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Purpose Whether androgens, distinct from estrogen, maintain bone health during male aging has implications for understanding osteoporosis. We assessed associations of different sex hormones with incidence of any bone fracture or hip fracture in older men. Participants and methods Analysis of 3307 community-dwelling men aged 76.8 ± 3.5 years, median follow-up period of 10.6 years. Plasma testosterone (T), dihydrotestosterone (DHT), and estradiol (E2) assayed by mass spectrometry, sex hormone-binding globulin (SHBG), and luteinizing hormone (LH) using immunoassay. Incident fractures determined via data linkage. We analyzed probability of fracture and performed Cox regression adjusted for age, medical comorbidities, and frailty. Results Incident fractures occurred in 330 men, including 144 hip fractures. Probability plots suggested nonlinear relationships between hormones and risk of any fracture and hip fracture, with higher risk at lower and higher plasma T, lower E2, higher SHBG, and higher LH. In fully adjusted models, there was a U-shaped association of plasma T with incidence of any fracture (Quartile 2 [Q2] versus Q1: fully adjusted hazard ratio [HR] = 0.69, 95% confidence interval [CI] 0.51–0.94, P = .020; Q3: HR 0.59, 95% CI 0.42–0.83, P = .002) and hip fracture (Q2 versus Q1: HR 0.60, 95% CI 0.37–0.93, P = .043; Q3: HR 0.52, 95% CI 0.31–0.88, P = .015). DHT, E2, and LH were not associated with fracture. Higher SHBG was associated with hip fracture (Q4 versus Q1: HR 1.76, 95% CI 1.05–2.96, P = .033). Conclusions Midrange plasma T was associated with lower incidence of any fracture and hip fracture, and higher SHBG with increased risk of hip fracture. Circulating androgen rather than estrogen represents a biomarker for hormone effects on bone driving fracture risk.

Osteoporosis resulting in bone fractures is a major cause of morbidity in older men. Previous studies implicated reduced exposure to estradiol (E2) with increased fracture risk in men. The extent to which circulating androgens contribute to maintenance of bone health is uncertain. We examined associations of different sex hormones with incidence of any bone fracture or hip fracture in older men. We analysed 3,307 community-dwelling men aged 76.8±3.5 years, median follow-up period of 10.6 years. Medical information was collected by questionnaire. Frailty was assessed using the FRAIL scale (1). Early morning plasma testosterone (T), dihydrotestosterone (DHT) and E2 were assayed by mass spectrometry, sex hormone-binding globulin (SHBG) and luteinising hormone (LH) by immunoassay. Incidence of any fracture and hip fracture were determined via data linkage to emergency department presentations and hospital admissions. Risk of fracture according to sex hormone concentrations was analysed. Hazard ratio of fracture according to sex hormone quartiles (Q1-4) was assessed using Cox regression models adjusted for age, medical comorbidities and frailty. In 30,355 participant-years of follow-up, the incidence of any fracture was 1.1% and hip fracture 0.5% per participant per year. Incident fractures occurred in 330 men, including 144 hip fractures. Probability plots suggested non-linear relationships between hormones and risk of any fracture and hip fracture, with higher risk at lower and higher concentrations of plasma T, lower E2, higher SHBG and higher LH. In fully-adjusted models, there was a U-shaped association of plasma T with incidence of any fracture (Q1: reference group, Q2: fully-adjusted hazard ratio [HR]=0.69, 95% confidence interval [CI]=0.51-0.94, p=0.020; Q3: HR=0.59, CI=0.42-0.83, p=0.002; Q4: 0.85, CI=0.62-1.18, p=0.335). A similar U-shaped association of T was found with incidence of hip fracture (Q1: HR=1.0; Q2: HR=0.60, CI=0.37-0.93, p=0.043; Q3: HR=0.52, CI=0.31-0.88, p=0.015; Q4: HR=1.04, CI=0.65-1.68, p=0.866). DHT, E2 and LH were not associated with incidence of any fracture or hip fracture (all p>0.050). SHBG was not associated with incidence of any fracture, but was associated with hip fracture (Q4 vs Q1: HR=1.76, CI=1.05-2.96, p=0.033). In conclusion, we found a non-linear or U-shaped association of T with fracture risk, with no association of E2. Mid-range plasma T was associated with lower incidence of any fracture and hip fracture, and higher SHBG with increased risk of hip fracture. Circulating androgen rather than estrogen may be a biomarker for hormone effects on bone driving fracture risk. A randomised controlled trial of T therapy powered for the outcome of fracture may be warranted and should recruit men with baseline T in the lowest quartile of values. Reference: (1) Hyde Z, et al. J Clin Endocrinol Metab 2010; 95: 3165-3172.

Background: Herbal remedies are widely available and whilst portrayed as generally harmless, they may contain a variety of potent medications that can cause unintended and potentially serious consequences. This danger is further amplified as their use is typically inadvertently not divulged to clinicians, such as in this case. Clinical Case: A 65-year-old gentleman was referred to Endocrinology for assessment of adrenal insufficiency (AI) as a cause of his lethargy. He was found to have serum cortisol levels of 38 nmol/L and 68 nmol/L (150 – 700 nmol/L) on two separate mornings. No identified recent use of inhaled, topical, intra-articular or oral exogenous steroid therapy was noted. He had an inadequate response to Synacthen stimulation with peak serum cortisol of 150 pmol/L at 60-minutes (Adequate response – Post-Synacthen cortisol >430 pmol/L). Baseline plasma ACTH was low - 1.1 pmol/L(2.0 – 10 pmol/L) and secondary (pituitary) AI was suspected. He had normal thyroid function and prolactin. The patient subsequently admitted to taking an herbal medication, “Montalin” for a few months. It was obtained over-the-counter in Indonesia for symptomatic relief of “muscle pains”. The tablet underwent analysis to investigate for presence of corticosteroid components. An intact Montalin capsule was partly dissolved and the soluble content was diluted and analysed via LCMS/MS. Presence of dexamethasone/betamethasone was confirmed at an approximate concentration of 69.8 nmol/L constituting 0.548mg and 0.81% of the tablet. At the time, betamethasone and dexamethasone could not be distinguished due to stereoisomerism, but since has been confirmed to be dexamethasone. The patient likely had suppression of the hypothalamic-pituitary-adrenal axis from unintended exogenous corticosteroid use, causing a picture of secondary AI. He requires ongoing corticosteroid replacement pending recovery of endogenous production. Although Montalin is marketed as an herbal over-the-counter medication it was confirmed to be adulterated with a potent corticosteroid. An alert issued by the Singaporean government details two further cases of adverse effects from Montalin and warns against its use. Conclusion: Herbal remedies with undisclosed potent ingredients may unknowingly cause serious adverse effects. With advances in LCMS/MS technology, laboratory analysis of either the tablets or the patient’s serum could be utilised to identify potential exogenous corticosteroid exposure.

SummaryContext Lower testosterone (T) is associated with poorer health outcomes in older men, however, the relationship between T, dihydrotestosterone (DHT) and estradiol (E2) with cardiovascular disease (CVD) in younger to middle-aged men remains unclear. Objectives We assessed associations between endogenous sex hormones with mortality (all-cause and CVD) and CVD events, in a cohort of men aged 17–97 years. Participants and methods Sex hormones were assayed using mass spectrometry in 2143 men from the 1994/5 Busselton Health Survey. Outcomes to December 2010 were analysed. Results Of the 1804 men included in the analysis, mean age was 50·3 ± 16·8 years and 68·9% of men were aged

SummaryObjectives Lower circulating androgens and poorer lung function are associated with increased cardiovascular risk and mortality in men. The association between androgens and lung function is unclear. We tested the hypothesis that circulating testosterone (T) and its metabolites dihydrotestosterone (DHT) and oestradiol (E2) are differentially associated with lung function in men. Methods Early-morning serum T, DHT and E2 were assayed using mass spectrometry in 1768 community-dwelling men from Busselton, Western Australia. Forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) were measured using spirometry. Linear regression models adjusting for age, height, smoking, exercise, body mass index, respiratory conditions and cardiovascular risk factors were used. Results Mean age was 50.1 ± 16·8 years. 16·0% were current smokers, 14·1% reported a history of asthma and 2·7% reported chronic obstructive pulmonary disease. Current smokers had higher T compared with never smokers (age-adjusted mean 14·5 vs 13·5 nmol/l, P = 0·002) and higher E2 (65·3 vs 60·1 pmol/l, P = 0·017). In fully adjusted analyses, T was associated with FEV1 (51 ml per 1 SD increase, P

SummaryObjectives Lower testosterone (T) levels are associated with poorer health outcomes in older men, but associations in younger or middle-aged men are uncertain, and data for dihydrotestosterone (DHT) and oestradiol (E2) are limited. We assessed the associations of circulating T, DHT and E2 with physical and health-related factors in a cohort comprising men aged 17–97 years. Participants and methods Serum from 2143 community-dwelling men from the 1994/95 Busselton Health Survey was assayed for T, DHT and E2 using liquid chromatography–tandem mass spectrometry. Men receiving hormonal therapy or reporting the use of testosterone, or with prostate cancer or orchidectomy were excluded. Results Of the men, 43% had never smoked, 6·1% had diabetes and 16·8% cardiovascular disease (CVD). Mean (±SD) age was 50·3 ± 17·0 years. Total T was moderately correlated with DHT (r = 0·56), E2 (r = 0·35) and sex hormone-binding globulin (r = 0·53). In age-, smoking-, body mass index (BMI)- and sex hormone-binding globulin (SHBG)-adjusted analyses, T was inversely associated with metabolic syndrome score, while DHT and E2 were not associated. In multivariable models, higher total T was associated with lower age, BMI and C-reactive protein, and with higher creatinine and haemoglobin, independently of SHBG. Higher DHT was associated with lower age, BMI and glucose level, and higher creatinine and haemoglobin. E2 was positively associated with age, BMI and haemoglobin. Conclusions In men spanning younger, middle and older ages, circulating androgens are more related to age and metabolic factors than CVD or chronic disease. Further investigation is required to clarify whether androgens and oestrogens have contrasting roles as risk predictors for CVD.

Background: Hereditary hemochromatosis resulting either from homozygosity for the C282Y polymorphism of the HFE gene, or compound heterozygosity for C282Y and H63D, manifests with liver disease and hypogonadism. However, it is unclear whether men who are heterozygotes for C282Y or H63D exhibit subtle abnormalities of sex hormone status. Aims: To evaluate whether heterozygosity for either of the HFE gene polymorphisms C282Y or H63D is associated with circulating testosterone and SHBG in men. Subjects and methods: We performed a cross-sectional analysis of 388 community-dwelling men. Men were genotyped for C282Y and H63D. Sera were analysed for testosterone and SHBG, and insulin resistance was estimated using a homeostatic model (HOMA2-IR). Results: Mean age of men in the cohort was 56.9 yr. Men who were heterozygous for the C282Y polymorphism in the HFE gene had higher SHBG levels than men who did not carry this polymorphism (mean±SE, 38.2±1.64 vs 32.8±0.71 nmol/l, p=0.006). Total and free testosterone levels did not differ in the two groups. In multivariate analysis adjusting for potential confounders including age, waist circumference, testosterone, and HOMA2-IR, C282Y heterozygosity remained associated with SHBG levels (p

Background: SoloSTAR® (SOL; sanofi-aventis, Paris, France) is a prefilled insulin pen device for the injection of insulin glargine and insulin glulisine. This is the first Australian survey to determine its usability, participant acceptance, and safety in clinical practice. Methods: A 3-month, nonrandomized, noncomparative, observational survey in Australia was conducted in individuals with diabetes. Participants were given SOL pens containing glargine, the instruction leaflet, and a toll-free helpline number. Training was offered to all participants. Safety data, including product technical complaints (PTCs), were gathered from ongoing feedback given by the participant or health care professional (HCP) and by independent interviews conducted 6–10 weeks after study start. Results: Some 2674 people consented to take part across 93 sites (150 HCPs), and 2029 participated in interviews. Of these, 52.6% had type 1 diabetes, 16.3% had manual dexterity problems, and 15.5% had poor eyesight not corrected by glasses. At the time of interview, 96.8% of participants were still using SOL. None of the eight PTCs reported were due to technical defects; most were related to handling errors. Some 62 participants reported 77 adverse events; none were related to a PTC. The vast majority of participants (95.4%) were “very satisfied” or “satisfied” with using SOL, and 89.7% of the participants had no questions or concerns using SOL on a daily basis. Similar positive findings were reported by participants with manual or dexterity impairments. Conclusions: In this survey of everyday clinical practice, SOL had a good safety profile and was very well accepted by participants.

BACKGROUND The development of prostate carcinoma is androgen-dependent. The coding sequence of the androgen receptor (AR) gene contains a CAG repeat polymorphism that has been shown to influence AR activity in vitro. Studies of this polymorphism as a prostate carcinoma risk factor have been conflicting. METHODS A matched case–control design was used in a clinic-based multicenter study of Australian prostate carcinoma subjects. Cancer subjects were matched by age and locality with controls, all of whom had a serum prostate specific antigen (PSA) level of less than 4 mg/L. Conditional logistic regression was used to determine the relative risk of prostate carcinoma dependent on AR gene CAG number. The association of disease characteristics at diagnosis with the polymorphism also was assessed. RESULTS Five hundred forty-five cases of prostate carcinoma and 456 matched case–control pairs were recruited. Association studies of disease characteristics at diagnosis showed age at diagnosis to be associated with AR CAG number by univariate (P = 0.004) and multivariate (adjusting for PSA, stage, and grade) linear regression (P = 0.018). No association was observed between the polymorphism and disease stage (TNM-based categories; P = 0.277), histologic grade (P = 0.41), or PSA level at diagnosis (P = 0.48). In the pairwise case–control analysis, the odds ratio of prostate carcinoma for a change of 5 CAG repeats gave an odds ratio of 0.9821 (95% confidence interval, 0.84–1.15). CONCLUSIONS In this Australian study population, the AR CAG repeat polymorphism was not a risk factor for prostate carcinoma, but a shorter repeat sequence was associated with earlier age at diagnosis. Cancer 2001;92:941–9. © 2001 American Cancer Society.

The action of androgens is essential for the development of benign prostatic hyperplasia and carcinoma of the prostate. The androgen receptor is a ligand-dependent nuclear transcription factor. The transcriptional activation domain of the androgen receptor gene contains a polymorphic CAG repeat sequence. A shorter CAG repeat sequence within the normal range has been reported to be associated with increased risk of prostate cancer and symptomatic benign prostatic hyperplasia. Here, we examine the in vitro transcriptional activity of the androgen receptor (AR) with different numbers of CAG repeats within the normal range in a number of different cell lines of prostatic (LNCaP, PC3) and non-prostatic (COS-1, MCF7) origin. We utilize a luciferase reporter driven by the rat probasin promoter (-286/+28) containing two androgen receptor binding sites. Transcriptional activation of the androgen responsive reporter was observed to be greater with the AR containing 15 vs 31 CAG repeats in COS-1 cells (123.2+/-16.6 vs 78.2+/-10.9, P value 0.01) and the well differentiated prostate cancer cell line LNCaP (103.4+/-17.7 vs 81.4+/-7.7, P value 0.045). No difference was observed in the poorly differentiated prostate cancer cell line, PC3 (106.9+/-21.9 vs 109. 6+/-21.4, P>0.5) or the breast cancer cell line MCF7 (120.4+/-39.4 vs 103.1+/-23.1, P value >0.5). Dose-response experiments with varying quantities of ligand (0.01, 0.1, 1 and 10 nM dihydrotestosterone) or AR cDNA did not demonstrate significant differences in transactivation of the androgen responsive reporter in PC3 cells by the different AR constructs. This suggests that the lack of influence of CAG number in this prostatic cell line is not related to dose of ligand or quantity of androgen receptor. Western immunoblot analysis of androgen receptor protein in transiently transfected COS-1 cells did not demonstrate a difference in the expression of the androgen receptor protein with different numbers of CAG repeats following incubation in the presence or absence of androgen. Gel shift assay did not demonstrate increased DNA binding by androgen receptor with a shorter CAG repeat sequence. These experiments using a relatively androgen- and prostate-specific reporter provide evidence for an inverse relationship between androgen receptor transcriptional activity and the number of CAG repeats in the transcriptional activation domain. The effect of CAG repeat number was cell specific suggesting the involvement of accessory factors expressed differentially between different cell lines.

Context Heterophilic antibodies are a well-described interferent but poorly appreciated and are often not a recognized problem affecting most immunoassays. We describe for the first time heterophilic antibodies interference affecting an adrenocorticotropic hormone (ACTH) assay in a patient with Cushing’s syndrome due to bilateral nodular adrenal hyperplasia. Case A 60-year-old retired female nurse underwent extensive invasive investigations, which were ultimately unnecessary, as a result of initial analytical interference in the ACTH assay, which could not be resolved using a proprietary heterophilic binding reagent. Results This case highlights the inherent difficulty of diagnosing Cushing’s syndrome and the large emphasis placed on laboratory tests. The consequence of not initially identifying interference in this patient’s laboratory test results led to unnecessary and costly investigations with potentially adverse outcomes. Conclusions Clinicians and the laboratory community need to be continuously vigilant and view laboratory results with caution when they are inconsistent with the clinical picture. This approach is paramount, especially at a time of increasing automation and ever-diminishing scientist involvement in sample processing.

Background : Microalbuminuria has been shown to be associated with cardiovascular mortality in type 2 diabetic subjects. It is unclear to what extent this is due to the increased prevalence of other cardiac risk factors. Aims : To examine the relationship of urine albumin excretion to cardiovascular mortality and to determine its status as an independent risk factor. Methods : In a prospective longitudinal study from 1986-1993 we followed 666 type 2 diabetic subjects from a diabetes outpatient service. Cardiovascular risk factors including urine albumin concentration were measured at study entry. Cox proportional hazards regression was used to determine risk factors for mortality. The hazard ratios of microalbuminuria and macroalbuminuria for all cause, cardiovascular and coronary heart disease mortality were determined after accounting for other cardiac risk factors including blood pressure, glycated haemoglobin, total cholesterol, HDL cholesterol, triglycerides, urea, smoking, body mass index, patient age and disease duration. Results : The prevalence of urine albumin of 30-300 mg/L at study entry was 31.7%. A total of 167 deaths occurred (80 from cardiovascular disease). Mortality hazard ratios in subjects with urine albumin of 30-300 mg/L as compared to

Background: Lower testosterone levels are associated with anaemia in older men and women. The relation between testosterone and haemoglobin (Hb) in younger and middle-aged men is less well defined. The aim of the study was to examine the association between testosterone and Hb levels in men spanning middle to older ages. Methods: A cross-sectional analysis of 492 men aged 30.7–94.5 years from the Busselton Health Survey, Western Australia, was carried out. Haemoglobin (Hb), early-morning serum total testosterone and sex hormone-binding globulin (SHBG) were measured. Free testosterone was calculated using mass action equations. Results: Haemoglobin correlated to total and free testosterone concentrations (r= 0.13, P= 0.003 and r= 0.20, P < 0.001, respectively). Hb and SHBG were inversely correlated (r=−0.14, P= 0.001). Hb increased across lowest to highest quartiles of total testosterone (P= 0.02) and free testosterone (P < 0.001), but not SHBG. After adjusting for age, waist circumference, smoking status, alcohol consumption, renal function and ferritin, total testosterone was associated with Hb (β= 0.037, P= 0.003) as was free testosterone (β= 2.32, P < 0.001), whereas SHBG was not associated. Conclusion: Testosterone concentration modulates Hb levels in community-dwelling men across a wide age range. Further studies are needed to clarify implications of this association between testosterone and Hb in men.

Background: Cross-sectional studies from different populations show a variable decline in blood testosterone concentrations as men age. Few population representative cohorts have been followed up over time. Objective: The objective of the study was to quantify longitudinally the change in serum testosterone and SHBG concentrations with age in two well-defined, representative but geographically widely separated regional Australian cohorts. Subjects and Setting: The Busselton cohort comprises individuals aged 18–90 yr residing in Western Australia assessed prospectively since 1981. Sera were assayed from 910 men, from whom further samples were available 14 yr later in 480. The Dubbo cohort involves individuals aged 61–90 yr living in Eastern Australia. Baseline sera were collected from 610 men and additional sera on a second (n = 370) and third (n = 200) occasion from 1989 to 2004. Men from both cohorts are community dwelling and of predominately European origin. Results: Longitudinal analyses show the following: 1) total testosterone declines comparably (P > 0.9) by 1.3% (Busselton) and 0.9% (Dubbo) per annum with the same rates of decline when analyses were restricted to men older than 60 yr of age; 2) annual changes in SHBG were also very similar in age-restricted analyses (2.3% vs. 2.5%, P = 0.48); and 3) the annual increase in SHBG was steeper in middle-aged and older men (P < 10−3vs. young men). These longitudinal changes were all up to 4-fold greater in magnitude, compared with cross-sectional analyses of baseline data. Conclusion: In two separate regional Australian populations, blood testosterone fell and SHBG increased comparably with age. Age-related changes in blood testosterone and SHBG previously described in urban-dwelling men are the same in men who reside in smaller regional cities of another continent.

We have investigated the possible link between androgen hyposensitivity caused by long androgen receptor (AR) CAG repeats, and breast carcinogenesis, in men. AR gene mutations have been described in men with androgen insensitivity syndrome and breast carcinoma, and some studies have shown long CAG repeats are associated with increased risk of breast cancer in women. DNA was isolated from male breast cancer biopsies, and the AR CAG repeat sized. Forty one male breast cancer samples were studied, including one sample from a man with spinal and bulbar muscular atrophy (SBMA), which is caused by an AR CAG repeat expansion. The man with breast cancer and SBMA had 49 CAG repeats (normal range 6-35), but all other breast cancer samples had repeats within the normal range. The frequency of CAG repeatsor =24 was significantly higher in the breast cancer group (excluding the SBMA subject) than in the normal population (p0.05), and was more marked in grade I and II tumors (p=0.001). There was no correlation between AR CAG repeat length and age at diagnosis. In conclusion, longer AR CAG repeats are more common in men with breast cancer than in the control male population. Androgen hyposensitivity, caused by long AR CAG repeats, may increase the risk of breast cancer in men.

Kennedy's disease (spinobulbar muscular atrophy) is an X-linked form of motor neuron disease affecting adult males carrying a CAG trinucleotide repeat expansion within the androgen receptor gene. While expression of Kennedy's disease is thought to be confined to males carrying the causative mutation, subclinical manifestations have been reported in a few female carriers of the disease. The reasons that females are protected from the disease are not clear, especially given that all other diseases caused by CAG expansions display dominant expression. In the current study, we report the identification of a heterozygote female carrying the Kennedy's disease mutation who was clinically diagnosed with motor neuron disease. We describe analysis of CAG repeat number in this individual as well as 33 relatives within the pedigree, including two male carriers of the Kennedy's mutation. The female heterozygote carried one expanded allele of the androgen receptor gene with CAG repeats numbering in the Kennedy's disease range (44 CAGs), with the normal allele numbering in the upper-normal range (28 CAGs). The subject has two sons, one of whom carries the mutant allele of the gene and has been clinically diagnosed with Kennedy's disease, whilst the other son carries the second allele of the gene with CAGs numbering in the upper normal range and displays a normal phenotype. This coexistence of motor neuron disease and the presence of one expanded allele and one allele at the upper limit of the normal range may be a coincidence. However, we hypothesize that the expression of the Kennedy's disease mutation combined with a second allele with a large but normal CAG repeat sequence may have contributed to the motor neuron degeneration displayed in the heterozygote female and discuss the possible reasons for phenotypic expression in particular individuals.

Introduction Since the description of the first immunoassay in 1959 by Nobel Laureates Berson and Yalow, this technology has matured and taken on a major role in clinical laboratories. However it has been plagued by a fundamental weakness. The use of non-human antibodies can produce erroneous results with serious clinical consequences if one does not maintain constant vigilance. We describe a patient who underwent extensive investigations for the cause of her Cushing’s syndrome as a consequence of an erroneous laboratory result. Case A 60-year-old female was found to have incidental bilateral adrenal gland enlargement on abdominal CT scan. She was subsequently noted to have classic symptoms and signs of Cushing’s syndrome including weight gain, increased bruising and increased roundness of her face. A bone mineral density scan confirmed severe osteoporosis. Initial biochemistry was as follows: 24 h urine free corticoids, 2100 nmol/d (reference Scantibodies tubes have specific binders which inactivate hetero-philic antibodies. The use of Scantibodies led to a misleading increase in the ACTH value to 21.1 mol/L. Measurements of ACTH by the alternate method lead to the correct ACTH result of Conclusions We have described a patient with ACTH independent Cushing’s syndrome due to bilateral adrenal hyperplasia who underwent extensive investigation as a result of heterophile antibody interference in the ACTH assay with misleading interaction with Scantibodies.