Anti-ganglioside antibodies are currently used in the differential diagnosis of suspected immune-mediated neuropathies. In-house and increasingly used commercial assays seem to perform suboptimally, and comparative information on their analytical performance are essentially lacking. Born within the frame of guidelines and standardization activities by the Italian Association of Neuroimmunology, this external quality assessment scheme (EQAS) is a real-life snapshot of the laboratory diagnostics in this field. The EQAS consisted of five surplus, anonymized serum samples from patients with clinically-defined neuropathies and two serum samples from healthy blood donors. Eight laboratories used commercial line-/dot-blots, seven in-house/commercial ELISAs (in addition, 13 laboratories tested a recently released ELISA by B\ufchlmann). Only high anti-ganglioside antibody reactivities were considered, in accordance with consolidated recommendations. Large variations in anti-ganglioside antibody profiles were observed, even, although to a lesser extent, within homogeneous classes of assays. Concordance between the profiles and clinical phenotypes was also partial. Although conducted on a relatively small, but representative number of Italian laboratories, this EQAS shows a critical between-laboratory disagreement in the test results of anti-ganglioside antibodies. Also considering the trend for using certified assays in generalist laboratories, strong efforts toward standardization and the identification of the best method(s) for their determinations are compellingly needed

C. Giannotta

D. Franciotta

E. Nobile-Orazio

E. Zardini

L. Benedetti

M. Gastaldi

R. Bedin

T. Biagioli

AIR Universita degli studi di Milano

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Anti-ganglioside antibodies: experience from the Italian Association of Neuroimmunology 
external quality assessment scheme 
 
 
Diego Franciotta*, Matteo Gastaldi, Tiziana Biagioli, Luana Benedetti, Claudia Giannotta, Roberta 
Bedin, Elisabetta Zardini and Eduardo Nobile-Orazio 
 
 
*Corresponding author:  Diego Franciotta, Laboratory of Neuroimmunology, IRCCS Mondino 
Foundation, Via Mondino 2, I-27100 Pavia, Italy, Phone: 0039-382-380365, FAX: 0039-382-380286, 
E-mail: diego.franciotta@mondino.it 
Matteo Gastaldi and Elisabetta Zardini: Laboratory of Neuroimmunology, IRCCS Mondino 
Foundation, University of Pavia, Pavia, Italy  
Tiziana Biagioli: AOU Careggi, Florence, Italy 
Luana Benedetti: IRCCS Ospedale Policlinico San Martino, University of Genova, Genoa, Italy 
Claudia Giannotta and Eduardo Nobile-Orazio:  Neuromuscular and Neuroimmunology Service, 
Department of Medical Biotechnology and Translational Medicine, Humanitas Clinical and 
Research Institute, Milan University, Rozzano, Milan, Italy 
Roberta Bedin: Ospedale Civile Azienda Ospedaliero-Universitaria di Modena, Modena, Italy 
Other centers and participants are listed in the Appendix 
  
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Abstract 
 
Background: Anti-ganglioside antibodies are currently used in the differential diagnosis of 
suspected immune-mediated neuropathies. In-house and increasingly used commercial assays 
seem to perform suboptimally, and comparative information on their analytical performance are 
essentially lacking. Born within the frame of guidelines and standardization activities by the Italian 
Association of Neuroimmunology, this external quality assessment scheme (EQAS) is a real-life 
snapshot of the laboratory diagnostics in this field. 
Methods: The EQAS consisted of 5 surplus, anonymized serum samples from patients with 
clinically-defined neuropathies, and 2 serum samples from healthy blood donors. Eight 
laboratories used commercial line-/dot-blots, 7 in-house/commercial ELISAs (in addition, 13 
laboratories tested a recently released ELISA by Bühlmann). Only high levels of anti-ganglioside 
reactivities were considered, in accordance with consolidated recommendations. 
Results: Large variations in anti-ganglioside antibody profiles were observed, even, although at a 
lesser extent, within homogeneous assays. Concordance between the profiles and clinical 
phenotypes was partial too. 
Conclusions: Although conducted on a relatively small, but representative number of Italian 
laboratories, this EQAS shows a critical between-laboratory disagreement in test results of anti-
ganglioside antibodies. Also considering the trend for using certified assays in generalist 
laboratories, strong efforts toward standardization and the identification of the best method(s) for 
their determinations are compellingly needed.  
 
Keywords: autoimmunity; dot-blot; ELISA; immune-mediated neuropathies; line-blot; medical 
diagnostics 
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Introduction 
After their discovery and for years, more than 20 antibodies to gangliosides, glycosphingolipid 
molecules densely expressed on the external side of neuronal membranes, have been 
representing possible pathogenetic agents, or biomarkers of disease at least, in inflammatory 
axonal neuropathies [1]. However, at present only a few anti-ganglioside antibodies associate with 
well-defined clinical phenotypes, and therefore good medical practice recommends to test these 
autoantibodies only when sufficiently typical inflammatory neuropathies are suspected [2-6]. On 
the other hand, there is a general trend for requesting anti-ganglioside antibodies in a wider 
spectrum of neuropathies by clinicians, and for testing extended autoantibody panels by 
laboratorists. Guidelines by the Italian Association of Neuroimmunology (AINI) have represented 
an attempt to rationalize such requests [6]. 
From the laboratory side, two methodological approaches are basically available: ELISA (either in-
house, or commercial) and line-/dot-blot (commercial), in the absence of a feasible standard 
technique. The glycosphingolipid nature of the antigens makes it difficult to extract, purify, and 
use them to devise completely reliable methods, so that it is accepted that they perform 
suboptimally [7]. In 1999, the so-called INCAT-ELISA was proposed by an international group of 
experts in neuropathies [8], and is still reckoned as an eligible test [3, 6, 9], notwithstanding the 
not negligible inter-laboratory variability reported since its publication [8]. It is likely that the 
shortcomings only partially depend on the in-house nature of the test. However, also to overcome 
these issues, laboratory diagnostics is long moving toward the use of certified commercial tests in 
generalist laboratories. The analytical performances of the commercial tests have been never 
compared with those of the in-house ELISA so far. Anyway, the trend toward test centralization in 
large laboratories urgently needs careful quality controls and, ultimately, standardizations in this 
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neuroimmunology area, given the reported intra- and between-method differences in both in-
house and commercial test results [8, 10]. 
We herein report our experience of external quality assessment (EQAS) on anti-ganglioside 
antibodies, which was promoted by AINI. The EQAS is a snapshot of the real-life diagnostics in this 
field, as serum samples from single patients with clinically-defined neuropathies were included, 
and as the participating laboratories used heterogeneous commercial or in-house assays. 
 
Material and methods  
Control samples 
Controls consisted of 5 anonymized and blinded-to-the-participants sera from 5 patients with the 
following neurological diseases (defined with clinical, electrophysiological, and cerebrospinal fluid 
investigations): sensory-motor Guillain-Barré syndrome (GBS), control #1; GBS, control #2; IgMκ 
monoclonal gammopathy-associated polyneuropathy (anti-MAG antibody negative), control #3; 
CANOMAD syndrome (Chronic Ataxic Neuropathy, Ophthalmoplegia, Monoclonal IgM protein, 
cold Agglutinins and Disialosyl antibodies), control #4; multiple mononeuropathy, control #5. 
Following routine analysis, the leftover samples were aliquoted into plastic cryogenic tubes, and 
frozen at -20 °C at the Florence or Pavia laboratories, before in-house analysis, or distribution to 
the participating laboratories. Two additional control sera were from healthy blood donors (gift by 
Bühlmann). 
Materials 
The antigens considered, tested for IgG and IgM reactivities, were: GM1, GM2, GD1a, GD1b, GD3, 
GQ1b, GT1a, GT1b. Sulfatides were not included, as not present in some commercial tests, and as 
testing for anti-sulfatide antibodies substantially lacks of clinical utility [5].  
Methods 
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The 15 participating laboratories, most of which being referral centers in Italy, are listed in the 
Appendix. The following tests were used (type and number of laboratories using each test in 
parentheses): Immunodot Dotzen-Zentec, Angleur Belgium (dot-blot, 3), Immunoline ganglio 
profile, Euroimmun, Lübeck, Germany (line-blot, 3), BÜHLMANN GanglioCombiTM Light (ELISA, 1), 
Immunoline Generic Assays, Dahlewitz, Germany (line-blot, 2), in-house INCAT-ELISA [8]. Thirteen 
laboratories had the opportunity of testing the same control samples with the recently released 
BÜHLMANN GanglioCombiTM MAG ELISA (rr-Bühlmann ELISA; lot, 0808; kits gently provided by 
Bühlmann). Commercial tests were performed in accordance with the manufacturer’s instructions. 
Following the local interpretative procedures, all the laboratories were expected to report ‘low’ 
and ‘high’ levels of positivity, but it was recommended to consider significant (positive test) only 
high reactivities (line-/dot-blot), or high levels of anti-ganglioside antibodies (ELISA), as low levels 
of positivity have non-specific meaning and should not be reported in routine diagnostics [2, 4, 6]. 
Each laboratory had a long-lasting expertise that allowed consistent interpretations of weak 
band/spot staining by visual inspection (line-/dot-blot), or appropriate cutoffs (ELISA), in order to 
recognize ‘low positive’ results (no consensus required for EQAS). As for rr-Bühlmann ELISA, 
samples with ODs corresponding to those within or higher than the ‘medium control’ range were 
considered as positive.  
 
Results 
Table 1 summarizes the results, taking into account only high levels of positivity; the 2 controls by 
healthy blood donors were not included, as they resulted negative on all the assays. Laboratory 
#14 was excluded from the analysis (EQAS not completed). On the basis of the main category of 
the tests used (solid-phase support), we analyzed the results between groups (line-/dot-blot vs 
ELISA), and within homogeneous groups. Sample #5 was interpreted as negative by all the 
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laboratories. A nucleus of agreement can be found for the IgM reactivity to GD1b in sample #3 
(12/14 laboratories). There was no substantial agreement in the autoantibody profiles for samples 
#1-4, even within the small subgroups using line-/dot-blots from the same manufacturer. 
As for the performance of rr-Bühlmann ELISA, two aspects were evaluated, namely, the inter-lab 
variability, and the comparison with the other ELISAs. At the given cutoff of positivity, all the 
laboratories were concordant in reporting the following samples as positive: #2 (anti-GM1 IgG), #3 
(anti-GD1b IgM), and #4 (anti-GD1b and anti-GQ1b IgM), except for laboratory #14 (sample #2, 
anti-GD1b IgG, and sample #3, anti-GQ1b IgM, as additional reactivities). Sample #5 was 
interpreted as negative by all the laboratories. When considering ‘low positive’ results, only slight 
between-laboratories differences emerged for samples #2-4, except for sample #1, which was 
judged anti-GM1 IgG-positive by 8 laboratories, and negative by 5 (not shown). When comparing 
the results by rr-Bühlmann ELISA with those by in-house ELISA, only partial agreements were 
found (sample #1, negative in 4 cases; sample #2, positive for anti-GM1 IgG in 3 cases; sample #3 
positive for anti-GD1b IgM in 5 cases; sample #4, positive for anti-GD1b and anti-GQ1b IgM in 2 
cases; sample #5, negative in all cases). 
As for the coherence between clinical phenotypes and autoantibody profiles, the EQAS main 
results show that a) anti-GM1 IgG found in sample #2 by 3/7 laboratories of the line-/dot-blot 
group (BG), 3/7 of the in-house/commercial ELISA group (EG), and 13/13 of the rr-Bühlmann ELISA 
group (BEG) was compatible with GBS [4]; b) at least one reactivity for anti-GD1b and anti-GQ1b 
IgM found in sample #3 by 7/7 of BG, 5/7 of EG, and 13/13 of BEG was among the most frequently 
detected high-titer anti-ganglioside IgM reactivities in anti-MAG antibody negative, IgM 
monoclonal gammopathy-associated polyneuropathy [11]; c) as for sample #4, anti-GQ1b IgM plus 
a reactivity for at least one disialylated ganglioside, fulfilling the criteria for CANOMAD diagnosis 
[7], was found in 5/7 of BG, 4/7 of EA, and 13/13 of BEG.  
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Discussion 
The main findings of the present EQAS on anti-ganglioside antibodies highlight a wide between-
laboratory heterogeneity in both methods and results. The diagnostic shortcomings in this 
neuroimmunology area are well-known and difficult to solve, as long as they rely on both the 
nature of the antigens and other analytical- and manufacturer-related factors (reviewed 
elsewhere [6]).  
This EQAS benefited from using single patient samples for comparison, thus allowing a real 
representation of the performance of each assay. Pooling samples can indeed alter the sample 
matrix and introduce bias. One fundamental point, previously established [2, 4, 6] and confirmed 
by our EQAS, is the recommendation of excluding ‘low positive’ results from the reports. If we had 
included ‘low positive’ results, further between-laboratory disagreement would have been 
emerged. On the other hand, a good test specificity, as sample #5, which was from a patient with a 
presumed non-autoimmune neuropathy, and the two samples from healthy controls were 
unanimously judged as negative. 
The choice of well-defined cutoff values still remains a matter of standardization, whether for line-
/dot-blot assays, or ELISAs. Indeed, notwithstanding that only ‘high positive’ results were 
considered, considerable variations in anti-ganglioside antibody profiles were observed, even, 
although at a lesser degree, within homogeneous assays. Slight variations were found even in the 
results of rr-Bühlmann ELISA, whose kits had the same lot for all the laboratories. As a possible 
interpretation of these differences, a very interesting study on between-laboratory variability of 
an ELISA for neurofilament measurement demonstrated that, in addition to relatively more-
difficult-to-control factors, such as analytical delays and reaction temperatures, the largely most 
8 
 
important cause of inaccuracy was preparation of standards [12]. The educational perspective of 
improving the human factor should be thus pursued. 
In the specialized literature, there is still no clear-cut evidence for the best solid-phase support for 
anti-ganglioside antibody testing. Some authors, more involved in laboratory practice, favor 
hydrophobic membranes (line-/dot-blot), as they might allow better antigen-antibody interactions 
[10], others, more involved in clinical practice, keep choosing polystyrene (ELISA) [4]. A reappraisal 
of the original INCAT-ELISA, which involved 1232 patients and controls tested for GM1 IgG and 
IgM, showed that the ELISA was reliable and added diagnostic value in selected clinical situations 
only when high antibody titers were considered [4]. This study confirm that tests for anti-
ganglioside antibodies should be requested only in selected populations of neuropathic patients. 
The most common associations between specific autoantibody reactivities and well-defined 
clinical phenotypes include multifocal motor neuropathy and GBS variants (for more details, see 
[6]). In turn, this view follows the emerging idea that the total quality in laboratory should shift 
from pure internal indicators to clinical parameters spanning from rational requests to the test 
relevance for clinical effectiveness and patient outcomes (brain-to-brain loop) [12]. 
Diagnostics for anti-ganglioside antibodies, as well as for other neuroimmunological tests, is 
moving from small specialized laboratories, which preferentially use in-house tests, toward large 
generalist laboratories using certified commercial tests. The transition is per se critical, but the 
here-reported inter- and intra-method disagreements urge extra-efforts for the identification of 
the best standardized method(s). The role for laboratory, neuroimmunology scientific societies, 
and, possibly, specialized manufacturers in this virtuous process is fundamental. 
 
Acknowledgements: The authors thank Renato Cotti for critical reading of the manuscript. 
9 
 
Author contributions: Scheme organizers: D.F, M.G, E.Z.; scheme administrator: D.F.; technical 
performance: T.B., E.Z., R.B., C.G., other participants listed in the Appendix; data analyses: D.F., 
E.N-O; writing of paper: D.F., M.G., T.B., L.B., R.B., E.Z., E.N-O. All the author accepted 
responsibility for the content of the submitted manuscript and approved submission. 
Research funding: This study was supported by the Italian Association of Neuroimmunology (AINI) 
and by the Italian Ministry of Health ‘Ricerca Corrente’ 2017-2019 Grant to IRCCS Mondino 
Foundation. 
Conflicts of interest: None declared. 
Employment or leadership: None declared. 
Honorarium: None declared. 
 
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Table 1 Anti-ganglioside antibody profiles as reported by the laboratories participating to the external 
quality assessment 
Lab  
Cntr #1 Cntr #2 Cntr #3 Cntr #4 Cntr #5 
qr Ag qr Ag qr Ag qr Ag qr Ag 
1 neg - neg - pos GD1b pos GD1b, GD3 GT1b, GQ1b neg - 
12 neg - pos GM1 pos GD1b pos GD1b, GD3 GT1b, GQ1b neg - 
13 neg - neg - pos GD1b GD3 GT1a pos 
GD1b, GD3 
GT1b, GQ1b neg - 
4 neg - pos GM1 GD1b pos 
GD1b, GT1b 
GQ1b pos GQ1b neg - 
6 neg - pos GM1 pos GD1b, GT1b GQ1b, GQ1b neg - neg - 
14 neg 
- 
pos GM1 GD1b nt nt nt nt pos 
GD1b, GT1b 
GQ1b 
2 neg - neg - pos GD1b pos GD1b, GQ1b neg - 
8 pos GT1a neg - pos GD1b, GT1a pos GD1b, GD3 GT1a, GT1b neg - 
3 pos GD1a GQ1b pos GM1 pos GD1b pos 
GD1b, GD1b 
GQ1b neg - 
5 neg - neg - pos GD1b, GQ1b pos GD1b, GQ1b neg - 
7 pos GQ1b neg - pos GD1b pos GQ1b neg - 
9 neg - pos GM1 pos GD1b, GD1b pos GD1b, GQ1b GD1b, GQ1b neg - 
10 neg - neg - neg - pos GD1b neg - 
11 pos GM1 pos GM1 pos GD1b, GQ1b pos GD1b neg - 
15 neg - neg - neg - pos GD1b neg - 
 
12 
 
Lab, laboratory number and type of laboratory test used: plain text, line-/dot blot (grouped by the 
same manufacturer); bold character, in-house/commercial ELISA. Results: plain text, IgG class; bold 
character, IgM class. Cntr, control sample; qr, qualitative result; Ag, antigen; nt, not tested 
 


Anti-ganglioside antibodies : experience from the Italian Association of Neuroimmunology external quality assessment scheme

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Anti-ganglioside antibodies : experience from the Italian Association of Neuroimmunology external quality assessment scheme

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