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Letters to the Editor |
Paraprotein
1 Department of Pathology, and Immunology, Division of Laboratory Medicine, Washington University, School of Medicine, 660 South Euclid Ave., Box 8118, St. Louis, MO 63110
aAuthor for correspondence. Fax 314-362-1461; e-mail gronowski{at}pathology.wustl.edu.
To the Editor:
Human anti-animal immunoglobulin or "heterophile" antibodies interfere in many immunoassays
(1)(2). Anti-animal immunoglobulin antibodies can occur idiopathically or after treatment with monoclonal antibodies. Those heterophilic antibodies that bind murine immunoglobulins are often referred to as human anti-mouse antibodies
(1). Two-site (sandwich) immunoassays are the assays most frequently reported to be subject to positive interference
(1). False-negative heterophile interferences, to our knowledge, have all been cases of endogenous anti-analyte (not anti-immunoglobulin) antibodies or anti-idiotype antibodies in patients treated with monoclonal antibodies
(3)(4)(5)(6). Here we describe a negative assay interference associated with an IgG
paraprotein.
The AxSYM Ultrasensitive hTSH II assay (Abbott Laboratories) is a sandwich assay for thyrotropin (TSH) that uses mouse monoclonal anti-TSH-coated microparticles and goat anti-TSH-alkaline phosphatase conjugate. In our laboratory, the functional detection limit of this assay is 0.1 mIU/L with a day-to-day CV of 12% (mean, 0.17 mIU/L).
Inconsistent thyroid function test results in an 80-year-old male patient suffering from a myelodysplastic syndrome (IgG monoclonal protein, 38 g/L) prompted our attention. TSH by the AxSYM was 0.1 mIU/L. In our laboratory, samples with TSH results 
0.1 mIU/L are routinely retested on the DPC Immulite 2000 Third Generation TSH assay [Diagnostic Products Corp.; day-to-day (total) CV at 0.077 mIU/L is 6.2%]. The TSH value obtained with this assay was 5.9 mIU/L (reference interval, 0.4–6.2 mIU/L). Further testing revealed nonpathologic free thyroxine (fT4; 10.9 ng/L; reference interval, 7–18 ng/L) and free triiodothyronine (fT3; 2.41 ng/L; reference interval, 1.4–3.5 ng/L). The lack of symptoms and the nonpathologic fT4 and fT3 results suggested that the Immulite 2000 result of 5.9 mIU/L was correct and that the AxSYM result was falsely low.
On a second sample, we performed dilution experiments with the AxSYM TSH diluent and a heterophilic blocking reagent (HBR; Scantibodies Laboratory, Inc.). The TSH diluent contains a proprietary mixture of Tris buffer and "bovine stabilizers", and the HBR contains a proprietary mixture of "immunoglobulins of murine origin with specific binders that neutralize by active attachment to the heterophilic antibody". The addition of 10 µL of AxSYM TSH diluent or HBR to 290 µL of sample had little effect on the TSH results. The addition of 30 µL of HBR produced a nonpathologic TSH result (4.0 mIU/L), whereas 30 µL of diluent had no effect. The addition of 50 µL of diluent or HBR brought the TSH into the nonpathologic reference interval. We were unable to repeat similar dilution experiments on the Immulite 2000 because of insufficient sample quantity and the refusal of the patient to provide additional material.
Immunoglobulins in the patient sample and a control sample were precipitated by the use of 40 g/L ammonium sulfate
(7). The precipitated immunoglobulins were then washed with phosphate-buffered saline and concentrated to 138 g/L (patient sample) and 100 g/L (control sample) by use of an Amicon YM10 filter unit (Millipore Corp.). Serum protein electrophoresis was performed on the concentrated immunoglobulins. The electrophoretic profile of the patient’s proteins (Fig. 1 in the Data Supplement, available with the online version of this Letter at http://www.clinchem.org/content/vol49/issue4/) demonstrated a prominent monoclonal peak in the 
region consistent with this patient’s previous analyses and known IgG paraprotein. The concentrated proteins were then added serially to a sample from a patient with a known TSH concentration, keeping the final volume at 300 µL. When a final immunoglobulin protein concentration of 34.5 g/L was added, there was no difference in TSH results between patient and control immunoglobulins (taking into account the dilution factor). When 79 g/L was added, however, the sample with patient immunoglobulin was approximately one-third of the value with control immunoglobulins added (6.3 vs 17.2 mIU/L). The TSH result was reduced even further (0.16 mIU/L) with 103.5 g/L (final concentration) of patient immunoglobulins.
These data suggest that the IgG paraprotein contained within the patient’s restricted immunoglobulin peak produced the false-negative results on the Abbott AxSYM TSH assay. We speculate that the patient’s antibody binds to an idiotope on one of the antibodies used in the AxSYM assay and sterically blocks the binding of TSH to that antibody. Because of this interaction, the values observed in the AxSYM are abnormally low. An anti-TSH antibody might be considered in the etiology of this interference, but such antibodies are rare
(8). Moreover, the fact that this interference is method specific argues against this mechanism. One explanation for the method specificity of the interference is that the patient has heterophilic activity that binds an idiotope present on the AxSYM antibodies but is absent on the antibodies used in the Immulite 2000 method. In addition, the presence of an endogenous anti-thyroid antibody is unlikely because the patient is clinically euthyroid.
Because this patient, to our knowledge, has received no immunoglobulin for treatment or diagnostic procedures, we believe that this report represents the first documentation of negative interference by an idiopathic heterophile antibody.
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References
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