Amadeo Sáez-AlquézarAmadeo Sáez-AlquézarSecond WHO Consultation: Development of a WHO Second WHO Consultation: Development of a WHO reference panel for the control of Chagas diagnostic reference panel for the control of Chagas diagnostic

teststests

GenevaGeneva - 2009 - 2009

Development of reference panels Development of reference panels for serological testingfor serological testing

Intended use, fitness for purposeIntended 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 PanelsPerformance Panels

Panels or Multipanels (EQAS)

Reference Panels

Low reactivity (OD/CO: 2,0 – 4,0)

Positive and Negative SamplesVery Well Characterized

To use in External Evaluation

Worldwide avaliable

PRODUCTION

CaCl2 Centrif. + Filtrat + Dialysis + Centrif.Preservative:Bronidox-L5

PlasmaUnits

SerumUnits

Testing

StorageSamples Selection According To The Purpose:MULTIPANELS (EQAS)SPECIFIC PANELSPERFORMANCE PANELSBORDERLINE CONTROL SERA

Using Plasma Units discarded in the serologicalScreening 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 testIHA

BioMérieux Chagatek BIOSchile Wiener rec Hemacruzi# OD/ CO OD/ CO OD/ CO OD/ CO Result

1 5,8 12,5 4,4 9,4 1/ 3202 4,2 10,3 3,6 8,7 1/ 1603 5,2 11,7 4,1 8,2 1/ 6404 4,0 10,0 4,3 7,5 1/ 6405 5,5 7,7 4,1 6,5 1/ 1606 5,9 11,5 4,1 9,1 1/ 3207 4,5 7,2 2,9 7,0 1/ 3208 3,1 6,5 2,6 3,7 1/ 320

Samples ELISA Anti-T.cruzi

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

testELISA Anti-T.cruzi IHA

Chagatek BIOSchile Wiener rec HemacruzyOD/CO OD/CO OD/CO Result

7,2 3,9 8,4 R 1/320

3,0 1,4 2,9 NR

6,8 3,8 7,5 R 1/320

9,0 4,6 8,8 R >= 1/640

2,2 1,9 2,1 NR

4,7 2,7 7,2 R 1/160

6,9 3,6 8,0 R 1/80

8,5 3,7 7,9 R >= 1/640

5,8 3,4 8,3 R 1/320

2,8 2,3 3,9 R 1/80

5,5 4,1 7,3 R 1/320

6,0 3,6 7,9 R >= 1/640

6,1 3,8 6,7 R 1/320

6,9 3,1 8,1 R 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 necessaryobtain adequate volume of each sample.

For this purpose should be necessary dilutesamples, with negative serum or by mixture

with other positive samples.

It is important to observe some criteria to make dilutions

Sample DilutionAnti-T.cruzi positive

DilCHAGATEKCHAGATEK BIOSCHILEBIOSCHILE WIENER recWIENER rec

SamplSamplee

11

SamplSamplee

22

SamplSamplee

33

SamplSamplee

11

SamplSamplee

22

SamplSamplee

33

SamplSamplee

11

SamplSamplee

22

SamplSamplee

33

1/13,73,7 10,10,

774,84,8 2,72,7 3,73,7 3,23,2 6,86,8 9,09,0 8,18,1

1/2 2,82,8 8,08,0 3,73,7 2,22,2 3,33,3 2,82,8 3,93,9 8,18,1 7,27,2

1/5 2,02,0 5,75,7 2,52,5 1,61,6 2,82,8 2,32,3 0,70,7 5,15,1 5,65,6

1/10 1,41,4 4,54,5 1,61,6 1,21,2 2,32,3 1,91,9 0,20,2 2,62,6 3,43,4

1/20 0,90,9 2,42,4 1,31,3 0,90,9 1,81,8 1,61,6 0,00,0 1,01,0 2,62,6

OD/CO values

Sample dilution to prepare anti-Sample dilution to prepare anti-T.cruziT.cruzi reference panels reference panels

Anti-T.cruzi ELISA - Chagatek

0,01,02,03,04,05,06,07,08,0

(1/1) (1/2) (1/5) (1/10) (1/20)

Dilution

OD

/CO

Initial OD/CO range (undiluted samples): 3,8-7,2

Acceptable dilution Unacceptable dilution

Sample dilution to prepare anti-Sample dilution to prepare anti-T.cruziT.cruzi reference panels reference panels

Anti-T.cruzi ELISA - Wiener rec

0,01,02,03,04,05,06,07,08,09,0

10,0

(1/1) (1/2) (1/5) (1/10) (1/20)

Dilution

OD

/CO

Initial OD/CO range (undiluted samples): 7,8 – 10,0

Acceptable dilution Unacceptable dilution

Sample dilution to prepare anti-Sample dilution to prepare anti-T.cruziT.cruzi reference panels reference panels

Anti-T.cruzi ELISA - BiosChile

0,0

1,0

2,0

3,0

4,0

5,0

(1/1) (1/2) (1/5) (1/10) (1/20)

Dilution

OD

/CO

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

MéthodsMéthods HIV*HIV* HTLV**HTLV** HBsAgHBsAg Anti-HBcAnti-HBcAnti-Anti-

HCV***HCV***ChagasChagas SyphilisSyphilis

ELISA 8 3 6 4 3 7-9 4

MEIA 2 - 1 1 - - -

ChLIA® 1 - 1 - - - -

HAI - - - - - 3 2

VDRL - - - - - - 5

RPR - - - - - - 3

Confirmatory WB WB Neutral. Anti-HBs IB WB FTA abs

Total 12 4 9 6 4 11-13 15

Total Total 61-6361-63

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 ProgramsTesting for anti-T.cruzi

Panel used in EQAS ProgramsTesting for anti-T.cruzi

OD/CO OD/CO OD/CO OD/CO OD/CO OD/CO OD/CO OD/CO

1 5,8 3,7 8,2 5,2 5,2 2,8 2,3 3,9

2 5,8 3,9 7,1 3,6 4,5 2,0 1,6 1,2

3 5,6 1,9 9,0 5,4 4,4 3,1 2,5 8,5

4 6,1 3,6 8,8 4,2 5,3 3,2 2,6 4,6

Samples Chagatek BiosChile Wiener rec BioKit recBioMérieux Imunoserum Wama Omega rec

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 negativeTesting

For at least 6 different ELISA testsTwo IHA testsIf possible, one complemmentary test

PROGRAMSPROGRAMS

PA1205PA1205 PA0606PA0606 PA0107PA0107 PA0707PA0707 PA0108PA0108

N Positive N Positive N

Positive Positive N Positive Positive

N N Positive N Positive

N N Positive N Positive

Positive Positive N Positive N

N N Positive N Positive

Positive Positive Positive Positive Positive

N N Positive Positive N

Positive N N N Positive

Positive Positive N Positive Positive

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

Serological Screening

WBChagatek Bioschile Wiener (rec) Wiener (Lys) Immunoserum Bio-Mérieux Omega Wama Bio-Mérieux Wiener TESA

(Lys) (Lys) (rec) (Lys) (Lys) (Lys) (rec) (Lys)Samples DO/CO DO/CO DO/CO DO/CO DO/CO DO/CO DO/CO DO/CO

1 0,3 0,2 0,1 0,1 0,4 0,1 0,2 NT Neg Neg2 0,3 0,2 0,2 0,1 0,4 0,1 0,2 NT Neg Neg3 5,5 3,5 7,9 6,2 6,1 7,7 6,6 6,0 1/80 1/160 POS4 2,5 2,0 4,2 1,8 2,2 1,8 2,9 2,6 1/160 1/40 POS5 0,3 0,1 0,1 0,1 0,3 0,1 0,2 NT Neg Neg6 7,0 3,8 6,7 5,8 4,5 6,9 5,4 5,1 1/160 1;160 POS7 8,2 3,7 8,0 6,9 5,7 8,3 5,8 5,9 1/320 1/60 POS8 4,9 2,9 5,8 6,9 6,5 6,6 3,1 3,1 1;320 1/80 POS9 0,3 0,3 0,1 0,1 0,3 0,2 0,1 NT Neg NT10 0,3 0,2 0,1 0,1 0,3 0,1 0,1 NT Neg NT

ELISA IHAAnti-T.cruzi tests used to carachterize samples of a EQAS sera panel

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

EQAS

1 2 3 4 5 6 87

109

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

screening PL results in positive samples for

anti-T.cruzi using an ELISA testanti-T.cruzi (Lysate)

Nº 2 Nº 3 Nº 4 Nº 6 Nº 7 Nº 9 Nº 100

1

2

3

4

5

Samples

OD

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

screening PL results in positive samples

anti-T.cruzi , using an ELISA testanti-T.cruzi (Recombinant)

Nº 2 Nº 3 Nº 4 Nº 6 Nº 7 Nº 9 Nº 100

1

2

3

4

5

Samples

OD

Panel sera with 20 samples• Positive samples N = 7• Negative samples N = 13

Internal Kits Evaluationbefore to be used and batch by

batch control

All samples are analyzed for all tests used in the serologicalscreening of blood donors + anti-HBsAlso for other tests when necessary (leishmania) Confirmatory tests are performed in positive samples

All samples are analyzed for all tests used in the serologicalscreening of blood donors + anti-HBsAlso for other tests when necessary (leishmania) Confirmatory tests are performed in positive samples

Anti-T.cruzi Performance Panel

Chagatek Bioschile Chagatest Imunoserum BMérieux Omega BMérieux WamaSamples OD/CO OD/CO OD/CO OD/CO OD/CO OD/CO Res Res

1 4,2 3,3 7,2 4,7 4,5 6,4 1/80 1/5122 5,0 3,6 8,9 5,1 6,4 7,7 1/160 1/20483 4,8 3,4 8,0 5,4 6,7 6,4 1/80 1/10244 5,6 4,0 9,6 5,6 8,4 7,7 1/640 1/10245 7,0 3,7 8,3 6,5 5,8 3,1 1/640 1/5126 6,1 3,4 7,2 6,2 4,7 3,0 1/640 1/5127 4,0 3,4 5,4 4,5 2,9 2,2 1/320 1/2568 0,2 0,2 0,0 0,3 0,1 0,3 NR NR9 0,2 0,2 0,0 0,3 0,1 0,2 NR NR10 0,3 0,2 0,0 0,3 0,1 0,2 NR NR11 0,6 0,1 0,0 0,3 0,1 0,1 NR NR12 0,3 0,1 0,1 0,3 0,0 0,1 NR NR13 0,3 0,2 0,0 0,4 0,1 0,1 NR NR14 0,3 0,2 0,0 0,3 0,1 0,2 NR NR15 0,3 0,2 0,1 0,3 0,0 0,4 NR NR16 0,3 0,2 0,0 0,3 0,1 0,1 NR NR17 0,3 0,2 0,0 0,3 0,1 0,1 NR NR18 0,3 0,1 0,0 0,3 0,0 0,1 NR NR19 0,3 0,2 0,0 0,3 0,1 0,2 NR NR20 0,3 0,1 0,0 0,3 0,1 0,1 NR NR

ELISA IHA

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)

Assays Antigenic fractions Sens Spec

RIPA Ab glucoproteins 72- 90 kD - -

Western blot * TESA (Excreted-secreted antigens) 100% 98,5%

Immunoblot** FP10, FP6, FP3, TcF 100% 100%

(*): 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