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Development of reference panels for serological testing Intended use, fitness for purpose. Amadeo Sáez-Alquézar Second WHO Consultation: Development of a WHO reference panel for the control of Chagas diagnostic tests Geneva - 2009. Serological Screening and Diagnostic of infectious diseases.

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Development of reference panels for serological testing Intended use, fitness for purpose


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    1. Development of reference panels for serological testingIntended use, fitness for purpose Amadeo Sáez-Alquézar Second WHO Consultation: Development of a WHO reference panel for the control of Chagas diagnostic tests Geneva - 2009

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

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

    4. Using Plasma Units discarded in the serological Screening of Blood Banks PRODUCTION CaCl2 Centrif. + Filtrat + Dialysis + Centrif. Preservative: Bronidox-L5 Serum Units Plasma Units Testing Samples Selection According To The Purpose: MULTIPANELS (EQAS) SPECIFIC PANELS PERFORMANCE PANELS BORDERLINE CONTROL SERA Storage

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

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

    7. 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%

    8. 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%

    9. 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%

    10. 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%

    11. 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

    12. Sample DilutionAnti-T.cruzi positive OD/CO values

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

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

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

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

    17. 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

    18. (*): 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 Different tests used in the characterization of Panels and Multipanels

    19. Multipanel used in EQAS ProgramsTesting for anti-T.cruzi

    20. Panel used in EQAS ProgramsTesting for anti-T.cruzi

    21. 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

    22. Sera Panels Used in External Quality Assessment Programs for anti-T.cruzi Serological Screening

    23. Anti-T.cruzi tests used to establish the samples features for a Sera Panel used in EQAS 1 2 3 4 5 6 7 8 9 10

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

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

    26. Internal Kits Evaluationbefore to be used and batch by batch control • Panel sera with 20 samples • Positive samples N = 7 • Negative samples N = 13 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

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

    28. In the absence of a universally accepted confirmatory test, how can the samples that were reactive in the serological screening be confirmed? (*): IMTSP/bioMérieux; Umezawa ES et al.1996 (**): Abbott: Cheng KY et al. 2007

    29. Conclusion • The best source for reference panels are plasma units from Blood banks (transformed in sera units) • Positive samples (anti-T.cruzi) could be diluted until 1:2 (no more) • 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) • Samples (anti-T.cruzi +) of reference panels must be positive by ELISA and IHA tests