Immunoassay interferences: laboratory pitfall in the diagnosis of adrenocortical carcinoma
Mafalda Martins Ferreira İD Carolina Moreno, Patrícia Oliveira, Isabel Paiva
Endocrinology, Diabetes and Metabolism, Centro Hospitalar e Universitário de Coimbra EPE, Coimbra, Portugal
Correspondence to Dr Mafalda Martins Ferreira; mafalda.ferreira@campus.ul.pt
Accepted 16 January 2024
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C BMJ Publishing Group Limited 2024. No commercial re-use. See rights and permissions. Published by BMJ.
To cite: Martins Ferreira M, Moreno C, Oliveira P, et al. BMJ Case Rep 2024;17:e257320. doi:10.1136/bcr-2023- 257320
SUMMARY
A woman in her late 50s with recent onset of hypertension, diabetes, lumbar pain and unintentional weight loss was diagnosed with a cortisol and androgen-producing adrenal mass. Despite this, serum adrenocorticotropic hormone (ACTH) concentration was inappropriately elevated, which was investigated thoroughly. Investigations included a brain magnetic resonance imaging to exclude concomitant pituitary adenoma, a corticotropin-releasing hormone stimulation test and a gallium-68 DOTATATE and 18F-FDOPA PET scan, both excluding ectopic ACTH production. Considering the disparity between clinical presentation and biochemical results, the ACTH was reanalysed using the Cobas immunoassay (Roche, Switzerland), ultimately unveiling the cause for ACTH elevation. ACTH levels had previously been measured with ACTH Immulite (Siemens, Germany), a two-site immunoassay which is prone to interferences causing falsely elevated ACTH concentrations. Inaccurate laboratory levels can lead to diagnosis delay and unnecessary diagnostic procedures and a close communication between the physicians and laboratorians is of utmost importance.
BACKGROUND
A definitive diagnosis requires attention to clin- ical presentation, differential diagnoses, laboratory and imaging findings and possible interferences to diagnostic procedures and techniques. A high clin- ical suspicion is essential every time a discordance between clinical and laboratory findings arise. A single equivocal laboratory result should not be relied on to make a definitive diagnosis.
Serum adrenocorticotropic hormone (ACTH) dosing is essential to diagnose and distinguish a variety of ACTH-related disorders, such as Cushing syndrome, Cushing disease, primary and secondary adrenal insufficiency, hypopituitarism and ectopic secretion of ACTH. Therefore, any incorrection in serum ACTH dosing can have a deleterious effect and a negative impact on diagnosis.
ACTH Immulite (Siemens, Germany) is an immu- nometric assay that has been implicated in ACTH dosing errors leading to misdiagnosis and health- related costs. 1 2
This paper describes the case of a woman with hypercortisolism and hyperandrogenism and a concomitant adrenal mass suspected of malignancy. She was found to have persistently elevated serum ACTH, then undergoing several unnecessary diag- nostic procedures to explain the elevation in ACTH levels.
CASE PRESENTATION
A woman in her late 50s presented to the endocri- nology clinic with a history of recent onset arterial hypertension and type 2 diabetes mellitus, as well as reported left lumbar pain and a 3-month long unintentional weight loss of more than 10% of her body weight.
Hypertension had begun 1 year earlier, for which she was treated with an ACE inhibitor, a calcium channel blocker and a thiazide diuretic. The patient regularly reported systolic blood pressure values higher than 160mm Hg. She was diagnosed with type 2 diabetes 3 months previously, managed with metformin 1000mg two times per day and sita- gliptin 100 mg daily, with a poor metabolic control (glycated haemoglobin of 8.5%). She additionally complained of moderate-intensity pain in the left lumbar region and a continual sensation of full- ness that hindered her appetite, resulting in weight loss. She did not report any urinary symptoms or B symptoms.
Her vital signs were blood pressure of 155/90 mm Hg, heart rate of 80 beats per minute and her body weight was 76 kg (BMI of 26.95 kg/m2). On exam- ination, she had an abdominal circumference of 97 cm with thin limbs, facial plethora and buffalo hump. No violaceous striae, skin bruising or lunar face was appreciated.
In order to investigate her marked weight loss and lumbar pain, a thoracic and abdominal CT scan was performed, revealing a well demarcated but hetero- geneous left adrenal mass measuring 106×121×70 mm (figures 1 and 2). The contrast washout was 70% and no lymph nodes or distant metastases were visible. Despite this, the adrenal mass was causing deviation of the body of the pancreas and left kidney and compressing the splenic and left renal vein, without thrombosis.
The biochemical investigation revealed a serum cortisol of 11 µg/dL post overnight 1 mg dexameth- asone suppression test (positive if >1.8 µg/dL). There was also androgen elevation: total testos- terone of 2.7 ng/ml (reference range: < 0.9ng/ mL), dehydroepiandrosterone sulphate of 11.6 µg/ mL (reference range: 0.3-1.8 µg/mL), androstene- dione of 39 ng/ml (reference range: 0.5-3.4 ng/ml) and 17 hydroxyprogesterone 2.64 ng/ml (refer- ence range: 0.1-2.3). Plasmatic metanephrines, 3-methoxytyramine, chromogranin A and aldoste- rone/renin ratio were normal.
At this moment, the most likely hypothesis would be that of an adrenal carcinoma producing cortisol and androgens. While weight gain is the most common situation in these cases, the size of the adrenal mass and the persistent sensation of fullness
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reported by the patient have contributed to reduced food intake and subsequent weight loss.
Unexpectedly, serum ACTH concentration measured with ACTH Immulite immunoassay (Siemens, Germany) was supe- rior to 500 pg/mL (reference range: 9-52 pg/mL) on repeated measurements at different time points.
INVESTIGATIONS
Given the unsolved aetiology of ACTH elevation in the presence of a cortisol and androgen-producing adrenal mass, the patient underwent brain MRI, which did not identify any pituitary adenomas or other sellar lesions. A CRH stimulation test was negative for ACTH and cortisol elevation. Therefore, a F-DOPA and gallium-68 DOTATATE PET were performed to exclude ectopic ACTH secretion, which were also unremarkable.
Serum ACTH dosing was then performed using another labo- ratory method, the Cobas immunoassay (Roche, Switzerland), which was found to be within the normal range: 8.1 pg/mL (reference range 9-52 pg/mL).
TREATMENT
Subsequently, a left adrenalectomy and nephrectomy were performed, and pathologic anatomy was compatible with an adrenocortical carcinoma with five out of nine points on the Weiss score (Ki67 of 2%)-ENSAT stage II.
OUTCOME AND FOLLOW-UP
A few months after the diagnosis, the patient had a local recur- rence that led to a new surgery with free resection margins. She is currently on mitotane 2.5 g per day, with no evidence of local or distant disease.
DISCUSSION
Immunoassays have a central role in clinical investigation and diagnosis and were first described in 1959 by Berson and Yalow earning them the 1977 Nobel Prize in Physiology or Medicine.1 Immunoassays detect the analyte of interest by using antibody- antigen reactions. Therefore, the presence of other antibodies in the patient’s serum can interfere with some of these reactions.
ACTH Immulite (Siemens, Germany) is a widely used two- site immunoassay that has been implicated in cases of misleading ACTH levels.2-5 The first description of erroneous ACTH dosing with ACTH Immulite was in 2013 by Grasko et al.2
False increases or (less frequently) false decreases in ACTH concentration caused by this immunoassay lead to misdiag- nosis, diagnostic delay and unnecessary diagnostic procedures. It has been reported that some patients have undergone inferior petrosal sinus catheterisation and pituitary surgery to explain ACTH elevations.4 Fortunately, when serum ACTH concentra- tion is repeated with the Cobas immunoassay (Roche, Switzer- land) or ACTH AIA (Tosoh, Japan), this showed normal levels, preventing any further invasive procedures.4
Two-site sandwich immunoassays are more sensitive and specific than competitive immunoassays. However, they are also more prone to cross-reactivity interference, as they use two antibodies, which bind to different sites on the antigen.67 These nonhuman antibodies can interfere with human antibodies, which might be autoantibodies (in patients with autoimmune disorders), directed to animal antigens, against antigens from iatrogenic exposure (antibody-targeted therapies or vaccination) or heterophilic (when there is no medical record of exposure to animal immunoglobulins or others known immunogenic anti- gens).6 Heterophilic antibodies, like rheumatoid factor (which can be present in up to 5% of the general population), have an unknown and variable prevalence. These antibodies are polyspe- cific, targeting poorly defined antigens and they can bind in a similar manner to the metabolite of interest, potentially causing inaccurate results.8
When a clinician is faced with an unexpected immunoassay- related laboratory result which is inconsistent with the clinical presentation, they should report the case to the laboratory. They can then assess if there were any pre-analytical issues (such as poor sample collection and storage) or rule out other potential clinical causes for interferences, including hyperbilirubinaemia, haemolysis and lipaemia.67 Regarding pre-analytical conditions, ACTH samples must be collected between 7 and 10a.m., in an EDTA tube and must be transported to the laboratory on ice, promptly centrifugated and analysed or frozen at -20℃ for a maximum of 30 days.’
After exclusion of pre-analytical errors, the ACTH dosing should be repeated with the same method.10 If the problem persists, it is suggested to proceed to serial dilutions or using heterophile blocking agents or precipitation of interfering immu- noglobulins with polyethylene glycol.1º However, in some cases, a normal result can only be obtained by retesting the sample with an alternative method.10
The other two immunoassays available to measure ACTH are ACTH Cobas immunoassay (Roche, Switzerland) or ACTH AIA (Tosoh, Japan), which are good options to retest ACTH, since they are less susceptible to interference.11-14 ACTH Cobas immunoassay uses murine monoclonal antibodies to capture and detect, whereas ACTH AIA (Tosoh, Japan) uses polyclonal anti- bodies from goat origin.13 14 ACTH Immulite uses polyclonal antibodies from rabbit for detection and murine monoclonal capture antibodies, which may explain a greater susceptibility to cross-reaction.12
Liquid chromatography-tandem mass spectrometry (LC-MS/ MS) was proposed in 2019 as a complementary method to measure intact ACTH (iACTH) after immunoprecipitation of biologically active ACTH molecules.15 This method allows the comparison and correlation between serum ACTH concentra- tion measured with an immunoassay and iACTH when discor- dant laboratory results occur.15 It was noted that LC-MS/MS
of iACTH correlated more strongly with ACTH Cobas immu- noassay results than with ACTH Immulite.15 Despite its higher sensitivity, specificity and lower susceptibility to cross-reactions, LC-MS/MS represents a more expensive method and may not always be readily available.
In conclusion, every physician specialist should be aware of the ACTH assay used by their reference laboratory and exercise a high index of suspicion in cases of clinical and laboratorial discordance. It is essential to collaborate closely with the labora- tory personnel to identify and correct any potential interferences.
Learning points
Adrenocorticotropic hormone (ACTH) measurement is performed mainly using immunoassays, which are relatively cheap and quick.
Immunoassays may however be prone to cross-reactions, leading to false-positive and false-negative results that can lead to misdiagnosis.
ACTH Immulite immunoassay (Siemens, Germany) is a widely used immunoassay to measure ACTH with some reported cases of falsely elevated results.
Knowing which immunoassay is used in the reference laboratory is essential, allied to a high index of suspicion when the laboratory results do not match the clinical picture, to avoid proceeding to unnecessary procedures.
Immunoassays ACTH Cobas (Roche, Switzerland) and ACTH AIA (Tosoh, Japan) may be used whenever there are discrepancies between the clinical picture and ACTH measurement. Liquid chromatography-tandem mass spectrometry also has a role in overcoming this problem.
Contributors The following authors were responsible for drafting of the text, sourcing and editing of clinical images, investigation results, drawing original diagrams and algorithms, and critical revision for important intellectual content: MMF, CM, PO and IP. The following authors gave final approval of the manuscript: MMF, CM, PO and IP.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None declared.
Patient consent for publication Consent obtained directly from patient(s). Provenance and peer review Not commissioned; externally peer reviewed.
Case reports provide a valuable learning resource for the scientific community and can indicate areas of interest for future research. They should not be used in isolation to guide treatment choices or public health policy.
ORCID İD Mafalda Martins Ferreira http://orcid.org/0009-0005-1260-5570
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