Immunoassay interference complicating management of Cushing’s disease: the onus is on the clinician and the laboratory

We read with interest the article by Grasko et al. ¹ on heterophilic antibody interference affecting adrenocorticotropic hormone (ACTH) immunoassay in a patient with Cushing’s syndrome. We describe a case of multiple immunoassay interferences complicating management of Cushing’s disease. A 54-year-old female was diagnosed with Cushing’s disease based on investigations on the Roche Cobas e602 and Siemens Centaur analyzers including an elevated plasma ACTH and high-dose dexamethasone suppression test that showed > 50% suppression of baseline serum cortisol. The patient underwent a transsphenoidal left hemi-hypophysectomy. Histopathology showed anterior pituitary gland tissue with moderate-to-strong staining for ACTH. Her blood pressure and glycaemic control improved with cessation of all antihypertensive agents and insulin. However, one week after the surgery, her serum cortisol concentrations on the Beckman Coulter DxI 800 analyzer were still within the normal range with lack of diurnal variation (cortisol results, 193–307 nmol/L). Given no biochemical cure, the patient underwent a completion hypophysectomy two weeks later. Histopathology showed anterior pituitary gland tissue with no abnormal cells. Plasma ACTH concentrations were unexpectedly high on the Siemens Immulite 1000 analyzer (ACTH, 20 pmol/L). Serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were also detectable on the Beckman Coulter DxI 800 analyzer (FSH, 2.4 IU/L; LH, 2.8 IU/L). The remaining pituitary hormones were low, consistent with total hypophysectomy. An ectopic ACTH-secreting lesion was excluded with negative whole-body CT and Gallium DOTATATE positron emission tomography (GaTATE PET) scans.

Serial dilution of patient’s plasma was non-linear for Siemens Immulite ACTH and Beckman Coulter DxI FSH and LH assays suggesting assay interference. ACTH result was 1.7 pmol/L on the Roche Cobas e602 analyzer. FSH and LH were undetectable on two alternative analyzers (Roche Cobas e602 and Siemens Centaur). Polyethylene glycol (PEG 8000) precipitation study to exclude macro-ACTH was not possible due to incompatibility with the Siemens Immulite analyzer. Similar to the case described by Grasko et al., incubation of patient’s plasma/serum with heterophilic-blocking tube (HBT) (Scantibodies Laboratory, Inc., Santee, CA, USA) resulted in an unexpected increase in ACTH, FSH and LH values (Table 1). There was no dilution incurred during the HBT study; therefore, the paradoxical rise in ACTH value was unlikely due to any dilution effect. Ellis and Livesery ² investigated the assay specificity of different methods which were available to detect heterophilic antibody interference in normal serum samples. They described post-HBT over-recovery in several assays on the Beckman Coulter Access analyzer (FSH, LH and growth hormone (GH)) and suggested that this over-recovery was assay specific and possibly dependent on the assay configuration. Those assays contained solid-phase goat-anti-mouse monoclonal antibody complexes. For assays that contained an anti-mouse component, it was suggested to check for false-positives by determining whether results were linear on dilution as the assays might prohibit the use of any blocker containing mouse components (e.g. HBT).³ Siemens Immulite ACTH and Beckman Coulter DxI FSH and LH contain an anti-mouse component in the assay format, and this may be the cause of over-recovery after HBT treatment. Cortisol measurements were comparable on three different analyzers (Beckman Coulter DxI 800, Roche Cobas e602 and Siemens Centaur). The cortisol results were likely true as a result of delayed remission.^3,4 Interestingly, in our case and all previously reported ACTH assay interference cases, the ACTH measurements were performed on the Siemens Immulite analyzers.^1,5,6

Table 1.

Investigations for assay interferences.

Procedures	Before dilution	10 × Dilution	Post-treatment with heterophilic-blocking tube (HBT)	Repeat measurement of neat plasma/serum on Roche Cobas
ACTH (pmol/L) (Siemens Immulite 1000)	20	310	35	1.7
FSH (IU/L) (Beckman Coulter DxI 800)	2.7	10.3	>250	0.4
LH (IU/L) (Beckman Coulter DxI 800)	3.3	15.7	>250	<0.1

All laboratories should adopt a clear diagnostic algorithm to identify and investigate immunoassay interferences. ⁷ While the laboratory may hold the necessary expertise to investigate, the onus of identifying potential cases of interferences is on the astute clinician as well as the laboratory.^8,9