Evaluation of five gentamicin assay procedures for clinical microbiology laboratories

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0095-1137/81/040742-08$02.00/0

Evaluation of Five

Gentamicin Assay Procedures For Clinical

Microbiology Laboratories

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

ClinicalPathologyDepartment, National Institutesof Health,Bethesda, Maryland20205 Received 10October1980/Accepted 13January1981

Fivegentamicin assayprocedures (a bioassay,anenzymeimmunoassay,alatex agglutinationinhibitiontest,afluorescenceimmunoassay,and a radioimmunoas-say) were evaluated to determine which was optimal for our laboratory. The evaluationwasbased onrecoveryandprecisionstudies and results ofanalysesof patientsamples,aswell as technicalassayperformance factors. The latex agglu-tinationinhibition testappearsuseful for laboratoriesperforming onlyoccasional assays for gentamicin; however, the fact that some rheumatoid factor-positive sera,as well as some other sera for unknownreasons, maygive falselylow values isapotentialdrawback to thisprocedure.Because of itsaccuracy,precision, rapid turn-aroundtime,and relativesimplicityofperformance,weselected theenzyme immunoassay procedurefor routine use forgentamicin assaysin ourlaboratory.

Gentamicin continues to be a widely used antimicrobial agentforthetreatment ofa vari-ety of serious infections. It is important to be able to determine quickly and accurately the concentration ofgentamicin in serum, and

oc-casionallyin otherbothfluids,because of its low therapeutic index. A variety of techniques is available for gentamicin measurement. It was

thepurpose of thisstudytoevaluatefive differ-ent techniques, a bioassay, an enzyme

immu-noassay(EMIT),alatexagglutinationinhibition (card) test, afluorescence immunoassay (FIA),

and a radioimmunoassay (RIA), to determine whichwasoptimalforourlaboratory.

MATERIALS AND METHODS Recovery studies. Gentamicin sulfate (Schering Corp., Kenilworth, N.J.) concentrated solution was

prepared by drying powderedgentamicinsulfate for3 h at 60°C under partial vacuum and then weighing and dissolving the gentamicin in 0.1 M potassium phosphate buffer (pH8.0) to a finalconcentration of 1,000

Mg/ml.

This solution was then diluted in 18 separate humansera togivefinal concentrations of 0, 1, 2, 4, 8, 12, and16dg/ml.Fourcategoriesofsera were selected frompatientsnotreceivinggentamicin: nor-maltovisualinspection (nine patients); lipemic (three patients); icteric (three patients); hemolyzed (three patients). Portions (250 ,l) of each serum at each concentration werestored at-70°C until just before assay. These studies were not performed using the bioassay or RIA methods. For statistical handling of the data, when anassay gave a result of"lessthan"

some lower limiton asampletowhichnogentamicin had been added, a concentration of0

Mg/ml

was as-signed to that sample for that determination. If any

gentamicin had been added andaresult less than some

lowerlimit wasobtained,aconcentration of0.5kg/ml

wasassigned tothat sample for that determination. If a result of "greater than" some upper limit was ob-tained on one or both of duplicate determinations, neither of thevalueswasused in the statisticalanalysis of thedata.

Precision studies. Concentrated gentamicin sul-fate solution (described above) was added to pooled human serum from Clinical Center patients to give final concentrations of 3, 6, and 12 ig of gentamicin perml. Portions of250fland5ml ofserum ateach concentration were storedat -70°C until just before assay. To determine intrarun precision, 30 assays at each concentration were performed on the same day. Precision studies were not performed by the bioassay procedure.

Patient samples. A total of 110 serum samples wereselected fromClinical Center patients who were being treated with gentamicin and other antimicrobial agents.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. After completion of the bioassay, the residual serum was

keptfrozen at -20°C in screw-capped glass vials for upto 7months beforebeing thawed and divided into

250-dl portions, which were refrozen and stored at -70°C. These were thawed just before assay by one of the non-bioassayprocedures. For statistical handling ofthe data, when an assay gave a result less than some lower limit (generally 1

tig/ml),

a gentamicin concen-tration of 0.5

lig/ml

was assigned to that specimen. If aresult greaterthansomeupper limit was obtained, that value was not used in the statistical analysis of thedata.

Bioassay procedure. Bioassay results were ob-tained by ourroutine procedure,performed as previ-ously described (2), except that the gentamicin sulfate was dissolved in 0.1 M potassium phosphate buffer 742

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(pH 8.0), standards were prepared on a weekly basis ratherthan daily,and specimens and standards were

storedinglass, rather than plastic, vials. The highest concentration measurable in ourroutineprocedureis 16,ug/ml.

EMIT. EMIT (Syva, Palo Alto, Calif.) was per-formed according to the directions of the reagents

manufacturer (Syva), except that calibrator curve

points were all established byduplicate rather than

single determinations. (The manufacturer recom-mends thatonly the0calibratorbe run induplicate.)

Theprinciple ofthis procedureinvolves competitive bindingbetweengentamicinin asampleand a known amountofenzyme-labeledgentamicin for an anti-gen-tamicinantibody. Theenzymeemployedisglucose

6-phosphate dehydrogenasederived from the bacterium Leuconostoc mesenteroides. Binding of the

enzyme-labeled gentamicin decreases the activity of the en-zyme. The enzymatic activity remaining results in

conversionof thecoenzyme nicotinamideadenine

di-nucleotidetoreducednicotinamide adenine dinucleo-tide; the resulting absorbance change is measured

spectrophotometrically. The instrumentation used was a model 1500automatic pipetter-diluter (CAVRO

Scientific Instruments, Los Altos, Calif.), a Gilford

Stasar III spectrophotometer (Gilford Instrument

Laboratories, Inc., Columbia, Md.) and a Syva CP-1000 timer-printer (manufactured for Syva by

Ox-bridge, Inc., Mountain View, Calif.). Values for the

absorbance changesof thecalibrator standardswere

plottedonspecial graphpaperprovidedwitheach kit

ofreagents;gentamicinconcentrations forthesamples

weredeterminedfrom theresultingcurves.The

high-estconcentration measurable bythisprocedureis 16

ig/ml.

Cardtest.The latex agglutination inhibition test

(Macro-Vue Card Test, Hynson, Westcott and

Dun-ning, Divisionof Becton Dickinson, Baltimore,Md.)

was done asspecified in the directionsofthe

manu-facturer, exceptthat all determinationsweredone in

duplicateand theresultswereaveraged.Theprinciple of thisprocedureinvolvesthe inhibitionof

agglutina-tionofgentamicin-sensitized latex particleswhichare

addedto amixtureofsample andaknownamountof anti-gentamicinantibody. Quantitationofgentamicin concentrationisachievedbymultiplyingthe recipro-cal ofthehighest dilution ofsampleshowing

aggluti-nationinhibitionbythe lowestconcentration of

gen-tamicin standard also showing agglutination inhibi-tion. The highest concentration measurable by this procedurevariesdependingupon theendpointof the

standard;it rangesfrom12.8to19.2,g/ml.

FIA.FIA(AmesDivision,MilesLaboratories,Inc., Elkhart,Ind.) wasdone asspecifiedinthe directions of the reagents manufacturer, except that up to 39

sampleswere run per curve; this wasspecified as a permissibleprocedure bythemanufacturer. The prin-ciple of this procedure involves competitive binding

betweengentamicininasampleandaknownamount of sisomicin labeled with a fluorogenic substrate (a derivativeof

umbelliferyl-,/-D-galactoside)

foran

anti-gentamicin-sisomicinantibody.Bindingof the labeled sisomicintoantibodypreventshydrolysisof the

fluo-rogenic substrate. /3-Galactosidase hydrolyzes

avail-able fluorogenic substrate to produce a fluorescent product,the amount of which is measured fluoromet-rically. Thefluorometer used was the Aminco Fluoro-Colorimeter (American Instrument Co., Savage, Md.). Fluorescence units of the standards were plotted againstgentamicinconcentrationon graph paper pro-vided by Ames; the gentamicinconcentrations of the samples 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., Newport Beach, Calif.) as specified in the directions of the reagents manufacturer. The highest concentration

measurable by thisprocedureis 16

tig/ml.

RESULTS

Table 1 shows the results of the recovery studies on normal, hemolyzed, icteric, and li-pemic sera. The results from only seven normal serafor the FIA are shown in the concentration rangeofOto 8

lig/ml

because the sera from the other two patients were used up in runs from which no results could be obtained because of unsatisfactory standard curves. Table 2 shows the results oflinear regression analysis of the data presented in Table 1 exceptthat the anal-ysis has been done only for the concentration range of O to 8

fig/ml.

The 12- and 16-ftg/ml

concentrations were not used in this analysis because a full set of data was not available from all the procedures at these concentrations. An ideal method for measuring gentamicin would give a slope (m) of 1.0 and a y-intercept (b) of zero for each of the different types of sera. In addition, the correlation coefficient (of measured concentrationscompared with concentrations of

gentamicin actually added) would be 1.00, and the standard error of the estimate (interpretable

as a standard deviation [5]) would be 0.00. All

threeprocedures evaluatedbyrecovery studies appearedto perform satisfactorilywith normal and hemolyzed sera. With icteric sera the FIA tendedtooverestimate theamountof

gentami-cin present. Withlipemic sera the EMIT proce-dure tended to underestimate the amount of gentamicin present. This can be seen more clearly from the results in Table1than from the linearregression analysisinTable 2, which does notinclude theresults obtainedat12and16

jug/

ml. The FIA appeared tooverestimate slightly

the amount of gentamicin present in lipemic

sera, but the number of measurements was

small.

Table 3 shows the results of the studies on

intra-runprecision.

Table4shows the correlation coefficients for the linear regression lines obtained by

plotting

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TABLE 1. Recoverystudies

Recovery bymethod

Gentamicin FIA EMIT Cardtest

Serum concn added

(,ugrnl No.of f o. No. of

Mean +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 9C

normal 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 9C

4 4.2±0.6 7 3.9±0.2 9 3.4±1.2 9C

8 8.3+0.8 7 7.8±1.4 9 8.0±0.9 9

12 11.0 4d 10.7 8e 10.9±1.6 9

16 -f 14.1 35 13.8 4

Hemolyzed O 0.0±0.0 3 0.0±0.0 3 0.0±0.0 3

1 1.4±0.3 3 1.4±0.1 3 0.7±0.4 3

2 2.1±0.4 3 2.3±0.1 3 2.2±0.6 3

4 4.4±0.5 3 4.3±0.3 3 3.6±0.0 3

8 8.8±0.4 3 8.6±0.8 3 7.9± 1.5 3

12 - 11.5 1' 13.2± 1.2 3

16 J J 14.4 2

Icteric 0 0.6±1.0 3 0.0±0.0 3 0.0±0.0 3

1 1.7±0.6 3 1.1±0.0 3 1.0±0.5 3

2 3.1 0.2 3 2.0±0.0 3 1.7±0.1 3

4 5.2 0.2 3 4.1±0.2 3 3.5±0.2 3

8 10.1±0.3 3 7.6±0.0 3 7.6±0.7 3

12 11.8±0.5 3 12.8±1.4 3

16 - 16.0 li 14.4 2'

Lipemic 0 0.4±0.7 3 0.0±0.0 3 0.0±0.0 3

1 1.7±0.6 3 1.0±0.4 3 0.9±0.4 3

2 2.9±0.8 3 1.8±0.3 3 2.0±0.3 3

4 4.9±0.8 3 3.6±0.2 3 3.7±0.5 3

8 9.0±1.5 3 7.1±0.8 3 7.4±0.3 3

12 11.2 lm 8.8±0.6 3 12.0±0.0 3

16 l 10.8±1.6 3 14.4 2

aMeanmeasuredgentamicin concentration (micrograms per milliliter) and standard

deviation

(SD).

bNumber ofvalues used for determining each mean and standard

deviation.

cSeetextfor discussion ofproblemswithtwo sera.

dBy FIA threesamplesatthis concentration were measured as>12

,g/ml.

eBy EMITonesampleatthis concentrationwasmeasuredas >16

jug/ml.

fBy FIA allsamplesatthisconcentrationweremeasured as>12,ug/ml. gBy EMIT sixsamplesatthisconcentration were measured as >16

jg/ml.

hBycardtestfivesamplesatthis concentration were measured as greater thanthe highest concentration measurablebyatleastoneof thetwocards used.

By EMITtwosamplesatthis concentration were measured as >16,ug/ml. By EMIT all three samplesatthis concentration were measured as >16,tg/ml.

kBy card test onesampleat thisconcentration was measured asgreater thanthe highest concentration measurable by one of the two cards used.

'By card test one sampleat this concentration was measured as greater than the highest concentration measurablebyboth of thecards used.

mByFIAtwosamples at thisconcentration were measured as >12

jg/ml.

gentamicin concentration measured by each method against that measured by each other method for ail thepatient samples tested.The best correlation was obtained between the EMITand the RIA.

Figures 1, 2, 3, and 4 show the results of

plottingthedata forpatientsamplesasobtained

by RIA, bioassay, FIA, and card test,

respec-tively, against the results obtained by EMIT.

The dataaredisplayed inthis fashion because weultimately selectedthe EMITfor routine use inourlaboratory.Note inFig.4that there were fourpatient samplesfor which thecard test gave avalue oflessthan 1, whereasthe EMIT gave

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EVALUATION

TABLE 2. Linearregression analysisof datafromTableI

Parameters ofequation

Serum Method rh SEE'

y m x b

Visually FIA FIA 1.0 Concn added +0.0 0.99 0.50

normal 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 0.36

EMIT EMIT 1.0 Concn added +0.2 0.99 0.37

Icteric FIA FIA 1.2 Conen added +0.6 0.99 0.51

Lipemic FIA FIA 1.1 Concn added +0.6 0.97 0.83

Card Card 0.9 Concn added +0.0 0.99 0.31

aEquation of

regression

line inthe form y=mx+b.

br,Correlation coefficient.

SEE,Standarderrorof the estimate.

TABLE 3. Intra-run precision

Recoveryby method

Gentamicin FIA EMIT Card

concn added

()4/mMean±

SD<a

No. of val- Mean±SD No.ofval- Mean±SD No. of

val--ea

SI)a uesh - ues ues

3 3.1 0.3 30 3.3±0.1 30 3.0±0.6 30

6 7.7+0.6 30 6.4±0.1 30 5.8±0.7 30

12 - 11.1±0.6 30 10.8±1.8 28

aMean measured gentamicinconcentration and standard

deviation

(SD) (micrograms per milliliter).

bNumber of values used fordetermining each mean and standard

deviation.

CNomean orstandard

deviation

wascalculated for FIA at this sample concentration because 20 of the 30 samplesweremeasuredas >12,tg/ml.

dBy cardtest twosamplesatthis concentration weremeasuredasgreater thanthehighestconcentration

measurablebyoneof thetwocards used for eachsample.

TABLE 4. Correlationcoefficients forresults obtainedonpatient samples

Method Bioassay EMIT RIA FIA Card

Bioassay 1.00

EMIT 0.88 1.00

RIA 0.86 0.95 1.00

FIA 0.86 0.90 0.89 1.00

Card 0.82 0.85 0.84 0.85 1.00

values for these

samples

of2.5to8.1,ug/ml. For

these

samples

the results of the other assay

procedures(except for thebioassayon one sam-ple) were in agreementwith the EMIT results. After this

discrepancy

was

noted,

these

samples

were checked for the presence of rheumatoid factor. Rheumatoid factorwaspresentintwoof thesamples,butnotinthesampleshowingthe greatest

discrepancy.

We retained thetwo rheu-matoidfactor-positive serainthe data base for

E

`.10

co

o8

.2 6 o

EO

:>4 0

<: o Z~~~

ce 2 ç/ o

O

I

0 2 4 6 8 10 12

EMIT Gentamicin Conc. (,ug/mI)

FIG. 1. Linearregression lineforvaluesobtained by RIA versus EMITfor108clinicalspecimens (12 data pointssuperimposedonothers). See Table5for regression statistics.

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E12

10

0

c,

8

E6

c6

o2

m

0>

1~~~~~

co

o

Zoo

0

0 0 2

0

o cb

0

00

0 o

~0O

0 2 4 6 8 10 12

EMIT Gentamicin Conc. (ug/mI)

FIG. 2. Linear regressionlineforvaluesobtained by bioassay versusEMITfor99 clinicalspecimens (eight data points superimposedonothers).See Table

5for regression statistics.

12

E

S10r

c,' 8

*o 6

<r E

4, 0 Lw -~

2

Ï7~

o" ~

0 2 4 6 8 10 12

EMIT Gentamicin Conc. (i>g/ml)

FIG. 3. Linear regression lineforvalues obtained by FIA versus EMITfor 100clinical specimens (12

datapoints superimposedonothers).See Table 5for regression statistics.

the card test because we felt that only very

rarely would a clinical laboratory performing

gentamicinassaysknowthataparticular sample waspositiveforrheumatoidfactor.

Table 5 presents the results of the linear regressionanalysesofthe data inFig.1to4.The last column in this table shows the 95% confi-dence interval forasample determinedtobe6.0

kg/ml

by

the EMIT

procedure,

when

assayed by

the otherprocedures (1). The numbersmay be

approximated bysubstituting the number6 for the EMIT value (x value) in the equations of the formy = mx + b andadding the quantity

twotimes the standarderrorof theestimate.

Table 6shows the reagent cost of measuring

a gentamicin serum concentration, either as a

single sample oras a single sample analyzed in abatch ofsixsamples,for theEMIT, FIA, and

cardtest.

14

12

a)

10

c,

r-8

E

2P 6

o

-4

0

2

o

o *9

ow 0°00

.0e-O

o oW

, o o

Xo

0 2 4 6 8 10 12 14

EMIT Gentamicin Conc. (,ug/mi)

FIG. 4. Linear regression lineforvaluesobtained by card test versus EMITfor109clinical specimens (16data pointssuperimposed on others). See Table 5

for regression statistics.

TABLE 5. Linearregressionanalysis ofdata inFig.

1, 2,3, and 4

Parameters ofequation" 95% confi-dence

in-Method rh SEE' terval for

y m x b predicted

value"

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

Bioassay Bioassay 0.9EMIT +0.3 0.88 1.08 3.8-8.1 value

FIA FIA 1.1EMIT +0.3 0.90 1.12 4.9-9.4 value

Card Card 1.0EMIT -0.1 0.85 1.39 3.3-8.9 value

"Equationof

regression

line intheformy=mx +b. h r,Correlationcoefficient.

'SEE,Standarderrorof theestimate.

" For asamplewithagentamicinconcentration of 6jug/ml asdeterminedbyEMIT.

TABLE 6. Costofmeasuringagentamicinserum

concentration'

Cost ofprocedure"

Method Single sample

Single sample inbatch of six samples

EMIT $20.46 $6.51

FIA $9.00 $2.75

Cardtest $4.00 $4.00

aIncludesonlycostof kits. Doesnotinclude cost of materials(e.g., testtubes) notprovided with kits, and does notinclude laborcost.

hCost based on

manufacturers'

recommendations

fornumber ofreplicatesofstandards, controls, and patient samples.

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TABLE 7. Timesfor measuring gentamicin serum concentrationsofasingle sample and of a batch of

30samples

Time (min) forprocedure' Single sample Batchof30 samples Method

Elapsed Tech- Elasped

Technol-time nologist_time time ogist_tirne

EMIT 14 14 170b 170b

FIA 45 30 160 160

Card test 12 4 225 220

aTime required to complete indicated

procedure

plus a standard curve when applicable.

bThis timecanbereduced to2hwhen the CP5000

(Clinical Processor) is used.

Table 7 shows times required for the deter-mination of gentamicin serum concentrations either as asingle specimen or as abatch of 30 specimens.

Technical considerations. (i) Bioassay.

Advantages of the bioassay include the lack of need for sophisticated and costly instrumenta-tion, relative simplicity of performance, and broadapplicabilityto

virtually

anyantimicrobial

agent.Thetechniques for

setting

up abioassay

arefamiliar tomost

microbiologists.

Disadvan-tagesof the

bioassay

arethe timerequired from specimen receiptto result availability (a mini-mumof5h)andinterferenceby other antibiotics which eithercannotbeinactivatedorfor which resistantassayorganismsarenotavailable.

(ii)

EMIT. Advantages of the EMITassay are

extremely rapidturn-around time (about2min perspecimen after a standard curve has been

established), relative ease ofperformance, and

establishment ofastandardcurve

(usable

forat

least 24 h)

independently

ofspecimen assays.

This last feature means that "stat" specimens

can easily be

handled,

and also means

that,

should there beany

problems

withthe

establish-mentofastandardcurve,therehas beenno

loss

of time, reagents, or

samples

from specimen processing. We also found the

quality

control features of the EMIT to be

especially

useful.

Specifically, precise

intervals are

given

within

which certain calibrator

points

should

fall,

and precise limits for

acceptability

oftheresults of

thegentamicincontrolarestated.

Only

100

pl

of serumis

required

fora

duplicate

determination.

According

to the

package

insert there is no

knowninterferencewith theassay

by

antibiotics other thansisomicin ornetilmicin.A disadvan-tage of the

procedure

isthat

occasionally

there was some problem

establishing

an

acceptable

standard curve; this

difficulty

was

usually

re-lated to the 16-,ug/ml calibrator and

generally

requiredonly re-assaying this calibrator one to

three times more to establish a satisfactory value. It wasourimpressionthat,after mostof the reagents in a given bottle (designated "A" or

"B" by the manufacturer)had been used up,it

wasoccasionallydifficult toestablishastandard

curve with these small volumes. This problem

mayhave been due to difficulties withaccurate

aspiration when onlysmall volumes ofreagent

remained in the bottles; we have since found

that the problem disappears when such smail volumes (with materialofthesame lotnumber)

arepooled.

(iii)

Cardtest.

Advantages

ofthis

procedure

includeitslack of dependence on complex

equip-ment, lack of need for a standard curve, and rapidity.Accordingto information received from

the manufacturer, sisomicin does cross-react withthe assay; it is not known whether

netil-micinalsocross-reactswiththe assay. According tothe packageinsert there are no other known

cross-reacting antibiotics. Disadvantages we

foundwereasfollows. Sincethereis no

"batch-ing" with thistechnique, the time involved for running large numbers of specimens becomes

considerable. Also, as noted by the manufac-turer, anelevated rheumatoid factormaycause afalsely lowassayresult. Wefoundthis to be a

problem up to a gentamicinconcentration of 4

,ug/mIin onepatient sample, inwhich the level

was<1.0,g/mIbylatex agglutinationinhibition

butatleast4.0

,ug/ml

byallthe otherprocedures.

In addition, as noted above, two other patient

samples gave inexplicably low results by this

procedure. In the recovery studies, in two sera

that were visually normal, an unusual type of

agglutinationoccurred in the lower dilutions of

all gentamicin concentrations. This agglutina-tion was presumed to be due to an interfering substance,since itdisappearedathigher serum

dilutions. The presence of this phenomenon

meant that in these two sera only the higher concentrations of gentamicin could be accu-ratelydetermined.Forthese two sera, gentami-cin concentrationsup to and including4,g/ml

wereall readasless than the lowest

concentra-tionmeasurable by each ofthe twocards used

ateach concentration. Therefore, forthe

sam-plestowhich 0, 1,2, and 4 ,ug ofgentamicin had

been added perml, valuesof 0, 0.5, 0.5, and 0.5 ,ug/ml, respectively, were assigned. Finally, there is sometimes difficulty in determiningthe

pre-ciseagglutinationinhibitionendpoint;this diffi-cultydecreases but does notdisappearwith fa-miliaritywith theprocedure. The

clarity

ofthe endpointswas also notedtovary somewhat

be-tweenlot numbers.

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

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748 SELEPAK ET AL.

advantages to this procedure ascompared with the others evaluated. According to the package insert there is no known interference with the assay by antibiotics other than sisomicin or ne-tilmicin. Aprincipaldisadvantage ofthis proce-dure is that the standards on which the standard curve for agivenrun isbased are incorporated inthe same run asthesamples. Ifthe standard curveis notsatisfactory,the entire run,including

both standardsand samples, must be repeated

from the step in which standards and samples had been initially diluted in buffer. Duringthe course of the evaluationwe used two different fluorometers; with the firstinstrument, slightly

over one-third of the runshad to be repeated.

Performance was considerably better with the second instrument. Anotherdisadvantageis that manualmixing of dilutions isrequired, increas-ingthe risk ofspillage. Inaddition,quality con-trol is not well defined for this procedure. For

example, acceptableranges offluorescent units for the standardsare notspecified.

Also,

dupli-cate readings are supposed to be done, but no statement is made about maximum allowable

differences, if any, between

duplicate

readings. Finally,since the standard curveis constructed from point to point, rather than

using

a "best-fit" line through ail thepoints, controls should

probablyberun at aconcentration in the inter-val between each two consecutive

standards,

rather thanusingonlytheone control,"a com-mercialcontrol,"recommended

by

the manufac-turer.

DISCUSSION

We have previously evaluated a number of different RIAprocedures (2). Althoughmany of theseare accurateandprecise, they requirethe use of expensive and sophisticated equipment

and are not cost-effective for low-volume oper-ation, as astandard curvemustbedevelopedfor eachrun.

The particular FIA procedure we evaluated

suffers fromanumber of technicalproblems, as detailed above. We attempted to evaluate the FIAprocedureof BioRad(BioRadLaboratories, Richmond,Calif.) but hadtoabandonthatpart of the study because of numerous technical problems with the reagents and instrumenta-tion.

The card testappears useful forlaboratories performing only occasional assays for gentami-cin; however, the fact that some rheumatoid

factor-positive sera, as

weil

as some other sera for unknownreasons, will give falsely low values is a potential source ofdifficulty with this pro-cedure.After thisstudy had been completed, we were informed that others have foundthat the

useof serum which has been repeatedly frozen andthawed may produce results with the card test thatcorrelate poorly with the results of both EMIT and RIAprocedures, whereas the use of fresh serum or serum which has been frozen only onceproduces results that correlate better with these procedures (D. Bernstein, of Hynson, Westcott and Dunning, personal communica-tion). Itshould be noted that the patient samples we used in this study were all frozen twice; hence, it is possible that a better correlation of the card test results with both EMIT and RIA results might be obtained with fresh serum or serumfrozen and thawedonly once.

Thebioassay procedure appears less accurate than either RIA or EMIT andalso has an exces-sivelylong 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-aroundtime, andrelativesimplicity of perform-ance as determined in ourstudy,weselected the EMIT procedure for routine use forgentamicin assays in ourlaboratory. Since the conclusion of thisstudy, Syva hasdevelopedacomputer pro-gram for use withthe assay; this program elim-inatesthe need for manual plotting of a standard curve and makesperformance of the assay pro-cedure still more simple. We should note that ourlaboratory has established the policy of di-luting any specimen and reassaying the dilution if theinitial determinationis -10 ig/ml.

Only small numbers of severely hemolyzed, icteric,andlipemic sera were tested, and all of these were sera to which gentamicin had been added in the laboratory. No patient sera that wereseverely hemolyzed, icteric, or lipemic were tested. Therefore, despite the fact that we de-tected noproblem with assaying hemolyzed sera, and no problem assaying icteric sera with the EMITprocedure, weare not prepared to state that one can accurately assay severely

hemo-lyzed oricteric sera by either the FIA or EMIT procedure. We do believe, however, that our data demonstrate that lipemic sera cannot be adequately assayed by the EMIT procedure.

With all oftheprocedures, it isimperativethat themanufacturer'sinstructions be followed pre-cisely.

There is considerable variation among the methods both inthe cost of reagents alone and intechnologist time, as noted in Tables 6 and 7. Actualreagent costs per test may differ greatly from these theoretical costs if standard curves or entire runs have to be repeated, as noted above. As withreagent costs per test, labor costs per testvary significantly with batch size for any

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(8)

procedure for which the number of standards and controls required is largelyindependent of the number ofclinical specimens inagivenrun.

It is important to note also that we have not

attemptedtoanalyze the effect ofcostsof

non-reagent materials, such as test tubes, nor the

effect of instrumentcosts onthe total costper test.

ACKNOWLEDGMENTS

We thank M. V.Ratnaparkhi and MarkH.Zweigforhelp with study design and data analysis,James E.Byrkitand Rita G. Minkerforhelpwith datahandling,andVirginiaM. Hol-combfor technical assistance.

LITERATURE CITED

1. Afifi, A. A.,andS. P. Azen.1972.Statisticalanalysis: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 ofserumgentamicin

as-sayprocedures foraclinical microbiology laboratory. J.

Clin.Microbiol. 12:583-589.

3. O'Leary, T. D., R. M. Ratcliff,andT.D.Geary. 1980. Evaluation ofanenzymeimmunoassay forserum

gen-tamicin.Antimicrob.Agents Chemother.17:776-778.

4. Phaneuf, D.,E.Francke,and H. C.Neu.1980.Rapid, reproducible enzymeimmunoassay for gentamicin. J.

Clin. Microbiol.11:266-269.

5. Westgard, J. O., and M. R. Hunt.1973.Use and inter-pretation ofcommonstatisticaltestsin method-com-parison studies. Clin. Chem. 19:49-57.