High Throughput Newborn Screening for Sickle Cell Disease –
                    Application of Two-Tiered Testing with a qPCR-Based Primary
                    screen

Joachim Janda; Sebastian Hegert; Jessica Bzdok; Rafael Tesorero; Ute Holtkamp; Siegfried Burggraf; Elfriede Schuhmann; Friedrike Hörster; Georg F. Hoffmann; Nils Janzen; Jürgen G Okun; Marc Becker; Jürgen Durner

doi:10.1055/a-2153-7789

CC BY-NC-ND 4.0 · Klin Padiatr 2023; 235(06): 366-372
DOI: 10.1055/a-2153-7789

Original Article

High Throughput Newborn Screening for Sickle Cell Disease – Application of Two-Tiered Testing with a qPCR-Based Primary screen

Hochdurchsatz-Neugeborenenscreening auf Sichelzellkrankheit – Anwendung einer zweistufigen Analytik mit einem qPCR-basierten Primärscreening

Joachim Janda ‡

¹Department of General Pediatrics, Division of Neuropediatrics and Metabolic Medicine, University Hospital Heidelberg Center of Paediatric and Adolescent Medicine, Heidelberg, Germany

,

Sebastian Hegert‡

²Screening-Laboratory Hannover, Hannover, Germany

,

Jessica Bzdok

³Laboratory Becker & Colleagues, Munich, Germany

,

Rafael Tesorero

¹Department of General Pediatrics, Division of Neuropediatrics and Metabolic Medicine, University Hospital Heidelberg Center of Paediatric and Adolescent Medicine, Heidelberg, Germany

,

Ute Holtkamp

²Screening-Laboratory Hannover, Hannover, Germany

,

Siegfried Burggraf

³Laboratory Becker & Colleagues, Munich, Germany

,

Elfriede Schuhmann

³Laboratory Becker & Colleagues, Munich, Germany

,

Friedrike Hörster

¹Department of General Pediatrics, Division of Neuropediatrics and Metabolic Medicine, University Hospital Heidelberg Center of Paediatric and Adolescent Medicine, Heidelberg, Germany

,

Georg F. Hoffmann

¹Department of General Pediatrics, Division of Neuropediatrics and Metabolic Medicine, University Hospital Heidelberg Center of Paediatric and Adolescent Medicine, Heidelberg, Germany

,

Nils Janzen

²Screening-Laboratory Hannover, Hannover, Germany

⁴Dept. of Clinical Chemistry, Hannover Medical School, Hannover, Germany

⁵Division of Laboratory Medicine, Kinderkrankenhaus auf der Bult, Hannover, Germany

,

Jürgen G Okun

¹Department of General Pediatrics, Division of Neuropediatrics and Metabolic Medicine, University Hospital Heidelberg Center of Paediatric and Adolescent Medicine, Heidelberg, Germany

,

Marc Becker

³Laboratory Becker & Colleagues, Munich, Germany

⁶Department of Conservative Dentistry and Periodontology, Ludwig Maximilians University Munich, Munich, Germany

,

Jürgen Durner

³Laboratory Becker & Colleagues, Munich, Germany

⁶Department of Conservative Dentistry and Periodontology, Ludwig Maximilians University Munich, Munich, Germany

› Author Affiliations

Abstract

Background Sickle cell disease (SCD) is a group of hemoglobinopathies with a common point mutation causing the production of sickle cell hemoglobin (HbS). In high-throughput newborn screening (NBS) for SCD, a two-step procedure is suitable, in which qPCR first pre-selects relevant samples that are differentiated by a second method.

Methods Three NBS centers using qPCR-based primary screening for SCD performed a laboratory comparison. Methods using tandem MS or HPLC were used for differentiation.

Results In a benchmarking test, 450 dried blood samples were analyzed. Samples containing HbS were detected as reliably by qPCR as by methods established for hemoglobinopathy testing. In a two-step screening approach, the 2^nd-tier-analyses have to distinguish the carrier status from pathological variants. In nine months of regular screening, a total of 353,219 samples were analyzed using two-stage NBS procedures. The 1^st-tier screening by qPCR reduced the number of samples for subsequent differentiation by>99.5%. Cases with carrier status or other variants were identified as inconspicuous while 78 cases with SCD were revealed. The derived incidence of 1:4,773, is in good agreement with previously published incidences.

Conclusion In high-throughput NBS for SCD, qPCR is suitable to focus 2^nd-tier analyses on samples containing HbS, while being unaffected by factors such as prematurity or transfusions. The substantial reduction of samples numbers positively impacts resource conservation, sustainability, and cost-effectiveness. No false negative cases came to attention.

Zusammenfassung

Hintergrund Die Sichelzellkrankheit (SCD) bezeichnet eine Gruppe von Hämoglobinopathien mit einer gemeinsamen Punktmutation, die zur Bildung von Sichelzell-Hämoglobin (HbS) führt. Für das Hochdurchsatz-Neugeborenenscreening (NGS) auf SCD bietet sich ein zweistufiges Verfahren an, in dem die qPCR HbS-haltige Proben vorselektiert, die mit einer zweiten Methode differenziert werden.

Methoden Drei NGS-Zentren, in denen ein qPCR-basiertes Primärscreening auf SCD durchgeführt wird, haben sich einem Laborvergleich unterzogen. Zur Differenzierung wurden Tandem-MS oder HPLC genutzt.

Ergebnisse In einem Laborvergleich mit 450 Trockenblutproben wurden HbS-haltige Proben mit qPCR ebenso zuverlässig erkannt, wie mit Methoden die zur Untersuchung von Hämoglobinopathien etabliert sind. Der Fokus der Folgeanalytik liegt beim zweistufigen SCD Screening somit auf der Unterscheidung zwischen Trägerstatus und pathologischen Varianten. In neun Monaten Regelscreening wurden insgesamt 353.219 Proben untersucht, wobei das 1^st-tier-NGS mittels qPCR die Probenzahl für die Differenzierung um>99,5% reduzierte. Fälle mit Trägerstatus oder andere Varianten wurden als unauffällig erkannt und 78 Fälle mit SCD diagnostiziert. Die abgeleitete Inzidenz von 1:4.773, stimmt gut mit bislang publizierten Inzidenzen überein.

Schlussfolgerung Im Hochdurchsatz-NGS auf SCD ist qPCR geeignet, um die Folgeanalytik auf Proben zu fokussieren, die HbS enthalten und dabei von Störkonstellationen wie Frühgeburtlichkeit oder Transfusionen unbeeinflusst zu sein. Die erhebliche Reduzierung der Probenzahl wirkt sich positiv auf Ressourcenschonung, Nachhaltigkeit und Wirtschaftlichkeit aus. Falsch negative Befunde sind nicht bekannt geworden.

Key words

newborn screening - sickle cell disease - two-tiered testing - qPCR - benchmarking - high-throughput screening

Schlüsselwörter

Neugeborenenscreening - Sichelzellkrankheit - zweistufige Diagnostik - qPCR - Laborvergleich - Hochdurchsatz-Screening

Full Text

References

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Supplementary Material

Supplementary Material