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Interference with nephelometric assay of C-reactive protein and antistreptolysin-O by monoclonal [IgM.sub.-[kappa]] from a myeloma patient.

To the Editor:

The serum concentrations of C-reacfive protein (CRP) and antistreptolysin-O (ASO) are measured by any of several rapid and reliable nephelometric and immunoturbidimetric methods. However, the presence of pathological concentrations of monoclonal immunoguloblin (Ig), glycosylated IgM, rheumatoid factor (RF), and anti-xeno Ig antibody [1-4] can interfere with their determination by certain methods with clinically important consequences.

We describe here interference by IgM-[kappa] from a patient with myeloma in the laser nephelometric assay for CRP and ASO. The patient, a Japanese woman, age 35 years, was referred to our hospital for treatment of IgM-[kappa]-type myeloma. The results of serological tests at the time of her admission (and reference range) were as follows: CRP, 0.465 g/L (0-0.003); ASO, 4.9 x [10.sup.6] IU/L (0166 000); IgG, 19.5 g/L (10.9-17.4); IgA, 3.78 g/L (1.63-3.25); and IgM, 70.0 g/L (1.31-2.83). The CRP, ASO, IgG, IgA, and IgM were measured by a Behring nephelometer II automated analyzer (Behringwerke AG) that used latex particles coated with anti-CRP rabbit antibody, streptolysin-O antigen, and anti-human IgG, IgA, and IgM rabbit antibody (Behringwerke AG), respectively. Although the patient exhibited no clinical signs or symptoms of inflammation or infection, the high concentrations of CRP and ASO persisted. We suspected the presence of a factor that interfered with the measurement of CRP and ASO by the nephelometric assay that used latex particles.

To resolve this question, we took a blood sample from the patient that showed a CRP of 0.274 g/L, an ASO of 5.35 x [10.sup.6] ICJ/L, and an IgM of 55.1 g/L measured by the nephelometric method and assayed it by different methods, i.e., the single radial immunodiffusion (SRID) method that uses a kit (Behringwerke AG) for CRP and IgM and the Rantz-Randall (RR) method that uses human type O erythrocytes (Eiken Chemical, Tokyo) for ASO. The results were as follows: CRP, <0.006 g/L; ASO, 50 x [10.sup.3] ICJ/L; and IgM, 60.0 g/L. The anti-CRP rabbit antibody used in SRID is the same as that used in the nephelometric assay. The disparate results suggested the presence of a factor that was interfering with measurement of CRP and ASO by the latter method. We used Sephadex G-200 (Pharmacia Fine Chemicals) (70 x 2.5 cm glass-jacketed column) gel filtration with a 67 mmol/L phosphate buffer (pH 7.4) to isolate fractions of the patient's serum in which values of CRP and ASO were detectable. The values of IgM, CRP, and ASO in each fraction were measured by the nephelometric assay. The elution pattern of IgM was normal, but some CRP and ASO were also eluted in the IgM fraction. This finding suggested that the patient's IgM itself might affect measurement of CRP and ASO by this method.

To exclude the possibility of nonspecific binding between the patient's IgM and latex particles, an RF assay was performed by nephelometry with the use of latex particles coated with human IgG (Behringwerke AG); the value for the patient's serum was within the reference range.

To verify the results obtained, we tested with the use of an equivolume mixture of purified patient's IgM (10 g/L) and normal human serum (NHS). As the control, IgM isolated from the sera of other myeloma patients with high IgM and high or low CRP values, was mixed with NHS. Two different methods (nephelometry and SRID or RR) for the quantitative determination of CRP and ASO were used to assay the mixture. Only the mixture of IgM of our patient and NHS interfered with the measurent of CRP and ASO by the nephelometry, yielding high values for CRP and ASO (Table 1).

Three possible hypotheses might explain the high values for CRP and ASO caused by the patient's IgM: the presence of IgM-CRP and ASO complex, the occurrence of a nonspecific reaction between IgM and latex particles, and the occurrence of nonspecific or specific reactions between IgM and anti-CRP antibody or streptolysin-O antigen coating the latex particles. The first hypothesis was ruled out by the finding that CRP and ASO were detected in the normal IgM fraction by Sephadex G-200 gel filtration. If CRP and ASO were bound to IgM, these fractions should show a deviation. CRP and ASO assay methods that used no latex particles (SIRD, RR) also did not show high values for CRP and ASO. The second hypothesis was ruled out by the finding that, in the RF assay by nephelometry using the same latex particle methodology (CRP latex and ASO latex), the RF value of the patient's serum was normal. However, our findings did not rule out the third hypothesis.

The results thus suggested that the IgM from the present patient may have bound to the latex particles coated with anti-CRP antibody and to those coated with the streptolysin-O antigen used in this study. We were unable to determine which part of the complex of the latex particles and anti-CRP antibody or streptolysin-O antigen had bound to the patient's IgM. This appears to be the first report of the occurrence of false-positive values for CRP and ASO, but not of RF, in the presence of monoclonal IgM-[kappa] from a patient with myeloma as determined with latex methodology, i.e., CRP latex, ASO latex, RF latex measured by the laser nephelometric method.

Accordingly, when high serum CRP and ASO are found in patients with myeloma in the absence of signs or symptoms of inflammation or infection, assays for these substances may require the removal of Ig from the serum sample to avoid interferences in the nephelometric assay and the resulting falsely increased values.


[1.] Muller W, Mietau R, Wohltmann D. Interference of IgM rheumatoid factor with nephelometric C-reactive protein determinations. J Immunol Methods 1985;80:77-90.

[2.] Sato M, Sugimoto M, Nanba S, Kasatori N, Urayama T. Hepatic cirrhosis showing false positive serum C-reactive protein reaction. intern Med 1993;32:498-501.

[3.] Ponge ThD, Le Carrer DL, Sagniez MM, Ponter M, Cottin SL. C-reactive protein in a patient with monoclonal IgM. Clin Chim Acta 1993;220: 101-6.

[4.] Gallou G, Legras B, Ruelland [A.sub.1c] Grosbois B, Cloarec L. Problems of C-reactive protein determination in patients with monoclonal immunoglobulins. Clin Chem 1993;39:918.

Koji Yamada (1) Atsuhito Yagihashi (1) Sayoko Ishii (1) Kuniko Tanemura (1) Takashi Kida (1) Naoki Watanabe (1) *

Yoshiro Niitsu (2)

(1) Dept. of Lab. Diagnosis

(2) Fourth Dept. of Intern. Med. Sapporo Med. Univ. School of Med. South-1, West-16, Chuo-ku Sapporo, 060, Japan

* Author for correspondence.
Table 1. Interference with measurement of CRP and ASOa by
serum treated (b) with anti-IgM antibody or purified IgM
from patient.

Sample Treatment CRP, g/L ASO, IU/L IgM, g/L

P.S. + saline solution 0.393 3 950 000 64.90
P.S. + anti-IgM 0.047 553 000 13.80
M.S. (1) + saline solution 0.163 54 000 40.50
M.S. (1) + anti-IgM 0.179 88 000 2.18
IgM <0.003 <58 000 52.00
N.S. + P.S. IgM 0.012 647 000 4.88
N.S. + M.S. (2) IgM <0.001 40 000 4.14
N.S. + M.S. (3) IgM <0.001 41 000 4.85
N.S. + saline solution <0.001 42 000 0.62

(a) CRP, ASO, and IgM were all measured by a Behring
nephelometer II using latex methodology.

(b) Serum was mixed with an equal volume of the
treatment solution.

P.S., patient's serum; N.S., normal human serum; P.S.
IgM, purified IgM from patient's serum; M.S. (1),
myeloma patient's serum containing high CRP; M.S.
(2) IgM and M.S. (3) IgM, purified IgM from serum of
myeloma patients with high or low CRP; IgM, purified
IgM from serum of a healthy volunteer.
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Title Annotation:Letters
Author:Yamada, Koji; Yagihashi, Atsuhito; Ishii, Sayoko; Tanemura, Kuniko; Kida, Takashi; Watanabe, Naoki;
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:Dec 1, 1997
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