The levels of evidence (class I-IV) supporting the recommendations and ratings of recommendations (A-C) are defined at the end of the "Major Recommendations" field.
Measurements of Binding and Neutralizing Antibodies
Binding Antibodies
There are no existing recommendations on binding antibody (BAB) assays. There is class I evidence that interferon-beta (IFN-beta) BAB assays have a very high sensitivity and specificity, and can be reliably used for IFN-beta antibody screening before performing a neutralizing antibody (NAB) assay (Level A recommendation). Different BAB assays should be evaluated and compared using a large number of serum samples in order to identify the method with the best sensitivity and specificity for NAB detection (Level B recommendation).
Neutralizing Antibodies
Measurements of binding and neutralizing antibodies against IFN-beta should be performed in specialized laboratories (Level A recommendation). Measurement of NABs with a validated cytopathic effect (CPE) assay is still the gold standard. It is recommended that A549 cells are used with a fixed amount of IFN-beta (the preparation used by the patient) for stimulation and serial dilution of the test sera. The stimulated cells can either be challenged with encephalomyocarditis (EMC) viruses or MxA production determined. Standard curves should be obtained using increasing amounts of IFN-beta until saturation is reached. The NAB titre should be calculated using the Kawade formula (Level A recommendation).
Titres above 20 to 60 (depending on the IFN-beta preparation used in the assay) are associated with a loss of IFN-beta bioactivity (class I evidence). As the European Medicines Association (EMEA) currently validates a NAB assay based upon the MxA production of A549 cells (MxA induction assay), it is recommended to use the EMEA protocol. (This recommendation is only based on class IV evidence, but consensus was reached to offer this advice as good practice.) Validation of simpler NAB assay methods is strongly recommended such as the in vivo biological response to IFN-beta administration (Level A recommendation).
Clinical Use of Measurements of Antibodies against IFN-beta
It is recommended that patients treated with IFN-beta are tested for the presence of NABs at 12 and 24 months of therapy (Level A recommendation). Measurements of NABs can be discontinued in those patients remaining NAB-negative during this period but should be resumed if disease activity increases (Level B recommendation). There is class I evidence that the presence of NABs significantly hampers the effect of IFN-beta on the relapse rate and on both active lesions and burden of disease seen on magnetic resonance imaging (MRI). In patients with NABs, NAB measurements should be repeated at intervals of 3 to 6 months and therapeutic options should be re-evaluated (Level A recommendation). Therapy with IFN-beta should be discontinued in patients with high titres of NABs (e.g., titres >100 in patients using IFN-beta-1b) sustained at repeated measurements with 3- to 6-month intervals (Level A recommendation).
Prevention and Treatment of NABs
Limited evidence is available on managements that reduce NAB formation to IFN-beta in multiple sclerosis (MS). Monthly 1 g intravenous (i.v.) methylprednisolone (MP) administration has been revealed to be safe and able to minimize the formation of NABs over time (Level C recommendation). However, no effect has been observed in reducing the amplitude of NABs titres once NABs have been formed. Further studies are warranted to strengthen these results and to expand the knowledge in such an intriguing matter.
Principal Recommendations Regarding Measurements of Antibodies against IFN-beta and the Clinical Use of NAB Measurements
- BAB assays can be reliably used for IFN-beta antibody screening before performing a NAB assay (Level A recommendation).
- Measurements of binding and neutralizing antibodies against IFN-beta should be performed in specialized laboratories (Level A recommendation).
- Measurement of NABs should be performed with a validated CPE assay or MxA production assay using serial dilution of the test sera. The NAB titre should be calculated using the Kawade formula (Level A recommendation).
- Tests for the presence of NABs should be performed at 12 and 24 months of therapy (Level A recommendation).
- Measurements of NABs can be discontinued in those patients remaining NAB-negative during this period but should be resumed if disease activity increases (Level B recommendation).
- In patient with NABs, measurements should be repeated after 3 to 6 months (Level A recommendation).
- Therapy with IFN-beta should be discontinued in patients with high titres of NABs sustained at repeated measurements with 3- to 6-month intervals (Level A recommendation).
Definitions:
Evidence Classification Scheme for a Diagnostic Measure
Class I: A prospective study in a broad spectrum of persons with the suspected condition, using a "gold standard" for case definition, where the test is applied in a blinded evaluation, and enabling the assessment of appropriate tests of diagnostic accuracy
Class II: A prospective study of a narrow spectrum of persons with the suspected condition, or a well-designed retrospective study of a broad spectrum of persons with an established condition (by "gold standard") compared to a broad spectrum of controls, where test is applied in a blinded evaluation, and enabling the assessment of appropriate tests of diagnostic accuracy
Class III: Evidence provided by a retrospective study where either persons with the established condition or controls are of a narrow spectrum, and where test is applied in a blinded evaluation
Class IV: Any design where test is not applied in blinded evaluation OR evidence provided by expert opinion alone or in descriptive case series (without controls)
Evidence Classification Scheme for a Therapeutic Intervention
Class I: An adequately powered prospective, randomized, controlled clinical trial with masked outcome assessment in a representative population or an adequately powered systematic review of prospective randomized controlled clinical trials with masked outcome assessment in representative populations. The following are required:
- Randomization concealment
- Primary outcome(s) is/are clearly defined
- Exclusion/inclusion criteria are clearly defined
- Adequate accounting for dropouts and crossovers with numbers sufficiently low to have minimal potential for bias
- Relevant baseline characteristics are presented and substantially equivalent among treatment groups or there is appropriate statistical adjustment for differences
Class II: Prospective matched-group cohort study in a representative population with masked outcome assessment that meets a–e above or a randomized, controlled trial in a representative population that lacks one criteria a–e
Class III: All other controlled trials (including well-defined natural history controls or patients serving as own controls) in a representative population, where outcome assessment is independent of patient treatment
Class IV: Evidence from uncontrolled studies, case series, case reports, or expert opinion
Rating of Recommendations for a Diagnostic Measure
Level A rating (established as useful/predictive or not useful/predictive) requires at least one convincing class I study or at least two consistent, convincing class II studies.
Level B rating (established as probably useful/predictive or not useful/predictive) requires at least one convincing class II study or overwhelming class III evidence.
Level C rating (established as possibly useful/predictive or not useful/predictive) requires at least two convincing class III studies.
Rating of Recommendations for a Therapeutic Intervention
Level A rating (established as effective, ineffective, or harmful) requires at least one convincing class I study or at least two consistent, convincing class II studies.
Level B rating (probably effective, ineffective, or harmful) requires at least one convincing class II study or overwhelming class III evidence.
Level C rating (possibly effective, ineffective, or harmful) requires at least two convincing class III studies.
