Chemiluminescent immunoassay

Serum hormone concentrations were measured using chemiluminescent immunoassay (Beckman, CA, USA) in the central clinical chemistry laboratory at the affiliated hospital. The detection ranges were 0.20–150 IU/L for FSH, 0.20–250 IU/L for LH, 20–10900 pg/ml for E2, 0.10–50 ng/ml for P and 1.2–250,000 IU/L for hCG. The sensitivities of the assay were 0.02 IU/L for FSH, 0.02 IU/L for LH, 5 pg/ml for E2, 0.01 ng/ml for P and 0.05 IU/ml for hCG. The intra-assay and inter-assay coefficients of variation were 3.0 and 6.0% for FSH, 3.0 and 6.0% for LH, 4.0 and 6.0% for E2, 4.0 and 6.0% for P, and 3.0 and 5.0% for hCG, respectively.

Serum glucose was measured by a clinical chemistry analyser (Beckman Coulter AU480).
Insulin was assayed using a paramagnetic particle, chemiluminescent immunoassay (Beckman).
The assay was not canine specific but was validated using canine serum (lower limit of
detection 0·03 µIU/ml, upper limit 300 µIU/ml, where 1 µIU/ml insulin = 6·945 pmol/l).
HPLC tandem MS (MS/MS) was utilised to determine plasma MH concentrations. Briefly, 200 µl
of plasma were diluted with 800 µl MeCN (methyl cyanide) and 10 µl of internal standard
solution (¹³C₇ D-MH; Toronto Research Chemicals, Inc.) and sonicated
for 5 min, followed by centrifugation for 5 min at 3750 rcf (relative centrifugal force).
The supernatant fraction (10 µl) was injected into a Shodex NH2P-40 3E column (Showa Denko
America Inc.), with guard on a Shimadzu LC10AVP HPLC (flow rate 300 µl/min, column
temperature 50°C). Mobile phase A was 61 % acetonitrile, 39 % water with 10 mmol/l formic
acid. Mobile phase B was 50 % acetonitrile, 50 % water with 10 mmol/l formic acid. The
gradient was 100 % A isocratic for 43 min followed by a 6-min gradient to 100 % B. After a
6-min hold at 100 % B, the system was equilibrated at 100 % A for 12 min. MS/MS was
performed in negative ionisation mode on a Sciex API 4000 MS/MS. For MH, the formic acid
loss was monitored at m/z 255 to 209. For C₇ MH, the formic
acid loss was monitored at m/z 262 to 216.

All biomarkers were measured on ICU days 1 and 3 except parathyroid hormone (PTH), which was measured on ICU day 1 only. Assays were performed at the Harvard Medical School Clinical and Translational Science Award core laboratory. Plasma hCAP18 levels were measured by enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (Hycult Biotech, Uden, Netherlands) which recognizes the 37 amino acid biologically active C-terminal fragment (LL-37) cleaved from hCAP18 [30 (link)]. Plasma 25D (combined D₂ and D₃, hereafter referred to as ‘total 25D’) and D-binding protein (DBP) levels were measured by ELISA using commercially available kits (Abbott Laboratories, Abbott Park, IL, USA and R&D Systems, Inc., Minneapolis, MN, USA, respectively). Plasma intact PTH was measured using a chemiluminescent immunoassay (Beckman Coulter, Fullerton, CA, USA). Interassay coefficients of variation, estimated using blinded replicate samples from ICU patients, were 5.5% for hCAP18, 3.5% for total 25D, 11% for DBP, and 3.1% for PTH.

TSH serum levels were measured by chemiluminescent microparticle immunoassay (Abbott Diagnostics, USA), with intra-assay coefficient of variation (CV) of 3.10% and an inter-assay CV of 3.50%. FT3 serum levels were measured by chemiluminescent microparticle immunoassay (Abbott Diagnostics, USA), with intra-assay CV of 2.80% and an inter-assay CV of 3.65%. FT4 serum levels were measured by chemiluminescent microparticle immunoassay (Abbott Diagnostics, USA), with intra-assay CV of 3.80% and an inter-assay CV of 5.70%.
Total testosterone serum levels were measured by chemiluminescent microparticle immunoassay (Architect, Abbott, Dundee, UK), with inter- and intra-assay coefficients of variation (CV) of 5.2 and 5.1%, respectively. FSH and LH were measured by chemiluminescent microparticle immunoassay (Architect, Abbott, Longford, Ireland) with inter- and intra-assay CV of 4.1 and 3.1% for LH, and 4.6 and 4.2% for FSH, respectively. Serum estradiol were measured by chemiluminescent microparticle immunoassay on the ARCHITECT platform (Abbott Laboratories), with a sensitivity of 0.6 pg/mL. PRL was measured by Chemiluminescent Immunoassay (Beckman Coulter, Brea, CA, USA) with inter- and intra-assay CV of 4.2% and 1.6%, respectively.

Total testosterone serum levels were measured by Chemiluminescent Microparticle Immunoassay (Architect, Abbott, Dundee, UK), with inter- and intra-assay coefficients of variation (CV) of 5.2 and 5.1%, respectively. FSH and LH were measured by Chemiluminescent Microparticle Immunoassay (Architect, Abbott, Longford, Ireland) with inter- and intra-assay CV of 4.1 and 3.1% for LH, and 4.6 and 4.2% for FSH, respectively. PRL was measured by Chemiluminescent Immunoassay (Beckman Coulter, Brea, CA, USA) with inter- and intra-assay CV of 4.2 and 1.6%, respectively.
The laboratory reference ranges were 2.2–8.7 ng/dL for testosterone, 1–9 IU/L for LH, 1–12 IU/L for FSH and 3–13 ng/mL for PRL. The assay methods and kits used did not change over the years for all hormones considered.