00/0

Evaluation of

Four

Gentamicin and Tobramycin

Assay

Procedures for Clinical Laboratories

FRANK G.WITEBSKY,l*CLARA A. SLIVA,2SALLY T. SELEPAK,' MARK E.RUDDEL,2JAMES D.

MAcLOWRY,' ERNESTINE E. JOHNSON,2ANDRONALDJ. ELIN2

Microbiology

Service'

and ClinicalChemistry Service,2ClinicalPathology Department, National Institutesof

Health, Bethesda,Maryland20205

Received 18 March1983/Accepted18 July 1983

Accuracy, precision, and clinical laboratory utility of the TDX(Abbott Labora-tories), Auto-ICS (Beckman Instruments, Inc.), COBAS-Bio (Roche Analytical Instruments,Inc.) with reagentkits(Syva), and EMIT (Syva) forgentamicin and tobramycinserumassay were assessed.TDX, COBAS-Bio, and EMIT analytical

systems showed aproportional bias of less than 10% forrecovery studies and a

coefficientof variation less than 5% for within-run precision. The results of the

recovery studies with the Auto-ICS showed a proportional bias of 25% with

gentamicin and 16% with tobramycin. The within-run precision expressedasthe

coefficient of variation for the Auto-ICS was 6.7% forgentamicin and 8.6% for

tobramycin. In comparisons involving gentamicin- and tobramycin-containing patient samples, the results with the TDX analytical system showed the best

agreement with theCOBAS-Bio. For the determination of thesetwoantibiotics,

the TDX analyticalsystemprovided the best overall accuracyand precision.

Aminoglycosides continue to be widely used

for the treatment of serious gram-negative rod infections. Because of their relatively low

toxic-to-therapeutic ratioand the relativeunreliability

ofexistent dosage nomograms, the monitoring

of serum aminoglycoside levels to assure ade-quate, nontoxic drug levels remains necessary.

New instrumentation capable of making such

measurements continues to be developed. Itis

the purposeofthis paper to comparethe

accura-cy, precision, and clinical laboratory utility of

theTDX(Abbott Laboratories, Diagnostics

Di-vision, Irving, Tex.), Auto-ICS (Beckman

In-struments, Inc., Brea, Calif.), COBAS-Bio

(Roche Analytical Instruments, Inc., Nutley,

N.J.), and EMIT(Syva,PaloAlto,Calif.)

proce-durefordetermination ofserumgentamicinand

tobramycin levels.

MATERIALS AND METHODS

Drug preparation. Gentamicin sulfate powder

(ScheringCorp.,Kenilworth, N.J.)wasdriedfor3hat

110°C anddissolved inastockhuman serumpool to

final concentrationsof5.0, 5.6, 6.7, and10.0,u.g/ml. A

pool containing 1.1 ,ugofdrug per ml was made by

dilutingthe5.6-,ug/ml poolwith the stockserumpool.

A3.4-,ug/mlpoolwasmadebycombiningaportion of

the 1.1-and

5.6-p.g/ml

pools1:1 (vol/vol).

Tobramycin powder (Eli Lilly & Co., Indianapolis,

Ind.) was dried for 3 h under vacuum at 60°C and

dissolved inastockhumanserumpooltofinal

concen-trations of10 and12,ug/ml.Apool containing2,ug of

drug per mlwas made bydilutingthe

10-p.g/ml

pool

with the stock serum pool. A6-,ug/ml pool was

pre-pared by combining portions of the 2- and 10-,ug/ml

pools 1:1 (vol/vol). All gentamicin and tobramycin

poolswereportioned into 3-ml samples and stored at

-70°C untiljust beforeuse.

Patientsamples. A total of 68 serumsamples were

selected from Clinical Center patients who were

re-ceiving gentamicin, and a total of50 serum samples

wereselected from Clinical Center patients who were

receiving tobramycin. Samples had been frozen at

-20°C fornot morethan3months and thenat-70°C

for not more than 9 months in screw-capped glass

vials. Samples were selected from stored patient sera

previously assayed byourroutine EMITprocedureto

includeanevenly distributed range ofgentamicin and

tobramycin concentrations from1.0 to10.0,ug/ml. All

sampleswereanalyzedontheday thespecimenswere

thawed,and allspecimenswereanalyzedonall

instru-ments,includingarepeatanalysis by the EMIT

proce-dure. Datafrom thisrepeatedEMIT procedurewere

utilized in thisstudy.

EMIT. EMIT (Syva) was performed according to

the directions ofthe manufacturer (Syva) of the

re-agents, exceptforestablishinganewstandardcurve at

thebeginning of eachworking day. In ourlaboratory,

thegentamicin standardcurveis putineveryotherday

unless the 6.0-,ug/ml control is out of range. The

tobramycin standard curve is put in once a week

unlessthe6.0-,ug/ml controlis outofrange. Nearlyall

of the recoveryandprecisionstudyvalueswere

deter-minedbyusinga new standard curve, and all of the

patient valuesweredeterminedbyusinga newcurve.

The principle of this procedure involves competitive

bindingbetween anaminoglycosidein asampleanda

known amountofenzyme-labeledaminoglycosidefor

anaminoglycoside antibody. Binding of the

enzyme-labeled aminoglycoside decreases the activity of

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GENTAMICIN AND TOBRAMYCIN ASSAY PROCEDURES 891

TABLE 1. Gentamicin recovery

Meana(CV)b

Method Slopec Interceptc

l.1d 3.4 5.0 6.7 10.0

TDX 1.1 (7.1) 3.5(7.1) 4.9(9.0) 7.2(7.2) 10.2(9.7) 1.04 -0.05

Auto-ICS 1.6(12.0) 4.3(16.8) 5.7(8.3) 8.9(12.7) 12.5(10.5) 1.25 0.00

COBAS-Bio 1.2(10.8) 3.4(7.0) 5.4(9.5) 7.2(4.3) 9.3'(8.5) 0.93 0.48

EMIT 1.3(13.4) 3.4(4.1) 5.1(5.1) 7.0(3.7) 9.4'(10.4) 0.93 0.34

a Meanmeasuredgentamicinconcentration(,Lg/ml)of determinationson10separatedays.

b

CV,

Coefficient of variation =

(1

standard deviation - mean) x 100.

cForthe equation ofregression line in the form y = mx + b, where y = method value,x = amountof

gentamicin added,m = slope,and b = intercept.

dAmountofgentamicinadded

(,ug/ml).

eMeanofdeterminations on9 separatedays.

the enzyme. The enzyme converts NAD to the re-duced form(NADH);the NADH is measured

spectro-photometrically. The instruments used were amodel

1500 automatic pipetter-diluter (CAVRO Scientific

Instruments, Los Altos, Calif.), Gilford Stasar III

spectrophotometer (Gilford Instrument Laboratories

Inc., Oberlin, Ohio), and a CP-5000 EMIT clinical

processor (manufactured for Syva by OXBRIDGE,

Inc., Mountain View, Calif.). It is our laboratory

policy to dilute all specimens with a gentamicin or

tobramycin concentration of 10 F±g/ml or higher 1:1

with thezero calibrator. The lowest gentamicin and

tobramycin concentration measurable by the EMIT

procedureis 1.0

Rg/ml.

COBAS-Bio.COBAS-Bio(Roche Analytical

Instru-ments) is acentrifugal analyzer with a built-in data

reduction system which was used according to the directions of the manufacturer. The instrumentwasset

for program model no. 2 data reduction, which is a

five-parameter logit/logarithm curve-fitting algorithm. The reagents used were those for the EMIT

proce-dure; thesereagents werereconstituted according to

the directions for the COBAS-Bio. The measurable

concentration range without dilution for these

ami-noglycosidesis 0.0to16.0,ug/ml.

Auto-ICS. Auto-ICS (Beckman Instruments) is an automatedratenephelometerwhichwasused

accord-ingtothe directions of the manufacturer. Theprinciple

of this procedure is a rate nephelometric inhibition

immunoassay, involving competitive bindingbetween

anaminoglycosideinasampleandaknownamountof

aminoglycoside-protein conjugateforan

aminoglyco-side antibody. At the normal instrument dilution of

1:21,the measurable concentrationrangeis 2.0to16.0

ug/ml.Ataninstrument dilutionof1:6,the measurable

concentrationrangeis extended downto0.6,ug/ml.A

higherinstrument dilution is also available formore

concentrated specimens. All studieswerecarriedout

withprogram tapeDA.

TDX.TDX(Abbott Laboratories), whichwasused

accordingtothe directions of the manufacturer,isan

instrument whichmeasuresfluorescencepolarization.

The principles of this procedure are (i) competitive

bindingbetween an aminoglycoside in a sample and

fluorescein-labeledaminoglycoside foran

aminoglyco-sideantibodyand(ii)greaterretention ofpolarization

of emitted light (fluorescence) by a slowly rotating

molecule (tagged antigen plus antibody) than by a

rapidly rotatingmolecule (tagged antigen alone).The

measurable concentrationrangewithoutdilutionis 0.0

to 10.01Lg/ml. Higher concentrationswere measured

without manual dilution by using the programmable

dilution protocolof the instrument.

RESULTS

Tables 1 and 2showthe results of the

gentami-cin recovery and precision studies. For the

recovery study (Table 1) determinations were

performed on 9 or 10 separate days. The TDX

analytical system showed the smallest

propor-tional bias (4%). The COBAS-Bio and EMIT

analytical systems had a negative proportional

bias of 7%, but the Auto-ICS had a positive

proportionalbias of 25%. TheAuto-ICSshowed

no systematic bias (intercept ofzero), and the

TDX showed a small systematic biascompared

with the EMITand theCOBAS-Bio. The TDX

showed the bestoverall among-day precision for

the five different concentrationsofgentamicin in

that the coefficient of variation did not exceed

10%. The other three analyticalsystemsshowed

a coefficient of variation greater than 10%o for

one or more of the gentamicin concentrations.

The within-runprecision (Table 2) was less than

TABLE 2. Within-runprecision

Gentamicin meana

Method

(CV)b

Tobramycinmeanc

6.7d 10.0" (CV)

6.0"

TDX 7.4(4.8) NTe 5.9(3.4)

Auto-ICS 8.9(6.7) NT 7.3 (8.6)

COBAS-Bio 7.7(2.6) NT 5.8 (4.1)

EMIT NT 9.8f(1.8) NT

aMeanmeasuredgentamicin concentration

(micro-grams permilliliter) of20consecutive within-run

de-terminations.

bCV, Coefficient of variation = (1 standard

devi-ation + mean) x 100.

CMeanmeasuredtobramycin concentration

(micro-grams permilliliter)of 20consecutive within-run

de-terminations.

dAmountofdrug added(micrograms per milliliter).

eNT,Nottested.

fMeanof nine consecutivedeterminations.

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892 WITEBSKY ET AL.

TABLE 3. Tobramycin recovery

Meana(CV)b

Method 2.0" 6.0 10.0 12.0 Slope' Intercept'

TDX 1.9(5.0) 5.6(4.5) 8.3(10.9) 12.7(13.6) 1.00 -0.41

Auto-ICS 2.8(16.6) 7.1(13.0) 11.1(9.3) 14.8(5.3) 1.16 0.25

COBAS-Bio 2.1 (11.1) 5.4(2.0) 10.0(12.4) 13.1 (10.7) 1.09 -0.50

EMIT 1.9(3.8) 5.5(4.0) 8.9(7.0) 12.9(0.8) 1.04 -0.46

aMeanmeasuredtobramycin concentration (,ug/ml) of determinations on4separatedays.

b

CV,

Coefficient of variation = (1 standard deviation +mean) x 100.

'Forthe equation ofregression line in the formy = mx + b, wherey = methodvalue,x = amount of

tobramycin added,m = slope,and b = intercept.

dAmountoftobramycin added(,ug/ml).

5% for the TDX, COBAS-Bio, and EMIT but

greaterthan 5% for the Auto-ICS.

Tables 2 and 3 show the results of the tobra-mycin recovery and precision studies. For the

recovery study (Table 3) determinations were

performed on 4 separate days. The TDX, CO-BAS-Bio, and EMIT showedaproportional bias

of less than10%, but the Auto-ICS hada

propor-tional bias of16%. The Auto-ICS had the

small-est systematic bias. The EMIT system showed the best among-day precision. The TDX and COBAS-Bio showedawithin-runprecision (Ta-ble2) less than 5%, but the Auto-ICS showeda

within-runprecisiongreater than 5%.

The TDX analytical system was compared

with the other three analyticalsystemsby using patient specimens containing either gentamicin

or tobramycin. The TDX was chosen as the

system forcomparison, since the results of the

recovery studies showed the best accuracy for

both antibiotics. Table 4 and Fig. 1 show the results of the statistical analysis of the data obtained from 68gentamicin-containing patient samples by using a debiased linear regression

analysis (assumes random error for both the abscissa andtheordinate) (1).The EMIT system

showed the greatest proportional bias (-19%) and thelargestintercept (0.68 ,ug/ml) compared with the TDX. The COBAS-Bio and Auto-ICS

hadaproportionalbias of less than 10% andan

TABLE 4. Statisticalanalysis'of 68

gentamicin-containing patient samples Regression analysisb Method

Slope Intercept Sy.x

COBAS-Bio 0.98 0.22 0.52

EMIT 0.81 0.68 0.47

Auto-ICS 1.07 0.21 0.70

aByadebiasedregressionmethod.

bFor theequationofregressionline in theformy=

mx+b,where y=methodvalue,x=TDXvalue,m=

slope,and b = intercept.

cSy.x= standarderroroftheregression.

intercept thatwasless thanone-thirdthat of the

EMIT system. The Auto-ICS had the largest

standarderrorof theregression

(Sy

.x,

measure

of thescatterof data points around the

regres-sion line) (2).

The results of the statistical analysis of data obtained from 50tobramycin-containingpatient samples byadebiased linearregression method

is shown in Table 5 andFig. 2. TheCOBAS-Bio and EMIT analytical systems show a

propor-tional bias of less than 10% compared with the TDX. The Auto-ICS hada proportional bias of

22%. The intercepts were acceptable for all

three systems. Thelargest standarderrorof the

regressionwasfound withtheAuto-ICSsystem.

There does appear to be an "outlier" for the

Auto-ICS (Fig. 2). If this value is deleted from theanalysis, the slope comparing the Auto-ICS with the TDX becomes 1.11, reducing the

pro-portional biasby 50%.

Theproblem with precision identified by this study for the Auto-ICS wasevaluatedby Beck-man Instruments. The Auto-ICS instrument

used byusfor this study washed the pipette tip with a solution containing polyethylene glycol.

Beckman Instruments determinedthat small but varying concentrations of polyethylene glycol

weredeliveredtothereactioncell, which affect-ed therateof the antigen-antibody reaction and led to the problem with precision. Beckman

TABLE 5. Statisticalanalysis"of50

tobramycin-containing patient samples Regressionanalysisb

Method

Slope Intercept Sy.

COBAS-Bio 0.99 0.10 0.44

EMIT 1.04 -0.19 0.15

Auto-ICS 1.22 0.18 0.58

aByadebiasedregression method.

b Equation ofregression line in the form y= mx +

b,where y=the methodvalue,x=the TDXvalue,m

= slope,and b = intercept.

cSy.x= standarderroroftheregression.

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GENTAMICIN AND TOBRAMYCIN ASSAY PROCEDURES 893

coefficient of variation of 3.8%. These results

show that the precision improved more than

twofold but the

analytical

bias still remained

(comparison with the results in Table 3).

DISCUSSION

Although radioimmunoassay has been well

established as aprecise and accurate methodfor

0s

a

02

/po

o

/8

R*

_~~~~~~~~~~~~ o

Z"

w o o 90 °o

I.9)

0 2 4 6 8 10 12

TDX GENTAMICIN

(pg/mi)

FIG. 1. Results of 68gentamicin-containing patient

samples. Regression linesare based ondebiased

re-gression.

Instruments made a modification ofthe

Auto-ICStowash the pipette tipwithbuffer solution

which is free of polyethylene glycol. With a

modifiedinstrument,werepeatedthewithin-run

precision study, using tobramycin at a

concen-tration of6.0 ,ug/ml. Twenty consecutive

deter-minations of thisspecimengave a meanvalue of

7.7 ,ug/ml, a standard deviation of 0.29, and a

12

E

10

0

6

m 4

0

Ln 2

0

: 12

E

T10

z 8 6

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m

0 0

12

E

N. 10

8

6

m 4

0

2

0

91

.: o -.1.0

ib. .r.--o

/

0 2 4 6 8

TDX TOBRAMYCIN (pg/mi)

FIG. 2. Results of 50 tobramycin-containing

pa-tient samples. Regressionlinesarebasedondebiased regression.

VOL.18, 1983

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894 WITEBSKY ET AL.

the measurement ofaminoglycoside serum

lev-els(3, 6), costand timeconstraintspreventedus

from performing radioimmunoassays on the

samples used in this

study.

AstheTDXshowed

thebestaccuracy(the least

proportional

bias)

of the systems testedfor

gentamicin

and

tobramy-cin measurement, we elected to compare the

results obtained on patient samples with the

other systems to the results obtained with the

TDX.Intherecoverystudies, among-day

preci-sion (coefficient of variation) was greater than

10% at some concentrations of gentamicin or

tobramycin for all thesystems. Asonly4

deter-minations were performed for tobramycin as

compared with 9 or 10 for

gentamicin,

the

among-day precision data for gentamicin are

more reliable than those for tobramycin. Our

results suggestthatfor

aminoglycoside

determi-nation it is not realistic to expect among-day

precision to be less than 10% throughout the

range of measurement. Only the TDX had

among-dayprecision values less than 10%atall

concentrations ofgentamicin assayed; only the

EMIThadamong-dayprecision valuesless than

10% at all concentrations of tobramycin

as-sayed. In the recovery

studies,

the Auto-ICS

showed the largest proportional bias and

overall showed the poorest

among-day

preci-sion, with valuesgreaterthan

10%

atfour of the

fiveconcentrations of

gentamicin

andtwoof the

four concentrations of

tobramycin assayed.

In

the within-run precision studies, the Auto-ICS

alsoshowed thelargest deviation from thetarget

values and the largest coefficient of variation.

The subsequent

modification

of the instrument

by

Beckman Instruments

improved

the

preci-sion (decreasedthe coefficient ofvariation), but

themagnitude ofdeviationfrom thetarget value

infact increased.

In the analysis ofthe data from the

patient

samples, the EMIT showed for

gentamicin

the

largest

proportional

bias

(-19%)

and the

largest

systematic bias

(intercept

of

0.68)

compared

withthe TDX. The reasonforthe

large

propor-tional and

systematic

biases with the EMIT is

not clear and is surprising in view ofthe good

correlation obtained with the EMIT versus

radi-oimmunoassayin other studies (4, 5). We have

previously evaluated the EMIT procedure (5),

although we did not use the CP-5000 EMIT

clinicalprocessor in that study; this instrument

considerably facilitates data handling, obviating

the need for manualcurveplotting and providing

a direct print-out of results. The COBAS-Bio,

which uses the EMIT reagents but in much

lower volumes per test, allows a considerable reagent cost-saving per test. The TDX is the

simplest instrumentto operate, and would seem

particularly suitable for high-volume

labora-toriesorthosewithalowvolume of

aminoglyco-side determinations butahighenough volume of

the other analyteswhich the instrumentis

capa-ble ofdetermining tojustify its purchase cost.

Since this evaluation, Abbott Laboratories has

employed a different curve fit procedure to

improve furthertheprecisionof the TDX at both

thehigher and lowerendsof the gentamicin and

tobramycin concentration ranges.

ACKNOWLEDGMENTS

Beckman InstrumentsloanedustheAuto-ICS andprovided the necessary reagents. Abbott Laboratories loaned us the TDX.

LITERATURE CITED

1. Cornbleet, P. J., and N. Gochman. 1979. Incorrect least-squaresregressioncoefficients in method-comparison

anal-ysis.Clin. Chem. 25:432-438.

2. Dixon, W. J., and F. J. Massey, Jr. 1969. Introduction to statisticalanalysis,p. 195-199. McGraw-Hill, New York. 3. Lantz, C. H., D. J. Lawrie, F. G. Witebsky, and J. D. MacLowry. 1980. Evaluation of serum gentamicin assay procedures foraclinical microbiology laboratory. J. Clin. Microbiol. 12:583-589.

4. Ngui-yen, J. H., T. Hofmann, M. Wigmore, and J. A. Smith. 1981.Comparative evaluation of three methods for measuring gentamicin and tobramycin in serum. Antimi-crob.Agents Chemother. 20:821-825.

5. Selepak,S.T.,F.G.Witebsky, E. A. Robertson, and J. D. MacLowry.1981.Evaluationof five gentamicin assay pro-cedures for clinical microbiology laboratories. J. Clin. Microbiol. 13:742-749.

6. Stevens,P.,L. S.Young,andW.L. Hewitt. 1975. Radioim-munossay, acetylating radio-enzymaticassay,and

micro-bioassayofgentamicin:acomparative study.J. Lab. Clin. Med. 86:349-359.

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