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Licensed Unlicensed Requires Authentication Published by De Gruyter March 15, 2021

Continuous reference intervals for leukocyte telomere length in children: the method matters

Analia Lesmana, Pei Tian, Vasiliki Karlaftis, Stephen Hearps, Paul Monagle, Vera Ignjatovic, Ngaire Elwood and The Harmonising Age Pathology Parameters in Kids Study Team



Children with very short telomeres commonly develop bone marrow failure and other severe diseases. Identifying the individuals with short telomeres can improve outcome of bone marrow transplantation, with accurate diagnosis requiring the use of age-matched reference intervals (RIs). This study aimed to establish RIs for telomere length (TL) in children using three commonly used methods for TL measurement.


Healthy children aged 30 days to 18 years were recruited for assessment using age as a continuous variable. Venous blood samples were collected and leukocyte TL was measured using terminal restriction fragment (TRF) analysis, quantitative PCR (QPCR) and flow cytometry with fluorescence in situ hybridization (Flow-FISH). Fractional polynomial model and quantile regression were performed to generate continuous RIs. Factors that might contribute to variation in TL, such as gender, were also examined.


A total of 212 samples were analyzed. Continuous RIs are presented as functions of age. TRF analysis and QPCR showed significant negative correlation between TL and age (r=−0.28 and r=−0.38, p<0.001). In contrast, Flow-FISH showed no change in TL with age (r=−0.08, p=0.23). Gender did not have significant influence on TL in children.


This study provides three options to assess TL in children by establishing method-specific continuous RIs. Choosing which method to use will depend on several factors such as amount and type of sample available and required sensitivity to age-related change.

Corresponding author: Prof. Vera Ignjatovic, Haematology Research, Murdoch Children's Research Institute, 50 Flemington Road, Parkville 3052, VIC, Australia; and Department of Paediatrics, The University of Melbourne, 50 Flemington Road, Parkville 3052, VIC, Australia, Phone: +61 (3) 9936 6520, E-mail:

Funding source: Royal Children’s Hospital Foundation

Award Identifier / Grant number: 2017-923


The authors thank staff of the Pathology Collection Department at The Royal Children’s Hospital for obtaining the consent of participants and the collection of samples. The authors thank staff of the Anaesthetic and Surgical Departments at the Royal Children’s Hospital.

  1. Research funding: This work was supported by grants from Royal Children’s Hospital Foundation [2017-923]. The funding organization played no role in the study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

  2. Author contributions: A.L. processed samples, performed research and wrote the manuscript. P.T. established methods, analyzed data and contributed to writing the manuscript. V.K. wrote the study protocol, obtained ethics approval for the study, reviewed the manuscript and is the coordinator of the study. S.H. performed statistical analysis and provided support on statistical analysis and reviewed the manuscript. V.I. contributed to the design of the study and was a major contributor in writing the manuscript. P.M. conceived the study and contributed to the design of the study and was a major contributor in writing the manuscript. N.E. conceived the study and contributed to the design of the study and was a major contributor in writing the manuscript. All authors read and approved the final manuscript. All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  3. Competing interests: Authors state no conflict of interest.

  4. Informed consent: Informed consent was obtained from parents or guardians of children who met study inclusion criteria.

  5. Ethical approval: The research related to human use has complied with all the relevant national regulations, institutional policies and in accordance with the tenets of the Helsinki Declaration. The study protocol has been approved by The Royal Children’s Hospital, Melbourne, Ethics in Human Research Committee (34183 A).

  6. Data sharing statement: Available data can be obtained by contacting the corresponding author.


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Supplementary Material

The online version of this article offers supplementary material (

Received: 2021-01-13
Accepted: 2021-02-25
Published Online: 2021-03-15
Published in Print: 2021-06-25

© 2021 Walter de Gruyter GmbH, Berlin/Boston

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