Hormone assays, quality control and sample collection

Peter A. Graham

doi:10.22233/9781910443866.3

British Small Animal Veterinary Association , 13 (2023); https://doi.org/10.22233/9781910443866.3

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Hormone assays, quality control and sample collection

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Author: Peter A. Graham
From: BSAVA Manual of Canine and Feline Endocrinology
Item: Chapter 3, pp 13 - 21
DOI: 10.22233/9781910443866.3
Copyright: © 2023 British Small Animal Veterinary Association
Publication Date: August 2023

This chapter focuses on hormone assays, quality control and sample collection. It provides an overview of immunoassay systems, covering the different formats and detection systems used. The chapter also discusses the importance of calibrators and the types of antibodies employed in hormone assays. Factors affecting validation of hormone assays are explored, and the need for quality assurance and quality control are emphasized. The final section contains a useful table detailing the sample type and handling requirements for hormone samples sent for external testing.

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Figures

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3.1

Classical immunoassays. (ai–ii) Radioimmunoassay: the amount of labelled hormone bound to the antibody is inversely proportional to the concentration of unlabelled hormone in the sample. (bi–ii) Immunoradiometric assay: the amount of labelled antibody remaining after washing is directly proportional to the concentration of hormone in the sample. In these examples the assay antibody binding sites are bound to the reaction vessel wall, but these could also be bound to a bead or a membrane. A wash step removes all unbound components prior to the detection stage.

= radiolabelled antibody (could be labelled with an enzyme in non-isotopic method);

= sample hormone;

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3.1 Classical immunoassays. (ai–ii) Radioimmunoassay: the amount of labelled hormone bound to the antibody is inversely proportional to the concentration of unlabelled hormone in the sample. (bi–ii) Immunoradiometric assay: the amount of labelled antibody remaining after washing is directly proportional to the concentration of hormone in the sample. In these examples the assay antibody binding sites are bound to the reaction vessel wall, but these could also be bound to a bead or a membrane. A wash step removes all unbound components prior to the detection stage.

= radiolabelled antibody (could be labelled with an enzyme in non-isotopic method);

= sample hormone;

= radiolabelled assay hormone.

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3.2

Glucose-6-phosphate dehydrogenase (G6PDH) homogeneous immunoassay for total thyroxine (T4). The conversion of nicotinamide adenine dinucleotide (NAD) to its reduced form (NADH) is monitored by ultraviolet light absorbance at 340 nm and compared with a calibration curve. (a) At low hormone concentrations, G6PDH cannot convert NAD to NADH because the enzymatic site is blocked by antibody interacting with enzyme-bound hormone. (b) At high hormone concentrations, G6PDH can convert NAD to NADH because the enzymatic site is not blocked by antibody, which is instead interacting with sample hormone. © 2023 British Small Animal Veterinary Association

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3.2 Glucose-6-phosphate dehydrogenase (G6PDH) homogeneous immunoassay for total thyroxine (T4). The conversion of nicotinamide adenine dinucleotide (NAD) to its reduced form (NADH) is monitored by ultraviolet light absorbance at 340 nm and compared with a calibration curve. (a) At low hormone concentrations, G6PDH cannot convert NAD to NADH because the enzymatic site is blocked by antibody interacting with enzyme-bound hormone. (b) At high hormone concentrations, G6PDH can convert NAD to NADH because the enzymatic site is not blocked by antibody, which is instead interacting with sample hormone.

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3.3

Example variation in canine total thyroxine (T4) results across six immunoassays. Nine serum sample pools of equidistant T4 concentration were measured in duplicate on each immunoassay. Concentrations reported for the highest values ranged from 40 to >60 nmol/l and those around the lower canine reference limit ranged from 10 to 20 nmol/l depending on the method used. Using pools of equidistant concentration also allows visualization of how well individual methods agree with themselves. A method with good linearity and precision should generate results through which a single straight line can be drawn. RIA = radioimmunoassay. (Reproduced from Graham (2010) ) © 2023 British Small Animal Veterinary Association

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3.3 Example variation in canine total thyroxine (T4) results across six immunoassays. Nine serum sample pools of equidistant T4 concentration were measured in duplicate on each immunoassay. Concentrations reported for the highest values ranged from 40 to >60 nmol/l and those around the lower canine reference limit ranged from 10 to 20 nmol/l depending on the method used. Using pools of equidistant concentration also allows visualization of how well individual methods agree with themselves. A method with good linearity and precision should generate results through which a single straight line can be drawn. RIA = radioimmunoassay. (Reproduced from Graham (2010) )

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3.6

Variation in cortisol concentration measured in a single external quality assessment canine pooled serum sample by 78 veterinary laboratories using 15 different analytical methods in 20 countries. Results range from 20 to 223 nmol/l (trimmed mean = 118 nmol/l, coefficient of variation = 20%). Although Method 14 yielded a low result compared with the consensus, it was reported against a lower reference interval than most other laboratories. Methods 8 and 11 were in-clinic analysers. CV = coefficient of variation (Data from European Society of Veterinary Endocrinology, 2020 ) © 2023 British Small Animal Veterinary Association

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3.6 Variation in cortisol concentration measured in a single external quality assessment canine pooled serum sample by 78 veterinary laboratories using 15 different analytical methods in 20 countries. Results range from 20 to 223 nmol/l (trimmed mean = 118 nmol/l, coefficient of variation = 20%). Although Method 14 yielded a low result compared with the consensus, it was reported against a lower reference interval than most other laboratories. Methods 8 and 11 were in-clinic analysers. CV = coefficient of variation (Data from European Society of Veterinary Endocrinology, 2020 )