A dried blood spot‐based method to measure levels of tartrate‐resistant acid phosphatase 5b (TRACP‐5b), a marker of bone resorption

Abstract

Objectives A number of basic questions about bone biology have not been answered, including population differences in bone turnover. In part, this stems from the lack of validated minimally invasive biomarker techniques to measure bone formation and resorption in field‐based population‐level research. The present study addresses this gap by validating a fingerprick dried blood spot (fDBS) assay for tartrate‐resistant acid phosphatase 5b (TRACP‐5b), a well‐defined biomarker of bone resorption and osteoclast number. Methods We adapted a commercially available enzyme‐linked immunosorbent assay (ELISA) kit from MyBiosource for the quantitative determination of TRACP‐5b levels in serum and plasma for use with DBS. We used a rigorous process of assay modification and validation, including the use of a matched set of 189 adult plasma, fDBS, and venous DBS (vDBS) samples; parameters evaluated included precision, reliability, and analyte stability. Results Plasma and DBS TRACP‐5b concentrations showed a linear relationship. There were no systematic differences in TRACP‐5b levels in fDBS and vDBS, indicating no significant differences in TRACP‐5b distribution between capillary and venous blood. Parallelism and spike‐and‐recovery results indicated that matrix factors in DBS do not interfere with measurement of TRACP‐5b levels from DBS using the validated kit. Intra‐ and interassay CVs were 5.0% and 12.1%, respectively. DBS samples should preferably be stored frozen but controlled room temperature storage for up to a month may be acceptable. Conclusions This DBS‐based ELISA assay adds to the methodological toolkit available to human biologists and will facilitate research on bone turnover in population studies.

Validation of an enzyme‐linked immunoassay assay for osteocalcin, a marker of bone formation, in dried blood spots

“…Very few of the DBS we assayed had osteocalcin levels below the level of detection we measured for this assay (0.34 ng/mL), indicating that it is feasible to use it for population‐level assays of osteocalcin from DBS, at least in the population our samples were drawn from (predominantly European ancestry). Consistent with some other DBS analytes (eg, TRACP‐5b [Eick et al, ], immunoreactive trypsinogen [Li et al, ], ferritin and the transferrin receptor [Cook, Flowers, & Skikne, ]), osteocalcin levels were fairly stable at −28°C and at room temperature for up to 28 days, but appeared to degrade at 37°C to produce fragments that were immunoreactive with the antibodies used by the manufacturer (ie, there was an apparent increase in osteocalcin levels over time at this elevated temperature). Osteocalcin levels also tended to increase with the number of freeze‐thaw cycles, but with variation among samples.…”

“…Total alkaline phosphatase, bone alkaline phosphatase, and osteocalcin are protein biomarkers that have been used as markers of bone formation (Eastell & Hannon, ). Recently, we validated a dried blood spot (DBS)‐based assay of one marker of bone resorption, namely TRACP‐5b (Eick et al, ); however, until now, no validated assay for measuring a marker of bone formation in DBS samples has been reported. Because DBS assays facilitate measurement of biomarkers from populations in remote, field‐based settings often characterized by limited diagnostic equipment, the development of such an assay has both clinical and nonclinical (eg, anthropological) value.…”

Current and future applications of biomarkers in samples collected through minimally invasive methods for cancer medicine and population‐based research

“…Medical research has thoroughly examined many molecules that are suitable for the detection of cancer at a population level, despite presently being of limited use in clinical settings due to more stringent requirements for sensitivity and specificity. Uniting oncology with human biology, which is a leader in the development of minimally invasive techniques for population-based studies (Eick et al, 2016(Eick et al, , 2017(Eick et al, , 2019(Eick et al, , 2020Gildner, 2021;McDade, 2014;McDade et al, 2007McDade et al, , 2020Rej et al, 2021;Valeggia, 2007), provides an opportunity to apply well-researched methods to a field that has recognized the need for greater biomarker sampling and less invasive techniques.…”

Measuring attack on self: The need for field‐friendly methods development and research on autoimmunity in human biology

“…In the last decade, biomarker analysis from field‐friendly (i.e., affordable; minimally‐invasively collected; easily‐stored) samples has come into focus within the fields of human biology and related disciplines (Eick et al, 2019; Eick et al, 2020, 2020; Goldman et al, 2017; Hoke, McCabe, Miller, & McDade, 2018; Ice & James, 2007; McDade, 2014; McDade, Williams, & Snodgrass, 2007; Mei, Alexander, Adam, & Hannon, 2001; Valeggia, 2007; Vitzthum, 2020). This shift in focus has opened doors for population‐based research (i.e., interdisciplinary, multi‐method human health research focused on specific groups of individuals) because in many cases, venous blood draw, magnetic resonance imaging, tissue sampling, and other more invasive, specialized, or expensive methods for assessing health‐ and physiology‐related metrics are difficult or impossible in non‐clinical settings (McDade et al, 2007).…”