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Multiplex analysis using a totally automated system for Newborn Screening of Congenital Hypothyroidism, Congenital Adrenal Hyperplasia and Cystic Fibrosis in the Northeast of Brazil.

Authors:

Abstract

Introduction: The Northeast of Brazil represents 27.7% of population with 9 States and approximately 690.000 newborn screening samples per year. The State of Bahia began to offer newborn screening in 1992. APAE-SALVADOR is the reference institution in the State processing 12.000 samples per month. During the last years, the inclusion of new markers by the Brazilian Newborn Screening Program, quantification of 17OH and analysis of Biotinidase activity has generated a significant increase in the number of assays. The impact is considered important inside the lab, since changes or limitations in infrastructure, human resources, punching sample process, data analysis can affect a critical parameter inside the Newborn Screening Program, the time from processing the sample to the results. Development of advanced technologies according the Brazilian Newborn Screening Program is needed to minimize the impact in the implementation of new markers. Materials and Methods : Lab of APAE-SALVADOR used a totally automated system (Nimbus NeoMAP) with multiplex assay (NeoMAP 4plex). Approximately 20.000 samples collected from 417 cities in State of Bahia have been analyzed during last 2 months in the routine. In the same study we evaluated the application of an additional feature offered with the use of the multiplex system, the analysis of the results by percentile instead the fixed cut-offs. DBS from positive samples (diagnostic confirmed) were used as controls in the study. Results: The time from receiving the sample to delivery of the results has been reduced 3 times using the multiplex technology. The use of NeoMAP 4plex Kits associated with totally automated equipment, Nimbus NeoMAP, reduced 75% the sample punching process. The use of percentiles has reduced the number of false positive samples and the potential false negatives in the routine compared to fixed cut-off. The results are presented in tables for each marker. Conclusion: The development of new technology that is focused on the requests and needs of the state newborn screening programs are essential for evolution of prevention in addition of new screening diseases and more sustainable public health system.
Multiplex Analysis Using a Totally Automated System for
Newborn Screening of CH, CAH and CF in The Northeast of Brazil.
Claudio Sampaio Filho (Jr)1, Fabiola Frias1, Raquel Veturiano1,
Simone Assunção do Amor Divino2, Maria Inês Andrade Sousa2,
Antônio Conceição da Purificação2
1-INTERCIENTIFICA, 2 APAE-SALVADOR
Introduction:
The Northeast of Brazil, wich includes 9 states, represents 27.7% of the Brazilian population and
accounts for approximately 690.000 newborns screening samples per year. During the last few
years the implementation of FASE IV of the National Newborn Screening Program (PNTN) has
increased the demand inside the labs to double the number of assays with little increment and/or
no modification in the infrastructure and human resources.
The implementation of new markers in newborn screening causes significant impact in State
Newborn Screening Programs in Brazil. Include new assays in the routine come to affect
processing time to analyse the samples, punches for spotting the sample, the assay procedure
itself and analysis.
The use of new technologies that could minimize any impact and take into account the limited
resources of investment and, at same time, increase the capacity to process samples, reduce the
spotting process, facilitate the analysis of the results and data management must be considered
essential to improve the newborn screening programs.
Fig. 1 Bahia is a northeastern Brazilian state with varied terrain from tropical coastline to the desertlike sertão. Its capital,
Salvador, is known for its iconic Pelourinho neighborhood, rich with 17th-century colonial architecture. The State has a
population of 15 milion people and 200.000 live births per year.
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Materials and Methods:
APAE-SALVADOR Lab used a totally automated system (Nimbus NeoMAP®) with assays in
multiplex format (NeoMAP®4plex). Approximately 20.000 samples have been processed
troughout the last 2 months in a routine compared to an automated system that runs one assay
individually per dot blood spot (DBS) and method using time resolved fluorescence method(TRF).
The processing times are compared to demonstrate the gain during the whole procedure, from
sampling to the results. In the same study we demonstrated an additional feature offered when the
multiplex technology is used: the analysis of the results by percentile instead the fixed cut-offs.
Fig. 1 The use of totally automated system involves an automatic puncher with data interface integrated to
the Nimbus NeoMAP®System, using the NeoMAP®4plex kits (TSH/T4/17OH/IRT). One equipment is able to
run almost 3072 analysis in 12 hours. The use of this system offered an increase of over 50% in the lab
production process comparing directly with TRF method/equipment. Additionally, The NeoMAP®system
offers a reduction in the time labor to punch DBS samples in the microplates in 75% and the same
percentage for sample consumption, since only one 3mm spot is sufficient to run 4 assays simultaneously in
the same well.
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Results:
The use of Nimbus NeoMAP®system that calculates the cut-off determined by Percentile per
routine has reduced significantly the number of false positive cases compared with TRF using
fixed cut-offs. The number of positive cases has been reduced for IRT in more than 60%. In this
study the positive samples determined in the primary screening were only 0.6%. The significant
reduction in the number of cases are explained by the difference in the use of fixed cut-off and
floating cut-off, but mainly because the characteristic of Bahia State population. Around 80% of the
population is said to be of black African ancestry. Data from IBGE (Brazilian Institute of Geography
and Statistics) from 2010 reported Bahia State contains the major population with African
descendants in Brazil.
According to the CLSI committee when discussing technical issues and challenges for newborn
screening using IRT, African descendants have IRT levels above the average observed in other
populations.
In Fact the use of NeoMAP®System with floating cut-off has reduced the number of false positive
samples in the routine compared the old TRF method using the fixed cut-off, even though the main
objective is to classify correctly the samples and reduce the number of false negatives. The use of
Fixed cutoff demonstrated to not be the correct form to identify CF cases since in the last few
years APAE-SALVADOR could detect at least 5 CF cases not previously identified in the newborn
screening.
Fig.2. The file printed from Nimbus NeoMAP®with percentile classification done by each routine for all 4
parameters. The positive samples are classified with values in red. The distribution of the samples are
plotted in a colored graph. The percentile or floating cut-off are not affected by external positive controls
since specific rules in the program can keep the analysis on the real samples/population.
The results observed in this study has demonstrated the importance of Percentile application to
correctly classify the samples in neonatal screening for Cystic Fibrosis. Since the NeoMAP®
system offers the Percentile for all the markers, the study includes the evaluation from fixed cut-off
against the floating cut-off for all the markers. Using the percentile resulted in a reduction in the
number of potential positives for CAH from 1,4 % to 0.6 %, T4 has been reduced from 6% to 1%
and TSH has increased from 0.1% to 0.5%. Samples included in this study are under evaluation to
confirm the diagnostic and if necessary, perform any adjustment of percentile for each marker.
The physical space to run the TSH, T4, 17OH and IRT has been reduced in 50% if compared with
the TRF equipment plus the reduction in the number of automatic punchers, since the NeoMAP®
system only need one DBS to run these 4 assays simultaneously.
The processing time from receiving the sample to delivering the results has been reduced 3 times
in comparison with time resolved fluorescence (TRF). The use of this system offered an increase
of 62.5% in the lab production process when compared. Besides, there was an additional gain of
75% in the time reduction and process to punch the samples.
Multiplex Analysis Using a Totally Automated System for
Newborn Screening of CH, CAH and CF in The Northeast of Brazil.
Claudio Sampaio Filho (Jr)1, Fabiola Frias1, Raquel Veturiano1,
Simone Assunção do Amor Divino2, Maria Inês Andrade Sousa2,
Antônio Conceição da Purificação2
1-INTERCIENTIFICA, 2 APAE-SALVADOR
Nº PO
References:
Conclusions:
The use of NeoMAP®system has defined new processing times inside the lab, reducing the
labor time to prepare the routine, the use of the samples, and increasing the production
capacity of the lab, by reducing use of physical space requirements.
The use of Percentile of Floating cut-off has reduced significantly the number of false positives
for CF and it seems to increase confidence in the correct classification of the samples in the
primary screening as recommended by CLSI, mainly because the population characteristics of
Bahia State.
The use of Nimbus NeoMAP®system and the respective NeoMAP®4plex kit to run newborn
screening for CH, CAH and CF in multiplex format and the results of this study translate the
advances and optimization of the lab process for newborn screening of CH, CAH and CF in
State of Bahia.
The development of new technology and solution that is focused on the requests and needs of
of Newborn Screening Program of each particular State are essential to expand the prevention
when the screening of new disease is added, minimizing impacts for limited investment
resources and more sustainable public health system.
Nimbus is a trademark of Hamilton
NeoMAP is a trademark of Intercientifica.
Multiplex Analysis Using a Totally Automated System for
Newborn Screening of CH, CAH and CF in The Northeast of Brazil.
Claudio Sampaio Filho (Jr)1, Fabiola Frias1, Raquel Veturiano1,
Simone Assunção do Amor Divino2, Maria Inês Andrade Sousa2,
Antônio Conceição da Purificação2
1-INTERCIENTIFICA, 2 APAE-SALVADOR
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