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The use of Percentiles for Determination of Cut-off Values in Newborn Screening Using Totally Automated System and Multiplex Assays.

Authors:

Abstract

Introduction : High numbers of false positives and identification of false negatives suggests adjustment of cut-off values is needed. Many studies about Cystic Fibrosis (CF) and Congenital Adrenal Hyperplasia (CAH) have been published recommending the use of percentiles to correctly classify negative and positives samples, mainly for CF when quantification of immunoreactive trypsinogen (IRT) is used. The use of percentiles has demonstrated benefits for the screening of CAH instead fixed cut-off point. For both diseases, other factors such as weight, sample collection date, and transportation conditions must be controlled to determine the correct classification of the samples, even using percentiles. INTERCIENTIFICA has developed a totally automated system for newborn screening for CH, CAH and CF in multiplex format that offers additionally the classification of the samples using percentiles per routine. Material and Methods: Positive confirmed samples were obtained from different labs in Brazil, plus external quality control materials, including internal controls with 3 different concentrations for each analyte (T4, TSH, IRT and 17OHP). The Nimbus NeoMAP associated to NeoMAP 4plex was used, with the software calculating and identifying samples using percentiles. The appropriate cut-off for each analyte is shown in the table with the sample results for each marker, against the classification using the fixed cut-off. Results: All the samples were corrected classified using the percentiles. The table shows the differences obtained between the use of fixed cut-off and cut-off determined by the percentile. The percentiles can be adjusted by the supervisor anytime and can directly affect the number of classified samples (positive/negative). Conclusion: The use of percentiles is offered as alternative method to the usual fixed cut-off for each marker and demonstrated advantages with reduction of false positives and false negatives in newborn screening. The use of a totally automated system, with multiplex format and automatic classification of the samples for each analyte, using percentiles per routine, is considered the most advanced and revolutionary solution for newborn screening programs.
The Use of Percentiles for Determination of Cut-off Values in Newborn Screening
Using Totally Automated System and Multiplex Assays.
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:
High numbers of false positives and identification of false negatives suggest that the adjustment of
cut-off values is needed. Many studies on Cystic Fibrosis (CF) and Congenital Adrenal
Hyperplasia (CAH) have been published recommending the use of percentiles to correctly classify
negative and positives samples, mainly for CF when quantification of immunoreactive trypsinogen
(IRT) is used. The use of percentiles, in instead of fixed cut-off, has demonstrated benefits for the
screening of CAH. For both diseases, other factors such as weight, sample collection date, and
transportation conditions must be controlled to determine the correct classification of the samples,
even when using percentiles. INTERCIENTIFICA has developed a totally automated system for
newborn screening for CH, CAH and CF in a multiplex format that additionally offers the
classification of the samples using percentiles per routine.
Materials and Methods:
Fig.1 Nimbus NeoMAP®a Totally Automated System for CH, CAH and CF, running assays in multiplex format, 8
microplates or 3072 results in 11 hours. The primary set of results is available in 3 hours for all the 4 markers
(TSH/T4/17OH/IRT).
The Totally Automated system, Nimbus NeoMAP® uses the NeoMAP® 4plex kit manufactured
by INTERCIENTIFICA. This product is based on the xMAP technology which uses magnetic
microspheres. The assay reaction for each one of the markers occurs in the surface of the
microspheres which are differentiated by their internal color. Each sample result is determined by
the reading process involving at least 50 microspheres for each marker, with an estimate precision
that has neither been obtained before nor can be compared to any other methodology available in
the market.
The positive confirmed samples were obtained from different labs in Brazil, plus external quality
control materials, including internal controls with 3 different concentrations for each analyte (T4,
TSH, IRT and 17OH). The Nimbus NeoMAP® associated to NeoMAP® 4plex was used, with the
software calculating and identifying samples using percentiles. The appropriate cut-off for each
analyte is presented in the percentile report with the sample results for each marker.
The Use of Percentiles for Determination of Cut-off Values in Newborn Screening
Using Totally Automated System and Multiplex Assays.
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:
All the samples were corrected classified using the percentiles. The table shows the differences
obtained between the use of fixed cut-off and cut-off determined by the percentile. The percentiles
can be adjusted by the supervisor anytime and can directly affect the number of classified samples
(positive/negative).
Fig 2. Typical Percentile report from a routine using NeoMAP®4plex. The primary section (top/left) defines the
statistics of the entire run. The second section (bottom/left) shows the results with numbers in red for outliers
calculated by the Percentile. The third section shows the sample distribution for the entire routine.
Fig. 3 Statistics, in details(left), from the Percentile report for the entire routine. The classification of the
samples by the software counts on specific adjustments to disconsider both internal or external controls
keeping the integrity of the classification. Any problem with the sample, e.g. no sample elution, is easily
identified by the system as shown above (right).
The Use of Percentiles for Determination of Cut-off Values in Newborn Screening
Using Totally Automated System and Multiplex Assays.
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 use of percentiles is offered as alternative method to the fixed cut-off for each marker and
demonstrated advantages with reduction of false positives and false negatives in newborn
screening.
The use of a totally automated system, with multiplex format and automatic classification of the
samples for each analyte, using percentiles per routine, is considered the most advanced,
appropriated and revolutionary solution for newborn screening programs of CH, CAH and CF.
The use of Percentile is recommended by CLSI and applied in many different labs running
newborn screening for CF and CAH. In Brazil, the State of Minas Gerais began to classify the
samples for CF using Percentile in January 2015.
INTERCIENTIFICA, historically know for its great number of innovations in Newborn Screening,
such as the multiplex technology and its related products, the totally automated system Nimbus
NeoMAP®and the NeoMAP®4plex kit, was the first company in the world to offer the Percentile
as an alternative method included in its software to classify the samples according to the
international recommendations.
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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