Introduction

The accurate measurement of parathyroid hormone (PTH) is essential in diagnosing primary and secondary hyperparathyroidism. The majority of patients with primary hyperparathyroidism are hypercalcaemic and have an elevated or non-suppressed PTH. However, a small proportion is normocalcaemic with a raised PTH in the absence of other causes of high PTH. In secondary hyperparathyroidism, an elevated PTH occurs in the presence of low or low/normal calcium [1]. In all these cases, correct evaluation of PTH is crucial for diagnosis. However, PTH measurements can rarely be falsely elevated due to immunoassay interference with heterophile, human anti-animal or other antibodies [2]. This could lead to unnecessary investigations, incorrect diagnosis and avoidance of parathyroid hormone analogue therapies. Here, we report a patient with osteoporosis and spuriously elevated PTH levels due to assay interference, most likely resulting from heterophile antibodies. We also review the literature on previously reported cases of falsely elevated PTH and their causes.

Case presentation

The patient was an 87-year-old woman who was initially seen at our bone health clinic in St James’s Hospital in 2019 and started on denosumab for severe osteoporosis with multiple vertebral fractures (L1, T8, T12). Table 1 illustrates her biochemistry results with PTH measured on the Roche Cobas e801 electrochemiluminescence immunoassay analyser. PTH was found to be consistently elevated (range: 383 to 784 pg/m) between 2019 and 2023 despite normal serum calcium, 25(OH)D (> 50 nmol/l) and eGFR (> 60 ml/min. A falsely elevated PTH was suspected, and a PTH sample in 2023 was subjected to polyethylene glycol (PEG) 6000 precipitation with the result corrected for dilution. Equal volumes of the PEG precipitation reagent and serum were combined, then incubated for 10 min at room temperature and centrifuged, with less than 10% of hormone being recovered resulting in a normal PTH (37 pg/ml). Serial dilutions were also performed, demonstrating linearity and raising the possibility of macro-PTH. To further investigate, serum PTH was tested on a different platform (Atellica Siemens) that returned a normal level of 27 pg/ml. The patient had kept a dog but had no exposure to other animals, no autoimmune disease and had never undergone treatment with animal monoclonal antibodies, suggesting heterophile antibodies as a cause of assay interference. Table 2 refers to previously published reports of falsely elevated PTH.

Table 1 Biochemical results
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Table 2 Case reports of falsely elevated PTH
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Discussion

This case adds to the rare reports of falsely elevated PTH in the literature. Most are due to interference with heterophile or human anti-animal antibodies (HAAA) [3,4,5,6, 8,9,10,11,12,13,14]. These bind to the animal antibodies in the immunoassay that attach to the PTH, resulting in higher reported levels. Specifically, HAAA is a monospecific, high-affinity antibodies (usually IgG, IgA or IgM) against animal immunoglobulins that may result from prior exposure to animals or from treatment with animal monoclonal antibody therapies [18]. Reported prevalence varies widely (up to 11.7%) [2] though interference with assays is rare with human anti-mouse antibodies (HAMA) most commonly implicated [18, 19]. Heterophile antibodies are previously referred to all interfering antibodies, but the term is now limited to those that develop in response to no clear immunogen [18, 19]. They are weakly polyspecific but can react with antibodies from different animal species though generally give less pronounced interference than HAMA [18].

In most cases of assay interference (as in our patient), PTH was elevated to multiple times the upper limit of normal to extremely high (> 5000 pg/ml). However, in one case attributed to heterophile antibodies, there was only a modestly raised PTH (110 pg/ml) [13]. Cavalier et al. in a series of 743 patients with elevated PTH found an interference rate of 4.5% due to HAMA or rheumatoid factor (RF) [20]. However, the effect on PTH levels in many patients was small (mean decline of 29%) when HAMA was removed and 49% (range 0–90%) after removing RF. Overall, in 52.9% of patients’ serum, PTH normalised, though the remaining abnormal results may not necessarily imply a pathological level [2].

Interestingly, spurious elevations in multiple hormones (prolactin, ACTH, FSH, βHCG, IGF-1, TSH and calcitonin) were found in one patient with falsely elevated PTH [8], while there were three cases of a coexisting spuriously high thyroglobulin level [11]. Prior drug therapy resulting in HAMA was implicated in five patients with a falsely elevated PTH. Two had a renal transplant and had received muromonab-CD3 (a murine monoclonal antibody) [5, 6], and three were treated with a natural killer (NK) therapy with exposure to the mouse antibody (NKp46) [11].

Parathyroid disease was present in five patients with falsely high PTH. Two had secondary hyperparathyroidism due to previous renal failure. In one, there was no decline in PTH after remedial parathyroidectomy though levels were normal after removing HAMA [6], while in the other PTH reduced from 3748 to 552 pg/ml on correction [5]. Extremely high PTH (> 2500 pg/ml) in one patient also did not completely correct due to underlying secondary hyperparathyroidism [19]. There was also failure of normalisation of PTH after parathyroidectomy in a case of proven primary hyperparathyroidism which was accounted for by assay interference [9], possibly resulting from monoclonal gammopathy. Finally, in a patient with idiopathic hypoparathyroidism with undetectable levels, a subsequent spuriously high PTH (636 pg/ml) was due to HAAA [3].

Only two cases of macro-PTH are reported where respective levels were 253 and 1846 pg/ml [15, 17]. This occurs when human immunoglobulin or unknown components bind to PTH to form a macro-hormone complex that is metabolically inactive, cannot be excreted by the kidneys and is measured by the assay [9]. While commonly seen with prolactin (9–42%), it is very infrequent with other hormones [17]. As the falsely high PTH is not due to assay interference, in these cases, using different platforms does not significantly change the result [15].

Finally, one patient had spuriously elevated PTH (1900 pg/ml) that was attributed to a high biotin (vitamin B7) intake of 2500 mcg/day that resolved after stopping supplements [16]. This was well above the recommended intake of 30 mcg per day [2, 16] and may cause interference with assays via biotinylated capture antibodies, though in most cases, this results in spuriously low PTH levels [2].

In summary, the finding of unexpectedly high PTH discordant with the clinical picture should raise the possibility of falsely elevated levels. No single test can accurately exclude erroneous results [18, 19]. A first step to consider would be to use PEG precipitation which removes macro-PTH and in the majority of cases interfering antibodies [18]. However, there are no diagnostic cut-offs for post-precipitation recoveries. Typically, a recovery rate of 40% is used for macro-prolactinaemia [8]. In our patient and in other cases, recovery ranged from < 1.4 to 13.5% [8, 10, 12] with assay interference to 10–20% for macro-PTH [17]. Secondly, to determine if the result could be affected by interfering antibodies, a serial dilution test should be performed which generally results in nonlinearity. However, this is not always reliable as we found in our patient and as reported elsewhere [18]. Thirdly, heterophile blocking tubes or addition of animal sera can be used to remove interfering antibodies though this was not available at our laboratory. Finally, doing a repeat test on a different assay (using other animal antibodies or with blocking agents) can confirm assay interference. Notably, the PTH result can also remain high using other platforms [5], reflecting the cross-reactivity of human antibodies.

A potential implication of falsely elevated PTH in normocalcaemic patients is the erroneous diagnosis of normocalcaemic primary hyperparathyroidism (NPHT). In a recent review, Shaker et al. advise that when NPHT is suspected, PTH should be tested on two different assays [1]. Of interest, Hassan et al. found in a normocalcaemic patient a PTH of 110 pg/ml, which was 56 pg/ml when retested on a different platform and was attributed to heterophile antibodies [13]. Importantly, in these and other cases, falsely elevated PTH levels resulted in unnecessary investigations and even surgery [2, 11, 16]. To conclude, PTH values discordant with the clinical picture (even in patients with parathyroid disease) should alert physicians to the possibility of erroneously high levels due to assay interference or macro-PTH. Good communication with testing laboratory to identify and investigate such cases is crucial.