Serum procollagen type I N-propeptide (PINP) is designated the reference marker of bone formation in osteoporosis; the reference marker for resorption is C-terminal telopeptide of type I collagen (CTX). PINP has very low circadian and biological variation, is not affected by food intake, and is very stable in serum after venepuncture. The two automated commercial assays for PINP provide similar results in subjects with normal renal function, allowing reference intervals to be used interchangeably. Bone turnover markers (BTM) are currently not recommended for fracture risk assessment and therefore not included in fracture risk calculators. In the management of osteoporosis, the main utility of BTM including PINP is for monitoring therapy, both antiresorptive as well as anabolic agents; monitoring is thought to help improve adherence. PINP as well as CTX may also be used in assessing offset of drug action following a pause in bisphosphonate therapy, to help decide when to re-instate therapy, or following cessation of denosumab therapy to assess efficacy of follow-on bisphosphonate therapy. PINP may also be used in the diagnosis of Paget's disease of bone as well as in monitoring response to therapy and for recurrence. Although BTM other than bone alkaline phosphatase are currently not recommended for use in metabolic bone disease of chronic kidney disease, PINP measured by assays specific to the intact molecule has potential in this condition. Further studies are needed to examine this area, as well as in malignant bone disease.

Direct measurement of the nonapeptide vasopressin has been limited by analyte instability ex vivo and in vivo rapid degradation, low serum concentrations requiring a sensitive assay and inherent secretory pulsatility. Copeptin is a 39 amino acid glycopeptide cleavage product of vasopressin synthesis with high stability, providing a marker of vasopressin secretion. Copeptin measurement has applications in diagnosis of diabetes insipidus and other diseases with altered vasopressin secretion. This review summarises our current understanding of serum copeptin measurement in diabetes insipidus and possible future applications of copeptin assays. As vasopressin is a stress hormone, there is emerging evidence on the use of copeptin for diagnosis and prognostication of disorders such as syndrome of inappropriate anti-diuretic hormone secretion, diabetes mellitus, critical illness, stroke, cardiovascular disease, respiratory disease, renal disease and thermal stress. Copeptin concentration measurement is likely to improve the diagnostic reliability of diabetes insipidus and, as a marker of stress, may have diagnostic or prognostic utility in specific clinical circumstances. Further studies are needed to determine if goal-directed therapy using plasma copeptin concentrations may improve patient outcomes.

Vitamin D is essential for bone health and is known to be involved in immunomodulation and cell proliferation. Vitamin D status remains a significant health issue worldwide. However, there has been no clear consensus on vitamin D deficiency and its measurement in serum, and clinical practice of vitamin D deficiency treatment remains inconsistent. The major circulating metabolite of vitamin D, 25-hydroxyvitamin D (25(OH)D), is widely used as a biomarker of vitamin D status. Other metabolic pathways are recognised as important to vitamin D function and measurement of other metabolites may become important in the future. The utility of free 25(OH)D rather than total 25(OH)D needs further assessment. Data used to estimate the vitamin D intake required to achieve a serum 25(OH)D concentration were drawn from individual studies which reported dose-response data. The studies differ in their choice of subjects, dose of vitamin D, frequency of dosing regimen and methods used for the measurement of 25(OH)D concentration. Baseline 25(OH)D, body mass index, ethnicity, type of vitamin D (D2 or D3) and genetics affect the response of serum 25(OH)D to vitamin D supplementation. The diversity of opinions that exist on this topic are reflected in the guidelines. Government and scientific societies have published their recommendations for vitamin D intake which vary from 400-1000 IU/d (10-25 μg/d) for an average adult. It was not possible to establish a range of serum 25(OH)D concentrations associated with selected non-musculoskeletal health outcomes. To recommend treatment targets, future studies need to be on infants, children, pregnant and lactating women.

Autoimmune glycaemic dysregulation and hyperinsulinaemic hypoglycaemia mediated by insulin autoantibodies is an increasingly recognised but controversial phenomenon described in both exogenous insulin naïve (insulin autoimmune syndrome) and exposed (exogenous insulin antibody syndrome) individuals. There has been a significant proliferation of case reports, clinical studies and reviews in the medical literature in recent years which have collectively highlighted the discrepancy between experts in the field with regard to the nomenclature, definition, proposed pathophysiology, as well as the clinical and biochemical diagnostic criteria associated with the condition. The essential characteristics of the condition are glycaemic dysregulation manifesting as episodes of hyperglycaemia and unpredictable hyperinsulinaemic hypoglycaemia associated with high titres of endogenous antibodies to insulin. Although the hypoglycaemia is often life-threatening and initiation of targeted therapies critical, the diagnosis is often delayed and attributable to various factors including: the fact that existence of the condition is not universally accepted; the need to exclude surreptitious causes of hypoglycaemia; the diverse and often complex nature of the glycaemic dysregulation; and the challenge of diagnostic confirmation. Once confirmed, the available therapeutic options are expansive and the reported responses to these therapies have been variable. This review will focus on our evolving understanding, and the associated diagnostic challenges - both clinical and laboratory - of this complex condition.

Group testing, also known as pooled sample testing, was first proposed by Robert Dorfman in 1943. While sample pooling has been widely practiced in blood-banking, it is traditionally seen as anathema for clinical laboratories. However, the ongoing COVID-19 pandemic has re-ignited interest for group testing among clinical laboratories to mitigate supply shortages. We propose five criteria to assess the suitability of an analyte for pooled sample testing in general and outline a practical approach that a clinical laboratory may use to implement pooled testing for SARS-CoV-2 PCR testing. The five criteria we propose are: (1) the analyte concentrations in the diseased persons should be at least one order of magnitude (10 times) higher than in healthy persons; (2) sample dilution should not overly reduce clinical sensitivity; (3) the current prevalence must be sufficiently low for the number of samples pooled for the specific protocol; (4) there is no requirement for a fast turnaround time; and (5) there is an imperative need for resource rationing to maximise public health outcomes. The five key steps we suggest for a successful implementation are: (1) determination of when pooling takes place (pre-pre analytical, pre-analytical, analytical); (2) validation of the pooling protocol; (3) ensuring an adequate infrastructure and archival system; (4) configuration of the laboratory information system; and (5) staff training. While pool testing is not a panacea to overcome reagent shortage, it may allow broader access to testing but at the cost of reduction in sensitivity and increased turnaround time.

Fibrosis prediction is an essential part of the assessment and management of patients with chronic liver disease. Traditionally the gold standard for assessment of fibrosis is liver biopsy, but it suffers from various limitations including risk of patient injury and sampling error. As a result, noninvasive tests of hepatic fibrosis have been used in patients with chronic liver disease due to conditions such as hepatitis B and C, and alcoholic and non-alcoholic fatty liver disease. With the advent of new direct-acting antivirals, hepatic fibrosis staging is an important component of treatment decisions in the care of patients with chronic hepatitis C virus infection. Current limitations of the noninvasive biomarker models include a significant indeterminate range, and a predictive ability that is limited to only a few stages of fibrosis. However newer technologies and novel proteins identified by proteomics and genomics offer the possibility for further refinement and individualisation of biomarker fibrosis models in the future.

The goal of screening programs for inborn errors of metabolism (IEM) is early detection and timely intervention to significantly reduce morbidity, mortality and associated disabilities. Phenylketonuria exemplifies their success as neonates are identified at birth and then promptly treated allowing normal neurological development. Lysosomal diseases comprise about 50 IEM arising from a deficiency in a protein required for proper lysosomal function. Typically, these defects are in lysosomal enzymes with the concomitant accumulation of the enzyme's substrate as the cardinal feature. None of the lysosomal diseases are screened at birth in Australia and in the absence of a family history, traditional laboratory diagnosis of the majority, involves demonstrating a deficiency of the requisite enzyme. Diagnostic confusion can arise from interpretation of the degree of residual enzyme activity causative of disease and is impractical when the disorder is not due to an enzyme deficiency per se. Advances in mass spectrometry technologies has enabled simultaneous measurement of the enzymes' substrates and their metabolites which facilitates the efficiency of diagnosis. Employing urine chemistry as a reflection of multisystemic disease, individual lysosomal diseases can be identified by a characteristic substrate pattern complicit with the enzyme deficiency. Determination of lipids in plasma allows the diagnosis of a further class of lysosomal disorders, the sphingolipids. The ideal goal would be to measure biomarkers for each specific lysosomal disorder in the one mass spectrometry-based platform to achieve a diagnosis. Confirmation of the diagnosis is usually by identifying pathogenic variants in the underlying gene, and although molecular genetic technologies can provide the initial diagnosis, the biochemistry will remain important for interpreting molecular variants of uncertain significance.

Establishing paediatric reference intervals (RIs) is a challenging task due to difficulties in subject recruitment, collection of adequate blood volume, and the inherent physiological changes of many biomarkers with age. Despite these challenges, several national and international initiatives have demonstrated: (a) the feasibility of prospectively designed paediatric RI studies; (b) the development of continuous RIs; and (c) the comparison of reference values across analyser types to harmonise paediatric RIs. Whilst these studies have improved the interpretation of paediatric test results and compliance with international accreditation (ISO15189) requirements, several gaps and challenges in translating current paediatric RIs into routine laboratory practice remain. Future priorities for paediatric RI studies include: (a) determination of the impact of discrete versus continuous RIs, analyser-specific versus harmonised RIs, and prospective collection versus data mining on the proportion of results outside the RIs; (b) understanding the clinical implications of analyser-to-analyser variation in reference values and use of evidence-based paediatric harmonised RIs where applicable; (c) adaptation of laboratory information systems to incorporate continuous RIs; (d) further understanding of the biological variation in paediatric biomarkers; (e) studies to address the paucity of accurate data for neonatal RI development; (f) periodic demonstration of RIs being clinically 'fit-for purpose'; and (g) agreement and policy updates for use of modern, best practice statistical methods in estimation of paediatric RIs. Furthermore, in vitro diagnostic manufacturers may require incentivised paediatric RI studies and publications through co-ordinated grants and collaboration at end-user sites to reduce the burden on sole users.

Osteoporosis (OP) is a condition where there is low bone density and microarchitectural deterioration which can predispose to fragility fractures. There is a wealth of literature on OP from the developed countries, but less so from Asia. This review will explore the field of OP research in South-East Asia with regard to the epidemiology, the diagnosis of OP and the role of laboratory tests in the management of OP, with emphasis on 25-dihydroxyvitamin D and bone turnover markers.

Hypophosphatasia (HPP) is a rare inherited systemic metabolic disease caused by mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. TNSALP is expressed in the liver, kidney and bone, and its substrates include TNSALP inorganic pyrophosphate, pyridoxal-5'-phosphate (PLP)/vitamin B6 and phosphoethanolamine (PEA). Autosomal recessive and dominant forms of the disease result in a range of clinical entities. Major hallmarks are low alkaline phosphatase (ALP) and elevated PLP and PEA levels. Very severe infantile forms of HPP cause premature death as a result of respiratory insufficiency and also present with hypo-mineralisation leading to deformed limbs with, in some cases, the near-absence of bones and skull altogether. Respiratory failure, rib fractures and seizures due to vitamin B6 deficiency are indicative of a poor prognosis. Craniosynostosis is frequent. HPP leads to an unusual presentation of rickets with high levels of calcium and phosphorus, resulting in hypercalciuria, nephrocalcinosis and low ALP levels. Hypercalcaemic crisis, failure to thrive and growth retardation are concerns in infants. Fractures are common in both infantile and adult forms of the disease, concomitantly occurring with unexplained chronic pain and fatigue. Dental clinical presentations, which include the premature loss of teeth, are also commonly found in HPP and specifically manifest as odontohypophosphatasia. A novel enzyme therapy for human HPP, asfotase alfa, which is specifically targeted to mineralised tissues, has been developed in the past decades. While this treatment seems very promising, especially for infantile HPP, many questions regarding its long-term effects, the management of treatment, and any potential secondary adverse effects remain unresolved.

The pressure on healthcare budgets including laboratory medicine is relentless and the focus on activities and costs remains the dominant funding model of laboratory medicine everywhere. The limitations of this model are well documented and for a decade or more laboratory professions worldwide have started looking at alternative models where the value of laboratory medicine and its impact on patient outcomes are the predominant driving force. There are multiple ways to determine the value of a medical test, particularly if one takes into consideration its impact upon the complete clinical pathway. Thus various approaches to value determination are being explored by a number of international organisations. These organisations will be reviewed below, including one which uses the concept of a value proposition that describes in detail how a test should be implemented by measuring its clinical, operational and economic impact. All approaches for determination of value require professional leadership. There is a need for research of varying types including that related to translating global evidence into local practice, a key challenge facing laboratory medicine and healthcare generally. Another challenge is to think and act beyond the silo of the laboratory to achieve greater collaboration with those colleagues more directly involved in patient care.

The diagnosis of acute myocardial injury requires a rise and/or fall of cardiac troponin (cTn) on serial testing, with at least one concentration above the 99[th] percentile value of a normal reference population according to the recently published Fourth Universal Definition of Myocardial Infarction.1 However, the magnitude of change in cTn that constitutes a significant rise and/or fall was again not specified in detail. High-sensitivity cardiac troponin (hs-cTn) assays can measure ten-fold lower concentrations of cTn with more precision than older assays, and can accurately quantify cTn in more than 50% of healthy individuals with a coefficient of variation of less than 10% at the 99[th] percentile. These hs-cTn assays are also able to detect the normal variations in cTn results that are due to biological variability. Understanding and quantifying the normal variations in cTn is important as this would allow significant changes to be better defined. Numerous studies have sought to investigate the biological variability of cTn over the last ten years. Such studies are usually conducted in healthy individuals, however individuals with chronic cardiac disease or chronic renal failure have also been examined. These studies have yielded varying results in regards to significant change values for cTn. In light of the recent redefinition for myocardial infarction, the purpose of this mini-review is to revisit the biological variability of cTn. In particular, we outline concepts for determining a significant change value, review the results of previous studies on the biological variation of cTn and discuss potential considerations for clinical practice.