Proposed candidate Proposed candidate materials and results of materials and results of the feasibility studies for the feasibility studies for a WHO serological reference a WHO serological reference panel panel T. T. cruzi cruzi type II type II Márcia Otani Márcia Otani Fundação Pró-Sangue Hemocentro de Fundação Pró-Sangue Hemocentro de São Paulo São Paulo
Proposed candidate materials and results of the feasibility studies for a WHO serological reference panel. T. cruzi type II. Márcia Otani Fundação Pró-Sangue Hemocentro de São Paulo. Raw material Epidemiological data. 4 y 5. 3. 1. 2. 3. Epidemiological Questionnaire. - PowerPoint PPT Presentation
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Proposed candidate materials and Proposed candidate materials and results of the feasibility studies for a results of the feasibility studies for a WHO serological reference panelWHO serological reference panel
T.T.cruzicruzi type II type II
Márcia OtaniMárcia OtaniFundação Pró-Sangue Hemocentro de São PauloFundação Pró-Sangue Hemocentro de São Paulo
Raw materialRaw material Epidemiological dataEpidemiological data
ID Age Gender State Collected date
Plasma volume
1 103362946 31 Female Minas Gerais
Januaria, for 28 years
2007-02-15 200
2 103350016 58 Male Chile
Canela,
2007-03-16 220
3 103402366 51 Female Pernambuco
Buique, for 40 years
2007-05-01 180
4 801603 43 Male Hidalgo, México 2008-02-08 202
5 801604 48 Male Hidalgo, México 2008-02-08 202
3
1
2
4 y 5
3
T. cruzi IT. cruzi IIaT. cruzi IIbT. cruzi IIcT. cruzi IIdT. cruzi IIe
Linajes filogenéticos deTrypanosoma cruzi
Adaptado de Souto et al. 1996;Fernandes et al. 1998; Brisse et al.
2000, 2004.
T. cruzi IT. cruzi IIaT. cruzi IIbT. cruzi IIcT. cruzi IIdT. cruzi IIe
Linajes filogenéticos deTrypanosoma cruzi
Adaptado de Souto et al. 1996;Fernandes et al. 1998; Brisse et al.
2000, 2004.
1 2 3
1-Have you ever heard about Chagas Disease? Y Y Y
2-Do you know how to recognize a kissing bug? Y Y Y
3-Have you ever lived in a house constructed of wood and mud? N N Y
4-Have you had contact with a kissing bug? N Y Y
5-Have any of your relatives ever had Chagas disease? Y N Y
6-Did you live in the same house with a relative who is positive for Chagas Disease?
N N Y
7-Do you know any case of Chagas Disease in the region where you have born or where you have lived?
N Y Y
8-Have you ever received blood transfusion? N N N
9-Have you ever been to any Chagas endemic region? N N N
10-Have you ever had any clinical symptoms for Chagas Disease ? N N Y
11-Are you under medical treatment ? N N Y
12-Have you ever done an electrocardiogram (EKG)? Y Y Y
Data from Latin America Reference Centers – PAHO Regional EQAS
Data from Latin America Reference Centers – PAHO Regional EQAS
Prototype X 2 times diluted April 2008
0
2
4
6
8
10
Test
Elis
a p/
Cha
gas
Elisa
cruz
i
Chaga
s Te
st E
lisa
III
Chaga
test
Elis
a
Chaga
test
Elis
a Rec
Bioelis
a Cha
gas
Chaga
s Te
st II
CS ELI
SA
Chaga
s EIA
Chaga
tek
ELISA
Chaga
s cr
een
Chaga
s Elis
a Per
oxida
sa
Chaga
s EIA
Colo
mbia
Test
Elis
a p/
Cha
gas
(UCR)
Chaga
s Pha
rmat
est
Imun
ocru
zi IF
I
Polich
aco
HAI Cha
gas
Kit
OD
/CO
Prototype Diluted 2x
Stability of Donor Sample (not pooled not diluted)
02468
101214
Test
Elis
a p/
Cha
gas
Elisa
cruz
i
Chaga
s Te
st E
lisa
III
Chaga
test
Elis
a
Chaga
test
Elis
a Rec
Bioelis
a Cha
gas
Chaga
s Te
st II
CS ELI
SA
Chaga
s EIA
Chaga
tek
ELISA
Chaga
s cr
een
Chaga
s Elis
a Per
oxida
sa
Test
Elis
a p/
Cha
gas
(UCR)
Chaga
s Colo
mbia
Chaga
s Pha
rmat
est
Imun
ocru
zi IF
I
Polich
aco
HAI Cha
gas
Kit
OD
/CO
Sep 2007 April 2008
Stability of prototype
0
2
4
6
8
10
12
14
Test
Elis
a p/
Cha
gas
Elisa
cruz
i
Chaga
s Te
st E
lisa
III
Chaga
test
Elis
a
Chaga
test
Elis
a Rec
Bioelis
a Cha
gas
Chaga
s Te
st II
CS ELI
SA
Chaga
s EIA
Chaga
tek
ELISA
Chaga
s cr
een
Chaga
s Elis
a Per
oxida
sa
Chaga
s EIA
Colo
mbia
Test
Elis
a p/
Cha
gas
(UCR)
Chaga
s Pha
rmat
est
Imun
ocru
zi IF
I
Polich
aco
HAI Cha
gas
Kit
OD
/CO
Sep 2007 April 2008
Data from Latin America Reference Centers PAHO Regional EQAS
Prototype stability by temperature / timeElisa cruzi
4,0
4,2
4,4
4,6
4,8
5,0
5,2
5,4
5,6
5,8
6,0
nov-07 jan-08 mar-08 mai-08 jul-08 set-08 nov-08
Assay Date
Me
an
O
D/C
O
-20ºC
4 ºC
22 ºC
37 º C
Mean
Mean
Mean
Mean
Prototype volume available = 1.4 liters
2 times dilution = 2.8 liters
It is possible to obtain a total of 6 liters
Units used mL available mL
1 80 120
2 120 100
3 60 120
4 2 200
5 2 200
Volume Volume
Mexico Units
M2 volume available = 400 mL
4 times dilution = 1.6 liters
ConclusionConclusion
• The mixture of 3 positive samples (from south of Latin America) diluted 4.8 times in negative samples have produced a prototype that presented stability at –20 ºC.
• We need to define the reactivity of the cut off value. Here we have used the dilution that is IIF = 1/20.
• Reactivity of the prototype diluted 2 times were 2 to 4 times de cutoff value in most of the tests evaluated and the IIF = 1/20.
• Perhaps we should submit this prototype to RIPA, Western Blot and Abbott Immunoassay.