Measurement of plasma ACTH concentration is the standard laboratory method used to support the diagnosis and monitoring of PPID cases (1) and serum insulin is an essential part of identifying and assessing insulin dysregulation (2). Several analytical methods are validated in horses including radioimmunoassay (RIA), immunofluorescent assay (Tosoh AIA) and chemiluminescent assay (Siemens Immulite) (3-5), with the latter being predominant historically in both publications and diagnostic laboratories. Interest has been growing in the immunofluorescent Tosoh AIA analysers since the above validation study and also work investigating possible cross reactivity between the Tosoh AIA and Immulite assays with other peptides (6,7).

In October 2023, further work began with the basic question of whether or not the Tosoh AIA analyser offered any diagnostic benefits over the incumbent Immulite analysers used within the VetPartners laboratories (Liphook, Rainbow and Rossdales). Although, admittedly, this work began with a somewhat sceptical mindset, we are all now convinced that the Tosoh AIA method offers significant benefits over the Immulite analysers and we will be changing to the former imminently. Please be clear at the outset that this change is made for purely scientific and clinical reasons and there is absolutely no cost saving to be made for the laboratories compared to staying with the Immulite analysers.

What changes will you see in results?

Insulin

  • Insulin measured on the new Tosoh AIA analyser will have relatively similar values to the old Siemens Immulites.

ACTH

  • ACTH measured on the new Tosoh AIA analyser will generally be lower than on the old Siemens Immulites.
  • However, the results from the two analysers do not have the same relationship across all concentrations.
  • Low ACTH values (in normal horses) will be similar between the two methods but as results get higher, the differences between the two analysers increases (with lower Tosoh results).
  • Thus, it will be highly unusual to see ACTH results measured by the new Tosoh AIA that are greater than 250-300 pg/mL, whereas the Immulite occasionally produced results >1000 pg/mL.
  • Reference intervals have been adjusted accordingly and will be lower with the new Tosoh AIA analysers, with the largest changes through late summer/autumn months. For example, for most of the year the Immulite cutoff was approx. 30 pg/mL versus mid to high 20s for Tosoh AIA; and the autumnal peak cutoff was around 70 pg/mL for the Immulite versus approximately 50 pg/mL for the Tosoh AIA.
  • TRH stimulation test results will differ and work is ongoing to clarify interpretation of this test.

Where is the advantage?

  • Diagnostically there is actually very little difference between the two methods. Comparison of ACTH results in a large group of clinically defined PPID cases indicated slightly better sensitivity with the Tosoh AIA and slightly better specificity with the Immulite, but overall not much difference.

So why change?

Insulin

  • Work done at the University of Pennsylvania by Professor Andrew van Eps (8) and also at Liphook has indicated that the Immulite Insulin assay is prone to generating spuriously high values in some horses that actually have normal serum insulin concentrations. A similar issue is also reported in human patients (9,10).

ACTH

  • Previous work has indicated that the Immulite ACTH assay also cross reacts with a further pituitary peptide known as CLIP (6). This peptide is known to circulate in PPID cases although the diagnostic implications of this remain unclear.
  • ACTH is prone to degradation in vitro post sampling. In practice degradation can be slowed, but not eliminated, by maintaining the sample in temperature-controlled packaging between sampling and analysis. Although this remains an issue when using either analytical method, ACTH values measured using the Immulite typically suffer about 3-4 times more degradation over 24 hours than when measured with the Tosoh AIA analyser.

Further information

Diagnosing Insulin Dysregulation & Equine Metabolic Syndrome

Diagnosing Pituitary Pars Intermedia Dysfunction (PPID)

See our sample requirements and turnaround times for our tests for ACTH (resting) and Insulin (fasting)

 

References

1               Stewart AJ, Ireland JL, Durham AE, McGowan CM. Diagnosis of equine pituitary pars intermedia dysfunction. The Veterinary Journal 2023;300-302:106036.

2               Frank N, Tadros EM. Insulin dysregulation. Equine Vet J 2014;46:103-112.

3               Irvine KL, Burt K, Hill AJ, Shaw S, Papasouliotis K. Initial analytic quality assessment and method comparison of an immunoassay for adrenocorticotropic hormone measurement in equine samples. Vet Clin Pathol 2016.

4               Couetil L, Paradis MR, Knoll J. Plasma adrenocorticotropin concentration in healthy horses and in horses with clinical signs of hyperadrenocorticism. J Vet Intern Med 1996;10:1-6.

5               Perkins GA, Lamb S, Erb HN, Schanbacher B, Nydam DV, Divers TJ. Plasma adrenocorticotropin (acth) concentrations and clinical response in horses treated for equine cushing’s disease with cyproheptadine or pergolide. Equine Vet J 2002;34:679-685.

6               Knowles EJ, Moreton-Clack MC, Shaw S, Harris PA, Elliott J, Menzies-Gow NJ. Plasma adrenocorticotropic hormone (acth) concentrations in ponies measured by two different assays suggests seasonal cross-reactivity or interference. Equine Vet J 2018;50:672-677.

7               Knowles EJ, Hyde C, Harris PA, Elliott J, Menzies-Gow NJ. Short communication: Identification of equine corticotropin-like intermediate lobe peptide (clip) binding to an adrenocortipcotrophic hormone (acth) assay capture antibody. Domest Anim Endocrinol 2023;83:106785.

8               van Eps AW, Kulp, J.C., Nolen-Walston,  R., Stefanovski, D. . A comparison of six immunoassays, including the tosoh aia-360,  for quantification of insulin in equine samples. Global equine endocrinology symposium p18. 2025.

9               Cavalier E, Huberty V, Carlisi A, Chapelle JP, Vroonen L, Beckers A. Human anti-animal antibodies interference in the siemens immulite chemiluminescent insulin immuno-assay: About one case. Clin Chim Acta 2011;412:668-669.

10            Iglesias P, Grande C, Cano AG, Maurí M. Anti-insulin antibodies interference in the siemens immulite chemiluminescent insulin immuno-assay. Clin Chim Acta 2013;425:186-187.