Limits...
AQP4 autoantibody assay performance in clinical laboratory service.

Fryer JP, Lennon VA, Pittock SJ, Jenkins SM, Fallier-Becker P, Clardy SL, Horta E, Jedynak EA, Lucchinetti CF, Shuster EA, Weinshenker BG, Wingerchuk DM, McKeon A - Neurol Neuroimmunol Neuroinflamm (2014)

Bottom Line: Six results were suspected false-positive: M23-FACS, 2; M1-ELISA, 2; and M23-FACS, M1-FACS, and M1-CBA, 2.M1/M23-cotransfected cells expressing smaller membrane arrays of aquaporin-4 yielded fewer false- positive FACS results than M23-transfected cells.High-order arrays of M23-aquaporin-4 may yield false-positive results by binding IgG nonspecifically.

View Article: PubMed Central - PubMed

Affiliation: Departments of Laboratory Medicine and Pathology (J.P.F., V.A.L., S.J.P., E.H., E.A.J., A.M.), Neurology (V.A.L., S.J.P., S.L.C., C.F.L., B.G.W., A.M.), Immunology (V.A.L.), and Health Sciences Research (S.M.J.), College of Medicine, Mayo Clinic, Rochester, MN; Institute of Pathology and Neuropathology (P. F.-B.), University of Tubingen, Germany; Department of Neurology (E.A.S.), College of Medicine, Mayo Clinic, Jacksonville, FL; and Department of Neurology (D.M.W.), College of Medicine, Mayo Clinic, Scottsdale, AZ.

ABSTRACT

Objective: To compare performance of contemporary aquaporin-4-immunoglobulin (Ig) G assays in clinical service.

Methods: Sera from neurologic patients (4 groups) and controls were tested initially by service ELISA (recombinant human aquaporin-4, M1 isoform) and then by cell-based fluorescence assays: fixed (CBA, M1-aquaporin-4, observer-scored) and live (fluorescence-activated cell sorting [FACS], M1 and M23 aquaporin-4 isoforms). Group 1: all Mayo Clinic patients tested from January to May 2012; group 2: consecutive aquaporin-4-IgG-positive patients from September 2011 (Mayo and non-Mayo); group 3: suspected ELISA false-negatives from 2011 to 2013 (physician-reported, high likelihood of neuromyelitis optica spectrum disorders [NMOSDs] clinically); group 4: suspected ELISA false-positives (physician-reported, not NMOSD clinically).

Results: Group 1 (n = 388): M1-FACS assay performed optimally (areas under the curves: M1 = 0.64; M23 = 0.57 [p = 0.02]). Group 2 (n = 30): NMOSD clinical diagnosis was confirmed by: M23-FACS, 24; M1-FACS, 23; M1-CBA, 20; and M1-ELISA, 18. Six results were suspected false-positive: M23-FACS, 2; M1-ELISA, 2; and M23-FACS, M1-FACS, and M1-CBA, 2. Group 3 (n = 31, suspected M1-ELISA false-negatives): results were positive for 5 sera: M1-FACS, 5; M23-FACS, 3; and M1-CBA, 2. Group 4 (n = 41, suspected M1-ELISA false-positives): all negative except 1 (positive only by M1-CBA). M1/M23-cotransfected cells expressing smaller membrane arrays of aquaporin-4 yielded fewer false- positive FACS results than M23-transfected cells.

Conclusion: Aquaporin-4-transfected CBAs, particularly M1-FACS, perform optimally in aiding NMOSD serologic diagnosis. High-order arrays of M23-aquaporin-4 may yield false-positive results by binding IgG nonspecifically.

No MeSH data available.


Related in: MedlinePlus

Fluorescence-activated cell sorting (FACS) employing cells singly transfected with M1-AQP4 or M23-AQP4 or cotransfected with both AQP4 isoformsFlow cytometry reveals nonspecific binding of control patients' serum IgG to live HEK293 cells expressing M23-AQP4, which is reduced when M1-AQP4 is coexpressed. IgG in sera of 2 positive control patients with neuromyelitis optica (NMO) binds to all AQP4-transfected cells but binds more avidly to cells expressing M23-AQP4 or both M23 and M1 (1:1 ratio) than to cells expressing M1 alone. IgG binding indices for the 14 control sera lacking NMOSD were all less than 2.00 for M1 single-transfected cells; for M23 single-transfected cells the median IgG binding index was 6.8 (range 2.98–25.8) and for M1/M23 cotransfected cells the median was 3.3 (range 1.98–14.7). The horizontal gray line indicates the cutoff for M23-FACS positivity (3.00). AQP4 = aquaporin-4; Ig = immunoglobulin; HEK = human embryonic kidney; NMOSD = NMO spectrum disorder.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4202686&req=5

Figure 3: Fluorescence-activated cell sorting (FACS) employing cells singly transfected with M1-AQP4 or M23-AQP4 or cotransfected with both AQP4 isoformsFlow cytometry reveals nonspecific binding of control patients' serum IgG to live HEK293 cells expressing M23-AQP4, which is reduced when M1-AQP4 is coexpressed. IgG in sera of 2 positive control patients with neuromyelitis optica (NMO) binds to all AQP4-transfected cells but binds more avidly to cells expressing M23-AQP4 or both M23 and M1 (1:1 ratio) than to cells expressing M1 alone. IgG binding indices for the 14 control sera lacking NMOSD were all less than 2.00 for M1 single-transfected cells; for M23 single-transfected cells the median IgG binding index was 6.8 (range 2.98–25.8) and for M1/M23 cotransfected cells the median was 3.3 (range 1.98–14.7). The horizontal gray line indicates the cutoff for M23-FACS positivity (3.00). AQP4 = aquaporin-4; Ig = immunoglobulin; HEK = human embryonic kidney; NMOSD = NMO spectrum disorder.

Mentions: We next tested serum from 16 patients (2 with NMO, 14 without NMOSD) by FACS assays, employing as substrate cells singly transfected with M1-AQP4 or M23-AQP4 or cotransfected with M1-AQP4 and M23-AQP4 (1:1 ratio). All substrates yielded similar results for the 2 control NMO sera (figure 3). IgG binding indices for the 14 non-NMOSD control sera were less than 2.00 for M1 single-transfected cells; for M23 single-transfected cells the median IgG binding index was 6.8 (range 2.98–25.8) and for M1/M23 cotransfected cells the median was 3.3 (range 1.98–14.7).


AQP4 autoantibody assay performance in clinical laboratory service.

Fryer JP, Lennon VA, Pittock SJ, Jenkins SM, Fallier-Becker P, Clardy SL, Horta E, Jedynak EA, Lucchinetti CF, Shuster EA, Weinshenker BG, Wingerchuk DM, McKeon A - Neurol Neuroimmunol Neuroinflamm (2014)

Fluorescence-activated cell sorting (FACS) employing cells singly transfected with M1-AQP4 or M23-AQP4 or cotransfected with both AQP4 isoformsFlow cytometry reveals nonspecific binding of control patients' serum IgG to live HEK293 cells expressing M23-AQP4, which is reduced when M1-AQP4 is coexpressed. IgG in sera of 2 positive control patients with neuromyelitis optica (NMO) binds to all AQP4-transfected cells but binds more avidly to cells expressing M23-AQP4 or both M23 and M1 (1:1 ratio) than to cells expressing M1 alone. IgG binding indices for the 14 control sera lacking NMOSD were all less than 2.00 for M1 single-transfected cells; for M23 single-transfected cells the median IgG binding index was 6.8 (range 2.98–25.8) and for M1/M23 cotransfected cells the median was 3.3 (range 1.98–14.7). The horizontal gray line indicates the cutoff for M23-FACS positivity (3.00). AQP4 = aquaporin-4; Ig = immunoglobulin; HEK = human embryonic kidney; NMOSD = NMO spectrum disorder.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4202686&req=5

Figure 3: Fluorescence-activated cell sorting (FACS) employing cells singly transfected with M1-AQP4 or M23-AQP4 or cotransfected with both AQP4 isoformsFlow cytometry reveals nonspecific binding of control patients' serum IgG to live HEK293 cells expressing M23-AQP4, which is reduced when M1-AQP4 is coexpressed. IgG in sera of 2 positive control patients with neuromyelitis optica (NMO) binds to all AQP4-transfected cells but binds more avidly to cells expressing M23-AQP4 or both M23 and M1 (1:1 ratio) than to cells expressing M1 alone. IgG binding indices for the 14 control sera lacking NMOSD were all less than 2.00 for M1 single-transfected cells; for M23 single-transfected cells the median IgG binding index was 6.8 (range 2.98–25.8) and for M1/M23 cotransfected cells the median was 3.3 (range 1.98–14.7). The horizontal gray line indicates the cutoff for M23-FACS positivity (3.00). AQP4 = aquaporin-4; Ig = immunoglobulin; HEK = human embryonic kidney; NMOSD = NMO spectrum disorder.
Mentions: We next tested serum from 16 patients (2 with NMO, 14 without NMOSD) by FACS assays, employing as substrate cells singly transfected with M1-AQP4 or M23-AQP4 or cotransfected with M1-AQP4 and M23-AQP4 (1:1 ratio). All substrates yielded similar results for the 2 control NMO sera (figure 3). IgG binding indices for the 14 non-NMOSD control sera were less than 2.00 for M1 single-transfected cells; for M23 single-transfected cells the median IgG binding index was 6.8 (range 2.98–25.8) and for M1/M23 cotransfected cells the median was 3.3 (range 1.98–14.7).

Bottom Line: Six results were suspected false-positive: M23-FACS, 2; M1-ELISA, 2; and M23-FACS, M1-FACS, and M1-CBA, 2.M1/M23-cotransfected cells expressing smaller membrane arrays of aquaporin-4 yielded fewer false- positive FACS results than M23-transfected cells.High-order arrays of M23-aquaporin-4 may yield false-positive results by binding IgG nonspecifically.

View Article: PubMed Central - PubMed

Affiliation: Departments of Laboratory Medicine and Pathology (J.P.F., V.A.L., S.J.P., E.H., E.A.J., A.M.), Neurology (V.A.L., S.J.P., S.L.C., C.F.L., B.G.W., A.M.), Immunology (V.A.L.), and Health Sciences Research (S.M.J.), College of Medicine, Mayo Clinic, Rochester, MN; Institute of Pathology and Neuropathology (P. F.-B.), University of Tubingen, Germany; Department of Neurology (E.A.S.), College of Medicine, Mayo Clinic, Jacksonville, FL; and Department of Neurology (D.M.W.), College of Medicine, Mayo Clinic, Scottsdale, AZ.

ABSTRACT

Objective: To compare performance of contemporary aquaporin-4-immunoglobulin (Ig) G assays in clinical service.

Methods: Sera from neurologic patients (4 groups) and controls were tested initially by service ELISA (recombinant human aquaporin-4, M1 isoform) and then by cell-based fluorescence assays: fixed (CBA, M1-aquaporin-4, observer-scored) and live (fluorescence-activated cell sorting [FACS], M1 and M23 aquaporin-4 isoforms). Group 1: all Mayo Clinic patients tested from January to May 2012; group 2: consecutive aquaporin-4-IgG-positive patients from September 2011 (Mayo and non-Mayo); group 3: suspected ELISA false-negatives from 2011 to 2013 (physician-reported, high likelihood of neuromyelitis optica spectrum disorders [NMOSDs] clinically); group 4: suspected ELISA false-positives (physician-reported, not NMOSD clinically).

Results: Group 1 (n = 388): M1-FACS assay performed optimally (areas under the curves: M1 = 0.64; M23 = 0.57 [p = 0.02]). Group 2 (n = 30): NMOSD clinical diagnosis was confirmed by: M23-FACS, 24; M1-FACS, 23; M1-CBA, 20; and M1-ELISA, 18. Six results were suspected false-positive: M23-FACS, 2; M1-ELISA, 2; and M23-FACS, M1-FACS, and M1-CBA, 2. Group 3 (n = 31, suspected M1-ELISA false-negatives): results were positive for 5 sera: M1-FACS, 5; M23-FACS, 3; and M1-CBA, 2. Group 4 (n = 41, suspected M1-ELISA false-positives): all negative except 1 (positive only by M1-CBA). M1/M23-cotransfected cells expressing smaller membrane arrays of aquaporin-4 yielded fewer false- positive FACS results than M23-transfected cells.

Conclusion: Aquaporin-4-transfected CBAs, particularly M1-FACS, perform optimally in aiding NMOSD serologic diagnosis. High-order arrays of M23-aquaporin-4 may yield false-positive results by binding IgG nonspecifically.

No MeSH data available.


Related in: MedlinePlus