The purpose of this study was to investigate the immunological and physical characteristics of IgM-λ type M-protein from patients who were measured low in the turbidimetric immunoassay (TIA) IgM ...assay without error codes for high concentration to determine the cause of the false low levels and to clarify the mechanism of their occurrence.
Materials were IgM patient samples and 8 serum samples from other IgM M-protein patients as controls. Patient samples were assayed by the TIA method, in which five manufacturers and six models (two reagent manufacturers) share the principle, and the BN ProSpec method (nephelometric method), which has a different principle. Dilution linearity tests, IgG addition experiments, isoelectric point electrophoresis, and hydrophobic chromatography were performed on patients and subjects. In addition, the binding capacity of γ-globulin by BIACORE was also examined.
The reaction curve of the patient IgM curved downward when the concentration of IgM exceeded 20 g/L, and no error code was obtained. In the measurement by the TIA method of five manufacturers and six models, patient IgM was measured at a false low level with no error code obtained in undiluted dilution by any of the instruments and reagents, but could be measured without any problem by the nephelometric method. In addition, in the patient IgG addition experiment, only patient IgM showed a false low level under high IgG concentration. Furthermore, the binding capacity of patient IgM to γ-globulin (IgG) by BIACORE was significantly higher than that of the control IgM-type M protein.
Patient IgM has an affinity (binding capacity) for IgG and forms an IgM-IgG complex under conditions of high IgG concentration. It was speculated that this complex inhibited the reaction with the anti-IgM antibody and the absorbance of the second reaction did not increase, suggesting a false low.
Tests for measuring immunoglobulins include quantitative immunoglobulin assays, serum protein fractionation tests, immunoelectrophoresis, and immunofixation electrophoresis. The presence of abnormal ...immunoglobulins is suspected when abnormal bands are detected in electrophoresis or when discrepant data appear between tests. Abnormal immunoglobulins include heavy chain disease proteins and half-molecule immunoglobulins that show molecular defects. Such abnormal proteins can be molecularly qualified by SDS-PAGE or immunoblotting. Bence Jones proteins with microheterogenicity can also be demonstrated as the migration changes by immunofixation electrophoresis of samples before and after enzyme treatment. Furthermore, after agarose membrane electrophoresis, the protein can be spontaneously transferred to a polyvinylidene difluoride (PVDF) membrane for easy immunoblotting. In other words, electrophoresis can be used for both detection and analysis of abnormal immunoglobulins.
The assay method that we use for measurement of about 11,000 AFP samples per year is the μTASWako i30. During our routine testing, we noticed that some AFP samples showed inconsistent results between ...measurements with and without 5-fold dilution. The overall rate of such inconsistency (more than 15% deviation) was 0.4% (44 samples out of 11,000), and the rate among samples containing AFP in the range of 100–2000 ng/mL was 5.7% (44 samples out of 770). In order to investigate the cause of the deviation, we checked the patient records of 18 patients whose samples showed the inconsistency and found that all patients had HCC, and 15 patients were in an advanced stage of the cancer. When serum samples from those patients were treated with PEG or protein A to remove immunoglobulin, their AFP values became closer to the diluted value. Chemical treatment using 4 M urea or 1 M acetic acid also restored the AFP values. Based on these observations, Wako Pure Chemical Industries modified the electrophoresis buffer of the reagent kit and successfully eliminated the deviation. The patients at an advanced stage of HCC appear to have abnormal immunoglobulin or some other serum protein that inhibits the electrophoretic concentration step of the assay by interacting with the immunocomplex of (DNA-antibody) – antigen (AFP) – (dye antibody) to cause the deviation. When we measure samples from patients with advanced stages of disease, we should consider the appearance of abnormal factors that could cause interference in the assays.
A serum protein fraction is employed as a screening for serum protein abnormalities widely and is essential for diagnosis and treatment of multiple myeloma through a detection of monoclonal protein ...in particular. The biggest significance, for which we conduct serum protein fraction in the hospital laboratory, is will be to let us to medical doctors an important view immediately. It is because we can read a patient’s case record with abnormal finding and consider the doubted clinical conditions. For example, characteristic β–γ bridging can suggest IgG4 related disease, it’s possible to consult other views and doubt IgG4 high price. When admitting nonspecific reactions biochemical and immunochemical tests, we can check the influence of monoclonal proteins. It is also useful for judgment of cryoglobulin type.
Temperature-dependent proteins are abnormal immunoglobulins, which exhibit varying properties with variation in temperature. Cryoglobulins are abnormal immunoglobulins that precipitate at cold ...temperature. Some types of cryoglobulins cause Raynaud’s phenomenon which develops severe damage of blood vessel in extremities by cold exposure. The proteins are also involved in nephropathy and hyperviscous syndrome of unknown origin. Cryoglobulins are divided into three types by the constituents of immunoglobulins: (1) type I, which has a monoclonal (M) protein only; (2) type II, which has an M protein and a polyclonal immunoglobulin (Ig); and (3) type III, which has an only polyclonal Ig. It is remarkably difficult to precisely distinguish among these variations because there are numerous factors that prevent clear observation of the M protein for cryoglobulin types II and III. In addition, this type of immunoglobulin is largely observed in a range of its standard values. Nonetheless, it has been confirmed that even a small amount of M protein can cause severe symptoms in patients. Therefore, it is useful to identify and analyze the specific type of cryoglobulin involved in the symptoms of a particular patient, and give the type as an additional comment to doctor in charge of the patient. We hope that the comment will provide incremental therapeutic value beyond the report of the clinical investigation itself.
We encountered a rare case of Waldenstrom macroglobulinemia with temporary appearance of 7S IgM half molecule and with monoclonal proteins binding to agarose gel.
The patient's serum and urine were ...analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The N-terminal amino acid sequences of the IgM with abnormal mass (68 kDa) were determined and compared with those of known immunoglobulin.
The 68 kDa IgM consisted of a defective μ chain (36 kDa) and an intact κ chain. N-terminal amino acid sequence analysis demonstrated that the defective μ chain had the variable region of IgM. The agarose gel-binding ability of the IgM-κ M-protein was lost after reduction or alkaline treatment of serum.
The 7S half molecule IgM in the present case may miss a large part of the constant region of the µ chain.
In systemic IgG4-related disease, an elevation of the serum IgG4 level(IgG4: 135 mg/dl or higher) and IgG4-positive plasma cell infiltration occurs. Since the total IgG and sum of subclasses, IgG1 ...through IgG4, were markedly different in a patient suspected of having Mikuliez's disease, we investigated the relationship between total IgG and sum of IgG subclasses. The subjects were healthy individuals, and low IgG4, high IgG4, hyper-gamma globulinemia and hypo-y globulinemia groups. Total IgG was measured using 'N-assay TIA IgG-SH' (Nittobo) and Hitachi 7700, and IgG subclasses were measured using BS-NIA reagent (Binding Site) and BN II (Siemens). Designation of total IgG and the sum of IgG subclasses was established in the healthy control subjects. However the total IgG level and sum of IgG1-4 levels were different when the balance among the IgG subclasses was lost. In case such as :1) the IgG4 level was high and 2) IgG1-type M protein was present. These results indicate that the reevaluation of measured data is necessary when the IgG4 concentration is high and the difference between total IgG concentration and the sum of IgG subclasses is large.
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