Center for disease control diagnostic immunology proficiency testing program results for 1976

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Copyright 01977 AmericanSociety forMicrobiology Printed inU.S.A.

Center for Disease Control

Diagnostic Immunology

Proficiency Testing Program Results

for 1976

ROGER N. TAYLOR,'* KAREN M. FULFORD,' VINCENT A. PRZYBYSZEWSKI,l AND

VICTORIA POPE2

DiagnosticImmunologySection, ProficiencyTestingBranch,'andVenereal DiseaseSerology Laboratory, Bacterial Immunology Branch,1 Center forDiseaseControl, Atlanta,Georgia 30333

Received forpublication4May1977

Over 900 laboratories participated inthe Diagnostic Immunology portionof

the 1976 Proficiency Testing Program, which was provided by the Center for

DiseaseControl under the authority of the Clinical Laboratories Improvement

Act of 1967. One hundredspecimens prepared bythe Centerfor DiseaseControl

for analysis were distributed on a quarterly schedule or in special surveys.

Feedback from participating laboratories included over 37,500 qualitative and

33,000 quantitative responses, which were analyzed to determine individual

laboratory proficiency levels. Inaddition, informationsuppliedby participants

in each survey helped to delineate trends in testingprotocols. The specimens

chosen for analysis called for a broad range of tests commonly performed in

diagnostic immunology laboratories, including those for rubella antibodies,

hepatitis B surface antigen, bacterial antibodies, rheumatoid factor,

immuno-globulins and other serum-specific proteins, and carcinoembryonic antigen. A

summary of the dataanalysisisprovidedsothat the laboratories can improve

theiroverall performance levels.

The Center forDiseaseControl (CDC)

Profi-ciency Testing Program was

developed

to

im-plement the Clinical Laboratories

Improve-mentActof1967 (4).Its function is tomeasure

laboratory performance with theintentof

eval-uating and improving that performance. The

program'sobjectivesareaccomplished by

iden-tifying deficiencies, evaluating methods, and

disseminating information.

Input tothisprogram isgainedinthe

follow-ing manner. CDC prepares specimens and

sends themtolicensed laboratories andtosome

nonlicensedparticipants(special study and

ref-erence laboratories) with the instruction that

they be tested in the manner routine to that

laboratory. This means that regular laboratory

staff membersareexpectedtoprocess the

speci-mens in the same manner and with the same

methods that they use for routine specimens.

Participation in the program is mandatory for laboratories that provide interstate testing ser-vices.

This report contains a summary of data

ob-tained from the 1976 Proficiency Testing

Pro-gramand discusses the implications of the

re-ported observations. Table 1lists types of tests

that the laboratories were requested to

per-form and tabulates the response levels of the

morethan 900 laboratories participating in the

Diagnostic Immunology portion of the

Profi-ciency Testing Program. Eachlaboratory was

asked to carry out onlythe tests that its staff

performs routinely.

Results ofspecificsurveys areprovided

else-where (5,6, 11,12,15, 16, 21-24).The purpose of

thisreport is todelineate the overall trends and

changes that becomeapparent when all of the

1976testdata wereevaluated.

MATERIALS AND METHODS

Most of the sera or plasma used for specimen preparationswerepurchasedfrom commercial sup-pliers on government contract. Other sources do-natedpools of humanserumofknown reactivityin

speciftictests.

Preliminary testing foracceptability of the sera wasdoneby the Diagnostic Immunology Section of the ProficiencyTestingBranch, by anappropriate CDCspecialty laboratory, orboth. HepatitisB sur-face antigen (HBsAg) reactivity for all sera was determined by the DiagnosticImmunology Section ortheVirology Section, Phoenix Laboratories Divi-sion.Onlysera negative for HBsAg by radioimmu-noassay wereusedfor specimens other than those to betested for HBsAg. Specimens obtainedasplasma weredefibrinated with calcium chloride or throm-bin.

Details ofspecimen preparationfor each survey areincludedinthesummaryanalyses(5, 6, 11, 12, 15, 16, 21-24). Briefly, specimens were adjustedto thedesired titer, filtered through sterile membrane filters, and dispensed into appropriate vials or 224

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CDC IMMUNOLOGY PROFICIENCY TESTING PROGRAM 225

TABLE 1. Summary of CDC Diagnostic Immunology Proficiency Testing Program 1976

No. of No.of Mean no. Determination chal-

sur-

of labs

re-Determnatioycl

sponding/

veys/yr

survey

Rubellaantibody ... 8 4 283

Streptococcal

antibod-ies

Antistreptolysin 0 6 2 354

Anti-deoxyribonucle-ase ... 6 2 23

Multiple

streptococ-cal antbodies ... 6 2 98

Rheumatoid factor .... 5 2 437

C-reactive protein .... 2 1 377

Infectious

mononucleo-sisserology ... 5 2 459

Antinuclear antibodies 4 2 337 Rickettsial antibodies. 1 1 359

Salmonellagroup D

an-tibody ... 1 1 365

Brucellaantibody... 2 1 393

Tularemiaantibody... 2 1 221

Toxoplasma antibody . 2 1 144

Immunoglobulin quan-titation

IgG... 7 2 333

IgA... 7 2 333

IgM ... 7 2 333 Complement

C3... 5 1 304

C4... 5 1 164

a-1-Antitrypsin ... 5 1 245

a-2-Macroglobulin .... 5 1 47

Haptoglobin... 5 1 213

Transferrin ... 5 1 82

Ceruloplasmin... 5 1 115

Carcinoembryonic

an-tigen ... 5 1 125 Syphilis... 40a 4a 404 HBsAg... 10 2 264

aDoes notinclude a replacement shipment of 10 samples

necessitatedby losses and delays in the mail.

tubes. Theadequacyofsampleswasconfirmed inde-pendently by the Diagnostic Immunology Section, by other CDClaboratories,andbyreference l1bora-tories. Anongoingquality control program used by theDiagnosticImmunologySectionensuresthat all specimenssatisfy preestablishedcriteria for

steril-ity, antibodytiter and stability, andbetween-vial

variability.

Each specimenshipmentwaspackagedin

accord-ancewithpostal regulationsandincluded

appropri-ate instructions and report forms. Completed

re-ports weretobepostmarkedwithin2 weeks of the initialshipping date. Responseswerecompiledand graded, and individualperformance rankings were reported to participants within 3 to 4weeks after responses werereceived. The acceptable responses weredetermined from referencelaboratoryresults. Overall-response data, which were evaluated and

compiledinsummaryanalysisorpublishedas

sepa-rate reports (18-20), were later sent toall partici-pants.

RESULTS

Rubella results were relatively constant

throughoutthe year (21-24). Geometric mean

titersfor samplessent outinconsecutive

quar-ters werenot significantly different (Table 2).

Slightchanges in methodologywerenoted. The

percentage of laboratories usingkaolin serum

treatment decreased slightly from 59.8 in the

firstquarterto 53.9 inthe fourth. Increases of

33.8 to 39.1% and 7.8 to 10.0% were noted in

those usingheparin-manganous chlorideserum

treatment and trypsinized human 0 cells,

re-spectively. Higher geometricmean titerswere

obtained with trypsinized human cells than

withother cells. Titers were about 24% lower

when chick cellswereused and 31% when

Ab-bott duracytes were used. Laboratories using

thekaolinserumtreatmenthadapercentageof

results outside the acceptable limits almost

twice aslargeasthose using the

heparin-man-ganouschlorideordextransulfateserum

treat-ment (Table 3).

The first-quarter shipment included four

specimens intendedtocomparethe variationin

test results for streptococcal antibodies

ob-tained withlyophilizedserum versuswhole

se-rumofhuman and horse antisera (21). Results

for thelyophilized specimens varied lessamong

respondents than those for whole sera.

Al-though the geometricmean antistreptolysin 0

titers and thepercentageofpositive qualitative

antistreptolysin 0 results were higher for the

horse serum specimens than for the human

serum specimens, the percentage of positive

multiple-enzymetestresultswas lower for the

horseserum(Table 4). Quality controltestshad

shown that the agglutination reaction ofthe

multiple-enzyme test was weaker with the

horse serumthan with the human serum, and

therefore the test withhorse seurm had to be

performed more carefullyfor accurate results.

Table 5 shows the participants' performance

with thestreptococcal antibodytests. The

aver-agepercentageofresponsesoutside the

accept-ablerange islargerfor the antistreptolysin 0

testthan forthe other twotests,but thehigher

TABLE 2. Resultsofrubellahemagglutination inhibitionfor repeatsamples

Geometricmean titer Sam- Survey Hepa- Dex- Kaolin

ples ea e-Koi

rin tran

A II, May 28 24 17

A II, May 29 23 17

A III, Aug. 26 25 15

B III, Aug. 56 49 50

B IV, Nov. 60 64 49

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TAYLOR ET AL.

TABLE 3. 1976rubellaproficiency testingresults

Reference _Accepta- No. of_N. Results_outside Sampleno. laboratoryrblerange patii acceptable

XG pants

~range

%

Heparin-MnCl2 or dextran sulfate

S6-001 8 .16 109 8

S6-002 111 64-256 108 20

S6-011 29 16-64 124 9

S6-012 29 16-64 124 9

S6-021 95 32-256 125 9

S6-022 35 16-128 122 11

S6-031 106 64-128 140! 49

S6-032 131 64-256 140 44

(20)b

Kaolin

S6-001 8 .16 155 I 19

S6-002 111 64-256 157 39

S6-011 29 16-64 148 36

S6-012 29 16-64 148 33

S6-021 95 32-256 159 12

S6-022 35 16-128 159 42

S6-031 106 64-128 157 69

S6-032 131 64-256 157 62

(39)b atG,Geometricmean.

bAverage.

TABLE 4. Results ofstreptococcalantibody tests

Antistreptolysin 0 Multiple

Serum enzyme

SrmGatiter| SDGb | %tive tive)

Human, whole 344 1.77 75 93 Human,lyophilized 205 1.50 68 85 Horse, whole 320 1.67 89 68 Horse, lyophilized 307 1.59 89 67

a'XG,Geometric mean.

bGeometricstandard deviation-antilog of the standard

deviationof thelog of titers.

percentages areresultsofsubstantially smaller

acceptable ranges for the antistreptolysin 0

tests. The number of laboratories reporting

anti-deoxyribonuclease Bandmultiple-enzyme

resultsis small but growing.

Because therehad beenexcessive variability

intherheumatoid factor test results from

pre-vious surveys, three specimens calibrated in

international

units (IU) for rheumatoid factor

were included in the second-quarter shipment

(19, 22). Results were reported in the usual

manner butwere then converted to IU by

com-paring results of the first two specimens

against the one containing 250 IU of

rheuma-toid factor per ml. The percentage of results

within one dilution of the median titer

in-creaseddramaticallyafterstandardization

(Ta-ble 6). Table 7 shows performance achieved

without the benefit ofstandardization in 1976.

Proceduresand distributions of results were

similar for the bacterial agglutination tests

(brucella, tularemia, salmonella group D, and

Weil-Felix-ProteusOX19)(21-24). Different

an-tigen sources apparently caused the greatest

variation in titers obtained. Geometric mean

titers ofslide test results were lower than of

tube test results, except for those from the test

for tularemia.Approximately 80% of the

partic-ipantsbased theirresultsonthe final dilution

of serum (including the antigen volume), and

about 65% used 2 + as the end-point reaction.

Titers of80 to 160 were considered significant

by mostlaboratories, but 70 to 80% considered a

changeintiterbetween acute and convalescent

sera to be more definitive. Table 8 shows the

performancewith these tests.

Few participant laboratories had difficulty distinguishing between negative and positive

samplesfor antinuclear antibodies by indirect

immunofluorescence methods; however, titers

were widely distributed (23, 24). In two

sur-veys, useofratliver cells resultedinthe

high-est geometric mean titers in these tests,

whereas the lowest titers were obtained with

TABLE 5. 1976streptococcalantibody proficiency testing results

Reference Results

Accepta- No.ofpar- outside

Sample no. laboratory blerange ticipants acceptable_{range (%)}

Antistreptoly-sin0

S6-003 180 160-250 340 22

S6-004 276 160-250 335 66

S6-005 287 250-333 344 38

S6-006 297 166-500 343 10

S6-033 443 256-625 364 49

S6-034 170 125-250 364 31

Anti-deoxyri- bonu-clease B

S6-003 254 166-680 12 17

S6-004 274 166-1360 13 23

S6-005 400 240-1360 13 15

S6-006 409 166-1360 13 8

S6-033 741 680-960 33 36

S6-034 202 170-240 31 26

(21J)b

Multiple enzymes

S6-003 156 .400 26 4

S6-004 113 s400 26 0

S6-005 115 c400 24 4

S6-006 155 .333 24 12

S6-033 342 200-500 25 44

S6-034 200 100-400 23 43

1

~~~~~(18)b

atG,Geometricmean. bAverage.

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TABLE 6. Effect of standardization on rheumatoid factortestresults Laboratories(%)reporting results equal to:

Test Median Median + 1tube Median + 2 tubes

Std" NotStdb Std Not Std Std Not Std

Slide 54.2 15.4 93.7 50.3 97.9 74.1

Tube 51.0 33.2 91.2 78.1 98.5 91.8

Total 41.0 27.1 91.0 61.3 97.6 75.1

aStd, Titers compared to reference preparation.

bNot Std, Raw titers reported.

TABLE 7. 1976 rheumatoid factor proficiency testing results

Reference

Results

laboratory Acceptable

No. ofpar-

outside

Sampleno. XZGaorqual- c

itative rane ticipants acceptable

sult range

Slide test

S6-013 Posb 1-640 73 3

S6-014 Pos 1-640 72 4

S6-015 Pos 1-2560 72 0

S6-023 Pos orNegcPosorNeg 44 0

S6-024 Pos Pos 79 38

(9)d

Latex tube

S6-013 346 160-640 171 22

S6-014 320 160-640 171 22

S6-015 691 640-2560 171 43

S6-023 254 Neg-320 146 11

S6-024 226 Neg-640 165 10

(22)d

at,Geometric mean.

bPos,Positive.

cNeg, Negative.

dAverage.

human leukocytes. Latex agglutination tests,

especially the Hylandtest, producedmore

neg-ative results on the lower-titered specimens

than did indirect immunofluorescence tests.

Only ninelaboratoriesreported usinga

peroxi-dase method fordetectingantinuclear

antibod-ies. Results of the antinuclear antibody and

toxoplasmatests areshown in Table 9.

Variables in the toxoplasma indirect

immu-nofluorescence results thatwerepointedoutin

the summary analysis include source of anti-gen, source of conjugate, and types of

micro-scopeocular andlightsourceused (23).

Table 10shows the infectious mononucleosis

test results. The percentage of results outside

the acceptable limits is greaterfor the oxcell

hemolysin tests than it is for the heterophile

tests. But, considering that the acceptable

ranges for the hemolytic testare smaller, the

performance is actuallybetter with theoxcell

hemolysin test. A detailed evaluation of tests

for infectious mononucleosis has been

pub-TABLE 8. 1976bacterial agglutininsproficiency testing results

Reference Aze ble lio Span Results Refe

n.abrencey

AcceptableNo. of par- outside

ac-Sample no. laboratory..

Ga range ticipants ceptable

range(%) Salmonella

Slide

S6-010 80 -160 300 17

Tube

S6-010 184 80-320 133 42

Weil-Felix Slide

S6-018 359 160-1280 293 15 Tube

S6-018 538 160-1280 129 9

Brucella Slide

S6-027 320 40-640 275 2

S6-028 226 40-640 271 1

Tube

S6-027 160 40-640 168 2

S6-028 180 40-640 168 5

Tularemia Slide

S6-035 160 160 138 60

S6-036 <20 <20 84 21

Tube

S6-035 135 80-160 131 26

S6-036 c40 c40 93 4

a

kG,

Geometricmean.

lished which compares sensitivity, specificity,

reproducibility, and cost of the mostfrequently

used tests (14).

Table 11 shows the immunoglobulin

quanti-tation results. Immunoglobulins (IgG, IgA,

IgM)arequantitatedby single radial

immuno-diffusion in most laboratories(22). Other

meth-ods usedbyparticipantsinclude automated

im-munoprecipitation, fluorometry,

electroimmu-noprecipitation, and lasarnephelometry. Most

results werereported inmilligramsper

decili-ter, but a few were in IU per milliliter. Results

(milligrams per deciliter) varied somewhat

among manufacturers of antiserum or plates. Usually more than half of the results were

outside the acceptable limits. The need for

standardization of this test isobvious.

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TABLE 9. Antinuclearantibodies andtoxoplasma proficiency testingresults

Reference No. of

Results

Samleno.laoraorAccepta-

paric- outside SamPle no. laboraty blerange pants acceptable

XGa pants range (%

Antinuclear antibody

S6-029 <20 <20 129 22

S6-030 212 80-512 130 28

S6-039 58 32-160 229 27

S6-040 <20 <20 161 6 (21)b

Toxoplasma Indirect

im-

munoflu-orescence

S6-037 108 32-256 113 24

S6-038 14 C32 106 37

(31)b Indirect

he- magglu-tination

S6-037 91 62-128 33 36

S6-038 45 32-64 31 23

(29)b

aXG, Geometricmean.

bAverage.

TABLE 10. 1976infectious mononucleosis proficiency testingresults

Reference No.of Results Sampleno. labOra- blerange

partici-

outside

toryXG Pants acceptable

_______ range (%)

Presumptive heterophile

S6-007 83 28-224 189 10

S6-008 <7 <7 117 71

S6-009 83 28-224 189 9

S6-025 22 <32 146 17

S6-026 96 10-224 175 9

(23)b

Oxcell

hemoly-sin

S6-007 135 80-160 24 33

S6-008 <10 <10 21 24

S6-009 135 80-160 24 33

S6-025 16 10-20 26 50

S6-026 80 40-160 28 21

(32)b

aXC,Geometricmean.

bAverage.

Because carcinoembryonic antigen (CEA) de-terminations are being performed in an in-creasing number of laboratories, a special

pro-ficiencytesting survey for CEA determinations

wasconductedinJune 1976 (20). Participation

was voluntary, and performance was evaluated

to assess inter- and intralaboratory variation

and toprovidebase-line data to help determine

whether CEA should be included in routine

surveys. Most of the participating laboratories

used the commercially available CEA-Roche

procedure, but a fewusedamethod in whicha

second antibody (anti-human globulin) is used

to precipitate the anti-CEA and bound CEA.

Nearly 25%of the results

reported

for the

sam-plesinthissurvey were

incorrectly

categorized

(normal, intermediate, elevated, or indicative

of metastasis). Differences in positivity

be-tweensamplesofnormal poolserum (1.1 ngof

CEA/ml)andserumwith aCEAconcentration

of 5.5 ng/dl were not detected in 16% ofthe

responses. Results reported for samples with

CEA levels -20 ng/ml showed that the direct

method produced significantly higher values

than the indirect method on either whole or

dilutVdplasma.

Third-generation tests for HBsAg are now

being usedin morethan95%ofthe laboratories

in the program,compared with68% a year ago

(18) (Fig. 1). Failure to detect HBsAg in

profi-ciencytesting samples has mostoften been

by

respondents using a second-generation test

only.False-positive resultsare most

frequently

obtained whenrespondents failtoconfirm

posi-tiveradioimmunoassay results. Table12shows

theresultsofthe hepatitisB surveys.

Table13shows the resultsofthreecommonly

usedtests fromthe syphilis serologyprogram.

Satisfactory performance percentages were

highforthese and for otherserologicaltests.

DISCUSSION

The Diagnostic Immunology portion of the

CDC

Proficiency

Testing Program

is intended TABLE 11. 1976immunoglobulin quantitation

proficiency testing

results

Reference No. of Results Sampleno.

_tory.

labora-_Ga0 Acceptable

patc-outside

range pants

acceptable

pnsrange

(%

Immuno-globulin G

S6-019 1,198b 1,120-1,300b 279 80

S6-020 802 750-850 279 87

(83)c

Immuno-globulin

A

S6-019 280 265-312 278 73

S6-020 124 80-150 277 46

Immuno-globulin

S6-019 442 330-532 278 54

S6-020 98 84-135 278 52

1

~~~~~~~~~(53)C

aXG,

Geometricmean.

bAllunitsinmilligramsperdeciliter.

cAverage.

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110

100

90

so

c 70 3rdGeneration

2 60

L 50 nGet

O 40

2'30

20

10

1stGeneration

0

1971 1972 1973 1974 1975 1976

FIG. 1. Tests used by participants in the CDC Proficiency TestingProgram for HBsAg. Multiple tests, Number of tests used expressed as the percentage in excess of 100% of the laboratories:

no.oftests -no.of laboratories

Multipletests=

100no.

of laboratories

TABLE 12. 1976hepatitis B proficiency testing resultsa

Acceptablere- No. ofpar- Incorrect Sample no.

Aceta

re-

pn.

op responses

L6-001 Neg 265 0

L6-002 Pos 265 12

L6-003 Pos 265 1

L6-004 Pos 265 12

L6-005 Pos 265 37

L6-006 Pos 262 8

L6-007 Neg 262 3

L6-008 Pos 262 0.4

L6-009 Pos 262 0.4

L6-010 Pos 262 9

(8)C

aL6-002 and L6-004 are

duplicates.

L6-005-HBsAglevelnearlower limitdetectable by

radioim-munoassay.

bPos, Positive;Neg, negative.

CAverage.

to measure laboratory proficiency for a broad

spectrumofserological

procedures.

Onecansee

from the list in Table 1 that the

samples

pro-vided encompass most of the tests commonly

used in aserology laboratory. Fourmajor

fac-tors are considered when the

samples

for the

diagnosticimmunology proficiency testing

pro-gram aredecidedupon: (i)thenumberof

labo-ratoriesthatprovidetherelevanttest, (ii)the

numberoftimesthoselaboratories

perform

the

test in ayear, (iii) theimportance ofthedisease

ordiseasestowhich thetestresultsare

related,

and (iv)the

degree

of

difficulty

thatis

usually

encountered in obtaining useful results. The

final samplemix isoften affected

by

other

fac-tors such as the availability of materials or

other resources and the regulatory

require-ments. Atpresent,the fourfactorslisted above

aresubjectively evaluated and

applied,

butan

effort is being made to

weight

them

numeri-callysothatamoreobjective

approach

to

plan-ning sample composition can be achieved.

Ef-forts arealso being madeto remove or modify

the otherconstraints.

The distribution oftests used and the

per-formancelevels for rubella testing

changed

lit-tle from previous years. There was some shift

towarduseof therecommendedmethodologies

(9, 10). It is strongly recommended that the

standardized methods be used.

They

were

de-veloped to minimize variation in results while

maintaining sensitivity and specificity. Poor

performance has

repeatedly

been correlated

with failure to adhereto the

established

stan-dards (21-24). The manuals

describing

these

methods can be obtained from the

Diagnostic

ImmunologyTraining Branch attheCDC.

Because the CDC selects reference

laborato-ries only from among those that use

recom-mended methodologies, participant

laborato-ries using cells or serum treatments that are

knownto

produce

lowerormorevariabletiters

are morelikelyto report

unacceptable

results.

Laboratories using kaolin serum treatment

hadalmost twice aslargeapercentageof

unac-ceptable results as did those

using

the

recom-mended serumtreatments. Previoussummary

analyses have shown that a similarsituation

exists when something other than fresh chick

ortrypsin human0 cellsareused

(23, 24).

To investigate the effects of

using

animal

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TABLE 13. 1976syphilisproficiencytesting results

FTA-ABSa VDRL° RPRC

Acceptable

_ceepartici-

No-t.

of % In-result correct

pants

2+ 126 9

B-2 + 126 21

2+-4+ 126 1

B-2+ 126 25

N 126 6

2+-3+ 126 6

N 126 9

2+-3+ 126 12

2+-3+ 126 0

2+-3+ 126 6

4+ 145 0

N 145 10

4+ 145 0

2+-4+ 145 0

3+-4+ 145 6

N 145 4

3+-4+ 145 2

4+ 145 0

N-B 145 14

2+-4+ 145 7

1+-3+ 144 58

4+ 144 1

3+-4+ 144 2

N 144 8

2+-3+ 144 12

3+-4+ 144 6

2+-3+ 144 13

4+ 144 3

N 144 9

4+ 144 1

4+ 154 2

4+ 154 0.6

N 154 7

4+ 154 2

2+ 154 19

4+ 154 1

2 + 154 19

N 154 7

4+ 154 1

(8)d

Acceptable No.o % In-result partici- correct

pants N-Wo N Rl-R2 N N N-Wo N N-Wo Rl-R2 N-Wo Rl-R2 N Wo-1 Wo N-Wo N N-Wo Wo-Rl N N N Wo Wo-Rl N N Wo N Wo-Rl N Ri-R2 Wo Ri Wo N RI N Wo N N Wo 165 2 165 11 165 5 165 7 165 1 165 3 165 4 165 3 165 5 165 5 201 2 201 1 201 3 201 16 201 1 201 2 201 1 201 6 201 2 201 14 195 4 195 30 195 2 195 0 195 42 195 22 195 3 195 4 195 2 195 1 205 26 205 23 205 35 205 2 205 24 205 3 205 27 205 3 205 0.5 205 36 (iO)d

AcetableAcceptbe partici-No. of %In

In-result pants correct

pants

N-Ri 131 0

N 131 5

R2-R4 131 2

N 131 4

N 131 2

N-Ri 131 2

N 131 2

N-Ri 131 2

R2-R4 131 2

N-Ri 131 1

R2-R4 153 4

N 153 4

R1-R2 153 4

Ri 153 21

N-Ri 153 2

N 153 1

N-Ri 153 3

R1-R2 153 3

N 153 3

N 153 10

N 164 4

R1-R2 164 2

R2 164 10

N 164 1

N-Ri 164 1

Ri 164 16

N 164 2

R2 164 10

N 164 1

R2-R4 164 3

R1-R2 172 4

R2-R4 172 3

Ri 172 48

N 172 2

R2-R4 172 3

N 172 5

R1-R2 172 5

N 172 5

N 172 1

Ri 172 48

(6)d

aFTA-ABS, Fluorescent treponemalantibody-absorptiontest.

bVDRL, VeneralDiseaseResearch Laboratorytest.

c RPR, RapidPlasmaReagintest.

dAverage.

serum versushumanserumandlyophilized se- Basedonproficiency testingexperience, itis

rum versus whole serum in a proficiencytest recommended that the anti-deoxyribonuclease

for streptococcal antibodies, combinations of Bandmultiple-enzymetests notbe usedasthe

each were included in the first shipment for onlyscreentodetect thepresenceof

antistrep-1976. The results revealed that, despite addi- tococcal antibodies. These tests are valuable

tional sources oferrorintroduced by lyophiliz- adjuncts to the antistreptolysin 0 test but

ing andreconstituting, therewaslessvariation should not be used in lieu ofit, especially for

in the results reported from lyophilized sam- measuringantibodiesintheseraof infants and

ples. The study also revealed that, when ani- olderpersonsinwhich the titersarefrequently

mal sera are used in proficiency testing, the nearthethreshold of thetests. Small laborato-resultsshouldbeinterpreted with caution. riesthatused onlythe multiple-enzymetest to Sampleno. T6-001 T6-002 T6-003 T6-004 T6-005 T6-006 T6-007 T6-008 T6-009 T6-010 T6-011 T6-012 T6-013 T6-014 T6-015 T6-016 T6-017 T6-018 T6-019 T6-020 T6-021 T6-022 T6-023 T6-024 T6-025 T6-026 T6-027 T6-028 T6-029 T6-030 T6-031 T6-032 T6-033 T6-034 T6-035 T6-036 T6-037 T6-038 T6-039 T6-040

230

TAYLOR ET AL.

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screen for antistreptococcal antibodies seemed

to have the most difficulty obtaining

satisfac-tory test results.

The largest source of variation in the

bacte-rial agglutination tests was the antigen (23). This reaffirms the necessity for using

standard-ized antigens when they are available, as for

the brucellatest (7), and emphasizes the need

todevelop standardantigens for the other

bac-terial agglutination tests. Recently, the CDC

reevaluated the reference antigen

recom-mended for use in the standardized brucella

agglutination tubetest and determinedthat it

should be diluted 1:50 instead of 1:100 to

con-form to the U.S. Department of Agriculture

standard (3, 7). The higher concentration

ap-pears to increase the reproducibility of results

byminimizing prozone and other erratic

reac-tionswhileproducingsharper end points.

Man-ufacturers have been notified of this change

and have been encouraged to use the same

brucella strain nowused by the National

Ani-mal Disease Laboratory.

Questions regardingthe sensitivity and

use-fulness of the slide agglutinationtests for

bru-cella have arisen many times, because many

laboratoriesusethemto screen or todetermine

titer. The CDC isconducting astudyinwhich

humanserum willbeusedto comparethetube

and slide tests. Results will bereported when

the studyiscompleted.

Standardization problems alsoexist withthe

antinuclear antibody tests. The various

sub-strates

produce

widely

varyingdegrees of

sensi-tivity. Because sensitivity and specificity are

usually inversely related, the latter probably

also varies widely. Until physical and

proce-dural standardsareformally developed, the

sit-uationcould be improvedby laboratories

volun-tarily using such reliabletests,substrates, and

reagents asthose listedby the CDC (2). To be

practical, standardization ofantinuclear

anti-body

and toxoplasma indirect

immunofluores-cence testsshould include specificationsof the

microscope oculars and the lightsources to be

used.

As a result of the poor performance in the

special survey, the CEA test will be

incorpo-rated into the routine proficiency testing

pro-gram for 1977 (20). Many of the aberrant CEA

results could have been eliminated by strict

adherence to the test

protocol

and to

quality

control procedures.

It waspreviouslyreported(18) that the most

reliable and economicalHBsAgresultsare

ob-tained by laboratories that usea

single

third-generation test

(preferably

radioimmunoas-say) andconfirm the

specificity

of

positive

reac-tionsby another method

(preferably

neutraliza-tion). Subsequentresults of

proficiency

testing

surveys indicate that many laboratories have

achieved good results by confirming positive

third-generation results with counterelectro-phoresis or some other second-generation test

andby reserving the neutralizationprocedures

forsamples containing antigen at levels below

thosedetectableby thesecond-generationtests.

By the end of 1976 more than 95% of the

laboratoriesin the Proficiency Testing Program

were using a third-generation test for HBsAg.

In light of their demonstrated superiority,

there can be little doubt that the increase in

numbers of laboratories using them represents

significant laboratoryimprovement. The rapid

shift toward third-generation tests is

gratify-ing, but, as their sensitivity increases, it

be-comes moreimportant to verify positive results

and thus eliminate false positives.

Therheumatoid factorstandardization

profi-ciency testing study (19) demonstrates that,

whereas a proficiency testing program can be

instrumental in improving laboratories by

evaluatingtestsandmethodologies(14), it can

also encourage standardization and estimate

*the amount of improvement thatcould be

ex-pected from standardization. To emphasize fur-ther the need for and value of standardization,

areference sampleforrheumatoid factorwillbe

included with future proficiency testing

sam-ples until otherstandardsarereadily available

and commonly used. The National Committee

for Clinical Laboratory Standards has

estab-lished a committee to study this problem and

developappropriate standards, but, until their

standards are available, better comparability

couldbe achieved by relatingtestresultstothe

international referencepreparation (1).

Although commercialstandards for

quantita-tive immunoglobulin determinations are

sup-posedly calibrated against the World Health

Organization Reference Preparation for

immu-noglobulins with valuesstatedinIUper

milli-liter, most laboratories continue to report in

milligrams per deciliter. These latter values

vary among manufacturers, as evidenced by

ourproficiency testing results and

by

the

var-ious conversion ratiosofmilligramsper

decili-ter toIUpermilliliter

reported

by the

manufac-turers themselves. Some of the variation in

immunoglobulin results could be avoided

by

using a reference preparation andreportingthe

results in IU per milliliter rather than

milli-grams per deciliter. If thelatter valuesare

pre-ferred,IUpermillilitercanbe convertedto

mil-ligrams per deciliter by the factors listed

by

Rowe etal. (13).

The major areas of

difficulty

with various

serological tests for

syphilis

occur with sera

having minimal levels of

reactivity.

Rarely

are

problems encounteredwith

clearly

reactiveand

VOL. 6, 1977

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(9)

nonreactive sera. Minimally reactive sera are

included to provide a morecritical evaluation of

laboratoryperformance. Withsuchserajudged

reactive by competent reference laboratories,

participants frequently report nonreactive

re-sults especially with theRapid Plasma Reagin

18-mmcircle cardtest. Asimilar problem has

been observed with the fluorescent

trepone-mal antibody-absorption test. Several factors

areinvolved, butamajor

problem

is feltto be

inexperience in test performance, since

well-standardizedreagents are

readily

available. A

testaddedtothe1977group,theReagin Screen

test, willprobably havesomeofthesame

vari-ation inresults becauseitscriticalareaisalso

the "minimally reactive" range. Acceptable

performance levels of the variouslaboratories

were high in syphilis serology in 1976. The

major change for the year was dropping the

automated fluorescent

treponemal

antibody

testfrom the programbecause equipment and

reagents are no longermanufactured. Any

re-sults sent by participating laboratories were

graded against the reference laboratories'

re-sults for the fluorescenttreponemal

antibody-absorption test.

Boththe artandscienceof proficiencytesting

are in their infancy. There is great need for

morebasic researchintolaboratory evaluation

methodology.Although theCDCandother

pro-ficiency testing agencies have a great deal of

experience withproficiency testing,some

ques-tions remain unanswered. Possible

mecha-nisms for achieving improved laboratory

per-formance by methods other than the traditional

approaches have been reviewed inthis paper,

butother potential roles of proficiency testing

needtobeexplored.

LITERATURE CITED

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2. Cavallaro, J. J., D. F. Palmer, and P. E. Bigazzi. 1976. Immunofluorescence detection of autoimmune dis-eases. Immunology series no. 7. Center for Disease Control, Atlanta, Ga.

3. Center forDiseaseControl. 1976.Bureauof Laborato-ries, current item 248. Recommended dilution of USDAbrucella reference antigen changed. Center for Disease Control, Atlanta, Ga.

4. Department of Health, Education, and Welfare. 1968.

42CFR part 74. Clinical laboratories; notice of pro-posed rule making, p. 15297-15303. Fed. Reg., vol. 33,

no. 201.

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