Clinical laboratories are a critical component of healthcare globally, providing an essential service through the assessment of patient specimens for important biomarkers of health and disease in blood, urine, and other body fluids. Laboratory test results contribute evidence-based objective data to patient charts and are fundamental to inform clinical decision-making in the assessment, prognostication, and monitoring of patients in tertiary care and community settings. Clinical laboratories are thus responsible for establishing appropriate reference standards to ensure the accuracy of test results reported to patient charts and also the accompanying interpretative recommendations. Reference standards are health-associated benchmarks against which blood test results can be compared, allowing clinical laboratories to flag abnormal results to notify clinicians of the need for follow-up testing and/or the initiation of treatment. Given their importance, most would assume comprehensive reference standards are readily available and implemented at clinical institutions worldwide. Unfortunately, critical gaps continue to exist in accurate and up-to-date reference standards, particularly in paediatrics.
Children are not small adults!
Children and adolescents undergo immense physiological changes throughout growth and development, influencing blood test result values for various markers of health and disease. For example, alkaline phosphatase changes significantly from birth to adolescence due to several physiological processes, including placental production at birth and increased muscle mass in adolescence. Reports have demonstrated that the application of reference standards derived in adults for alkaline phosphatase interpretation in children inappropriately flags over 80 per cent of test results. This further emphasises the importance of using age- and sex- appropriate paediatric reference standards for clinical decision making and avoiding the potential risk of erroneous or missed diagnosis.
While the historic gap in the availability of paediatric reference standards is concerning, it is not necessarily surprising. Reference standard establishment guidelines by recognised bodies require the recruitment of 120 healthy volunteers for appropriate derivation by nonparametric methods, considered the ‘gold standard’ approach. In paediatrics, this target sample size is incredibly challenging particularly given that biomarkers commonly need to be partitioned by age and sex, substantially increasing required sample size and subsequent resources. This is simply not feasible for most paediatric clinical laboratories. While recognised guidelines also allow the verification of previously derived reference standards from manufacturers or other research groups using smaller sample sizes, manufactures often do not include paediatric reference standards in their documentation. Thus, the majority of reference standards used in paediatric clinical laboratories worldwide were either determined in the adult population, derived using samples from hospitalised patients without appropriate statistically considerations, or determined decades ago on older/less accurate laboratory instruments or methodologies. In recent years, a number of international initiatives have begun to address these gaps, contributing to significant improvements in paediatric laboratory medicine.
Canadian laboratory initiative on paediatric reference intervals – our experience
The Canadian Laboratory Initiative on Paediatric Reference Intervals (CALIPER) programme is a nationwide health initiative to improve the diagnosis and monitoring of children and adolescents with medical concerns. Our main objective is to establish a comprehensive database of paediatric reference standards for various blood tests, in order to fill existing gaps that prevent the accurate test result interpretation with the ultimate goal of improving the care of children and adolescents around the world. Children from birth to 18 years of age are eligible to take part in CALIPER. Through a community-based approach, children and adolescents are recruited from schools, community centres, and summer camps. Participation involves informed consent, completion of a health questionnaire, anthropometric measurements, and blood collection. Since 2009, more than 12,000 healthy children and adolescents have participated, resulting in a robust biobank of paediatric blood specimens for reference standard establishment. Blood specimens are then analysed for various biomarkers of health disease on several analytical platforms to ensure utility in paediatric clinical institutions globally. Initial reports focused on routine biochemical parameters on the Abbott ARCHITECT platform. This was subsequently expanded through transference and de novo efforts to other common analytical platforms for test measurement (e.g. Beckman, Ortho, Roche, Siemens, and DiaSorin assays). New studies have also expanded to other laboratory areas, including critical care assays on point-of-care devices and hematology parameters.
To date, comprehensive age- and sex-specific reference standards for over 200 laboratory biomarkers of health and disease on analytical instruments have been established and published in over 70 peer-reviewed papers in high-impact journals. Adhering to CLSI statistical guidelines, our direct approach utilises the robust or nonparametric methods for the derivation of upper and lower reference limits. Based on these peer-reviewed data, an online database and mobile application was developed (www.caliperdatabase.org). The CALIPER database is easily and freely accessible and has been adopted by thousands of healthcare institutions worldwide, highlighting the quality and richness of these data and global reach of this programme.
CALIPER is not alone in its work to improve paediatric test result interpretation. Several international initiatives have contributed to the availability of paediatric reference standards, including the German Health Interview and Examination Survey for Children and Adolescents (KiGGS), the Scandinavian Initiative for the Establishment of Paediatric Reference Intervals (NORICHILD), the Children’s Health Improvement through Laboratory Diagnostics (CHILDx) in the U.S., Harmonising Age Pathology Parameters in Kids (HAPPI Kids) in Australia, and the Australasian Harmonised Reference Intervals for Paediatrics (AHRIP). Most initiatives use similar approaches by recruiting from healthy children and adolescents in community or outpatient settings, but often focus on select biomarkers of interest. Interestingly, new studies have harnessed high level statistical techniques to derive reference standards using patient data from Laboratory Information Systems. Key groups in Germany led by Zierk et al. have contributed immensely to establishing this indirect approach, which may serve as a useful option for paediatric clinical institutions worldwide moving forward, avoiding need to recruit hundreds of healthy children by using their own patient data. Together, the collective efforts of these groups have moved the field forward immensely, but the work still continues.
Current state and future directions
Key evidence gaps still remain in the field of paediatric reference standards. First, analytical platforms and assays are constantly being modified/newly developed. Changes to procedures in test measurement or calibration can significantly impact test results and thus reference standards. Leaders in the field should continue to monitor changes in analytical test measurement and conduct new studies when significant changes in assay technology occur.
With this in mind, the CALIPER programme recently derived age- and sex-specific paediatric reference standards for biochemical and immunochemical parameters on the new Alinity and Attellica assays from Abbott Diagnostics and Siemens Healthineers, respectively. Second, most paediatric reference standard studies have focused on routine blood tests that are commonly ordered as part of the standard of care. Critical gaps remain in specialised markers of health and disease, including markers of inflammatory disease (cytokines, calprotectin, autoimmune), nutritional deficiency (essential trace elements), and environmental toxicity (heavy metals). Our team has recently launched sub studies in these areas of specialised testing and will continue to expand the programme to other relevant diagnostic areas, particularly as new biomarkers emerge.
Finally, health-associated data for both routine and specialised parameters in neonates is lacking. It is very challenging to recruit and successfully collect an additional blood sample from neonates and children within the first year of life. Thus, most available data in the literature consists of residual specimens from outpatients. To address this significant gap, CALIPER has begun the new CALIPER Mother & Child Initiative, which is aimed at closing the gaps in both neonatal and maternal reference standards for important biomarkers of neonatal and maternal health (www.caliperproject.ca/mchi).
We have seen immense progress in the field of paediatric reference standards in the past decade as part of a global collective effort. However, we still have far to go to ensure every paediatric clinical institution has access to appropriate reference standards for their population and analytical instrumentation. We look forward to embarking on new studies to address remaining gaps and contributing to the field of paediatric reference standards alongside our colleagues globally.
This article appears in the latest issue of Omnia Health Magazine. Read the full issue online today.