Doreen Dillaerts

Fifth Percentile Cutoff Values for Antipneumococcal Polysaccharide and Anti-Salmonella typhi Vi IgG Describe a Normal Polysaccharide Response

Background Serotype-specific antibody responses to unconjugated pneumococcal polysaccharide vaccine (PPV) evaluated by a World Health Organization (WHO)-standardized enzyme-linked immunosorbent assay (ELISA) are the gold standard for diagnosis of specific polysaccharide antibody deficiency (SAD). The American Academy of Allergy, Asthma and Immunology (AAAAI) has proposed guidelines to interpret the PPV response measured by ELISA, but these are based on limited evidence. Additionally, ELISA is costly and labor-intensive. Measurement of antibody response to Salmonella typhi (S. typhi) Vi vaccine and serum allohemagglutinins (AHA) have been suggested as alternatives. However, there are no large cohort studies and cutoff values are lacking. Objective To establish cutoff values for antipneumococcal polysaccharide antibody response, anti-S. typhi Vi antibody, and AHA. Methods One hundred healthy subjects (10–55 years) were vaccinated with PPV and S. typhi Vi vaccine. Blood samples were obtained prior to and 3–4 weeks after vaccination. Polysaccharide responses to 3 serotypes were measured by WHO ELISA and to 12 serotypes by an in-house bead-based multiplex assay. Anti-S. typhi Vi IgG were measured with a commercial ELISA kit. AHA were measured by agglutination method. Results Applying AAAAI criteria, 30% of healthy subjects had a SAD. Using serotype-specific fifth percentile (p5) cutoff values for postvaccination IgG and fold increase pre- over postvaccination, only 4% of subjects had SAD. One-sided 95% prediction intervals for anti-S. typhi Vi postvaccination IgG (≥11.2 U/ml) and fold increase (≥2) were established. Eight percent had a response to S. typhi Vi vaccine below these cutoffs. AHA titer p5 cutoffs were ½ for anti-B and ¼ for anti-A. Conclusion We establish reference cutoff values for interpretation of PPV response measured by bead-based assay, cutoff values for S. typhi Vi vaccine responses, and normal values for AHA. For the first time, the intraindividual consistency of all three methods is studied in a large cohort.

Effect of previous vaccination with pneumococcal conjugate vaccine on pneumococcal polysaccharide vaccine antibody responses

During the past 10 years, pneumococcal conjugate vaccine (PCV) has become part of the standard childhood vaccination programme. This may impact upon the diagnosis of polysaccharide antibody deficiency by measurement of anti‐polysaccharide immunoglobulin (Ig)G after immunization with unconjugated pneumococcal polysaccharide vaccine (PPV). Indeed, contrary to PPV, PCV induces a T‐dependent, more pronounced memory response. The antibody response to PPV was studied retrospectively in patients referred for suspected humoral immunodeficiency. The study population was divided into four subgroups based on age (2–5 years versus ≥ 10 years) and time tested (1998–2005 versus 2010–12). Only 2–5‐year‐old children tested in 2010–12 had been vaccinated with PCV prior to PPV. The PCV primed group showed higher antibody responses for PCV–PPV shared serotypes 4 and 18C than the unprimed groups. To a lesser extent, this was also found for non‐PCV serotype 9N, but not for non‐PCV serotypes 19A and 8. Furthermore, PCV‐priming elicited a higher IgG2 response. In conclusion, previous PCV vaccination affects antibody response to PPV for shared serotypes, but can also influence antibody response to some non‐PCV serotypes (9N). With increasing number of serotypes included in PCV, the diagnostic assessment for polysaccharide antibody deficiency requires careful selection of serotypes that are not influenced by prior PCV (e.g. serotype 8). Further research is needed to identify more serotypes that are not influenced.

Clinical autoantibody detection by microarray

Abstract Background: AMiDot is a microdot array-based immunoassay that allows simultaneous detection of multiple autoantibodies on a single patient. We evaluated the AMiDot “Systemic Autoimmune Disease” (SAD) panel, which detects antibodies to 17 different antigens. Methods: AMiDot was performed on 184 samples from blood donors and on 280 randomly selected clinical samples containing antibodies to extractable nuclear antigens or to dsDNA. The results obtained by AMiDot on the clinical samples were compared to results obtained by EliA (Thermo Fisher) for anti-Ro60, anti-La, anti-RNP, anti-Scl-70, anti-CENPB, anti-Sm, and anti-Jo-1 and by Farr assay for anti-dsDNA. Discordant results were further analyzed by immunodot (D-tek). Results: Concordance between AMiDot and EliA was ≥87% and κ agreement ≥0.44. When compared to EliA and immunodot (in case of discordance between AMiDot and EliA), concordance improved to ≥91% and κ agreement to ≥0.77. The sensitivity of AMiDot (compared to EliA and immunodot, in case of discordance between AMiDot and EliA) was ≥93%, except for anti-Ro60 (84%). The concordance and κ agreement of AMiDot with the Farr assay (for dsDNA antibodies) was, respectively, 84% and 0.33. The sensitivity of AMiDot for dsDNA (compared to Farr assay) was 25%. The specificity was ≥97% (in blood donors as well as in clinical samples). The within-run imprecision was 9%–27% and the between-run imprecision 29%–39%. Conclusions: AMiDot offers an alternative to line immunodot assay. Individual antibody assays might suffer from low sensitivity.

FRI0393 Prevalence of myositis-specific antibodies in idiopathic inflammatory myopathy compared to disease and healthy controls

Background Myositis-specific autoantibodies (MSA) are increasingly recognized as important diagnostic and prognostic markers in idiopathic inflammatory myopathies (IIM) (polymyositis, dermatomyositis, sporadic inclusion body myositis and necrotizing autoimmune myositis). The prevalence of these MSAs in other systemic autoimmune rheumatic diseases and neuromuscular diseases is unclear. Objectives To compare positivity of MSA in a cohort of IIM patients to positivity in healthy controls and different systemic autoimmune rheumatic diseases (SARD) or chronic inflammatory demyelinating polyneuropathy (CIDP). Methods A line immunoassay (Myositis 12 IgG DOT for BlueDiver) for IgG autoantibodies against Jo-1, PL-7, PL-12, EJ, SRP, Mi-2, MDA-5, TIF1-γ, HMGCR, SSA/Ro52kD, SAE1/2 and NXP-2 antigens was performed in patients with IIM (n=146), healthy controls (blood donors, n=40) and disease controls (n=200). The disease control group consisted of patients with other SARD (rheumatoid arthritis, RA; systemic sclerosis, Ssc; Sjögrens syndrome, SjS; and systemic lupus erythematosus, SLE) (n=40 for each disease group) and CIDP (n=40). A result >10 arbitrary units was considered significantly positive. Results 50% of 146 patients with IIM tested positive for an MSA (table 1), compared to 3,5% of 200 disease controls (table 1). 1 SSc patient was positive for Jo-1, 1 CIDP patient was positive for PL12, 2 SSc patients were positive for TIF1-gamma, 2 patients (1 SSc and 1 SjS patient) were positive for SAE1/2, and 1 SjS patient was positive for NXP2. The prevalence of SAE1/2 and TIF1-gamma positivity was similar in the IIM and disease control group. No healthy control had a significantly positive MSA. Antibody detected IIM Disease controls Healthy controls (n=146) (n=200) (n=40) 1 positive (>10 AU) MSA detected 68 (47%) 7 (3,5%) 0 (0%)  Jo-1 28 (19%) 1 (0,4%) 0 (0%)  PL12 3 (2%) 1 (0,4%) 0 (0%)  PL7 1 (1%) 0 (0%) 0 (0%)  EJ 2 (1%) 0 (0%) 0 (0%)  Mi-2 7 (5%) 0 (0%) 0 (0%)  MDA-5 7 (5%) 0 (0%) 0 (0%)  SAE1/2 3 (2%) 2 (0,8%) 0 (0%)  TIF1-gamma 2 (1%) 2 (0,8%) 0 (0%)  NXP2 5 (3%) 1 (0,4%) 0 (0%)  SRP 3 (2%) 0 (0%) 0 (0%)  HMGCR 7 (5%) 0 (0%) 0 (0%) >1 positive (>10 AU) MSA detected 4 (3%) 0 (0%) 0 (0%)  Jo-1 + EJ 1  Jo-1 + NXP2 1  SAE1/2 + NXP2 1  Jo-1 + SAE1/2 + NXP2 1 Conclusions MSA positivity in patients with a clinical non-IIM diagnosis (other SARD or CIDP) is infrequent compared to positivity in the IIM group. For TIF1-gamma and SAE1/2 assay performance may need to be optimized. The distribution of subtypes of MSA in this IIM cohort is consistent with data of previous studies.1 References Allenbach Y, Benveniste O. Diagnostic Utility of Autoantibodies in Inflammatory Muscle Diseases. J Neuromuscul Dis. 2015; 2:13–25. Disclosure of Interest None declared