A 68 year-old woman presented to her general practitioner with fatigue and 5kg of weight loss. Her past medical history was significant for a hiatus hernia and hypercholesterolaemia. Her regular medications included rabeprazole and rosuvastatin. She intermittently took cholecalciferol 1000IU/day. Initial investigations revealed a haemoglobin of 87g/L (115-160), corrected calcium of 2.83mmol/L (2.1-2.6), and serum globulins of 61g/L (24-41). Her physical examination was unremarkable except for pallor. Further investigations in hospital showed a parathyroid hormone (PTH) of 4.9pmol/L (2.0-9.5), 25-hydroxy vitamin D (25OHD, Abbott Architect) of >400nmol/L (50-150nmol/L), phosphate of 1.44mmol/L (0.9-1.6), 1,25-dihydroxyvitamin D (1,25(OH)2D) of 152pmol/L (48-190), and an IgM kparaprotein in the gamma region of 44g/L (figure 1). A positron emission tomography/computer tomography scan showed extensive mildly fluorodeoxyglucose (FDG) avid lymphadenopathy above and below the diaphragm, with large FDG avid mass lesions in the pelvis, and widespread bone marrow infiltration. A bone marrow aspirate showed moderate to heavy marrow involvement with a mature lymphoproliferative disorder, consistent with a diagnosis of lymphoplasmacytic lymphoma/Waldenstrom’s macroglobulinaemia (LPL/WM). Bone densitometry was normal.
The 25OHD level was felt to be inconsistent with clinical findings and the possibility of an aberrant result was considered.
Vitamin D results using different assays are summarised in table 1. The 25OHD result of >400 nmol/L was confirmed on repeat analysis using the Abbott Architect instrument. A subsequent 1-in-2 dilution of the sample returned a result of 84nmol/L – highly suggestive of the presence of an interferent. The presence of rheumatoid factor, a known potential interferent, was excluded with a measurement below the limit of quantitation of the assay. Treatment of the sample with antibody blocking reagent (Scantibodies) was not consistent with the presence of heterophile antibody interference. Analysis on a liquid chromatography tandem mass spectrometry (LC-MS/MS) vitamin D method returned a result of 82nmol/L. Measurement of the sample on a Siemens Centaur immunoassay platform produced a result of 75nmol/L, which was in agreement with the LC-MS/MS result. The sample analysed was collected prior to the patient starting chemotherapy and we concluded that the most likely interferent was the presence of the monoclonal IgM kappa paraprotein. Following chemotherapy, and in the setting of normal globulins, the patient’s vitamin D result was 64nmol/L. Falsely elevated vitamin D levels due to assay interference have previously been reported with LPL/WM and myeloma (1).
DISCUSSION
The most striking feature of this patient’s initial investigations was the 25OHD level of >400nmol/L. Possible causes of hypervitaminosis D include prolonged ingestion of large doses of cholecalciferol, deficiency or variants in CYP24A1, and an artefactual result due to assay interference.
Hypervitaminosis D due to excessive oral or intramuscular supplementation is rare. Development of hypercalcaemia due to excessive oral cholecalciferol requires prolonged ingestion of doses in the order of 40-50 000 IU daily for at least six months. A case series from India reported on 15 patients with hypercalcaemia secondary to intramuscular administration of cholecalciferol (2). The shortest period of time to develop hypercalcaemia was five weeks, during which time the patient received 3 000 000 IU of vitamin D. Hypervitaminosis D due to excessive exogenous intake is associated with hypercalcaemia, low PTH, and normal phosphate levels.
CYP24A1 deficiency is a rare cause of an elevated 25OHD and hypercalcaemia. The CYP24A1 gene encodes vitamin D 24 hydroxylase which metabolises both 25OHD and 1,25(OH)2D to inactive metabolites 24,25-dihydroxyvitamin D (24,25(OH)2D) and calcitroic acid (3). Expression of CYP24A1 is usually induced by both hypercalcaemia and 1,25(OH)2D, thereby preventing vitamin D-induced hypercalcaemia. CYP24A1 deficiency was first described in infants but can present at any age. CYP24A1 variants/deficiency are characterised biochemically by hypercalcaemia, hypercalciuria, undetectable PTH and parathyroid-hormone related peptide, low 24,25(OH)2 D3, and elevated 1,25(OH)2 D3. Clinical manifestations include nephrolithiasis and nephrocalcinosis. A definitive diagnosis can be made by genetic testing. Seven cases of hypercalcaemia with onset during pregnancy have been described in women with CYP24A1 variants/deficiency, in the setting of 25OHD 1-alpha hydroxylase expression by the placenta and enzyme upregulation in the maternal kidney(3).
Once pseudo-hypervitaminosis D from paraprotein interference was confirmed, the cause of the hypercalcaemia in this patient needed to be determined. Along with primary hyperparathyroidism (PHPT) and familial hypocalciuric hypercalcaemia (FHH), pseudohypercalcaemia (4)was also considered. Factitious or pseudohypercalcaemia may occur due to hyperalbuminaemia, thrombocytosis, or assay interference by paraproteins. Pseudohypercalcaemia leading to the incorrect diagnosis of PHPT has been reported with LPL/WM, myeloma, monoclonal gammopathy of uncertain significance, mixed cryoglobulinaemia with Sjogren’s syndrome, and markedly elevated IgE levels. Pseudohypercalcaemia may be identified by the demonstration of normal ionised calcium levels. In the case presented, the patient’s ionised calcium was mildly elevated at 1.33mmol/L (1.13-1.30), excluding pseudohypercalcaemia.
While PHPT is usually characterised by an elevated PTH level, 10 to 20% of patients will have an inappropriately “normal” PTH level, making it difficult to distinguish from FHH (5). The prevalence of FHH in the west of Scotland was estimated to be 1 in 78 000, compared with the estimated incidence of PHPT of 50 cases per 100 000 patient years (6,7). FHH is an inherited autosomal dominant condition with almost complete penetrance. The majority of mutations are linked to the gene encoding the calcium-sensing receptor on the long arm of chromosome 3(8). New mutations are relatively rare. Differentiating FHH from asymptomatic PHPT may be difficult because of considerable overlap in urine calcium excretion, serum calcium, serum phosphate, serum magnesium, and PTH between the two disorders. The best investigation to try to distinguish PHPT from FHH is the calcium:creatinine clearance ratio (CCCR). A CCCR of <0.01 is unusual for PHPT, but is seen in 80% of individuals with FHH (8). The CCCR may also be low with vitamin D deficiency and low dietary calcium intake. Pregnancy leads to an elevated CCCR in the setting of physiological hypercalciuria. In cases where results are repeatedly equivocal, testing of first-degree relatives or genetic testing may be useful.
On questioning previous pathology providers, multiple corrected serum calcium levels between 2011 and 2016 had been normal. A 24 hour urine collection demonstrated calcium excretion of 1.8mmol/day, with a CCCR of 0.005. While the patient’s CCCR is low, the previous normal corrected serum calcium results make it most likely the patient has PHPT. In view of the patient’s normal bone density, mild degree of hypercalcaemia, the absence of relatives for testing, and her diagnosis of LPL/WM it was decided not to pursue gene testing to differentiate between the two disorders as it would not change management. On review of the literature, there does not appear to be an association between WM/LPL and PHPT.
In conclusion, this case highlights the potential for immunoassay interference when measuring 25OHD, and the potential for paraproteins to cause interference. The patient’s history and presentation were not in keeping with vitamin D intoxication, which prompted consideration of other causes of an elevated 25OHD level. The presence of an elevated ionised calcium confirmed hypercalcaemia, as pseudohypercalcaemia should be considered in patients with WM. The most likely cause of hypercalcaemia in this patient is PHPT, a condition which is not typically associated with WM/LPL.
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