laboratory. II. Internal quality control
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1 198 1 Clin Pthol 1995;48: Clinicl Microbiology nd Public Helth Lbortory, Addenbrooke's Hospitl, Cmbridge CB2 2QW J J Gry T G Wregitt T A McKee P McIntyre C E Roth D J Smith G Sutehll G Higgins R Gerghty R Whetstone U Desselberger Correspondence to: Dr J J Gry. Accepted for publiction 18 July 1994 Internl qulity ssurnce in clinicl virology lbortory. II. Internl qulity control J J Gry, T G Wreghitt, T A McKee, P McIntyre, C E Roth, D J Smith, G Sutehll, G Higgins, R Gerghty, R Whetstone, U Desselberger Aims-In April 1991 dditionl qulity control procedures were introduced into the virology section of the Clinicl Microbiology nd Public Helth Lbortory, Cmbridge. Internl qulity control (IQC) smples were grdully included in the serologicl ssys performed in the lbortory nd supplemented kit controls nd stndrd ser. Methods-From April 1991 to December 1993, 2421 IQC procedures were crried out with reference ser. Results-The IQC smples were evluted ccording to the Westgrd rules. Violtions were recorded in 6 of 188 (3.3%) controls nd were highest in the IQC smples of complement fixtion tests ( (8%) of controls submitted for complement fixtion tests). Conclusions-The inclusion of IQC smples in the serologicl ssys performed in the lbortory hs highlighted btch to btch vrition in commercil ssys. The setting of cceptble limits for the IQC smples hs incresed confidence in the vlidity of ssy results. (J Clin Pthol 1995;48:198-22) Keywords: Internl qulity control, qulity ssurnce, virology. Qulity control mesures permit the ssessment of ccurcy nd precision in ssys performed in the dignostic lbortory. Their inclusion is designed to increse the probbility tht ssy results reported by the lbortory re vlid nd tht clinicins my confidently use those results when mking dignostic or therpeutic decisions. For mny yers internl qulity control (IQC) procedures hve plyed n essentil prt in monitoring the dy to dy performnce of ssys used in clinicl biochemistry deprtments. To dte, most dignostic microbiology lbortories (including microbil serology) hve relied on control mteril supplied s n integrl prt of commercil ssys. Although kit controls cn be used to determine whether prticulr ssy run is cceptble they re not cpble of monitoring btch to btch vrition. Internl qulity control opertes by detecting errors which my be rndom or systemtic. Rndom errors re difficult to eliminte but my be minimised through trining nd supervision. System bsed errors my be relted to the sensitivity, specificity, reproducibility, or stbility of n ssy. Although the results of serologicl ssys my be expressed in terms of positivity or negtivity, most re expressed s ntibody titres or rtios (for exmple, in enzyme linked immunosorbent ssys (ELISAs) s the rtio of the opticl density (OD) of the test smple to the OD of the cut off control) or unit vlues or concentrtions (for exmple, when interntionl, ntionl or rbitrry stndrds re included). Therefore, most results re expressed s numericl vlues mking them menble to simple sttisticl nlysis. Internl qulity control smples re included in the ssys performed in the lbortory nd, when the results lie within pre-determined limits, re used to vlidte the test results.' IQC smples re lso useful to monitor the performnce of equipment nd the stbility of regents indirectly. IQC smples cn be interntionl, ntionl or locl stndrd ser or pools of serum smples, well chrcterised in previous ssys, exhibiting vlues within cliniclly significnt rnges. A sufficient volume of ech control is required so tht IQC smples cn be included in ech test run enbling intrnd inter-btch ssy vrition to be monitored continuously. From April 1991 to December 1993, 2421 IQC smples (ssy controls), in ddition to nd independent of kit controls nd stndrd ser, were grdully included in 17 serologicl ssys performed t the Clinicl Microbiology nd Public Helth Lbortory, Cmbridge. Methods Locl ser, well chrcterised "in house" in previous ssys, or interntionl or ntionl serum stndrds with vlues within cliniclly significnt rnges were included in number of ssys performed in the lbortory (tble 1). The results obtined with ptients' serum smples in these ssys were only considered vlid if the control vlues ly within their expected rnges. The men or trget vlue nd the stndrd devition (SD) of the proposed ssy control were clculted from the results obtined fter testing the smple on 2 seprte occsions. The SD is mesure of the sctter of vlues round men nd is used to set cceptble limits for results subsequently obtined with the ssy control. Control serum smples exhibit norml stochstic distribution of results, therefore 95-5% of the vlues should lie within J Clin Pthol: first published s /jcp on 1 Mrch Downloded from on 2 September 218 by guest. Protected by copyright.
2 Internl qulity ssurnce in clinicl virology lbortory II 199 Co -o. Q- E t, C 4-o E Co. 1 _ A SD 7 +2SD _ +1SD 6 Trget vlue 5 4 SD _-1SD 3 l3sd 2 1 nu II c +3SD _+2SD +1SD rget vlule _2SD L-3SD 1 B 9 _ SD 7 - SD ;1SD rget vlue 5 El7SD 4 _ -SD SD 2 1 _ I I I I I I nl 1 E 9 v SD 6 _ 1SD Trget vlue 5-1s S Trget vlue =51 AU/ml D 9 _ SD SD 6 -+S 5 - Trget vlue 4 [95lS 3-2SD 2 3SD 1 _ I F 9 +3SD 88_+2SD 7 +1SD 6 - Trget vlue SD 4 L2SD 3 L3SD 2-1 / I II II Trget vlue = 51 AU/ml Figure 1 Violtions of the Westgrd rules. A-C: Wrning rules (A: 12SD; B: 22SD; C: 41sD). D-F: Mndtory rules (D: 13sD; E: R4SD; F: 1.J + 2SD of the men or trget vlue nd 99 7% within +3SD.2 Shewhrt plots3 were drwn, with the trget Tble 1 Assy IQC mteril used for ssy controls Control ser HIV 1+2 ntibody ELISA HIV 1 low positive (DMR)* HIV ntibody ELISA HIV 2 low positive (DMR) HAV totl ntibody ELISA Pool of positive ser HAV IgM ELISA Pool of positive ser HBsAg ELISA Serum contining 1 IU/ml HBsAg ELISA Serum contining 1 IU/ml HBsAg ELISA Serum contining 1 IU/ml nti-hbs ELISA Serum contining 1 mlu/ml nti-hbs ELISA Serum contining 12 mlu/ml nti-hbs ELISA Serum continingg 6 mlu/ml nti-hbc ELISA Pool of positive ser HCV ntibody ELISA Positive control serum (DMR) Rubell SRH 15 IU/ml serum (DMR) Rubell ltex gglutintion 25 IU/ml serum T gondii ltex gglutintion Antibody positive serum (blood ** Pul-Bunnell ntibody Pool of positive ser L pneumophil RMAT Pool of positive ser HSV IgG ELISA Antibody positive serum (blood CMV IgG ELISA Antibody positive serum (blood VZV IgG ELISA Antibody positive serum (blood Respirtory CFT Blood dontion A Respirtory CFT Blood dontion B * DMR Division of Microbiologicl Regents, Centrl Public Helth Lbortory, London. ** Regionl Blood Trnsfusion Centre, Cmbridge. All ser not designted DMR or blood dontion were locl "in-house" ser. CMV = cytomeglovirus; CFT = complement fixtion test; HAV =heptitis A virus. vlue, nd the limit vlues of + 1 SD, ± 2SD nd + 3SD delineted for ech control used. Subsequent vlues obtined with the ssy controls were plotted nd Westgrd rules were pplied to determine the vlidity of ech ssy run. Westgrd rules define specific performnce limits nd re designed to detect both rndom nd systemtic errors.3 Three of the six commonly used Westgrd rules re wrning rules whose violtion should trigger review of test procedures, regent performnce nd equipment clibrtion, nd three re mndtory rules which, if broken, should result in the rejection of the results obtined with ptients' serum smples in tht ssy. Figures 1A to 1 C illustrte the three wrning rules nd figs 1D to 1F the three mndtory rules. Wrning rule 12SD (fig 1A) is violted if the control vlue exceeds the men by ± 2SD (n event likely to occur normlly in less thn 5% of cses). Wrning rule 22SD (fig 1B) detects systemtic errors nd is violted when two consecutive control vlues exceed the trget vlue on the sme side of the men by + 2SD. Wrning rule 41SD (fig 1 C) is violted if four consecutive control vlues exceed the sme limit (men + 1 SD or men -1 SD) nd my indicte the need to perform instrument mintennce or regent clibrtion. Mndtory rule 13SD (fig 1D) pplies when J Clin Pthol: first published s /jcp on 1 Mrch Downloded from on 2 September 218 by guest. Protected by copyright.
3 2 Gry, Wreghitt, McKee, McIntyre, Roth, Smith, et l 1 9 E 8 7., 6 > 5 X 4. 3 < 2 1 I -7 o 1.7 r_ n o -9 t- 7 A +3SD +2SD -+1SD Trget vlue - -1SD -2SD I-3SD n Trget vlue = 49 AU/ml I._ 7- B SD 4- +~~~~~~~~~~~~~2SD 1-3~~~~~~~~~~+SD Trget vlue = f- D New test btch introduced 3 H v \If IF Trget vlue IF -- - N v1 IF. it 2 k %\., Figure 2 Shewhrt plot ofa: VZV IgG ELISA IQC (violtions of the Westgrd rules not detected); B: HIV ntibody ELISA IQC (violtions of the Westgrd rules not detected); C: HCV ntibody ELISA IQC (violtions of the 13sD rule detected); D: HBsAg ELISA IQC (violtion of the 1, rule detected). 1 [- the control vlue exceeds the trget vlue by + 3SD; the ssy run is then regrded s out of control. Mndtory rule R4SD (fig 1E) is rnge rule within run nd is only pplied when the control is tested in duplicte. The rule is violted when the difference in SD between the duplictes exceeds 4 SD. The 1X (mndtory) rule (fig IF) detects systemtic error nd is violted when the lst 1 consecutive control vlues re on the sme side of the men or trget vlue. In the IQC scheme reported here the R4SD rnge rule ws not pplied s the ssy controls were tested singly. Results Exmples of Shewhrt plots illustrting the performnce of the ssy controls, used in the vricell zoster virus (VZV) IgG ntibody ELISA, HIV 1+2 ntibody ELISA, heptitis C virus (HCV) ntibody ELISA, nd heptitis B virus surfce ntigen (HBsAg) ELISA over 2 consecutive ssy runs, re shown in figs 2A to 2D, respectively. No violtions of the Westgrd rules were found with the ssy controls in the VZV IgG ELISA or the HIV ntibody ELISA during the period illustrted in figs 2A nd 2B. In the HCV ntibody ELISA the 13SD rule ws violted on two occsions triggering the rejection of those prticulr ssy runs (fig 2G). A reduction in the sensitivity of the HBsAg ELISA fter the introduction of new btch of regents ws indicted by violtion of the 1x rule (fig 2D). %%.j %%%% 2 15, A totl of 2421 ssy controls were included in the serologicl ssys performed between 1991 nd The Westgrd rules were pplied to the results obtined with 188 ssy controls included in 891 ssys. A totl of 29 (1-6%) violtions of the 13SD rule nd 31 (1 71 %) violtions ofthe 1iX rule were detected (tble 2). On 18 occsions, both the 1 3SD nd the 1X rules were violted t the sme time. Tble 3 illustrtes the results obtined with the HIV 1 IQC smple in HIV 1+ 2 (combined) ntibody ELISA when new btch of ssy regents ws introduced. The incresed sensitivity of the new btch chnged the expected men or trget vlue of the IQC smple from 2-8 (OD of the smple:od of the cut offvlue) to 4-4. Therefore, if the trget vlue nd the performnce limits hd not been reclculted, the results of two ssy runs, performed with Tble 2 Assy controls nd violtions of the Westgrd rules, 13sD nd lox Rule Voltion Number Assy control tested 13SD 1x HBsAg (1 IU/ml) (1 IU/ml) (1IU/ml) Anti-HBs (1 mlu/ml) 22 1 (12 mlu/ml) 22 (5 mlu/ml) 22 Anti-HBc 25 Anti-HCV Anti-HIV I Anti-HIV1+2b VZV IgG CMV IgG 23 HSV IgG 23 1 Totl (1 6%) (1*71%) 1 O I g1 ng/ml control J Clin Pthol: first published s /jcp on 1 Mrch Downloded from on 2 September 218 by guest. Protected by copyright.
4 Internl qulity ssurnce in clinicl virology lbortory II Tble 3 Violtion of the Westgrd rule 13SD, cused by introducing new btch of ssy regents: HIV 1 control in the HIV ntibody ELISA Acceptbility Control prmeter Rtio of OD If control If control smple to OD Acceptble prmeters prmeters NOT number cut off Assy btch Men SD rnge reclculted reclculted 2-8* A Yes Yes 2 16 A Yes Yes A Yes Yes 4 18 A Yes Yes A Yes Yes 4.4** B Yes Yes B Yes Yes 8 5 B Yes Yes B Yes Yes B Yes Yes B Yes Yes B Yes Yes 13 4 B Yes Yes B Yes Yes B Yes Yes B Yes No* B Yes Yes B Yes Yes B Yes No* B Yes Yes A Yes Yes A Yes Yes A Yes Yes A Yes Yes A Yes Yes A Yes Yes A Yes Yes * Clculted from the results from 2 ssy runs with btch A. ** Control prmeters reclculted using results obtined with btch B. * >3SD bove the men. the new btch of regents, would hve been were tested with influenz A, influenz B, drejected through violtions of the 13SD rule. enovirus, Chlmydi sp, Coxiell burnetii, nd In the remining 613 ssys the Westgrd Mycoplsm pneumonie ntigens. The results rules were not pplicble s the results were obtined with these controls show tht 25 of expressed either s n ntibody titre or s posi- 312 (8 %) procedures were outside their extive or negtive. In the Legionell pneumophil pected rnges (tble 4). RMAT (57 ssys), Toxoplsm gondii ltex gglutintion test (174 ssys), heptitis A virus totl ntibody ELISA (7 ssys), heptitis A Discussion virus IgM ntibody ELISA (37 ssys), nd The inclusion of IQCs with the bility to detect the Pul-Bunnell ssy (57 ssys), the results rndom nd systemtic errors permits the dy obtined with the ssy controls were within to dy monitoring of ssy performnce inthe expected rnges (dt not shown). In the dependently of kit controls nd increses the rubell virus ntibody ssys, single rdil he- probbility tht results obtined with clinicl molysis (79 ssys) nd the ltex gglutintion smples re vlid. ssy (113 ssys) the controls were outside The combintion of Westgrd rules used'4 their expected rnges in one of 79 (1 26%) nd nd their wrning or mndtory functions four of 113 (3 53%) ssys, respectively (dt should be governed by the need to reject true not shown). In the complement fixtion tests errors but not to reject vlid test results. If ll two ser were included in 26 ssy runs nd test results were rejected when the ssy control Tble 4 Results obtined with ssy controls in the complementfixtion tests Number outside Control Antigen Titre Acceptble rnge Number tested cceptble rnge Per cent A Influenz A 8 < Influenz B <8 < Chlmydi C Burnetti <8 < Adenovirus M pneumonie <8 < Subtotl B Influenz A Influenz B 8 < Chlmydi <8 < C burnetti <8 < Adenovirus <8 < M pneumonie 8 < Subtotl Totl J Clin Pthol: first published s /jcp on 1 Mrch Downloded from on 2 September 218 by guest. Protected by copyright.
5 22 vlue ws greter thn the men + 2SD, then filure to llow for pproprite control vlues (for exmple, chnging between btches of regents) would result in proportion of ssy runs being flsely rejected. Dt obtined with the ssy controls cn lso be used to set cceptble limits in ssys in which nonymous smples re tested s prt of n internl qulity ssessment scheme (IQAS) (see Prt I). This cn be ccomplished by clculting the coefficient of vrition (CV) from the men nd SD of the ssy control. The CV cn then be used to determine the rnge of cceptble vlues when smple is repeted. This is prticulrly useful in ssys where numericl or unit vlues re obtined. A discrepncy would be recorded if the difference between the two vlues ws greter thn the CV Ṫhe CV should be clcuted from n ssy control with concentrtion of ntigen or ntibody tht is close to tht of the test smple, s the CV will chnge t different concentrtions. In the nti-hbs ssy the intermedite control hd CV of 15-2% nd discrepncy would be recorded if there ws greter thn 15-2% difference between the results of the nmed nd nonymous smple, wheres the high control hd CV of 13-5%. Internl qulity control opertes by detecting errors. Rndom errors re impossible to eliminte but my be minimised through trining nd dherence to stndrd operting procedures. System bsed errors my be relted to the sensitivity or specificity of n ssy, the use of non-stndrd methods, poor equipment clibrtion, or the instbility of regents. Gry, Wreghitt, McKee, McIntyre, Roth, Smith, et l Although errors will inevitbly occur, they cn be significntly reduced through the introduction of IQC s n integrl prt of comprehensive IQAS. Trining must be continully updted with regulr meetings to discuss qulity control filures nd successes, chnges in stndrd operting procedures, the introduction of new ssys, nd the opertion ofnew equipment. Publiction ofresults within the lbortory nd encourgement of stff to discuss technicl problems nd possible solutions cn significntly improve the overll performnce. Problems nd inconsistencies in lbortory procedures cn be identified through the IQAS nd the use of IQC will increse the confidence in test results. In conclusion, IQC procedures were performed s prt of comprehensive IQAS where some specimens received in the lbortory were resubmitted for testing nonymously. The conduct of nd the results chieved with the IQAS re presented in Prt I. We thnk ll stffof the Virology section of Clinicl Microbiology nd Public Helth Lbortory for their support of the IQA Scheme nd IQC mesures nd for fruitful discussion. Thnks re lso due to M Frrington, H Ludlm, M Wilcox, D Brown, MAT Coles, nd M Amphlett for criticl reding of the mnuscript. 1 Westgrd JO, Koch DD, Oryll JI, Qum EF, Feldbruegge DH, Dowd DE, et l. Selection of mediclly useful qulity control procedures for individul tests done in multitest nlyticl system. Clin Chem 199;36: Tylor RN, Juong AY, Fulford KM, Przybyszewski VA, Hern TL. Qulity control for immunologic tests. Helth Eduction nd Welfre Publiction No. (CDC) Atlnt: Centre for Disese Control, 1979: Westgrd JO, Hunt MR, Groth T. A multi-rule Shewhrt chrt for qulity control in clinicl chemistry. Clin Chem 1981;27: Gry JJ. Internl qulity ssurnce in dignostic virology lbortory. ACM News 1994; J Clin Pthol: first published s /jcp on 1 Mrch Downloded from on 2 September 218 by guest. Protected by copyright.
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