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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  ‡
1   Department of General Pediatrics, Division of Neuropediatrics and Metabolic Medicine, University Hospital Heidelberg Center of Paediatric and Adolescent Medicine, Heidelberg, Germany
,
Sebastian Hegert‡
2   Screening-Laboratory Hannover, Hannover, Germany
,
Jessica Bzdok
3   Laboratory Becker & Colleagues, Munich, Germany
,
Rafael Tesorero 
1   Department of General Pediatrics, Division of Neuropediatrics and Metabolic Medicine, University Hospital Heidelberg Center of Paediatric and Adolescent Medicine, Heidelberg, Germany
,
Ute Holtkamp
2   Screening-Laboratory Hannover, Hannover, Germany
,
Siegfried Burggraf
3   Laboratory Becker & Colleagues, Munich, Germany
,
Elfriede Schuhmann
3   Laboratory Becker & Colleagues, Munich, Germany
,
Friedrike Hörster
1   Department of General Pediatrics, Division of Neuropediatrics and Metabolic Medicine, University Hospital Heidelberg Center of Paediatric and Adolescent Medicine, Heidelberg, Germany
,
Georg F. Hoffmann
1   Department of General Pediatrics, Division of Neuropediatrics and Metabolic Medicine, University Hospital Heidelberg Center of Paediatric and Adolescent Medicine, Heidelberg, Germany
,
Nils Janzen 
2   Screening-Laboratory Hannover, Hannover, Germany
4   Dept. of Clinical Chemistry, Hannover Medical School, Hannover, Germany
5   Division of Laboratory Medicine, Kinderkrankenhaus auf der Bult, Hannover, Germany
,
Jürgen G Okun
1   Department of General Pediatrics, Division of Neuropediatrics and Metabolic Medicine, University Hospital Heidelberg Center of Paediatric and Adolescent Medicine, Heidelberg, Germany
,
Marc Becker
3   Laboratory Becker & Colleagues, Munich, Germany
6   Department of Conservative Dentistry and Periodontology, Ludwig Maximilians University Munich, Munich, Germany
,
Jürgen Durner
3   Laboratory Becker & Colleagues, Munich, Germany
6   Department of Conservative Dentistry and Periodontology, Ludwig Maximilians University Munich, Munich, Germany
› Author Affiliations Funding Information Dietmar Hopp Stif tung — ht tp://dx.doi.org/10.13039/ 501100005941; 2311220 and 1DH1911376, granted to Georg F. Hoffmann.
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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 2nd-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 1st-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 2nd-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 1st-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

Supplementary Material



Publication History

Article published online:
25 September 2023

© 2023. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial-License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).

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  • References

  • 1 Brandow AM, Liem RI. Advances in the diagnosis and treatment of sickle cell disease. J Hematol Oncol 2022; 15: 20
    CrossrefPubMedSearch in Google Scholar
  • 2 An R, Huang Y, Rocheleau A. et al. Multispectral imaging for MicroChip electrophoresis enables point-of-care newborn hemoglobin variant screening. Heliyon 2022; 8: e11778
    CrossrefPubMedSearch in Google Scholar
  • 3 Kato GJ, Piel FB, Reid CD. et al. Sickle cell disease. Nat Rev Dis Primers 2018; 4: 18010
    CrossrefPubMedSearch in Google Scholar
  • 4 Ware RE, de Montalembert M, Tshilolo L. et al. Sickle cell disease. The Lancet 2017; 390: 311-323
    CrossrefPubMedSearch in Google Scholar
  • 5 Ryan K, Bain BJ, Worthington D. et al. Significant haemoglobinopathies: guidelines for screening and diagnosis. Br J Haematol 2010; 149: 35-49
    CrossrefPubMedSearch in Google Scholar
  • 6 Tanhehco YC, Nathu G, Vasovic LV. Development of curative therapies for sickle cell disease. Front Med 2022; 9: 1055540
    CrossrefPubMedSearch in Google Scholar
  • 7 Lobitz S, Telfer P, Cela E. et al. Newborn screening for sickle cell disease in Europe: recommendations from a Pan-European Consensus Conference. Br J Haematol 2018; 183: 648-660
    CrossrefPubMedSearch in Google Scholar
  • 8 Loeber JG, Platis D, Zetterström RH. et al. Neonatal Screening in Europe Revisited: An ISNS Perspective on the Current State and Developments Since 2010. Int J Neonatal Screen 2021; 7
    CrossrefPubMedSearch in Google Scholar
  • 9 Daniel Y, Elion J, Allaf B. et al. Newborn Screening for Sickle Cell Disease in Europe. Int J Neonatal Screen 2019; 5: 15
    CrossrefPubMedSearch in Google Scholar
  • 10 González-Irazabal Y, Hernandez de Abajo G, Martínez-Morillo E.. Identifying and overcoming barriers to harmonize newborn screening programs through consensus strategies. Crit Rev Clin Lab Sci 2021; 58: 29-48
    CrossrefPubMedSearch in Google Scholar
  • 11 Chace DH, Hannon WH. Impact of second-tier testing on the effectiveness of newborn screening. Clin Chem 2010; 56: 1653-1655
    CrossrefPubMedSearch in Google Scholar
  • 12 Lobitz S, Klein J, Brose A. et al. Newborn screening by tandem mass spectrometry confirms the high prevalence of sickle cell disease among German newborns. Ann Hematol 2019; 98: 47-53
    CrossrefPubMedSearch in Google Scholar
  • 13 Czibere L, Burggraf S, Fleige T. et al. High-throughput genetic newborn screening for spinal muscular atrophy by rapid nucleic acid extraction from dried blood spots and 384-well qPCR. Eur J Hum Genet 2020; 28: 23-30
    CrossrefPubMedSearch in Google Scholar
  • 14 Kunz JB, Awad S, Happich M. et al. Significant prevalence of sickle cell disease in Southwest Germany: results from a birth cohort study indicate the necessity for newborn screening. Ann Hematol 2016; 95: 397-402
    CrossrefPubMedSearch in Google Scholar
  • 15 Brockow I, Blankenstein O, Ceglarek U. et al. Nationaler Screeningreport Deutschland 2020. DGNS; . 2022
    PubMedSearch in Google Scholar
  • 16 Tesorero R, Janda J, Hörster F. et al. A high-throughput newborn screening approach for SCID, SMA, and SCD combining multiplex qPCR and tandem mass spectrometry. PLOS ONE 2023; 18: e0283024
    CrossrefPubMedSearch in Google Scholar
  • 17 Frömmel C. Newborn Screening for Sickle Cell Disease and Other Hemoglobinopathies: A Short Review on Classical Laboratory Methods—Isoelectric Focusing, HPLC, and Capillary Electrophoresis. Int J Neonatal Screen 2018; 4: 39
    CrossrefPubMedSearch in Google Scholar
  • 18 Boemer F, Cornet Y, Libioulle C. et al. 3-years experience review of neonatal screening for hemoglobin disorders using tandem mass spectrometry. Clin Chim Acta 2011; 412: 1476-1479
    CrossrefPubMedSearch in Google Scholar
  • 19 Pattloch D. Sichelzellkrankheit unter Neugeborenen in Deutschland: Eine Studie an Routinedaten der AOK. Gesundheitswesen 2019; 81: 986-992
    Thieme ConnectPubMedSearch in Google Scholar
  • 20 Frömmel C, Brose A, Klein J. et al. Newborn screening for sickle cell disease: technical and legal aspects of a German pilot study with 38,220 participants. Biomed Res Int 2014; 2014: 695828
    CrossrefPubMedSearch in Google Scholar
  • 21 Grosse R, Lukacs Z, Cobos PN. et al. The Prevalence of Sickle Cell Disease and Its Implication for Newborn Screening in Germany (Hamburg Metropolitan Area). Pediatr Blood Cancer 2016; 63: 168-170
    CrossrefPubMedSearch in Google Scholar
  • 22 Lobitz S, Frömmel C, Brose A. et al. Incidence of sickle cell disease in an unselected cohort of neonates born in Berlin, Germany. Eur J Hum Genet 2014; 22: 1051-1053
    CrossrefPubMedSearch in Google Scholar
  • 23 GBD 2019 Diseases and Injuries Collaborators. Global burden of 369 diseases and injuries in 204 countries and territories, 1990-2019: a systematic analysis for the Global Burden of Disease Study 2019. Lancet 2020; 396: 1204-1222
    Search in Google Scholar
  • 24 Olatunya OS, Babatola AO, Ogundare EO. et al. Perceptions and Practice of Early Diagnosis of Sickle Cell Disease by Parents and Physicians in a Southwestern State of Nigeria. ScientificWorldJournal 2020; 2020: 4801087
    CrossrefPubMedSearch in Google Scholar
  • 25 Ranque B, Kitenge R, Ndiaye DD. et al. Estimating the risk of child mortality attributable to sickle cell anaemia in sub-Saharan Africa: a retrospective, multicentre, case-control study. Lancet Haematol 2022; 9: e208-e216
    CrossrefPubMedSearch in Google Scholar
  • 26 Arishi WA, Alhadrami HA, Zourob M. Techniques for the Detection of Sickle Cell Disease: A Review. Micromachines (Basel) 2021; 12
    CrossrefPubMedSearch in Google Scholar