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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 C-reactive protein (CRP)
A protein present in blood serum in various abnormal states, like inflammation.

Mentioned in: Pelvic Inflammatory Disease

CRP, See C-reactive protein.
) and antistreptolysin-O (ASO ASO arteriosclerosis obliterans.
ASO 1 Administrative services organization, see there 2 Allele-specific–oligonucleotide hybridization 3 Anti-streptolysin O, see there
) 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 rheumatoid factor
n. Abbr. RF
Any of the immunoglobulins found in the serum of individuals with rheumatoid arthritis that enhance the agglutination of suspended particles that are coated with pooled human gamma globulin and that are used
 (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 myeloma /my·elo·ma/ (mi?e-lo´mah) a tumor composed of cells of the type normally found in the bone marrow.

giant cell myeloma  see under tumor (1).
 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 Serological tests
Tests of immune function that are performed using the clear yellow liquid part of blood.

Mentioned in: Scrub Typhus
 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 nephelometer /neph·e·lom·e·ter/ (nef?il-om´it-er) an instrument for measuring the concentration of substances in suspension by means of light scattering by the suspended particles.

 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 SRID Spatial Reference ID
SRID Standards/Requirements Identification Document
SRID Simple Radial Immunodiffusion
SRID Senior Rater Identification
SRID Supplier Request for Information Disposition (aerospace)
SRID Starting Row Identifier
) 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 elution /elu·tion/ (e-loo´shun) in chemistry, separation of material by washing; the process of pulverizing substances and mixing them with water in order to separate the heavier constituents, which settle out in solution, from the  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 nonspecific /non·spe·cif·ic/ (non?spi-sif´ik)
1. not due to any single known cause.

2. not directed against a particular agent, but rather having a general effect.


 binding between the patient's IgM and latex particles, an RF assay was performed by nephelometry nephelometry

measurement of the concentration of a suspension by means of a nephelometer.
 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 NHS
National Health Service

NHS (in Britain) National Health Service
). 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 SIRD Support Instrumentation Requirements Document
SIRD Southern Institute for Rural Development
SIRD Summer Institute on Research Design
SIRD Survey of Industrial Research and Development
SIRD Supersites Integrated Relational Database
, 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.
COPYRIGHT 1997 American Association for Clinical Chemistry, Inc.
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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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