Case Report Annals of Clinical Biochemistry 2014, Vol. 51(4) 490–494 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0004563214520751 acb.sagepub.com

A possible analytical and clinical role of endogenous antibodies causing discrepant adrenocorticotropic hormone measurement in a case of ectopic Cushing’s syndrome Leonard Saiegh1, Majed Odeh2, Limor Chen-Konak1, Nizar Elias2, Mohammad Sheikh-Ahmad1, Maria Reut1, Gleb Slobodin2, Jacob Bejar3 and Carmela Shechner1

Abstract Heterophilic antibodies are well described, but poorly appreciated interferents and is often not a recognized problem affecting most immunoassays. We report a patient presented with ectopic Cushing’s syndrome (CS), but repeated plasma adrenocorticotropic hormone (ACTH) concentrations conducted by immunoassay were inappropriately within the reference range and not elevated, most probably as a result of antibody interference. A 36-year-old woman, presented with large gastric neuroendocrine carcinoma and severe ectopic CS, while repeated plasma ACTH concentrations conducted by immunoassay were inappropriately within the reference range. As we expected ACTH concentration to be higher, we performed several tests to evaluate whether there was any assay interference causing falsely lower than expected ACTH results. We measured ACTH using a different immunoassay, assayed the sample in dilution, assayed the sample after being incubated in heterophilic antibody blocking agent tube and performed recovery studies. Tests indicated the presence of interfering compounds, most probably heterophilic antibodies. When clinicians find ACTH concentrations to be lower than expected, we recommend the laboratory investigate antibody interference.

Keywords Adrenocorticotropic hormone, antibody, false, Cushing’s, immunoassay Accepted: 30th December 2013

Introduction Endogenous antibodies (Abs) are a heterogeneous and unpredictable mix of immunoglobulin of different classes (IgG, IgM, IgA, etc.), with vastly different affinities to various antigens.1 In some cases, these endogenous Abs are known to cause analytical interference in immunoassays leading to falsely high or falsely low results.1 In this report we describe a unique case of inappropriately normal, not elevated adrenocorticotropic hormone (ACTH) concentrations in ectopic

1

Endocrinology Department, Bnai-Zion Medical Center, Haifa, Israel Department of Internal Medicine A, Bnai-Zion Medical Center, Haifa, Israel 3 Pathology Department, Bnai-Zion Medical Center, Haifa, Israel 2

Corresponding author: Leonard Saiegh, Endocrinology Department, Bnai-Zion Medical Center, 47 Golomb St., P.O.B 4940, Haifa 31048, Israel. Email: [email protected]

Saiegh et al. Cushing’s syndrome (CS). The cause of inappropriately normal ACTH concentrations seems to be attributed to interfering compounds, most probably to heterophilic Abs.

Case report A 36-year-old previously healthy woman, a mother of three children, with no history of blood transfusion, was admitted to hospital with a 2 months history of weight gain, face and legs swelling, hirsutism, skin hyperpigmentation and abdominal pain. A computed tomography scan carried out before admission revealed a large gastric mass with retroperitoneal involvement. Physical examination upon admission revealed central obesity, face swelling, mild pitting oedema of legs, hirsutism, mild diffused skin hyperpigmentation and normal blood pressure. Other physical findings were unremarkable. Laboratory studies revealed normal glucose concentrations, severe hypokalaemic metabolic alkalosis, serum potassium was 2.2 meq/L (reference range 3.5–5.1 meq/L), venous pH was 7.51 (reference range 7.34–7.42), serum bicarbonate was 35.8 meq/L (reference range 21–24 meq/L), normal kidney and liver function tests, negative rheumatoid factor and anti-nuclear Ab. Urinary free cortisol was 14,158 nmol/day (reference range up to 317 nmol/day), serum cortisol taken at 8:00 was 2870 nmol/L (reference range 138–690 nmol/L) and serum cortisol taken at 8:00 after dexamethasone 8 mg given midnight was 2456 nmol/L. Morning plasma ACTH concentration was 6.85 pmol/L (reference range 1.1–10 pmol/L) with no increment after corticotropin releasing hormone stimulation. Gastroscopy revealed large polypoid gastric mass, and endoscopic biopsy specimen was consistent with neuroendocrine carcinoma (NEC) with proliferation index (KI 67) of 10–15%, stained positive for synaptophysin, chromogranin and ACTH. Given the profile above, it was apparent that the patient had suffered from ectopic CS caused by large gastric NEC. Ectopic ACTH-dependent CS caused by indolent tumours, usually bronchial carcinoids, may present with reference range ACTH concentration.2,3 Yet, not elevated plasma ACTH concentration in severe ectopic CS caused by large gastric NEC as in our case is not a typical finding.2,3 Therefore, the possibility of false results due to immunoassay interference has been investigated.

491 analyser Immulite 2000 (Siemens). The method’s sensitivity was 1.1 pmol/L and reference range was 1.1– 10 pmol/L. Intra- and interassay coefficients of variations were 8.7 and 10.0%, respectively. In the reference laboratory, using a different ACTH immunoassay, plasma ACTH was measured by a two-site immunoradiometric assay that measures ACTH (1–39), (reference range up to 22 pmol/L).4 ACTH precursors were measured separately by two-site immunoradiometric assay that measures pro-opiomelanocortin (POMC) and proACTH, whereas other peptides derived from POMC, such as N-terminal POMC, ACTH and -lipotropic hormone, are not recognized.5 Heterophilic antibody blocking agent tubes (HBT) were purchased from David Cantor, Office of Development, Scantibodies Laboratories Inc., San Diego, CA. Plasma samples (0.5 mL) were incubated in HBTs for 60 min at 4 C and were then assayed in the same manner (with no dilution) as untreated plasma in the usual chemiluminescent immunoassay for ACTH in our laboratory. Recovery studies were performed by adding to the patient’s sample a high concentrated ACTH control solution (Immulite ACTH Control Module), with ACTH concentration of 79 pmol/L. We predicted that in this admixture the measured ACTH concentration would be significantly lower than expected and, this would most probably, be an indication for the existence of interfering agents.

Results Several measures were taken in order to verify whether the inappropriately normal and not elevated ACTH concentration was a false result. Plasma ACTH precursor concentration was not found to be elevated 78 pmol/L (reference range up to 100 pmol/L). Then the following tests were performed to evaluate whether there was any assay interference. 1. Plasma ACTH concentration measured by a different immunoassay was elevated 36 pmol/L (reference range up to 22 pmol/L); 2. The plasma was assayed in dilution (Table 1); 3. When the patient’s sample was incubated in HBT, ACTH concentration was higher 33 pmol/L (reference range for ACTH 1.1–10 pmol/L); 4. Recovery studies were performed (Table 2).

Methods

Discussion

In our laboratory plasma ACTH concentrations were assayed by solid-phase two-site sequential enzyme chemiluminescent immunoassay using an automated

Approximately 20% of ectopic ACTH-dependent CS is caused by indolent tumours, usually bronchial carcinoids, which may present with not elevated ACTH

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Annals of Clinical Biochemistry 51(4)

Table 1. Assaying sample in dilution. Patient’s sample

Not diluted

1:10 Diluted and multiplied by 10

1:20 Diluted multiplied by 20

1:100 Diluted multiplied by 100

Number 1 ACTH concentration (pmol/L) Number 2 ACTH concentration (pmol/L)

5.3 7.5

49 36

67 63

ND ND

ND: not detectable. In both samples taken from our patient, number 1 and 2, when plasma was diluted and then multiplied, ACTH concentrations were higher, indicating the presence of interfering substance that had a weaker impact when diluted. Dilution 1/100 gave no detectable result, most probably because ACTH concentration was below kit limits even though the interfering substance was highly diluted (reference range for ACTH 1.1–10 pmol/L).

Table 2. Recovery studies.

Tube No.

Expected ACTH concentration (pmol/L)

Measured ACTH concentration (pmol/L)

1a 2b 3 4c 5

– 4 5.3a þ 4b ¼ 9.3 – 5.2c þ 4b ¼ 9.2

5.3 4 6.5 5.2 8.8

a

Our patient’s plasma. Known concentrated ACTH solution. c Healthy subject’s plasma. In tube no. 1, we measured ACTH concentration in our patient’s plasma. In tube no. 2, we measured ACTH concentration in the known concentrated ACTH solution (measured result was similar to the calculated expected). In tube no. 3, we performed the spiking of the sample from our patient with the known concentrated ACTH solution, and we showed only (6.5/9.3) 70% recovery (i.e. only 70% of the expected ACTH concentration was measured, demonstrating interfering agents in the sample that gave 30% lower concentration). In tube no. 4, we measured plasma ACTH concentration in a healthy subject, and in tube no. 5, we performed the spiking of sample from a healthy subject with the same known concentrated ACTH solution. In comparison to tube no. 3, this spiking showed (8.8/9.2) 96% recovery (i.e. the result was very similar to the expected, showing no interfering substance). b

concentrations, and may yield misleading results in dynamic tests.2,3 Lower than expected and not elevated plasma ACTH concentration is not a typical finding in severe ectopic CS caused by large gastric NEC. In such cases, one might suspect that the non-elevated ACTH concentration is a result of defective tumour ACTH processing, and that the severe clinical and biochemical CS is due to the presence of high concentrations of biologically active ACTH precursors.6 However, similar to cases with other hormone concentration determination by immunoassay methods, the possibility of false results due to antibody (Ab) interference should always be considered. Interference in immunoassays by Abs is a wellknown phenomenon. Interfering Abs can include heterophilic Abs (non-specific Abs), anti-animal Abs

(specific Abs) or auto-Abs formed against one of the body’s own components and often associated with an autoimmune disease.7 Heterophilic Abs are Abs produced against poorly defined antigens and are generally weak Abs with multispecific activities.8 These heterophilic Abs may be associated with autoimmune, as well as other inflammatory diseases. Yet, they are also present, albeit to a lesser extent, in apparently healthy people.9 Rheumatoid factor is an example of a heterophilic Ab.9 Assay interference caused by heterophilic Abs may occur in both competitive and non-competitive assays, yet the latter is more common, and interference may be the result of a number of mechanisms causing either falsely high or low values.10 Some studies have shown that interfering endogenous Abs are present in up to 30– 40% of patients’ samples,11 but other estimates vary widely.12,13 One report, however, suggests that interfering Abs of various types only produce clinically discrepant results in about 0.5% of immunoassays,14 although others have reported a higher15,16 or a lower incidence.17 Even with such low prevalence, the significance of interference must be appreciated given the millions of immunoassay tests that are performed every year. The first step for eliminating interference from an immunoassay is recognizing its existence. Most commonly this occurs through inconsistency with the clinical profile, and testing is required in order to obtain evidence supporting immunoassay interference. Due to the heterogeneity of different interfering Abs, no single routine test is available to identify heterophilic Abs. Not elevated ACTH concentration in severe ectopic CS due to large NEC, as in our patient, is not a typical finding. Thus, several biochemical tests were conducted in order to verify whether the observed lower than expected plasma ACTH concentration was a false result. First, as it is well known that inadequate blood collection and storage can affect ACTH concentration, we excluded any technical problems. Second, because falsely low ACTH concentration in ectopic gastric NEC due to high concentrations of ACTH precursors has been previously reported,18 we measured ACTH precursor concentration and found it to be within the reference range.

Saiegh et al. Next, several biochemical tests were conducted in order to identify whether there was any assay Ab interference that can explain the inappropriately normal ACTH results. As it is known, the exact effect of Ab interference depends on the type of assay.1 In our patient, when we used an alternative different method, we measured a higher plasma ACTH concentration. Moreover, when the patient’s plasma was diluted we achieved higher plasma ACTH concentrations. When ACTH concentration was measured in the patient’s plasma spiked with a known ACTH concentration solution, a significantly lower ACTH concentration was measured (30% than expected). HBT blocking reagent is a unique formulation of immunoglobulins targeted against heterophilic antibodies to neutralize their interference in immunoassays. HBT was used to neutralize heterophilic Abs that are sometimes known to yield falsely low values of hormones measured by immunoassay method.1 Higher plasma ACTH concentrations indicated the existence of interfering Abs, most probably heterophilic Abs, that were most probably inactivated by HBT. Whilst we cannot exclude the possibility that the inference was due to something other than heterophilic antibodies, the fact that the use of HBT altered the results indicates to us that heterophilic antibody-based interference is most likely. Very recently, a case of ACTH independent bilateral nodular adrenal hyperplasia has been described, in which heterophilic Abs resulted in falsely measurable and higher than expected ACTH concentration. In that case the HBT failed to detect heterophilic Ab interference, but using alternative ACTH immunoassay method led to the correct diagnosis.19 Usually, agreement in more than one of the abovementioned tests does not exclude interference, though the likelihood is significantly reduced. However, based on disagreement in one or more such tests, the possibility of interference can be inferred.14 In our case, we had disagreement among all tests we used, which indicated with high certainty the existence of interfering substances, which are most likely heterophilic antibodies. It is known that ACTH fragments like ACTH (1–24) can bind to one of the two antibodies in the sandwich immunometric assay and falsely lower ACTH concentrations.20 So, it is possible that the tumour was releasing fragments of ACTH and that should also be considered. It also should be noted that the insuppressible and massively elevated serum and urine cortisol concentrations associated with consistently modest ACTH concentrations should raise the possibility of an ‘idiotypic immune response’.21 In this phenomenon, known also as the ‘idiotypic network’, an endogenous

493 immunoglobulin antibody can occur against an antigen such as ACTH or any of its related compounds. The antibody produced in the first instant can itself be antigenic, eliciting a secondary immune response producing ‘anti-idiotypic’ antibody, which in some cases can mimic in vivo the action of the antigen, i.e. receptor ACTH antibody, without being necessarily measurable by immunoassays. The simultaneous presence of circulating antibodies which can bind the antigen, i.e. ACTH, as well as anti-idiotypic antibody which can mimic its action, i.e. ‘mimetic antibody’, is a known immunological phenomenon called ‘double whammy effect’.22 It is not therefore inconceivable that two or even more endogenous antibodies can occur in the same patient, some of which can bind the antigen causing analytical interference while another (receptor antibody) simultaneously exerting a pathological role in its own right. In our case, this ‘double whammy effect’ could be also considered, but as the ACTH concentration after excluding heterophilic antibodies by HBT was elevated and could explain the very high cortisol concentrations, the sole effect of analytical interference was the most probable. When suspicion of immunoassay interference is encountered, it is also mandatory to rule out the existence of high molecular mass immunoreactivity, and investigation should include polyethylene glycol precipitation (PEG) or gel filtration immunosubtraction study.23 Although in our case this kind of interference was not highly suspected, in order to rule that out, we intended to perform PEG or gel filtration immunosubtraction study tests, but unfortunately these tests were not performed due to technical issues.

Summary To the best of our knowledge, this is the first case that describes falsely lower ACTH concentration through an immunoassay interfering agent, most probably heterophilic antibodies. Therefore, when clinicians suspect an ectopic CS but find ACTH results to be lower than expected, we recommend ruling out antibody interference. We suggest using one or two screening tests, starting with multiple dilutions or using HBT, as these tests are available and not costly. If there is disagreement with at least one of these tests, we recommend complementation with other screening tests mentioned earlier. Conflict of interests None declared.

Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

494 Ethical approval Patient consent was not obtained as the patient passed away. No ethical approval required as all tests performed were part of routine investigation and management.

Guarantor LS.

Contributorship LS wrote the first draft of the manuscript, LC analysed the samples and data, JB analysed the pathological specimens, MO, MS and CS conceived the study. All authors reviewed and edited the manuscript and approved the final version of it.

References 1. Jones AM and Honour JW. Unusual results from immunoassays and the role of the clinical endocrinologist. Clin Endocrinol (Oxf) 2006; 64: 234–244. 2. Limper AH, Carpenter PC, Scheithauer B, et al. The Cushing syndrome induced by bronchial carcinoid tumors. Ann Intern Med 1992; 117: 209–214. 3. Odell WD. Bronchial and thymic carcinoids and the ectopic ACTH syndrome. Ann Thorac Surg 1990; 50: 5–6. 4. White A, Smith H, Hoadley M, et al. Clinical evaluation of a two-site immunoradiometric assay for adrenocorticotrophin in unextracted human plasma using monoclonal antibodies. Clin Endocrinol (Oxf) 1987; 26: 41–51. 5. Crosby SR, Stewart MF, Ratcliffe JG, et al. Direct measurement of the precursors of adrenocorticotropin in human plasma by two-site immunoradiometricassay. J Clin Endocrinol Metab 1988; 67: 1272–1277. 6. White A and Gibson S. ACTH precursors: Biological significance and clinical relevance. Clin Endocrinol (Oxf) 1998; 48: 251–255. 7. Levinson SS. Antibody multispecificity in immunoassay interference. Clin Biochem 1992; 25: 77–87. 8. Kaplan IV and Levinson SS. When is a heterophile antibody not a heterophile antibody? When it is an antibody against a specific immunogen. Clin Chem 1999; 45: 616–618. 9. Selby C. Interference in immunoassay. Ann Clin Biochem 1999; 36: 704–721. 10. Kricka LJ. Human anti-animal antibody interferences in immunological assays. Clin Chem 1999; 45: 942–956.

Annals of Clinical Biochemistry 51(4) 11. Boscato LM and Stuart MC. Incidence and specificity of interference in two-site immunoassays. Clin Chem 1986; 32: 1491–1495. 12. Thompson RJ, Jackson AP and Langlois N. Circulating antibodies to mouse monoclonal immunoglobulins in normal subjects – incidence, species specificity, and effects on a two-site assay for creatine kinase-MB isoenzyme. Clin Chem 1986; 32: 476–481. 13. Bjerner J, Nustad K, Norum LF, et al. Immunometric assay interference: Incidence and prevention. Clin Chem 2002; 48: 613–621. 14. Ismail AA, Walker PL, Barth JH, et al. Wrong biochemistry results: Two case reports and observational study in 5310 patients on potentially misleading thyroid stimulating hormone and gonadotropin immunoassay results. Clin Chem 2002; 48: 2023–2029. 15. Marks V. False-positive immunoassay results: A multicenter survey of erroneous immunoassay results from assays of 74 analytes in 10 donors from 66 laboratories in seven countries. Clin Chem 2002; 48: 2008–2016. 16. Ward G, McKinnon L, Badrick T, et al. Heterophilic antibodies remain a problem for the immunoassay laboratory. Am J Clin Pathol 1997; 108: 417–421. 17. Levinson SS and Miller JJ. Towards a better understanding of heterophile (and the like) antibody interference with modern immunoassays. Clin Chim Acta 2002; 325: 1–15. 18. Tsuchiya K, Minami I, Tateno T, et al. Malignant gastric carcinoid causing ectopic ACTH syndrome: Discrepancy of plasma ACTH levels measured by different immunoradiometric assays. Endocr J 2005; 52: 743–750. 19. Grasko J, Willliams R, Beilin J, et al. A diagnostic conundrum: Heterophilic antibody interference in an adrenocorticotropic hormone immunoassay not detectable using a proprietary heterophile blocking reagent. Ann Clin Biochem 2013; 50: 433–437. 20. Raff H, Findling JW and Wong J. Short loop adrenocorticotropin (ACTH) feedback after ACTH-(1-24) injection in man is an artifact of the immunoradiometric assay. J Clin Endocrinol Metab 1989; 69: 678–680. 21. Ismail AA, Walker PL, Cawood ML, et al. Interference in immunoassay is an underestimated problem. Ann Clin Biochem 2002; 39: 366–373. 22. Ismail AA. The double whammy of endogenous insulin antibodies in non-diabetic subjects. Clin Chem Lab Med 2008; 46: 153–156. 23. Ismail AA. Testing for insulin antibodies is mandatory in the differential diagnosis of hypoglycaemia in nondiabetic subjects. Clin Endocrinol (Oxf) 2012; 76: 603–604.

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