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Letters |
UCLA Medical Center, Department of Pathology, and Laboratory Medicine, 10833 Le Conte Ave., Mailroom A2-179 CHS, Los Angeles, CA 90095-1713, Fax 310-794-4864, E-mail abutch{at}mednet.ucla.edu
To the Editor:
A large number of serum proteins are routinely measured by automated immunonephelometric assays utilizing antisera derived from rabbits, goats, or sheep. Because this type of immunoassay uses polyclonal reagents derived from animals other than mice, it is free from interference by the human anti-mouse antibodies that commonly plague monoclonal antibody-based sandwich assays and competitive immunoassays (1). Nonspecific antibodies against murine immunoglobulins can be detected in the serum of a significant proportion of patients, with a prevalence estimate as high as 80% (2). The incidence of anti-rabbit antibodies in serum samples is considerably lower, with estimates between 0.1% and 5% (1)(3)(4)(5)(6). Interference from human anti-rabbit antibodies has been documented for two-site immunoassays and radioimmunoassays for several hormone assays as well as for creatine kinase MB (3)(4)(5)(6)(7)(8). It recently was reported that anti-rabbit antibodies produced falsely increased C-reactive protein values when measured by nephelometric methods utilizing rabbit reagents (9). In this study, two patients developed anti-rabbit antibodies during treatment with rabbit anti-lymphocyte globulin for immunosuppression. Interestingly, other nephelometric assays utilizing rabbit antisera such as IgG and albumin were not affected, indicating that the interference was unique to the C-reactive protein assay (9).
Our clinical laboratory recently identified a sample with an abnormally
high serum transthyretin value of 1406 mg/L (reference interval,
200–400 mg/L) that was being requested for assessment of nutritional
status. The serum sample was analyzed by nephelometry using the
automated BN II analyzer (Dade Behring), which
uses rabbit anti-transthyretin antibodies. Other samples that were
subsequently submitted for testing had transthyretin values of
1038–1107 mg/L (Table 1
, specimens A–D). The increased transthyretin values
could not be explained by the patient’s clinical history or
medications. The patient was a 56-year-old female, status post
chemotherapy for malignant B-cell lymphoma with an IgM monoclonal
protein of 15 g/L. She was taking erythropoietin and furosemide. To
investigate the presence of an interfering substance, transthyretin was
remeasured using the Array automated nephelometer (Beckman Coulter).
Results were considerably lower on reanalysis and were slightly
increased or within the reference interval (Table 1
). Because the Array
uses antisera derived from goats whereas the BN II uses antisera from
rabbits, the interference may have been attributable to anti-rabbit
antibodies.
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To determine whether the interference was limited to the transthyretin
assay, haptoglobin was measured using the BN II and found to be within
the reference interval of 750-3500 mg/L (Table 1
). This was unexpected
because the patient had received blood transfusions for a steadily
declining hemoglobin and should have had extremely low or undetectable
haptoglobin concentrations. When haptoglobin was remeasured using the
Array, undetectable values were obtained (Table 1
) that were more
consistent with the clinical picture. Thus, the interference was not
restricted to transthyretin and occurred with another BN II assay
utilizing rabbit antisera.
To confirm that the false increase in transthyretin was the result of
human antibodies against rabbit immunoglobulins, various classes of
immunoglobulin were removed from the serum samples by affinity
chromatography. After IgG was removed by passage through a protein
G-Sepharose column, the transthyretin values were only slightly higher
than results obtained by the Array (Table 1
). A similar
reduction in transthyretin was observed when a rabbit
immunoglobulin column was used. When both IgG and IgM were removed by a
protein L-Sepharose column, transthyretin values were comparable to
those obtained by the Array, suggesting that IgM antibodies also
contributed to the interference. Similar results were obtained when
haptoglobin was examined (Table 1
). As expected, IgG and IgM
concentrations (measured by the Array) were substantially reduced after
column chromatography using protein G and protein L, confirming that
the expected immunoglobulin fractions had been depleted.
Because animal sera and immunoglobulins are routinely used as blocking agents to prevent anti-animal antibody interferences in other types of immunoassays (1), this approach was examined to determine whether it would eliminate the interferent. The addition of rabbit serum or purified rabbit immunoglobulin to serum samples failed to reduce the falsely increased results for transthyretin and haptoglobin (data not shown).
In summary, this report demonstrates that human anti-rabbit antibodies can interfere with BN II nephelometric assays for transthyretin and haptoglobin that use rabbit antisera. Because the patient had no history of medicinal treatment with animal immunoglobulins or occupational/social exposure to rabbits, the interference would usually be classified as heterophile antibodies (10). However, the interfering antibodies were not multispecific, a feature of heterophile antibodies, because the interference was not observed in nephelometric assays using goat antisera. Nevertheless, I believe that this is the first report describing a human anti-rabbit interference in an automated immunonephelometric assay in the absence of a well-documented animal exposure. It is recommended that all unexplained increases in transthyretin and other nephelometric assays should be verified by repeat analysis using an assay that utilizes antisera from a different animal species.
References
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