Key Points
Question
What is the clinical utility of current commercially available antiretinal antibody testing?
Findings
In this cross-sectional study, 13 of 14 samples from patients without autoimmune retinopathy tested positive for retinal antibodies, with a median of 5 antibodies per patient.
Meaning
This study suggests that most retinal antibodies included in antiretinal antibody testing evaluations available on a clinical service basis have limited clinical utility in the evaluation of a patient for suspected autoimmune retinopathy.
Abstract
Importance
The clinical utility of most antiretinal antibodies (retina antibodies) currently available for testing remains unclear and unproven. Despite this, the presence of retinal antibodies is included in current diagnostic autoimmune retinopathy criteria.
Objective
To evaluate the clinical significance of comprehensive retinal antibody evaluations currently offered in North America.
Design, Setting, and Participants
In this cross-sectional study, 14 patients without autoimmune retinopathy were recruited into the Mayo Clinic Neuroimmunology Biorepository for this study between January 1, 2019, and October 1, 2019. These serum samples without autoimmune retinopathy were sent in masked fashion to a Clinical Laboratory Improvement Amendments–certified laboratory. Using similar methods, the Mayo Clinic Neuroimmunology Research Laboratory independently assessed the same sample to ascertain reproducibility of the findings.
Main Outcomes and Measures
Results of the autoimmune retinopathy and cancer-associated retinopathy panels.
Results
Thirteen of 14 (93%; 95% CI, 66%-100%) serum samples tested positive for retinal antibodies, with a median of 5 retinal antibodies (range, 0-8) per patient at the Clinical Laboratory Improvement Amendments–certified laboratory, which provides a specificity of 7% (95% CI, 0%-34%). Confirmatory immunohistochemistry staining in human retina was present in 12 of 14 samples (86%). α-Enolase was found in 9 (64%). The only retinal antibody not present was recoverin. These nonspecific retinal antibody results were replicated at the Mayo Clinic Laboratory on Western blot using pig retina proteins as substrate.
Conclusions and Relevance
The presence of retinal antibodies in 93% of the patients without autoimmune retinopathy indicates a lack of specificity and that most detectable retinal antibodies have limited clinical relevance in the evaluation of patients for suspected autoimmune retinopathy. Current retinal antibody testing, other than recoverin, should be interpreted with caution, especially for cases of low clinical suspicion. The poor specificity is important to recognize to prevent the potentially unnecessary commencement of systemic immunosuppressants that may result in significant extraocular adverse effects. Identification of biomarkers that have a high predictive value for inflammatory or autoimmune retinal diseases is needed to move the field forward.
This cross-sectional study evaluates the clinical significance of comprehensive retinal antibody evaluations currently offered in North America.
Introduction
Autoimmune retinopathy is a retinal process characterized by immune-mediated retinal degeneration that encompasses paraneoplastic autoimmune retinopathy, including cancer-associated retinopathy (CAR) and melanoma-associated retinopathy, and nonparaneoplastic autoimmune retinopathy. Antibodies against recoverin were the first to be associated with CAR, found to be a reliable marker of the disease, and often detected in patients with underlying cancers.1 Multiple other putative antiretinal antibodies (retinal antibodies) have been identified during the past 2 to 3 decades,2,3 and the presence of retinal antibodies is one of the major diagnostic criteria in the expert consensus on the diagnosis and management of autoimmune retinopathy published in 2016.4 A proposed 2-tier system of Western blot followed by confirmatory immunohistochemistry has been advocated as the gold standard to identify retinal antibodies.4
Although the presence of retinal antibodies has been called the sine qua non of autoimmune retinopathy,5 increasing literature has questioned the significance and specificity of retinal antibody testing.3,6,7,8 Prior studies9,10,11 have found that retinal antibodies can be found in approximately one-third of healthy individuals in research laboratories. In addition, Faez et al12 found a poor concordance rate of only 36% in laboratories testing for retinal antibodies among patients suspected of having autoimmune retinopathy.
The use of nonstandardized laboratory practice has been proposed as a potential cause of poor specificity and concordance.13 This limitation has been believed to be overcome using standardized protocols with appropriate controls at the only Clinical Laboratory Improvement Amendments (CLIA)–certified laboratory in the US for retinal antibody testing.13,14 However, CLIA approval refers to good laboratory techniques but does not mean that the testing is validated or clinically meaningful.8 A comparison laboratory study14 among patients without autoimmune retinopathy has not been conducted, which is important to determine the specificity of these antibodies. Therefore, we aimed to prospectively test patients who lacked autoimmune retinopathy to investigate the frequency of false-positive results at the CLIA-certified laboratory and investigate whether these findings could be replicated in the Mayo Clinic Neuroimmunology Research Laboratory.
Methods
Patients without autoimmune retinopathy were recruited into the Mayo Clinic Neuroimmunology Biorepository for this study between January 1, 2019, and October 1, 2019. These patients consisted of 4 healthy controls and 10 patients with the following diagnoses without autoimmune retinopathy: nonarteritic anterior ischemic optic neuropathy in 2 patients, multiple sclerosis in 3 patients, age-related macular degeneration in 3 patients, aquaporin 4 IgG–positive neuromyelitis optica in 1 patient, and myelin oligodendrocyte glycoprotein IgG–associated disorder in 1 patient. None of these patients had symptoms concerning for autoimmune retinopathy or history of cancer. These serum samples were sent in a masked fashion to the Ocular Immunology Laboratory at Oregon Health Sciences University, currently the only CLIA-certified laboratory in the US that offers testing for retinal antibodies using line-blot analysis along with confirmatory immunohistochemistry on human retina. The Mayo Clinic Institutional Review Board approved this study, and participants provided informed written consent. All data were deidentified. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.
These serum samples were also tested in the Mayo Clinic Neuroimmunology Research Laboratory with Western blot using pig retina as substrate. For each sample, 20 μg of lysate was used. Human serum was diluted 1:100. Commercially available antibodies to aldolase, 1 μg/mL (Abcam); α-enolase, 1:5000 (Boster Biological); and recoverin, 1:1000 (Millipore) were used as positive controls. Proteins were predicted based on molecular weight estimates from migration of molecular weight standards and in reference to positive controls.
Results
Serum samples from 13 of 14 patients without autoimmune retinopathy (93%; 95% CI, 66%-100%) tested positive for retinal antibodies at the CLIA-certified laboratory, with a median of 5 (range, 0-8) of the 12 antibodies that are tested on the autoimmune retinopathy and CAR panels. This finding provides a specificity of 7% (95% CI, 0%-34%). The number (percentage) of antibodies in the positive samples were as follows: carbonic anhydrase II, 6 (43%); heat shock protein 27, 4 (29%); aldolase, 9 (64%); enolase, 9 (64%); arrestin, 3 (21%); tubulin, 3 (21%); pyruvate kinase M2, 6 (43%); glyceraldehyde 3-phosphate dehydrogenase, 4 (29%); recoverin, 0; RAB6, 4 (29%); heat shock protein 60, 7 (50%); and TUB-like protein 1, 6 (43%). Confirmatory immunohistochemistry staining of the retina was present in all but 2 patients.
In the Mayo Clinic Neuroimmunology Research Laboratory, we attempted to replicate the findings reported by the CLIA-certified laboratory. Using pig rather than human retina, we found positive retinal antibody bands in all 14 patients with Western blot, with a median of 7 bands (range, 4-13), confirming the lack of specificity reported by the CLIA-approved tests (Figure). The positive antibodies in each patient for the CLIA-certified laboratory and Mayo Clinic Neuroimmunology Research Laboratory are given in the Table. There was an overall concordance of 57%.
Figure. Western Blot at the Mayo Clinic Research Laboratory.
The Western blots show multiple bands in most patients, which largely correspond to the molecular weight (MW) of antibodies that were positive at the Clinical Laboratory Improvement Amendments–certified laboratory.
Table. Comparison of the CLIA-Certified Laboratory and Research Laboratory Retinal Antibody Positivitya.
| Patient No. | Disease status | No. of positive antibodies | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Carbonic anhydrase II (29 kDa) | HSP27 (27 kDa) | Aldolase (44 kDa) | Enolase (48 kDa) | Arrestin (45 kDa) | Tubulin (50 kDa) | PKM2 (58 kDa) | GADPH (36 kDa) | Recoverin (23 kDa) | Rab6 (24 kDa) | HSP60 (60 kDa) | TULP1 (70 kDa) | ||
| 1 | Healthy | 3 | 1 | 3 | 3 | 2 | 1 | 3 | 3 | 3 | |||
| 2 | Healthy | 3 | 3 | 3 | 2 | 1 | 1 | 1 | 3 | 2 | |||
| 3 | Healthy | 2 | 2 | 3 | 2 | ||||||||
| 4 | Healthy | 2 | 2 | 2 | |||||||||
| 5 | NAION | 3 | 1 | 3 | 3 | 2 | 1 | 1 | |||||
| 6 | NAION | 3 | 2 | 1 | 1 | ||||||||
| 7 | AMD | 2 | 2 | 3 | 1 | 2 | |||||||
| 8 | AMD | 1 | 3 | 1 | 2 | ||||||||
| 9 | AMD | 3 | 3 | 2 | 1 | 1 | 1 | 1 | |||||
| 10 | MS | 1 | 2 | 3 | 2 | ||||||||
| 11 | MS | 3 | 3 | 3 | 2 | 1 | 1 | 3 | |||||
| 12 | MS | 1 | 3 | 2 | 1 | 3 | 1 | 1 | 3 | ||||
| 13 | NMOSD | 3 | 3 | 3 | 1 | 3 | 3 | ||||||
| 14 | MOGAD | 3 | 3 | 1 | 3 | ||||||||
Abbreviations: AMD, age-related macular degeneration; CLIA, Clinical Laboratory Improvement Amendments; GADPH, glyceraldehyde 3-phosphate dehydrogenase; HSP, heat shock protein; MOGAD, myelin oligodendrocyte glycoprotein antibody–associated disorder; MS, multiple sclerosis; NAION, nonarteritic anterior ischemic optic neuropathy; NMOSD, neuromyelitis optica spectrum disorder AQP4-IgG+; PKM2, pyruvate kinase M2; TULP1, TUB-like protein 1.
The number 1 indicates positive at the CLIA-certified laboratory using line blot analysis; 2, positive at the Mayo Clinic Research Laboratory on Western blot; and 3, positive at both laboratories.
Discussion
The findings of this cross-sectional study raise concern regarding the clinical significance of most retinal antibodies in the diagnosis and evaluation of patients with suspected autoimmune retinopathy under the current method. The potential lack of specificity of commonly reported putative retinal antibodies (such as α-enolase) warrants emphasis, and ophthalmologists need to be aware of the pitfalls of interpreting the results of this testing. Because these antibodies (other than recoverin) were detected in both a research and CLIA-certified laboratory (the latter using line-blot), the assays are likely correct, but most of the antibodies are not specific enough to be used as biomarkers of autoimmune retinopathy. Furthermore, the findings indicate that the use of confirmatory immunohistochemistry as part of the 2-tier design is not sufficient to improve specificity because all but 2 patients without autoimmune retinopathy had immunohistochemical staining of the human retina at the CLIA-certified laboratory.
The presence of retinal antibodies in a high percentage of the patients without autoimmune retinopathy suggests that testing should not be routinely performed in cases of low suspicion. Incorrectly equating the presence of any retinal antibody with the diagnosis of autoimmune retinopathy could subject patients to the unnecessary risks of chronic immunosuppression to treat a disease they may not have.
Antibodies to recoverin and α-enolase are the 2 most commonly cited antibodies in CAR, and both have been suggested to have direct pathogenic effects on the retina.3,5 In this study, recoverin was the only retinal antibody that was not found in these patients without autoimmune retinopathy, which suggests that it may be specific for CAR. In contrast, α-enolase was present in two-thirds of patients, which strongly suggests that α-enolase is not specific for autoimmune retinopathy. Other studies10,15 have found that recoverin is specific for CAR, whereas α-enolase antibodies have been reported to be found in 13% to 33% of healthy individuals and 43% to 74% in patients with autoimmune retinopathy, leading authors to suggest that these antibodies can be helpful in the correct clinical context, but their presence alone is not sufficient to make the diagnosis of autoimmune retinopathy.
Limitations
This study has limitations, including the small sample size, which makes the 95% CI quite broad. Laboratory techniques and species substrate differed between the 2 laboratories, which likely contributed to some of the nonconcordant results. In addition, some of the patients had nonautoimmune retinal disease, such as age-related macular degeneration, which may elicit an immune response and retinal antibody production. Although the patients in the study did not clinically demonstrate signs of autoimmune retinopathy, subclinical retinal antibody–mediated disease cannot be excluded. However, autoimmune retinopathy is rare, and the presence of retina antibodies should not automatically lead to a diagnosis of autoimmune retinopathy.
Conclusions
The results of this study indicate that antibodies included in current antiretinal antibody testing evaluations have limited clinical utility in the evaluation of a patient for suspected autoimmune retinopathy. Identification of biomarkers that have a high predictive value for inflammatory and autoimmune retinal diseases appears to be warranted to move the field forward.
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