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