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Reliable detection of sex chromosome abnormalities by quantitative fluorescence polymerase chain reaction

  • Camilla Mains Balle ORCID logo EMAIL logo , Dorte L. Lildballe ORCID logo , Ivonne Bedei ORCID logo , Ruth Luschka , Anne Skakkebæk ORCID logo , Simon Chang ORCID logo , Zeynep Agirman , Jan Keller , Axel Weber , Ramón E. Schäfer , Johannes Becker-Follmann and Claus H. Gravholt ORCID logo
Published/Copyright: March 20, 2025
Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 63 Issue 8

Abstract

Objectives

Many patients with sex chromosome abnormalities (SCAs) are diagnosed late in life or remain undiagnosed, leading to delayed or inadequate medical intervention and care. This study aimed to develop a reliable, rapid and cost-effective test for identifying SCAs using a blood sample – an essential step toward establishing a neonatal screening program.

Methods

A total of 360 blood samples (180 SCA patients, and 180 controls) were obtained from four cross-sectional studies of adult patients with SCAs and age-matched controls. Informed consent was collected, and all procedures followed the Declaration of Helsinki. Multiplex quantitative fluorescence polymerase chain reaction (QF-PCR) utilizing short tandem repeat (STR) and X-linked segmental duplication (SD) markers was performed. Results were analyzed using an automated algorithm. Deviant results were manually reviewed to differentiate errors in the PCR process from those in automated data analysis.

Results

Following automated data analysis of QF-PCR results, the method accurately identified 174 SCA patients (sensitivity: 96.7 %) and 171 controls (specificity: 95.0 %). Mosaic karyotypes were particularly challenging to diagnose. Manual reanalysis of the QF-PCR results corrected all false positives, achieving 100 % specificity.

Conclusions

This method is promising for reliable SCA detection in blood samples, offering cost-effectiveness and scalability. The specificity following automated data analysis was not satisfactory. The underlying PCR technique, however, demonstrated 100 % specificity, indicating that refining the automated analysis algorithm would significantly reduce false positive results. With further refinements, we believe this test would be highly suitable for further evaluation in a newborn screening setting.

Keywords: postnatal diagnosis; sex chromosome anomalies; quantitative fluorescence polymerase chain reaction
Corresponding author: Camilla Mains Balle, MD, PhD-student, Department of Endocrinology, Aarhus University Hospital, Palle Juul-Jensens Boulevard 99, 8200, Aarhus N, Denmark; and Department of Clinical Medicine, Aarhus University, 8000, Aarhus C, Denmark, E-mail:

Funding source: Danish Diabetes and Endocrine Academy (DDEA; funded by the Novo Nordisk Foundation)

Award Identifier / Grant number: NNF22SA0079901

Acknowledgments

Turner-Syndrom-Vereinigung Deutschland e.V. and Deutsche Klinefelter-Syndrom Vereinigung e.V donated blood and buccal smears for establishing the method.

  1. Research ethics: All clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. The samples in this study were derived from four cross-sectional studies on patients and healthy, age-matched controls (ClinicalTrials.gov identifier: NCT00624949, NCT00999310, NCT02526628, NCT01678261). The study on KS was approved by The Danish Data Protection Agency (1-16-02-472-15) and the Scientific Ethics Committee for the Central Denmark Region (1-10-72-131-15). The study on 46,XX males was notified to the Danish Data Protection Agency and to the Legal Department of Central Denmark Region (1-16-02-51-18) and approved by Scientific Ethics Committee for the Central Denmark Region (1-10-72-25-18).

  2. Informed consent: Informed written consent was obtained for all participants.

  3. Author contributions: Development of the method (JBF, RES, IB); laboratory work and analysis of samples (ZA, JBF); development of the automated algorithm (JK); karyotyping controls used to calibrate the algorithm (AW); collecting blood samples from patients and healthy controls (SC, AS); data processing and statistical analysis (CMB); writing up the initial draft for the manuscript (CMB, JBF, RES, CHG); critical review of the manuscript (CMB, DLL, IB, RL, AS, SC, ZA, REM, JBF, CHG). All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  4. Use of Large Language Models, AI and Machine Learning Tools: Large language models were used to a small extend to improve grammar.

  5. Conflict of interest: The authors state no conflict of interest.

  6. Research funding: Danish Diabetes and Endocrine Academy (DDEA; funded by the Novo Nordisk Foundation (NNF22SA0079901)).

  7. Data availability: The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Supplementary Material

This article contains supplementary material (https://doi.org/10.1515/cclm-2024-1400).

Received: 2024-12-02
Accepted: 2025-03-10
Published Online: 2025-03-20
Published in Print: 2025-07-28

© 2025 Walter de Gruyter GmbH, Berlin/Boston

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https://doi.org/10.1515/cclm-2024-1400
Keywords for this article
postnatal diagnosis; sex chromosome anomalies; quantitative fluorescence polymerase chain reaction

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. Setting analytical performance specification by simulation (Milan model 1b)
  4. Reviews
  5. Unveiling the power of R: a comprehensive perspective for laboratory medicine data analysis
  6. Clostebol detection after transdermal and transmucosal contact. A systematic review
  7. Opinion Papers
  8. A value-based score for clinical laboratories: promoting the work of the new EFLM committee
  9. Digital metrology in laboratory medicine: a call for bringing order to chaos to facilitate precision diagnostics
  10. Perspectives
  11. Supporting prioritization efforts of higher-order reference providers using evidence from the Joint Committee for Traceability in Laboratory Medicine database
  12. Clinical vs. statistical significance: considerations for clinical laboratories
  13. Genetics and Molecular Diagnostics
  14. Reliable detection of sex chromosome abnormalities by quantitative fluorescence polymerase chain reaction
  15. Targeted proteomics of serum IGF-I, -II, IGFBP-2, -3, -4, -5, -6 and ALS
  16. Candidate Reference Measurement Procedures and Materials
  17. Liquid chromatography tandem mass spectrometry (LC-MS/MS) candidate reference measurement procedure for urine albumin
  18. General Clinical Chemistry and Laboratory Medicine
  19. Patient risk management in laboratory medicine: an international survey to assess the severity of harm associated with erroneous reported results
  20. Exploring the extent of post-analytical errors, with a focus on transcription errors – an intervention within the VIPVIZA study
  21. A survey on measurement and reporting of total testosterone, sex hormone-binding globulin and free testosterone in clinical laboratories in Europe
  22. Quality indicators in laboratory medicine: a 2020–2023 experience in a Chinese province
  23. Impact of delayed centrifugation on the stability of 32 biochemical analytes in blood samples collected in serum gel tubes and stored at room temperature
  24. Concordance between the updated Elecsys cerebrospinal fluid immunoassays and amyloid positron emission tomography for Alzheimer’s disease assessment: findings from the Apollo study
  25. Novel protocol for metabolomics data normalization and biomarker discovery in human tears
  26. Use of the BIOGROUP® French laboratories database to conduct CKD observational studies: a pilot EPI-CKD1 study
  27. Reference Values and Biological Variations
  28. Consensus instability equations for routine coagulation tests
  29. Hematology and Coagulation
  30. Flow-cytometric lymphocyte subsets enumeration: comparison of single/dual-platform method in clinical laboratory with dual-platform extended PanLeucogating method in reference laboratory
  31. Cardiovascular Diseases
  32. Novel Mindray high sensitivity cardiac troponin I assay for single sample and 0/2-hour rule out of myocardial infarction: MERITnI study
  33. Infectious Diseases
  34. Cell population data for early detection of sepsis in patients with suspected infection in the emergency department
  35. Letters to the Editor
  36. Lab Error Finder: A call for collaboration
  37. Cascading referencing of terms and definitions
  38. Strengthening international cooperation and confidence in the field of laboratory medicine by ISO standardization
  39. Determining the minimum blood volume required for laboratory testing in newborns
  40. Performance evaluation of large language models with chain-of-thought reasoning ability in clinical laboratory case interpretation
  41. Vancomycin assay interference: low-level IgM paraprotein disrupts Siemens Atellica® CH VANC assay
  42. Dr. Morley Donald Hollenberg. An extraordinary scientist, teacher and mentor
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