new reference reagent for the quality assurance of anti-d antibody detection

New reference reagent for the quality assurance of anti-Dantibody detectionP. Phillips,* D. Voak,† M. Downie,† N. Clark,† J. Miguta,‡ J. Rawlings,§ B. Patel§ and M.Redman§*National Institute for Biological Standards and Control (NIBSC), Potters Bar, UK,†East Anglia Blood Centre, Cambridge, UK,

‡South Thames Blood Centre, Tooting, UK,§North London Blood Centre, Colindale, UK

Received 11 March 1998; accepted for publication 21 May 1998

SUMMARY. A UK BTS-NIBSC freeze-dried anti-D pre-paration has been prepared and used to monitor theperformance of routine antibody detection tests and ofthe test operators. With the day-to-day use of this pre-paration, adverse changes in test performance and in testoperator may be detected and appropriate action takenbefore the effect becomes significant. Two dilutions ofthis preparation have been defined, one which should bedetected unequivocally in every test; the other, moredilute, may not be detected in every test but is used tomonitor changes in performance. Experience with theuse of this preparation is reported from three test centres

undertaking routine antibody detection tests. By mon-itoring results over a series of working days, significantdifferences were noted in operator performance withinone test centre, as was a reduced sensitivity of a giventest system within one test centre compared with thesame system in the other test centres. These differenceswere detected only by monitoring the results obtainedwith this preparation.

Key words: antibody screening, monitoring of test opera-tor, monitoring of test performance, quality assurance.

Quality Systems compliant with ISO Guide 25, such asBS EN 45001, define systems for test laboratories,particularly those performing tests for third parties.These Quality Systems address not only the laboratory’smanagement system but also its technical ability, includ-ing training and qualifications of personnel, validity oftest methods and procedures, uncertainty of test measure-ments, the suitability of test equipment and of the testenvironment. Such ISO Guide 25 compliant QualitySystems define the technical standard to be achievedusing internationally agreed criteria, assuring the techni-cal competence of the test laboratory’s services and thevalidity of its test data. This is unlike quality manage-ment systems compliant with BS EN ISO 9001/2 whichensure consistency of operation to some quality objectiveof the organization’s own selection (Phillipset al., 1993).

With ISO Guide 25 compliant quality systems, prior toundertaking tests, operators need to undertake a definedtraining programme and to be assessed using definedobjective criteria for satisfactory performance. In addi-tion, there needs to be a mechanism for assuring the

continued satisfactory performance of the test and testoperator. By this means, deterioration in test or operatorperformance may be detected and acted upon before itbecomes significant to the test results.

One such method is for tests to include aliquots of atest material, stored prior to testing at a suitable tem-perature to ensure stability. The test response with anindication of the particular test operator is tabulated orplotted. When the test is under control, the test responsewill be a scatter, indicating the reproducibility of the testmethod, about some mean response. A deterioration intest or operator performance will be indicated by a shiftin test response and appropriate action instigated.

This report details the usefulness of selected dilutionsof a freeze-dried anti-D preparation, prepared by the UKTransfusion Service-NIBSC Standing Advisory Groupfor Immuno-haematological Reagents, used to assureroutine antibody detection tests by a daily internal datamonitoring procedure. This principle is already in usewithin UK Blood Centres to monitor the performance ofHBV, HCV and HIV viral marker detection tests (LiasonGroup of the UK Blood Transfusion Services – NationalInstitute for Biological Standards and Control, 1995).This report is the first example of its application toroutine antibody screening.

Transfusion Medicine, 1998,8, 225–230

225q 1998 Blackwell Science Ltd

Correspondence to: National Institute for Biological Standards andControl, Blanche Lane, South Mimms, Potters Bar, Herts. EN6 3QG,UK.

MATERIALS AND METHODS

A 4-L pool of anti-D plasma (coded 95/784), eachconstituent unit being less than 5 IU mL–1, was freeze-dried in nominal 1-mL quantities in neutral glassampoules, using procedures operating under a qualitymanagement system certified to ISO 9001.

A number of ampoules were opened and reconstitutedin 1 mL distilled water. Master dilutions (1 in 8, 1 in 16and 1 in 32) were prepared using inert pooled plasma.Aliquots of these master dilutions and of the diluentplasma were coded, stored frozen and distributed to threetest centres (East Anglia, North London and South

Thames Transfusion Centres) for storage at – 208C orbelow prior to testing.

The test methods (Table 1) were those in routine usefor antibody screening within the test centres. Pools of4–6 group OR1r red cells were used to minimize effectsdue to variation in D antigen site-density. Material wastested at least daily over a series of working days.

RESULTS

A previous anti-D preparation (91/608) (Phillipset al.,1992) was used to assure the efficacy of red cell washers

226 P. Phillips et al.

q 1998 Blackwell Science Ltd,Transfusion Medicine, 8, 225–230

Table 1. Test methods.

Test centre and test methods used Description of test method.

Test Centre ALISS Low-ionic-strength (suspension) spin tube antiglobulin methodDiamed gel (manual) Antiglobulin methodBioVue bead (manual) Antiglobulin method

Test Centre BLIM Low-ionic-strength addition antiglobulin method (i.e. a low-ionic-strength medium

added to a red cell/plasma mixture)NISS Normal-ionic-strength antiglobulin methodLIP Low-ionic-strength addition, polybrene addition, antiglobulin methodDiamed gel (manual) Antiglobulin method

Test Centre CDiamed gel (automated) Automated (Sampler II) antiglobulin methodDiamed gel (manual) Antiglobulin method

Table 2. Comparative sensitivity of routine antibody screen methods in the detection of aliquots of dilutions of anti-D 95/784using pooled OR1r red cells.

Percentage positive reactions (þ þ þ to w) by various routine antiglobulin test methods

Aliquot Column technology Spin tube methodsof anti-Dat dilution Gel Auto Gel Manual Bead Manual LISS NISS LIM LIP

1 in 8 100 100 100 100 100 98 100 100 1001 in 16 89 98 100 89 79 95 100 100 921 in 32 0 95 42 0 15 79 33 21 8Inert diluent 0 0 0 0 0 7 9 0 0No. ofresults 39 42 50 39 33 42 48 48 48Test site C A B C A A B B BNo. oftest operators 2 5 1 2 5 9 1 1 1

and of test operators undertaking spin-tube antiglobulintests. Anti-D 95/784 supersedes that preparation.

Comparative sensitivity

Table 2 shows the comparative sensitivity of the variousantibody detection methods used by the three test centresusing their routine procedures, in the detection of ali-quots of the master dilutions of anti-D 95/784 usingpooled OR1r red cells.

It is seen that all test methods in all test centres detectaliquots of the 1 in 8 dilution with almost 100% detec-tion. The single failure to detect the anti-D aliquot of adilution of 1 in 8 is clearly not acceptable.

With aliquots of the 1 in 16 dilution, differences areapparent between the test methods, which are even morestriking at 1 in 32. In particular, the detection rate in thethree test centres using the same method (Diamed gelmanual) ranged from 0% to 95%. Clearly, test site C isoperating to a lower sensitivity in the Diamed gel(manual) test than the others, bearing in mind that allthree test sites are using one type of test kit, pooled testred cells and aliquots of the same master dilutions.

With aliquots of the 1 in 32 dilution, the lower sensi-tivity of low-ionic-strength addition methods (LIM, LIP)is seen compared with a low-ionic-strength suspensionmethod (LISS). A small percentage of false positivereactions were seen with the LISS and NISS methods.

Quality assurance of anti-D antibody detection227


Table 3. Day-to-day variation within onetest centre in the test result obtained withaliquots of dilutions of anti-D 95/784using pooled OR1r red cells.

Test method

Diamed gel Diamed gelAutomated Manual

Dateof aliquot of anti-D at dilution: Testtest 1 in 8 1 in 16 1 in 8 1 in 16 operator

27 May 1997 þ þ þ þ þ þ þ 128 May 1997 þ* w* þ w 230 May 1997 þ þ þ þ þ þ þ 102 June 1997 þ þ w* þ w 103 June 1997 þ þ þ þ þ 104 June 1997 þ þ þ þ þ 105 June 1997 þ þ þ þ þ þ 106 June 1997 þ þ þ þ þ þ þ þ 109 June 1997 þ þ þ þ þ þ þ 113 June 1997 þ þ w þ þ þ 116 June 1997 þ þ w þ þ þ þ 117 June 1997 þ þ þ þ þ þ þ 118 June 1997 þ þ w þ þ w 119 June 1997 þ þ w þ þ þ 120 June 1997 þ þ þ þ þ þ 123 June 1997 þ þ w 0† þ 124 June 1997 þ þ w þ þ þ 126 June 1997 þ þ w þ þ w 127 June 1997 þ þ w þ þ þ 130 June 1997 þ þ þ þ þ þ þ 207 July 1997 þ þ þ þ þ þ 215 July 1997 þ 0 þ 0 217 July 1997 þ 0 þ 0 221 July 1997 þ 0 þ 0 222 July 1997 þ þ w þ þ þ 224 July 1997 þ þ w þ þ þ 225 July 1997 þ þ w þ þ þ 2

*Result indeterminate by automated reader, human reading of result indicated. †A review of theresults of this study indicates a mix-up of test samples with the diluent sample.

Monitoring of day-to-day variation withina test centre

All the test methods used in the three test centres showedday-to-day variation in the test results obtained withaliquots of the dilutions. An example is shown in Table 3.

With aliquots of the 1 in 8 master dilution, the resultswith the gel test (manual) ranged fromþ þ þ to 0. Onreview of the results, the negative test result was due to amix up of the test samples by the test operator. With theautomated gel test, the results ranged fromþ þ to þ.One of theþ test results was obtained after operatorintervention of a machine-indeterminate result.

It is clear there is a wide variation in the resultsobtained, even when the variables are minimized by onetest operator in a test centre, using pooled OR1r red cellsand aliquots of the same master dilutions. This reflects theinherent variability of the antibody detection system. On3 days, a negative result was obtained with the aliquot at 1in 16 despite a positive reaction on the other 24 days.

DISCUSSION

Value of performance monitoring

The use of performance monitoring is a simple procedureto permit small changes in performance to be detected.Using a larger set of data than reported here would permitdifferences in performance between test operators or indifferent batches of reagents/kits to be detected. Wherethere is a deterioration in performance, it may well bedetected before the deterioration becomes significant tothe test results and hence contribute to maintaining thequality objectives of the test laboratory.

Performance monitoring can be used to demonstrate atest centre’s ability to test consistently to a given sensi-tivity. Where a weak monitoring sample is consistentlydetected by a test operator, a negative test result on a testspecimen has a far greater credence than a negative testresult in the absence of such data monitoring. This isparticularly relevant to antenatal antibody detectionwhere the majority of test samples would be expectednot to have alloantibody. The reduced sensitivity observedin this study with test centre C, detected only by the use ofthisperformance monitoring, wassubsequently shown(D.Voak, personal communication) to be due to the use of‘ID-Cellstab’ red cell suspension medium. Change to ‘ID-Diluent 2’ as used in test centre A improved test perfor-mance in test centre C. (A comparison of test methodol-ogies also shows that test centre B used an ‘in-house’method comprising a different low-ionic-strength addi-tion medium, serum volume, red cell suspension strength,and incubation period than recommended for use by themanufacturer. This may be a factor in the lesser sensitivityof test centre B, compared with centre A, in the detection

of aliquots of the 1 in 32 dilution, although the sensitivitiesare comparable at the lesser dilutions.)

Perhaps hospital accreditation bodies (which are notISO Guide 25 compliant) and managers placing contractsfor antibody screening should determine what measuresare in place for ensuring consistent and adequate sensi-tivity of antibody detection procedures. Such measuresbecome more important when viewed against suchchanges as the trend to remove the cross-match in certaincircumstances and the use of less well trained staff. Itshould be noted this study indicates that test kits, includ-ing automated readers, are not excluded from the need toassure continued satisfactory performance. Test andoperator data monitoring may be useful in objectivelydemonstrating laboratory performance when adversetransfusion-related incidents are being analysed, suchas is being undertaken with the UK ‘Serious Hazardsof Transfusion’ (SHOT) programme.

A suitable, stable material could be prepared for usewithin a test centre. However, the use of a nationalpreparation permits comparison to be made betweendifferent test centres. The use of pooled R1r test cellswith the dilutions of anti-D 95/784 and its diluentminimizes effects due to variations in D antigen site-density between different batches of red cells, allowingany variation in test operator or test method to be moreapparent. The anti-D 95/784 dilutions and diluent couldbe tested with the red cell samples used in the antibodyscreening tests but the day-to-day variation in results willbe compounded by variations between the red cellsamples. However, if used in this manner, the anti-D95/784 dilutions and diluent could have an additional usein replacing existing positive and negative red cellcontrols, minimizing addition workload.

Performance monitoring is a simple quality assurancemeasure that is inexpensive to implement; the materialcould replace an existing positive control. Experiencewith internal data monitoring in other test areas, forexample EN 45001 accredited test laboratories, hasshown that the test operators themselves are bestplaced to record results, particularly in graphical form,which can be a visible indication of their performanceand contribution to the test procedure. Where test opera-tors under-achieve, this indicates a need for retrainingwhich should be undertaken in a constructive manner toensure their continued motivation.

Dilutions of anti-D 95/784 recommended for use

The use of aliquots at the 1 in 16 dilution is moredemanding of the test methods than aliquots at the 1 in8 dilution. Since there is no clinical evidence to suggestthat the routine tests used in this study are failing to detectantibodies of clinical significance, it is recommended by



UK Transfusion Service-NIBSC Standing Advisory Groupfor Immuno-haematological Reagents* that on each work-ing day, every test operator includes anti-D 95/784 at 1 in8 and 1 in 16, together with the diluent pooled plasma, intheir routine antibody screening testing. The use of codedsamples would eliminate bias but introduces a complex-ity into the test procedure, since it would involve adifferent preparation code for each day and, ideally, foreach test operator.

All test laboratories should be able to detect unequi-vocally anti-D 95/784 at 1 in 8 in every routine antibodyscreening test. Results should not be reported unless anunequivocally positive test result with anti-D 95/784 at 1in 8 is obtained. If an operator fails to detect anti-D 95/784 at 1 in 8, this should trigger an immediate investiga-tion to determine the cause. If the failure is because ofoperator performance, individual confidential tuition andassessment should be given to restore operator confi-dence and performance. If the failure is because of someother reason, appropriate action should be taken torestore satisfactory performance. If the failure is obtainedduring ‘on-call’ testing, the whole antibody screeningtest should be repeated and, if a repeat failure is obtained,assistance should be sought from a more experiencedoperator.

Test laboratories should expect not to detect unequi-vocally anti-D 95/784 at 1 in 16 in every test; however, itis expected that it will be detected in at least 50% of testsover a period of time. Furthermore, by monitoring thegrade of reaction with this dilution, any changes in testand/or operator performance can be detected and appro-priate action taken. The use of the 1 in 8 dilution wouldbe unlikely to enable changes in performance to be seen.

The use of these dilutions needs to be put in contextwith other controls routinely used in antibody screening.External Quality Assessment Schemes have a role to playin assessing performance but exercises tend to be infre-quent, perhaps 4–6 times a year, and, unlike the perfor-mance monitoring reported here, do not assess everyoperator on each of their working days. Replicate testswere initially designed to test red cell washer efficacyand operator performance in the manual spin-tube anti-globulin tests. Performance monitoring is an extension ofthis principle. The use of IgG-coated red cells to demon-strate adequate reactivity of antiglobulin reagent in testswith negative results is a key control in manual tests. Itsuse is limited by such cells, in general, being too stronglycoated that a gross loss in anti-IgG reactivity is needed toeffect noticeable reduction in red cell agglutination(Voak et al., 1985). Furthermore, the use of coated redcells is not always applicable to antiglobulin tests other

than spin-tube tests. A weak antibody control is oftenused with each batch of antibody screening test todemonstrate adequate reactivity. In principle, analysisof data from such a control could provide data to monitoroperator and test performance. The advantage of anti-D95/784 is as a nationally available preparation which testlaboratories can use to demonstrate satisfactory andcomparable sensitivity.

Preparation 95/784 has anti-D activity. New antibodydetection technologies differ in their comparative sensi-tivity to anti-D, anti-K and anti-Fya (Pooleet al., 1995).Some solid-phase antibody detection systems have failedto detect anti-K due to the quality of the red cells used(Chapman, personal communication). Antibody detec-tion systems are vulnerable in the detection of anti-Kbecause of the logistical difficulty in consistently produ-cing a matched-pair of R1R1 and R2R2 reagent red cellsbetween them with homozygous expression of all anti-gens of probable clinical significance, including Kell(Phillips & Voak, 1995, 1996), and because of the highproportion of Kk red cells with reduced site density (D.Voak, personal communication). The use of internal datamonitoring with an anti-K preparation should enablethe ready detection of under-performing test systems.Studies are in place to investigate the usefulness ofextending internal data monitoring to include anti-Fya

and anti-K, in addition to anti-D.As test systems progress from manual fully automated

operation with automated sample handling, data capture,interpretation and report generation, assurance of satis-factory test performance by data monitoring will benecessary, particularly when staff have little serologicalknowledge or skill. Anti-D 95/784 is of value not only tothe user but to the manufacturer, in the developmentstages of new technologies in order to demonstratesatisfactory performance and in post-marketing demon-stration of continued satisfactory performance, particu-larly if the proposed Europeanin vitro DiagnosticMedical Device Directive becomes operative.

ACKNOWLEDGMENT

We thank the staff of Standards Division, NIBSC, fortheir expertise in processing anti-D 95/784.

REFERENCES

British Standards Institution. (1989) BS EN 45001.GeneralCriteria for the Operation of Testing Laboratories.

British Standards Institution. (1994) BS EN ISO 9001.QualitySystems. Model for Quality Assurance in Design, Develop-ment, Production, Installation and Servicing.

British Standards Institution. (1994) BS EN ISO 9002.QualitySystems. Model for Quality Assurance in Production, Instal-lation and Servicing.

Quality assurance of anti-D antibody detection229


*Anti-D 95/784 is available from the National Institute for BiologicalStandards and Control.

Liaison Group of the UK Blood Transfusion Services –National Institute for Biological Standards and Control.(1996)Guidelines for the Blood Transfusion Services in theUnited Kingdom, 3rd edn.

Phillips, P.K. & Voak, D. (1996). Can pooled red cells be usedfor antibody screening of patients’ specimens?TransfusionMedicine, 6, 307–309.

Phillips, P.K. & Voak, D. (1997) Can pooled red cells be usedfor antibody screening of patients’ specimens.TransfusionMedicine, 7, 319–324 (letter).

Phillips, P.K, Voak, D, Smith, K, Rowan, R.M. & Lewis, S.M.(1994) The illusion of quality in quality management sys-tems: meaningful accreditation.Transfusion Medicine, 4,179–183.

Phillips, P.K, Voak, D, Whitton, C.M, Downie, D.M. &Bebbington, C. (1993) BCSH-NIBSC anti-D referencereagent for antiglobulin test: the in-house assessment of redcell washing centrifuges and of operator variability in thedetection of weak, macroscopic agglutination.TransfusionMedicine, 3, 143–148.

Poole, G.D, Evans, R.G, Voak, D, Scott, M.L, Chapman, J.F. &Phillips, P.K. (1996).Evaluation of Six Systems for theDetection of Red Cell Antibodies. MDA Evaluation Report96/14. Medical Devices Agency. Surbiton. UK.

Voak, D, Downie, D.M, Moore, B.P.L, Ford, D.S, Engelfriet,C.P. & Case, J. (1986). Quality control of anti-humanglobulin tests: use of replicate tests to improve performance.Biotest Bulletin, 1, 41–52.



new reference reagent for the quality assurance of anti-d antibody detection

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