Amadeo Sáez-Alquézar Second WHO Consultation: Development of a WHO reference panel for the control of Chagas diagnostic tests Geneva Development of reference panels for serological testing Intended use, fitness for purpose

Serological Screening and Diagnostic of infectious diseases Quality Control Procedures Serum Panels as a Reference An indispensable tool

Serum Panels as a Reference Tool Internal Control sera (ICS) Specific Panels Performance Panels Panels or Multipanels (EQAS) Reference Panels Low reactivity (OD/CO: 2,0 – 4,0) Positive and Negative Samples Very Well Characterized To use in External Evaluation Worldwide avaliable

PRODUCTION CaCl 2 Centrif. + Filtrat + Dialysis + Centrif. Preservative: Bronidox-L5 Plasma Units Serum Units Testing Storage Samples Selection According To The Purpose: MULTIPANELS (EQAS) SPECIFIC PANELS PERFORMANCE PANELS BORDERLINE CONTROL SERA Using Plasma Units discarded in the serological Screening of Blood Banks

Selection of plasma units from blood banks to prepare anti-T.cruzi positive samples of the reference panels SEROLOGICAL SCREENING FOR CHAGAS USING EIA / ELISA TEST, SHOWING: OD/CO 3,0 [Chagatek or Wiener rec] OD/CO 2,0 [bioMérieux] CRITERIA:

Testing Sera Obtained from Plasma Units Several ELISA Tests (Lys and rec) 1 – 3 IHA Tests

Testing Plasma Units from Blood Banks descarted with positive anti-T.cruzi screening test Concordance between ELISA tests: 100% Concordance between ELISA and IHA tests: 100%

Testing Plasma Units from Blood Banks descarted with positive anti-T.cruzi screening test ELISA Anti-T.cruziIHA ChagatekBIOSchileWiener recHemacruzy OD/CO Result 7,23,98,4R 1/320 3,01,42,9NR 6,83,87,5R 1/320 9,04,68,8R >= 1/640 2,21,92,1NR 4,72,77,2R 1/160 6,93,68,0R 1/80 8,53,77,9R >= 1/640 5,83,48,3R 1/320 2,82,33,9R 1/80 5,54,17,3R 1/320 6,03,67,9R >= 1/640 6,13,86,7R 1/320 6,93,18,1R 1/160 Concordance between ELISA tests: 100% Concordance between ELISA and IHA tests: <100%

Testing Plasma Units from Blood Banks descarted with positive anti-T.cruzi screening test Concordance between ELISA tests: 100%, but different of the screening test Concordance between ELISA and IHA tests: <100%

Testing Plasma Units from Blood Banks discarded with positive anti-T.cruzi screening test Concordance between ELISA tests: <100% Concordance between ELISA and IHA tests: <100%

Sample Dilution To prepare sera panels must be necessary obtain adequate volume of each sample. For this purpose should be necessary dilute samples, with negative serum or by mixture with other positive samples. It is important to observe some criteria to make dilutions

Sample Dilution Anti-T.cruzi positive Dil CHAGATEKBIOSCHILE WIENER rec Sample1Sample2Sample3Sample1Sample2Sample3Sample1Sample2Sample3 1/1 3,710,74,82,73,73,26,89,08,1 1/2 2,88,03,72,23,32,83,98,17,2 1/5 2,05,72,51,62,82,30,75,15,6 1/10 1,44,51,61,22,31,90,22,63,4 1/20 0,92,41,30,91,81,60,01,02,6 OD/CO values

Sample dilution to prepare anti-T.cruzi reference panels Initial OD/CO range (undiluted samples): 3,8-7,2 Acceptable dilution Unacceptable dilution

Sample dilution to prepare anti-T.cruzi reference panels Initial OD/CO range (undiluted samples): 7,8 – 10,0 Acceptable dilution Unacceptable dilution

Sample dilution to prepare anti-T.cruzi reference panels Initial OD/CO range (undiluted samples): 1,8 - 4,4 Acceptable dilution Unacceptable dilution

Samples Characterization We consider the more important step to assess the quality of panels

Testing samples Tests used in the serological screening are qualitative tests determines whether the substance being tested for is present or absent Results obtained by the PL will be compared with a reference panel sent by de Organizer Center. So the the reference panel must be very well tested, to assure the certainty of the results

MéthodsHIV*HTLV**HBsAgAnti-HBc Anti- HCV*** ChagasSyphilis ELISA MEIA ChLIA® HAI VDRL RPR ConfirmatoryWB Neutral.Anti-HBsIBWBFTA abs Total Total61-63 Different tests used in the characterization of Panels and Multipanels (*): anti-HIV 1+2, anti-HIV 1+2+O y HIV Ag/Ab (**): anti-HTLV 1+2 (***) Anti-HCV y HCV Ag/Ac ®: Architect – Vitros - Lyason

Multipanel used in EQAS Programs Testing for anti-T.cruzi

Panel used in EQAS Programs Testing for anti-T.cruzi

External Quality Assessment Blind Panels For a single screening test (f.instance: anti-T.cruzi) N = 5 to 10 samples 5-7 positive and 3-5 negative Testing For at least 6 different ELISA tests Two IHA tests If possible, one complemmentary test

PROGRAMS PA1205PA0606PA0107PA0707PA0108 NPositiveN N N NN N NN N N N NN N NN N NNN N Sera Panels Used in External Quality Assessment Programs for anti-T.cruzi Serological Screening

Anti-T.cruzi tests used to establish the samples features for a Sera Panel used in EQAS

Participant Laboratories (PL) results in a EQAS for anti-T.cruzi screening

Panel sera with 20 samples Positive samples N = 7 Negative samples N = 13 Internal Kits Evaluation before to be used and batch by batch control All samples are analyzed for all tests used in the serological screening of blood donors + anti-HBs Also for other tests when necessary (leishmania) Confirmatory tests are performed in positive samples All samples are analyzed for all tests used in the serological screening of blood donors + anti-HBs Also for other tests when necessary (leishmania) Confirmatory tests are performed in positive samples

Anti-T.cruzi Performance Panel HCV (+) HIV (+) HBsAg (+) HTLV (+)

In the absence of a universally accepted confirmatory test, how can the samples that were reactive in the serological screening be confirmed? Suplemental Tests (confirmatory) AssaysAntigenic fractionsSensSpec RIPA Ab glucoproteins kD-- Western blot * TESA (Excreted-secreted antigens)100%98,5% Immunoblot** FP10, FP6, FP3, TcF100% (*): IMTSP/bioMérieux; Umezawa ES et al.1996 (**): Abbott: Cheng KY et al. 2007

Conclusion 1.The best source for reference panels are plasma units from Blood banks (transformed in sera units) 2.Positive samples (anti-T.cruzi) could be diluted until 1:2 (no more) 3.Positive samples (anti-T.cruzi) must be tested by, at least: 6 differente ELISA tests (Lys and rec) 1 IHA test 1 suplemmental test (TESA blot, Immunoblot or RIPA) 4.Samples (anti-T.cruzi +) of reference panels must be positive by ELISA and IHA tests