Laboratory Identification of Pseudohypercalcemia Induced by Hyperglobulinemia in Multiple Myeloma: A Case Report

ABSTRACT

Background

Pseudohypercalcemia is easily misdiagnosed in clinical practice, potentially leading to unnecessary interventions. Multiple myeloma (MM) presenting with concurrent hypergammaglobulinemia represents a rare etiology of pseudohypercalcemia, and there are few reports of systematic laboratory validation of this pathological entity.

Methods

We report a patient with MM who presented with markedly elevated serum total calcium on routine biochemical testing. The hypercalcemia persisted despite 1 month of calcitonin therapy administered at an external hospital. After admission, the clinical laboratory team performed a panel of confirmatory assays, including direct ionized calcium measurement, post‐dilution linearity evaluation, inductively coupled plasma mass spectrometry (ICP‐MS) verification, and polyethylene glycol (PEG) precipitation testing.

Results and Conclusions

The results confirmed that the elevation in serum total calcium was attributed to enhanced calcium binding to circulating globulins, secondary to hypergammaglobulinemia, rather than analytical interference in the detection system. During clinical follow‐up, serum total calcium levels normalized progressively in parallel with the decline in globulin levels following targeted anti‐myeloma therapy. This case demonstrates that clinicians and laboratory professionals should maintain a high index of suspicion for pseudohypercalcemia when encountering patients with marked paraproteinemia or abnormal circulating macromolecules. A multimodal differential diagnostic approach using complementary laboratory indicators is warranted to avoid unnecessary examinations and interventions, thereby reducing the waste of medical resources and preventing potential adverse events associated with inappropriate treatment.

Pseudohypercalcemia induced by calcium‐binding monoclonal immunoglobulin in multiple myeloma is verified via laboratory assays and validated by therapeutic response.

Abbreviations

25‐OH‐VD

25‐hydroxyvitamin D

Ca

ionized calcium

Ca

total calcium

DFHH

Familial hypocalciuric hypercalcemia

ICP‐MS

Inductively coupled plasma‐Mass Spectrometry

MM

Multiple myeloma

PEG

polyethylene glycolt

PTH

Parathyroid hormone

1. Background

Multiple myeloma (MM) is a hematologic malignancy driven by clonal proliferation of neoplastic plasma cells. Its clinical manifestations are diverse and commonly include bone pain, anemia, renal impairment, and hypercalcemia [1]. Hypercalcemia in MM typically arises from calcium release by osteolytic bone lesions. In healthy individuals, approximately 50% of serum total calcium exists as the biologically active ionized form; 40%–45% is pH‐dependently bound to albumin and immunoglobulins, with the remainder complexed to anions [2]. While globulins exert negligible effects at physiological concentrations relative to albumin, this balance is disrupted in severe hyperglobulinemia [3, 4].

Pseudohypercalcemia induced by hyperglobulinemia is frequently observed in MM, characterized by elevated total serum calcium but normal or low ionized calcium in the absence of hypercalcemic symptoms [5]. Most cases are misdiagnosed as true hypercalcemia and receive unwarranted calcium‐lowering therapy, incurring avoidable clinical risks [5, 6, 7]. Currently, evidence supporting globulin‐mediated pseudohypercalcemia is largely restricted to case reports, and standardized diagnostic strategies remain to be established. We report a case of MM with hyperglobulinemia‐induced pseudohypercalcemia, highlighting the critical role of laboratory medicine in its identification and confirmation.

2. Case Presentation

A 48‐year‐old woman with IgG κ‐type MM was referred to our Hematology Department for persistent hypercalcemia refractory to standard therapy. She had a 1‐year history of recurrent gingival bleeding and was previously diagnosed with MM, hyperglobulinemia, and hypercalcemia at an external hospital. Her prior treatment included one cycle of VRD induction chemotherapy (bortezomib, lenalidomide, dexamethasone), salmon calcitonin for hypercalcemia, zoledronic acid for bone lesions, and a single session of plasma exchange. However, 1 month after the initiation of calcium‐lowering therapy, her total serum calcium levels were persistently elevated, ranging from 3.68 to 3.90 mmol/L (reference interval: 2.15–2.55 mmol/L).

On admission, she was in stable general condition and asymptomatic for hypercalcemia. The routine biochemical testing (colorimetry, Roche, cobas c702) revealed markedly elevated total protein (120.6 g/L, reference interval: 65.0–85.0 g/L) and globulin (86.7 g/L, reference interval: 20.0–40.0 g/L), accompanied by hypoalbuminemia (33.9 g/L, reference interval: 40.0–55.0 g/L). Serum total calcium was elevated at 3.68 mmol/L (reference interval: 2.11–2.52 mmol/L), with a corrected calcium level of 3.82 mmol/L. In contrast, ionized calcium measured by direct ion‐selective electrode (Roche, cobas b123) was low at 0.96 mmol/L (reference interval: 1.12–1.32 mmol/L). Serum protein electrophoresis (Sebia, CAPILLARYS 3 TERA) identified a monoclonal IgG kappa M‐protein of 61.8 g/L, accounting for 71.3% of total globulin. Parathyroid hormone (PTH) and 25‐hydroxyvitamin D (25‐OH‐VD), measured by chemiluminescent immunoassay (Roche, cobas 8000 e801), were 6.54 pmol/L (reference interval: 1.60–6.90 pmol/L) and 29.6 nmol/L (reference interval: ≥ 50 nmol/L), respectively. Imaging revealed multiple osteolytic lesions. The patient had no history of exogenous calcium supplementation, and 24‐h urinary calcium excretion (colorimetry, Roche cobas c702) was 3.9 mmol/24 h (reference interval: 2.5–7.5 mmol/24 h). Familial hypocalciuric hypercalcemia (FHH) was deemed unlikely given the absence of hypercalcemic symptoms and negative family history.

During hospitalization, repeated testing confirmed persistent total calcium elevation (3.06–3.68 mmol/L) with consistently low ionized calcium (0.92–0.96 mmol/L). Given both the significant discrepancy between total and ionized calcium levels and the absence of hypercalcemia symptoms, the laboratory medicine team was consulted to investigate potential analytical interference. A panel of validation assays was performed, including: (1) confirmation of total calcium levels by Inductively coupled plasma‐Mass Spectrometry (ICP‐MS), which ionizes samples via inductively coupled plasma and is considered the gold standard for serum calcium detection; (2) assessment of linearity via serial dilution at ratios of 1:3, 1:6 and 1:12; and (3) evaluation of immunoglobulin‐mediated interference using a polyethylene glycol (PEG) precipitation protocol. For PEG precipitation, equal volumes of patient or control serum were mixed with 25% PEG solution, incubated at room temperature for 15 min, and centrifuged; the supernatants were then collected for total calcium measurement using a routine biochemical analyzer. ICP‐MS results showed excellent concordance with routine colorimetric measurements, ruling out systematic analytical error (Figure 1A). Serial dilution showed satisfactory linearity (Figure 1B). Notably, the total calcium recovery rate after PEG precipitation was significantly reduced in the patient sample compared with three independent controls (Table 1), indicating that elevated total calcium was tightly associated with circulating globulin levels.

FIGURE 1.

The confirmatory assay and time‐dependent changes of the patient's serum calcium results. (A) Correlation between serum calcium levels measured by colorimetry and ICP‐MS in samples from three different controls (with definite etiology and symptoms of hypercalcemia) and from the patient at three different time points. (B) The linearity evaluation of the patient's serum calcium at three dilution ratios: 1:3, 1:6, and 1:12. The x‐ and y‐axes for each point are as defined in parentheses; the line shows the linear correlation between measured and theoretical values after dilution. (C) Time‐dependent changes in the patient's globulin and calcium concentrations.

TABLE 1.

Results of globulin and calcium concentration tests before and after PEG precipitation in patient and control samples.

	Globulin (g/L)			Calcium (mmol/L)
	Pre‐PEG	Post‐PEG	Recovery rate (%) a	Pre‐PEG	Post‐PEG	Recovery rate (%) a
Control 1	77.2	5.4	7.0	2.10	1.86	88.6
Control 2	75.2	4.6	6.1	2.32	1.78	76.7
Control 3	78.7	2.2	2.8	2.08	1.68	80.8
Patient	88.7	2.4	2.7	3.68	1.54	41.8

^a The PEG precipitation recovery rate was calculated by the formula: Recovery rate = post‐PEG/pre‐PEG × 100%.

Based on these findings, calcium‐lowering therapy was discontinued and treatment with the VRD regimen for MM was continued. Over 9 months of follow‐up, serial laboratory monitoring showed gradual normalization of serum calcium, which closely paralleled the reduction in globulin levels (Table 2, Figure 1C).

TABLE 2.

Table of results from blood tests for patient.

Date	Albumin (g/L)	Globulin (g/L)	Ionized calcium (mmol/L)	Calcium (colorimetry) (mmol/L)	Calcium (ICP‐MS) (mmol/L)
2025/02/14	33.9	88.7	0.96	3.68	3.58
2025/02/26	/	67.8	0.95	3.11	/
2025/02/28	28.1	68.3	/	2.91	/
2025/04/01	33.1	66.5	0.92	3.06	2.93
2025/06/20	/	30.7	/	2.67	/
2025/08/16	42.4	26.4	0.94	2.42	/
2025/11/08	42.9	21.8	/	2.35	2.28

3. Discussion and Conclusions

This case illustrates the classic features of hypergammaglobulinemia‐induced pseudohypercalcemia in MM. The patient presented with markedly elevated total serum calcium, normal PTH, low 25‐OH‐VD, and crucially, reduced ionized calcium, with no clinical manifestations of true hypercalcemia. This phenotype differs mechanistically from typical MM‐related true hypercalcemia, which is driven primarily by osteolytic bone destruction [8].

The patient exhibited abnormal calcium profiles, manifesting as decreased ionized calcium and elevated total calcium. The reduction in ionized calcium was likely due to 1 month of calcitonin therapy administered at the external hospital, whereas the elevation in total calcium likely resulted from increased calcium binding to the abnormally high concentrations of circulating immunoglobulins (i.e., paraproteins), rather than from analytical interference caused by these paraproteins. To date, several cases of pseudohypercalcemia mediated by calcium‐binding paraproteins have been reported, and this phenomenon has been predominantly observed in patients with elevated monoclonal immunoglobulins, with paraprotein subtypes including IgG λ, IgG κ, and IgA [5, 9, 10, 11]. In addition, IgM paraproteins associated with Waldenström's macroglobulinemia can also cause this condition [12]. Studies have shown that the enhanced calcium‐binding property of monoclonal immunoglobulins is due to calcium‐binding sites located within their Fab region. However, elevated polyclonal immunoglobulins do not exhibit such calcium‐binding capacity [13].

We first verified the accuracy of routine colorimetric total calcium measurements using ICP‐MS, and confirmed assay linearity via serial dilution. PEG precipitation, a widely used method for precipitating immunoglobulins and other biological macromolecules [14, 15], further demonstrated a direct association between circulating globulins and elevated total calcium. Notably, serum total calcium normalized in parallel with declining globulin levels as myeloma was controlled, indicating a causal link between hypergammaglobulinemia and pseudohypercalcemia.

It's worth noting that the albumin‐corrected total serum calcium formula is invalid in this case. This formula only applies to calcium binding disturbances caused by albumin abnormalities and cannot correct the calcium binding abnormality induced by massively elevated abnormal globulins, leading to calcium overestimation and diagnostic confusion. In addition, low 25‐OH‐VD, common in MM patients due to malnutrition and renal impairment, has no direct correlation with pseudohypercalcemia but helps exclude vitamin D‐mediated true hypercalcemia. Normal PTH and urinary calcium levels further exclude primary hyperparathyroidism and FHH, respectively.

Laboratory medicine specialists played an indispensable role in the diagnosis and management of this case. Identifying the marked discrepancy between total and ionized calcium led to the suspicion of pseudohypercalcemia and prompted targeted confirmatory assays. Clinicians followed these recommendations, discontinued unnecessary calcium‐lowering therapy, and focused on MM treatment, thus avoiding the risks of misdiagnosis. This case highlights the core value of laboratory medicine, which extends beyond reporting accurate results to include interpreting abnormal findings and guiding key clinical decisions.

The true prevalence of the increased calcium‐binding property mediated by monoclonal immunoglobulins remains unclear, as most evidence is derived from case reports [3, 4, 5, 9, 10, 11, 12, 13, 16]. Since hypercalcemia is an expected feature of MM, patients who appear hypercalcemic but have normal or low ionized calcium levels are at high risk of misdiagnosis and unnecessary anti‐hypercalcemic interventions. A clinical screening process for globulin‐induced pseudohypercalcemia is recommended: simultaneous measurement of total calcium (tCa) and ionized calcium (iCa); assessment of globulin and albumin levels; PEG precipitation, serial dilution and ICP‐MS validation when hyperglobulinemia coincides with tCa‐iCa dissociation; and discontinuation of calcium‐lowering therapy once pseudohypercalcemia is confirmed.

In conclusion, we report a case of pseudohypercalcemia secondary to IgGκ MM, in which serum total calcium remained persistently elevated despite exogenous calcitonin therapy. Systematic laboratory validation confirmed that the hypercalcemia stemmed from calcium binding to pathologically elevated globulin fractions, rather than analytical interference, rendering the albumin‐corrected calcium formula invalid. Discontinuation of calcium‐lowering therapy and initiation of VRD anti‐myeloma treatment led to progressive normalization of total calcium alongside declining globulin levels. This case highlights the clinical value of laboratory medicine in resolving diagnostic dilemmas, and the proposed screening workflow may help prevent misdiagnosis and inappropriate management of similar cases.

Author Contributions

Anjiang Zhao analyzed all examinations, wrote the original draft, and was a major contributor in writing the manuscript. Fan Zhong interpreted all examinations. Yankui Liu was responsible for graphics. Guixing Li was responsible for conceptualizing and reviewing the manuscript.

Funding

The authors have nothing to report.

Ethics Statement

Written informed consent was not required because all the information is anonymized, and the submission does not include images that may identify the person. This article has been written in accordance with the ethical standards of the institution and considering the case report guidelines.

Consent

Consent was not required due to the anonymized information that is used in this case report.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author.