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Newborn Screening for Biotinidase Deficiency Using a Totally Automated System and Enzymatic Analysis With Results Expressed in nmol/min/mL.

Authors:

Abstract

Introduction : Newborn Screening for Biotinidase Deficiency has been implemented in the Brazilian Newborn Screening Program (PNTN) since 2012. Since 1999 INTERCIENTIFICA offers the NeoLISA BIO kit using an enzymatic colorimetric method that differentiates the enzyme's activity by the color produced in the end of the reaction. The national legislation accepts the use of qualitative methods in Newborn Screening for Biotinidase Deficiency but the market requests new protocols with reduced times, totally automated processes and results reported in numeric values. Materials and Methods : The NeoLISA BIO Kits associated with Nimbus NeoLISA have been used to develop a totally automated system to determine the enzymatic activity of Biotinidase in numeric values (nmol/min/mL). Standards and Internal Controls with different levels of Biotinidase activity, plus external quality control materials, have been used for this study. The protocol of the assay takes 4 hours instead the 18 hours of the qualitative protocol. The results are compared between the protocols and are presented in tables. Results : The new protocol demonstrates the same confidence and quality results expected from a product used by the Brazilian market for more than 15 years. Therefore, the protocol with 4 hours reduces substantially the time process of the samples in the routine. The analysis demonstrated comparable results using the protocol for 18 hours in a manual process against the protocol for 4 hours in totally automated system, Nimbus NeoLISA. Conclusion :The NeoLISA BIO kit using an automated system, Nimbus NeoLISA, with a protocol of 4 hours has shown excellent concordance compared with traditional method in 18 hours protocol. The new assay protocol considers the needs of the Brazilian market to promote innovation according market needs. The results presented can be considered evidence that this product keeps the same level of confidence and quality expected for newborn screening programs.
Newborn Screening for Biotinidase Deficiency Using a Totally Automated
System and Enzymatic Analysis With Results Expressed in nmol/min/mL.
Claudio Sampaio Filho (Jr)1, Fabiola Frias1, Raquel Veturiano1,
Vanessa Silva1, Bruna Gallo1, Fabio Bernardes1, Jonatas Boalento1,
Anakele Massi1, Josias Lira1,
1-INTERCIENTIFICA, S.J.Campos - Brasil
Introduction:
Newborn Screening for Biotinidase Deficiency was implemented in the Brazilian Newborn
Screening Program (PNTN) in 2012. Since 1999 INTERCIENTIFICA has been offering the
NeoLISA® BIO kit using an enzymatic colorimetric method that differentiates the enzyme's activity
by the color produced in the end of the reaction. The national legislation accepts the use of
qualitative methods in Newborn Screening for Biotinidase Deficiency but the market requests new
protocols with reduced times, totally automated processes and results reported in numeric values.
The main objective is to offer alternative solution for newborn screening of Biotinidase Deficiency
for the Brazilian Newborn Screening Programs are introducing this new assay in the routine.
Fig.1 Qualitative analysis (normal and deficient samples) Fig.2 Nimbus NeoLISA®
Nº PO
Materials and Methods:
The NeoLISA® BIO Kits associated with Nimbus NeoLISA® have been used to develop a totally
automated system to determine the enzymatic activity of Biotinidase using numeric values
(nmol/min/mL). Standards and Internal Controls with different levels of Biotinidase activity, plus
external quality control materials, have been used for this study. The assay protocol takes 4 hours
instead of the 18-hour qualitative protocol. The results are compared between the protocols and
are presented in tables below :
The method is based on an enzymatic reaction using the artificial substrate of Byocitin. Microplates
with dot blood spots (DBS) are placed in the equipment and all steps of protocol are performed
automatically. The absorbance measured is directly proportional to the Biotinidase Activity in the
sample.
Kit Reagents :
BIO Standards & Controls
BIO R1
BIO R2
BIO R3
BIO R4
BIO R5 Fig.3 NeoLISA® BIO
Assay Procedure :
1. Dispense R1 - 100ul - incubate for 4 hours at 37 degrees Celsius.
2. Dispense R2 - 30ul and transfer by vacuum system.
3. Dispense R3 - 30ul - incubate 3 minutes.
4. Dispense R4 - 30ul - incubate 3 minutes.
5. Dispense R5 - 30ul - incubate 3 minutes.
6. Measurement (570nm).
All the reagents are ready for use in both quantitative and qualitative assay formats. The
enzymatic colorimetric method is simple, does not involve antibodies or wash steps, reduces any
potential interference or possibilities of cross reactivity.
Newborn Screening for Biotinidase Deficiency Using a Totally Automated
System and Enzymatic Analysis With Results Expressed in nmol/min/mL.
Claudio Sampaio Filho (Jr)1, Fabiola Frias1, Raquel Veturiano1,
Vanessa Silva1, Bruna Gallo1, Fabio Bernardes1, Jonatas Boalento1,
Anakele Massi1, Josias Lira1,
1-INTERCIENTIFICA, S.J.Campos - Brasil
Nº PO
Results:
The new protocol demonstrates the performance and quality results expected from a product used
by the Brazilian market for more than 15 years. Therefore, the protocol with 4 hours reduces
substantially the time process of the samples in the routine.
Table 1 : Qualitative and Quantitative options of NeoLISA BIO
The analysis demonstrated comparable results using the manual 18-hour protocol versus 4-hour
protocol in the totally automated system, Nimbus NeoLISA®.:
Table 2 : 3 levels of Biotinidase activity controls evaluated in 21 runs over 20 days.
The screening performance was evaluated using samples (n=6.000) from routine newborn
screening labs using the qualitative assay format for the same product. Retrospective confirmed
Biotinidase deficiency samples were used (n=2) plus inactivated normal samples as external
positive controls (n=10). As expected the concordance was 100% since this is the same product
differentiated by the assay format and it uses the totally automated system Nimbus NeoLISA®.
The normal Biotinidase activity for newborns was determined as 9.7 nmol/min/mL of PABA,
intermediate >2.9 and <0.9 nmol/min/mL of PABA and Deficient >0.9 nmol/min/mL of PABA.
Fig.3 Typical Standard Curve for NeoLISA® BIO Quantitative assay format.
BIO Controls nmol/min/mL DP CV
DEFFICIENT 1 0.36 8.09
INTERMEDIATE 4 1.38 8.29
NORMAL 12 0.84 3.67
NeoLISA® BIO QUALITATIVE QUANTITATIVE
Results Normal/Intermediate/Defficient nmol/min/mL of PABA
Protocol Manual Totally Automated
Standard Curve No Yes (6 Stds/3 Ctls)
Assay Procedure Time 18h 4h
Kit Format 300/3000 tests 3000 tests
Newborn Screening for Biotinidase Deficiency Using a Totally Automated
System and Enzymatic Analysis With Results Expressed in nmol/min/mL.
Claudio Sampaio Filho (Jr)1, Fabiola Frias1, Raquel Veturiano1,
Vanessa Silva1, Bruna Gallo1, Fabio Bernardes1, Jonatas Boalento1,
Anakele Massi1, Josias Lira1,
1-INTERCIENTIFICA, S.J.Campos - Brasil
Nº PO
Conclusions:
The most significant advances to the new Quantitative assay format, in a totally automated
equipment, compared to the Qualitative assay format, include :
1. Simple 6 step protocol, more robust procedure for automated system.
2. Reduced time for assay – 4 hours.
3. Optimized space with possibilities to integrate PKU, GAL and MSUD into the same equipment
simultaneously.
4. Units expressed as common in literature (nmol/min/mL of PABA).
The NeoLISA® BIO kit using an automated system, Nimbus NeoLISA®, with a protocol of 4 hours
has shown excellent concordance compared to the traditional 18-hour protocol. The new
Quantitative assay protocol considers the Brazilian market demands to develop innovation
according to market needs. The results observed can be considered evidence that this product
keeps the same level of confidence and quality expected for newborn screening programs.
Contact :
Claudio Augusto Sampaio Filho
csampaio@intercientifica.com.br
Av. Sishima Hifumi, 2911 – Urbanova
Pq. Tec. UNIVAP – mod. 306-309
12244-000 S.J.Campos, SP
Brazil
www.intercientifica.com.br
ic@intercientifica.com.br
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Sishima Hifumi, 2911-Urbanova Pq. Tec. UNIVAP-mod. 306-309 12244-000 S
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Av. Sishima Hifumi, 2911-Urbanova Pq. Tec. UNIVAP-mod. 306-309 12244-000 S.J.Campos, SP