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A Totally Automated System for Newborn Screening of PKU, GAL and MSUD using 384 well microplates.

Authors:

Abstract

Introduction: The search for lab innovation in newborn screening is a constant process. The implementation of new markers, the necessity to obtain a robust process, cost effectiveness while minimizing steps, reduce infrastructure adjustments, reduce investments in human resources, keep or improve quality in the results are considered essential points. The Brazilian industry, with the objective to offer better solutions for newborn screening labs has developed a new solution for quantification of Phenylalanine, Total Galactose and Leucine using a totally automated system and microplates with 384 wells. Materials and Methods : An automatic puncher was configured for the use of 384 wells, spots of 3mm in diameter, associated to a totally automated equipment ( Nimbus NeoLISA 384 ) and the kits NeoLISA PKU 384, NeoLISA GAL 384 and NeoLISA MSUD 384 to run in one batch/routine 1940 samples. A run with 1940 samples takes 7 hours. For the same number of samples using the 96 well in automated system the same process takes 12 hours. Samples from different concentrations of each analyte, external quality control material and internal controls have been used for comparison analysis and reproducibility. The results are directly compared to the 96 well format in a totally automated system. A demonstrative graphics and tables are used to present the data. Results : Excellent reproducibility was observed between 384 well analysis and 96 well analysis. While a routine workflow with 1940 samples is rare, since most labs run 500-1000 samples a day, demonstrating the capacity of the equipment, the reduction of time process and the possibility to run simultaneously the 3 products in the same run is important. Conclusion : The use of 384 wells associated with a totally automated equipment, Nimbus NeoLISA 384 and kits NeoLISA PKU, GAL and MSUD 384 are considered an innovation for newborn screening labs in the quantification of Phenylalanine, Total Galactose and Leucine.
A Totally Automated System for Newborn Screening of PKU, GAL and MSUD
Using 384 Well Microplates.
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:
The search for lab innovation in newborn screening is a constant process. The implementation of
new markers, the necessity to obtain a robust process, cost effectiveness while minimizing steps,
reduce infrastructure adjustments, reduce investments in human resources, keep or improve
quality in the results are considered essential points. The Brazilian industry, aiming to offer better
solutions for newborn screening labs has developed a new solution for quantification of
Phenylalanine, Total Galactose and Isoleucine/Leucine using a totally automated system with 384-
well microplates.
Fig. 1 The 384-well microplates have the same dimensions of the standard 96-well microplates. The volume for
each well is reduced producing less contaminant material, use of water for reaction, using less water and
reagents, which is considered a more sustainable process. The 4-fold increase in the production reduces the
number of steps in the automated system and makes the process more robust.
Nº PO
Materials and Methods:
An automatic puncher was configured for the use of 384-well microplates, spotting 3mm
associated to an automated system ( Nimbus NeoLISA® 384 ) and the kits NeoLISA® PKU 384,
NeoLISA® GAL 384 and NeoLISA® MSUD 384 to run in one batch/routine 1940 samples. The
total time to run those samples was 7 hours. For the same number of samples using the 96-well
microplate format, in the same automated system, the run would take 12 hours. Samples from
different concentrations of each analyte, external quality control material and internal controls have
been used for analysis comparison and reproducibility. The results are directly compared to the
96-well format.
Fig. 2 The Nimbus NeoLISA® Automated System is exactly the same equipment for 96 well format but with a
different setup in the plate reader mode. The equipment is integrated with an automatic puncher and LIS to load
sample id
´
s and deliver full data results, including different bar code systems and communication protocols.
The Nimbus NeoLISA® does not require water reagent source attached as other automated equipments offered
in the market. The NeoLISA® Kits (PKU/GAL and MSUD) were configured to obtain the better performance using
the 384-wells microplate and consists in small adjustments to the reduced volume of the reaction in 384 well
microplates. For this study the routines were prepared using 6 microplates per run divided in 2 plates each for
PKU, GAL and MSUD for the same samples.
A Totally Automated System for Newborn Screening of PKU, GAL and MSUD
Using 384 Well Microplates.
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:
Excellent reproducibility was observed between analysis of equipment using 384-well and 96-well
microplates. A routine of 1940 is unusual, since most labs run 500-1000 samples a day, which
demonstrates the capacity of the equipment, the reduction of processing time and the possibility to
run simultaneously the 3 products NeoLISA® PKU / NeoLISA® GAL / NeoLISA® MSUD in the
same run. This is possible since the protocol of the assays are similar.
Protocol :
1. Dispense Elution Reagent - incubate 30 minutes
2. Transfer Eluate - filtration system
3. Add Work Reagent - incubate 30 minutes
4. Add Color Reagent – incubate 2 minutes
5. Read 570/690nm
There is a high possibility that the Brazilian Minister of Health will introduce the Newborn
Screening for Galactosemia and Maple Syrup Urine Disease in the Brazilian Newborn Screening
Programs is imminent since Galactosemia has demonstrated strong evidences with high frequency
in our population. The same scenario is observed for MSUD with very well organized network
group (Rede DXB) around Brazil and with successful therapy to cure the disease.
Table 1. Overall Agreement Nimbus NeoLISA® 96 versus 384-well microplate. Nimbus NeoLISA® 96-well
microplates has used with the INTERCIENTIFICA products produced for more than 20 years to run (NeoLISA®
kits) PKU / GAL and MSUD assays in the Brazilian Newborn Screening Programs. Nimbus NeoLISA® 384 is the
new automated system offered by INTERCIENTIFICA as a continuous improvement innovation process, mainly
to the use of 384 well microplates. The Overall Agreement study was carried out using 4825 samples from
routine labs in Brasil. The samples were directly compared in a primary screening assay for each marker. No
pre-selection of samples has been done, instead, only usual routine samples, some few previously positive (10-
20 samples ) samples/internal controls of PKU / GAL and MSUD.
Fig. 3 Typical timeline for Nimbus NeoLISA® 384, 2 plates (750 samples) for each one of the markers (PKU / GAL /
MSUD) in a total of 6 plates simultaneously or 2250 samples in a run. Reagents ready for use and specific position in the
deck, barcode id of the reagents, remote access and control with restrictions access rules determined by supervisor.
Overall Agreement Nimbus NeoLISA 96/ 384 Sample size
PKU 99.62% 4825
GAL 99.58% 4825
MSUD 99.62% 4825
A Totally Automated System for Newborn Screening of PKU, GAL and MSUD
Using 384 Well Microplates.
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
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
Conclusions:
The development of new solutions for newborn screening must consider basic concepts and
limitations of health investment system, reduce time labor, increase cost-benefit and maintain or
increase quality assignments.
Use of 384 well microplates reduce substantially the number of mechanical steps by the
automated equipment (punchers and automated processors), reduce potential errors in the
manipulation of the microplates by the users, increase productivity by the additional microplates
that could integrate the automated systems.
Use of 384 well microplates generate more sustainable product and solution for the market, since
less water reagents production is necessary, reduce plastic consume, reduce production of
waste/contaminant reagents, better utilization of the automation system increasing the production
capacity of the equipment in 75% when compared with the same equipment using 96 well
microplates..
Since the equipment is configured to run enzymatic colorimetric methods other NeoLISA®
products can be integrated as BIO and G6PD.
The use of 384 wells associated with a totally automated equipment, Nimbus NeoLISA® 384 and
kits NeoLISA® PKU, GAL and MSUD 384 are considered an innovation for newborn screening
labs in the quantification of Phenylalanine, Total Galactose and Leucine/Isoleucine.
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