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An algorithm for acetylcholine receptor antibody testing in patients with suspected myasthenia gravis.

To the Editor:

Myasthenia gravis (MG) [1] is a well-characterized autoimmune disease with an estimated prevalence of 1 in 5000 individuals (1). The clinical presentation varies from mild weakness of limited muscle groups (class I or ocular MG) to severe weakness of multiple muscle groups (class V or severe generalized MG). Detection of autoantibodies to the neuromuscular nicotinic acetylcholine receptor (ACHR) has proved useful in assisting in the diagnosis of MG; however, the complexity of this disease, combined with the variety of antibodies associated with MG, has led to multiple attempts to correlate disease severity with antibody detection and concentration. In general, the ACHR antibody concentration is directly proportional to disease severity, but neither the presence nor the absolute concentration of ACHR antibodies correlates with disease severity in any individual patient. ACHR antibodies are specific for MG because they are not detected in healthy individuals or in patients with other autoimmune or neuromuscular disorders; however, their absence does not rule out disease, because only about 85% of confirmed MG patients with generalized disease possess ACHR antibodies (2,3). ACHR antibodies are less frequently detected in MG patients with mild disease or restricted muscle weakness (2, 3). MG patients without detectable ACHR antibodies often have antibodies to other neuromuscular junction proteins, such as muscle-specific kinase, which is detected in about 70% of seronegative MG patients (3).

The heterogeneous nature of the ACHR antibody response has led to the categorization of ACHR antibodies into 3 types: binding, blocking, and modulating. Assays of binding antibody, the most common antibody, measure antibody binding to [sup.125]I-[alpha]-bungarotoxin-labeled ACHR. Blocking antibodies interfere with receptor-ligand interaction and are measured by measuring the inhibition by patient serum of [sup.125]I-[alpha]-bungarotoxin labeling of ACHR or by serum displacement of [sup.125]I-[alpha]-bungarotoxin from bungarotoxin-receptor complexes. The dissociation constants reported for ACHR antibody [KD, approximately 2.35 x [10.sup.-11] mol/L (4)] and [alpha]-bungarotoxin [[K.sub.D], approximately 2.6 x [10.sup.-10] mol/L (5)] explain not only the ability of these antagonists to prevent the association of acetylcholine [[K.sub.D], 6.2 x [10.sup.-6] mol/L (4)] with its receptor but also the ability of blocking antibodies to displace [alpha]-bungarotoxin from the ACHR. Modulating antibodies accelerate the rate of ACHR internalization by cross-linking adjacent receptors and are detected by measuring the amount of internalized, processed [sup.125]I-[alpha]-bungarotoxin-labeled ACHR released from cultured cells (2, 3). In addition to being more technically demanding owing to the requirement of viable cell culture, modulating antibody assays cannot adequately distinguish between blocking antibody-released radioactivity and modulating antibody-released radioactivity and therefore cannot truly distinguish these 2 types of ACHR antibodies.

Different ACHR antibody testing algorithms have been proposed to elucidate the relative importance of each ACHR antibody subtype with respect to diagnosis and disease severity, but a comparison of the various studies is complicated by the use of alternative methods in different patient populations. In this study, to determine the prevalence and frequency of each type of ACHR antibody, we retrospectively evaluated the presence and concentration of ACHR binding, blocking, and modulating antibodies in 39 380 samples of patient sera submitted from throughout the US to ARUP Laboratories for the assessment of all 3 ACHR antibodies.

Most samples (n = 34 640, 88%) did not possess detectable ACHR antibodies, whereas one or more ACHR antibody types were detected in 12% (n = 4740) of the clinical samples tested. The clinical status of our patient population was not available to us; however, these data suggest that physicians most often use ACHR antibody testing to rule out rather than to confirm the diagnosis of MG (Fig. 1). In agreement with previous reports (2, 3), our most sensitive assay was the ACHR binding antibody assay, which was positive in 4178 (88%) of the 4740 ACHR antibody-positive serum samples. Modulating antibodies were detected in 70% (n = 3297) of the samples, and blocking antibodies were least prevalent, detected in 65% (n = 3074) of ACHR antibody-positive sera (Fig. 1). Combining binding and blocking ACHR antibody testing identified 97% of the patient population with detectable ACHR antibodies, whereas testing for binding and modulating ACHR antibodies identified only 93% of the ACHR antibody-possessing population. Of the 39 380 samples submitted, only 0.4% (n = 160) tested positive for modulating antibodies in the absence of binding and blocking antibodies.

Approximately 15% of MG patients fail to demonstrate any ACHR antibodies; consequently, treatment does not change according to the type of ACHR autoantibody present. These data suggest that the most cost-effective algorithm in the diagnosis of MG is testing for ACHR binding and blocking antibodies with reflex testing for modulating antibodies only in the presence of one or both of these other ACHR antibodies.

[FIGURE 1 OMITTED]

Previously published online at DOI: 10.1373/clinchem.2009.140392

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures of Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: All financial support was provided by the ARUP Institute for Clinical and Experimental Pathology, ARUP Laboratories (an enterprise of the University of Utah) and its Department of Pathology, or the University of Utah Department Of Pathology.

Expert Testimony: None declared.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

References

(1.) Phillips LH. The epidemiology of myasthenia gravis. Semin Neurol 2004;24:17-20.

(2.) Howard FM, Lennon VA, Finley J, Matsumoto J, Elveback LR. Clinical correlations of antibodies that bind, block, or modulate human acetylcholine receptors in myasthenia gravis. Ann N Y Acad Sci 1987;505:526-38.

(3.) AgiusMA, Richman DP, Vincent A. Autoantibody testing in the diagnosis and management of autoimmune disorders of neuromuscular transmission and related disorders. In: Kaminski HJ, ed. Myasthenia gravis and related disorders. 2nd ed. Totowa, NJ: Humana Press; 2009. p 143-56.

(4.) Sine SM. Functional properties of human skeletal muscle acetylcholine receptors expressed by the TE671 cell line. J Biol Chem 1988;263:18052-62.

(5.) Vincent A, Newsom-Davis J. Acetylcholine receptor antibody characteristics in myasthenia gravis. I. Patients with generalized myasthenia or disease restricted to ocular muscles. Clin Exp Immunol 1982;49:257-65.

Thomas R. Haven [2] *

Mark E. Astill [2]

Brian M. Pasi [3]

James B. Carper [3]

Lily L. Wu [2,4]

Anne E. Tebo [2,4]

Harry R. Hill [2,4]

[1] Nonstandard abbreviations: MG, myasthenia gravis; ACHR, acetylcholine receptor.

[2] ARUP Institute for Clinical and Experimental Pathology Salt Lake City, UT

[3] ARUP Laboratories, Inc. Salt Lake City, UT

[4] Department of Pathology University of Utah Salt Lake City, UT

* Address correspondence to this author at: ARUP Institute for Clinical and Experimental Pathology

500 Chipeta Way

Salt Lake City, UT 84108-1221

Fax 1-801-584-5048

E-mail haventr@aruplab.com
Fig. 1. Number and percentage of samples in patient population
submitted forbinding (BIN), blocking (BLO), and modulating (MOD)
ACHR antibody testing.

 No. of samples

BIN (-) 34640 (88.0%)
BLO (-)
MOD (-)

BIN (+) 2412 (6.1%)
BLO (+)
MOD (+)

BIN (+) 840 (2.1%)
BLO (-)
MOD (-)

BIN (+) 666 (1.7%)
BLO (-)
MOD (+)

BIN (-) 343 (0.9%)
BLO (+)
MOD (-)

BIN (+) 260 (0.7%)
BLO (+)
MOD (-)

BIN (-) 160 (0.4%)
BLO (-)
MOD (+)

BIN (-) 59 (0.1%)
BLO (+)
MOD (+)

Note: Table made from bar graph.
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Title Annotation:Letters to the Editor
Author:Haven, Thomas R.; Astill, Mark E.; Pasi, Brian M.; Carper, James B.; Wu, Lily L.; Tebo, Anne E.; Hil
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:Jun 1, 2010
Words:1295
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