Immunoreactive insulin stability in horses at risk of insulin dysregulation

Abstract Background Diseases associated with insulin dysregulation (ID), such as equine metabolic syndrome and pituitary pars intermedia dysfunction, are of interest to practitioners because of their association with laminitis. Accurate insulin concentration assessment is critical in diagnosing and managing these diseases. Hypothesis/Objectives To determine the effect of time, temperature, and collection tube type on insulin concentrations in horses at risk of ID. Animals Eight adult horses with body condition score >6/9. Methods In this prospective study, subjects underwent an infeed oral glucose test 2 hours before blood collection. Blood samples were divided into ethylenediaminetetraacetic acid, heparinized, or serum tubes and stored at 4 or 20°C. Tubes were centrifuged and analyzed for insulin by a chemiluminescent assay over 8 days. Changes in insulin concentrations were compared with a linear mixed effects model. Results An overall effect of time, tube type and temperature was identified (P = .01, P = 0.001, and P = 0.001, respectively). Serum and heparinized samples had similar concentrations for 3 days at 20°C and 8 days at 4°C; however, after 3 days at 20°C, heparinized samples had significantly higher insulin concentrations (P = .004, P = .03, and P = .03 on consecutive days). Ethylenediaminetetraacetic acid samples had significantly lower insulin concentrations regardless of time and temperature (P = .001 for all comparisons). Conclusions and Clinical Importance These results suggest an ideal protocol to determine insulin concentrations involves using serum or heparinized samples with analysis occurring within 3 days at 20°C or 8 days at 4°C.


| INTRODUCTION
An aging equine population and increased prevalence of obesity together with a better understanding of equine endocrine disorders have led to a rise in the recognition of endocrinopathies in horses. [1][2][3] Equine metabolic syndrome (EMS) and pituitary pars intermedia dysfunction are of particular importance because of their association with laminitis. 4,5 A fundamental feature of EMS is insulin dysregulation (ID) which describes alterations in insulin metabolism, namely hyperinsulinaemia, excessive insulin secretion after a carbohydrate challenge, peripheral tissue insulin resistance, or a combination of those elements. 6 As excessive insulin concentrations have been associated with an increased risk of developing laminitis, an accurate diagnosis of ID is therefore critical for ensuring optimal patient health and welfare outcomes. 7,8 Diagnosis of ID involves demonstration of resting (basal) hyperinsulinaemia, demonstration of excessive insulin response to an oral carbohydrate stimulation, and/or demonstration of peripheral tissue insulin resistance. 6 Convenient dynamic tests to detect peripheral tissue insulin resistance and enteroinsular axis activity have been developed for use in practice. The 2-step insulin sensitivity test, measures glucose in response to an IV insulin challenge whereas the oral glucose test (OGT) and the oral sugar test (OST) measure insulin after an oral glucose challenge. 9,10 The OGT and the OST, which are equivalent to identify horses with ID, are frequently used in practice and make insulin a commonly measured analyte. 5,10 In remote equine practice, accuracy of laboratory results can be affected by prolonged time to analysis because of considerable distances between horses and testing laboratories. The effects of handling on sample stability for multiple analytes have been researched in horses. 11,12 However, information regarding the most appropriate techniques and protocols for maintaining stability of immunoreactive insulin in horses is poorly described. Human studies have identified improved immunoreactive insulin stability in whole blood stored in ethylenediaminetetraacetic acid (EDTA) tubes; as such EDTA has replaced serum as the conventional storage medium for insulin analysis in people. 13 Serum is still the mainstay for insulin quantification in equine practice; however, it remains to be seen whether the potential benefits of EDTA are compatible with equine endocrinological testing. 14 Previous studies have concluded that storage of equine serum or plasma samples at room temperature over 3 days had minimal effect on immunoreactive insulin concentration when analyzed via a radioimmunoassay (RIA) technique. 14 However, with the RIA no longer available, practitioners and laboratories have since relied on a variety of assays, with the chemiluminescence immunoassay (CLIA) being the most common. 15 The RIA and the CLIA use different antibodies to detect insulin and the type of assay used greatly impact results when measuring immunoreactive insulin concentrations. 16,17 Therefore, results found previously using the RIA might not be as relevant to practitioners now utilizing the available CLIA as each assay measures different antigens with possibly different stabilities.
Inconsistencies between assays and a lack of in-depth research leave little information available for equine practitioners on the best methods available to ensure stability of equine insulin. The aim of the study was therefore to determine the effects of sample storage conditions (time, temperature, and tube type) on measurements of immunoreactive insulin concentration using the CLIA in a clinically relevant context using horses at risk of ID.

| Horses
Eight adult horses (4 geldings and 4 mares) of varying breeds (3 Warmbloods, 2 Standardbreds, 2 Australian Stock Horses, and 1 Quarter Horse) were selected from the institution's equine research herd. Specifically, inclusion criteria for horses consisted of a body condition score (BCS) ≥6/9 so as to increase the likelihood of selecting horses with ID. 4 Horses averaged an age of 15 ± 4 years with a mean body weight of 583 ± 40 kg, and a median BCS of 7/9 (6-8). 18 Each animal was deemed healthy before the study by physical examination.
Ethical approval was obtained from the institutional animal care and ethical use committee.

| Design
Horses were stabled and fasted for 10 hours, with free access to water before testing. 19 After collection of baseline blood samples, Blood was collected into lithium heparin, EDTA (BD, Belliver Industrial Estate) and silicate-containing serum (BD, Belliver Industrial Estate) tubes. To reduce variation in results, samples were taken within a 5-minute time period, and tube selection randomized. 20 For each horse, 12 serum, 12 heparin, and 12 EDTA tubes were obtained and stored unseparated at 4 C or at 20 C for up to 8 days.
Samples were centrifuged at 3000g and analyzed daily over the course of 5 days, with Day 1 being the day of collection, and with an additional analysis conducted on Day 8. On days of analysis, a 4 and 20 C tube from each tube type was randomly selected for each horse and, after centrifugation, the resulting serum or plasma sample was assessed for hemolysis. Hemolysis was graded visually on a scale from 0 to 3, with 0 being non-hemolyzed, transparent serum or plasma, and 3 being completely hemolyzed, dark red fluid (Supplementary Figure S1). The serum or plasma was pipetted using a 1-mL plastic disposable pipette into 1.5-mL aliquot tubes (Volume 1.5 mL Graduated Microcentrifuge Tube, QSP Scientific Plastics, San Diego, California). Samples were then analyzed using a commercially available CLIA (Immulite 1000 Chemiluminescent Assay, Siemens, Bayswater, Victoria, Australia) to determine serum or plasma immunoreactive insulin concentrations.
In order to determine the inter-and intra-assay coefficient of variation of the CLIA in serum, heparinized, and EDTA samples, additional blood samples were collected from 8 additional horses with insulin concentrations ranging from 3.0 to 197.0 μIU/mL. Samples were processed at 4 C as described above and run twice on kits with the same lot number (intra-assay coefficient of variation) and an additional time on kits with a different lot number (inter-assay coefficient of variation).

| Data analysis
A Shapiro-Wilk test was conducted to assess for a normal distribution.
Normally distributed data for age and weight were presented as mean ± SD, whereas BCS, as an ordinal variable, was presented as median (range).
A linear mixed effects model was used to determine changes in immunoreactive insulin concentrations. The variables "time," "temperature," "tube," and "hemolysis" were included as fixed effects, whereas "horse" was included as a random effect. Transformation of the immunoreactive insulin concentrations into percentage of baseline was required to satisfy residual normality; the baseline used for comparison among individual horses was the value collected from serum samples stored at 4 C on Day 1, as this reading most closely resembles the patient's true insulin concentration at the time of sampling. 14 Then a 1-way repeated measures ANOVA was performed to detect conditions significantly different from Day 1, 4 C, and serum sample. Statistical analysis was carried out using commercial statistical software (Prism, GraphPad Software, Inc. La Jolla, California; IBM SPSS Statistics 24, IBM Corp. Armonk, New York).
A P-value <.05 was considered significant.

| RESULTS
Overall, there was a significant effect of temperature (P = .001), collection tube (P = .001), and the time to centrifugation (P = .01) on equine immunoreactive insulin concentration.
For serum samples, the intra-assay coefficient of variation was 6.5% and the inter-assay coefficient of variation was 7.9%. For heparinized samples, the intra-assay coefficient of variation was 6.1% and the inter-assay coefficient of variation was 12.5%. For EDTA samples, the intra-assay coefficient of variation was 4.0% and the inter-assay coefficient of variation was 14.4%.  Figure 1 and Table 1)  for heparin, and P = .04 for EDTA, Table 1) and in serum samples at 20 C, immunoreactive insulin concentrations were significantly lower on Day 8 than on Day 1 (P = .001, Table 1). Multiple studies have concluded that samples collected and stored in EDTA tubes have no effect on insulin degradation. 13,21 This suggests that the low values and the larger variation are likely related to the assay used, not specifically to EDTA. Unlike samples used to determine ACTH concentrations, the manufacturer does not recommend analysis of EDTA samples with the insulin CLIA, further substantiating this point.

| DISCUSSION
Immunoreactive insulin detected using this method from EDTA samples is inaccurate, variable, and as low as 10% of the concentration of immunoreactive insulin detected in serum samples; to the authors' knowledge, there is no prior evidence of this occurring in equid-derived samples. However, the fact that insulin is analogous among species would explain how the effect was replicated when measuring equine immunoreactive insulin. 22 As such, the authors do not recommend the use of EDTA tubes for collection of equine insulin blood samples to be analyzed using this assay.
Immunoreactive insulin concentrations in heparinized plasma were more variable and consistently higher than in serum after Day 4 at 20 C which has been observed previously in human studies. 23 A larger range of values was also observed in heparinized samples compared to serum. The influence of heparin itself is likely the cause of the overestimation in values, but this interaction has not been thoroughly documented. 23,24 Although the purpose of this study was not to investigate the ability of the OGT to detect ID in obese horses, these unpredictable increases could potentially lead to false positive cases of ID.
In contrast, serum sample ranges were generally lower and less dispersed, even at 20 C and the intra-and inter-assay coefficients of variation were consistent with other studies. 15