Spurious laboratory results associated with immunoglobulin M gammopathy in a dog with multiple myeloma

Abstract An 11 year old female‐neutered Labrador presented for facial swelling. Clinicopathological abnormalities included hyperglobulinemia, azotemia, hypercalcemia, nonregenerative anemia, thrombocytopenia, and spurious hypoglycemia. Normoglycemia was subsequently confirmed using a cage‐side analyzer (AlphaTRAK, Zoetis, UK). Serum and urine protein electrophoresis documented monoclonal (immunoglobulin M) gammopathy with Bence‐Jones proteinuria. Computed tomography imaging revealed a monostotic osteolytic bone‐lesion, and bone marrow cytology and histopathology documented plasmacytosis with multiple myeloma oncogene 1 / interferon regulatory factor 4 positivity, consistent with multiple myeloma. Infectious disease testing initially indicated seropositivity for Leishmania, Borrelia, and Anaplasma spp.; however, Leishmania PCR (splenic and bone marrow aspirates), and paired serological titers for Borrelia and Anaplasma were negative. Consequently, initial serological results were considered to be false positive because of paraproteinemia‐associated assay interference. Chemotherapy (prednisolone and melphalan combination therapy) was initiated, but the dog was euthanased 30 days later because of the development of pericardial effusion. This is a report of spurious serological (and other laboratory) results occurring secondary to monoclonal gammopathy in a dog.


| INTRODUCTION
Hyperglobulinemia and monoclonal gammopathy in dogs is most commonly seen secondary to either neoplasia (multiple myeloma, Waldenström's macroglobulinaemia, chronic lymphocytic leukemia, and lymphoma) or infectious diseases (such as ehrlichiosis and leishmaniasis), 1 and has also been reported secondary to coccidioidomycosis in dogs from endemic regions. 2 Despite similarities in clinical presentation, treatment and prognosis varies considerably, therefore cases presenting with hyperglobulinemia and monoclonal gammopathy require thorough investigation to ensure a robust diagnosis. Rigorous criteria are used in people to help diagnose multiple myeloma 3 and are often utilized in veterinary cases, including the presence of abnormal antibodies or antibody fragments (referred to as paraproteins or M-proteins) in serum or urine (or a combination of both), bone marrow plasmacytosis, and evidence of subsequent organ or tissue impairment (end-organ damage, including bone lesions). 3 Circulating paraproteins in human patients cause spurious laboratory results, 4 including false-positive serological tests, [5][6][7][8] pseudohypoglycemia, [9][10][11] pseudohypophosphatemia, 12 pseudohyperphosphatemia, 13,14 pseudohypercalcemia, 15 falselyelevated C-reactive protein, 16 and altered bilirubin assays. 17

| DISCUSSION
The pitfalls of paraproteinemia-induced spurious laboratory results are well known and recognized in human medicine, leading to clinically impossible results, 24 erroneous diagnoses, unnecessary testing, and incorrect therapies. 25 Although the exact incidence of paraproteinemiainduced spurious laboratory results is unknown, up to 35% of samples can be affected. 26 These are often analytical errors, defined as the effect of a substance present in the sample that alters the correct value of the result because of precipitation, inappropriate binding to assay reagents, or high viscosity and increased turbidity. 4 In the described case, routine serum biochemistry analysis on the Beckman Coulter AU840 chemistry analyzer initially documented hypoglycemia below the limit of quantification (<12.6 mg/dL) despite repeated assessment on a cage-side analyzer confirming normoglycemia, with no applicable clinical signs. This is likely attributable to the difference in assay methodology. The former analyses glucose via the hexokinase method, which is the preferred method for glucose analysis. Paraprotein interference can occur because of antibody cross-reactivity against antigens involved in the hexokinase reaction, [9][10][11]24,27 or increased precipitation and turbidity 27 impacting on the optical change in the assay. This is common in people, particularly with IgM, because of its high molecular weight and ability to form polymers, thus reducing its solubility. 4 Conversely, cageside glucose monitors (such as the AlphaTRAK glucometer) measure glucose via a coulometric electrochemical sensor, which is a dry chemistry method allowing measurement of the analyte concentration in a protein-free filtrate, 9 and is therefore unaffected by paraproteinemia.
Along with spurious chemistry results, false-positive serological results occur in humans secondary to paraproteinemia, both because of hematological malignancies (such as multiple myeloma) and secondary to benign age-related increases in immunoglobulin levels. This includes false-positive Galactomann antigen tests for aspergillosis, 8,28,29 falsepositive serological tests for syphillis, 5,29,30 and false-positive rK39 rapid diagnostic tests for leishmaniasis. 6 There are also false-positive results for a wide range of infectious agents (viral, bacterial, and protozoal) in human patients receiving immunoglobulin therapy IV. [31][32][33][34][35] The mode of interference in the Leishmania assay in the described case could be associated with the paraprotein cross-linking the antigen with the conjugate antibody, 28 or an inability to remove unbound antibody conjugate by the washing procedure. Alternatively, increased turbidity and precipitation could have affected measurement of the optical density and absorbance. Determining the exact mechanism is complex and would require further studies. Given that veterinary serological testing often relies on similar ELISA technology, paraprotein interferences could plausibly occur in animals secondary to paraproteinemia. Clinicians should be aware of these potential analytical errors as many clinical and clinicopathological abnormalities (anemia, thrombocytopenia, hyperglobulinemia) occur secondary to both infectious etiologies and multiple myeloma, and serological testing is therefore commonly performed in the investigation of these cases.
In this case, although there was no history of foreign travel, infectious disease was still initially considered a possible etiology, given that anaplasmosis, 36  An alternative method to avoid interference includes deproteination of the sample. 27 Polyethylene glycol (PEG) is a polymer of ethylene glycol that precipitates proteins by steric hindrance without denaturing or interfering with them. 13 Protocols have been published describing PEG precipitation to minimize or reduce paraprotein interference, 43 and studies have assessed PEG precipitation for multiple chemistry analytes, including creatinine 44 and phosphate 13 in human medicine, and could also be considered in veterinary medicine.
Use of a different analytical method (eg, dry chemistry) could also be used, which eliminates the effects of large molecules such as paraproteins.
A limitation in this case was the lack of cytological or histopathological evaluation of the heart-base mass, and as such we cannot conclude the underlying etiology of this lesion and whether it was an incidental comorbidity in an older animal (such as chemodectoma), or a rare presentation of extramedullary plasmacytoma.
Mediastinal plasmacytoma occurring in people with multiple myeloma is very rarely documented, with only 20 cases in the literature, 45 and was thus considered unlikely in this case. Another limitation is the short follow-up time because of euthanasia, thus precluding follow-up infectious disease testing in the face of normalized globulin concentrations.

ACKNOWLEDGMENT
No funding was received for this study.