Diagnostics of autoimmune encephalitis associated with antibodies against neuronal surface antigens

Abstract

This document presents the guidelines for testing antibodies against neuronal surface antigens that have been developed following a consensus process built on questionnaire-based surveys, internet contacts, and discussions at workshops of the sponsoring Italian Association of Neuroimmunology (AINI) congresses. Essential clinical information on autoimmune encephalitis associated with antibodies against neuronal surface antigens, indications and limits of testing for such antibodies, instructions for result interpretation, and an agreed laboratory protocol (Appendix A) are reported for the communicative community of neurologists and clinical pathologists.

Appendix

• 1.0 Preanalytical procedures

• 1.1 Blood is collected in tubes without anticoagulant. Fasting samples are required. No necessity of food restrictions.

• 1.2 Blood sample is centrifuged, after clot formation, as soon as possible (3000 g for 10 min).

• 1.3 Serum samples are tested after centrifugation, or stored in aliquots at −20 °C until analysis (1 month, or preferably at −80 °C for longer periods).

• 1.4 Frozen serum samples should not be thawed and frozen again.

• 1.5 Grossly hemolyzed or lipemic samples should be discarded. Centrifuge the serum prior to assay to remove any particulate matter.

• 1.6 Plasma samples are allowed.

• 1.7 In the case of CSF testing, refer to the document on cerebrospinal fluid analysis and the determination of oligoclonal bands.

• 2.0 Analytical procedures

• 2.1 Cell-based assay. The commercial CBA provided by Euroimmun is currently the only available standardized test. Differently from in-house tests, for which the research purpose is specified in the report, standardized tests allow a formally valid use of diagnostic results.

• 2.1.1 Preparation of reagents

• 2.1.1.1 Slides on which transfected cells are attached are ready for use and sealed in a sachet that should be opened when it has reached room temperature (18–25 °C) to avoid condensation. If the sachet is damaged, slides are unusable.

• 2.1.1.2 Secondary antibody: shake before use and protect from direct light.

• 2.1.1.3 Washing solution: dissolve the buffer with distilled water, add Tween-20 and shake for at least 20 min (for assays performed after more than 1 week, a fresh washing solution must be prepared at each analytical session, in appropriate volumes).

• 2.1.2 Preparation of samples

• 2.1.2.1 Dilute 1:10 serum in PBS-Tween 20 (11 μL of sample in 100 μL of PBS-Tween-20). CSF should be tested undiluted. Shake the samples with vortex after thawing.

• 2.1.2.2 For sample titration, continue with dilutions of 1:10 (1:100 to 1:1000 to 1:10,000 and so on, until titration has been reached).

• 2.1.3 Analytical procedure

• 2.1.3.1 Method

• i) Dispense 30 μL of previously diluted serum or undiluted CSF in the well of the glass holder, avoiding bubble formation, using the polystyrene base as a reference.

• ii) Dispense all the samples of the series before starting the incubation.

• iii) Initiate the incubation by placing the biochips with transfected cells on the glass support, ensuring that the serum is in contact with the biochip and avoiding cross-contamination; incubate for 30 min at room temperature.

• iv) Washing: immerse the slides with biochips in a beaker containing PBS-Tween-20, and then immerse them in the appropriate PBS-Tween-20 containing cuvette for 5 min (if available, gently shake with a rotary stirrer).

• v) Dispense 30 μL of conjugated antibody (fluorescein anti-human IgG antiserum) in the wells.

• vi) Remove the biochip slides from the cuvettes one at a time and quickly dry the back and sides of the slide with absorbent paper, then immediately place it in the appropriate slots of the glass holder. Check that the biochip and conjugated antibody are in contact.

• vii) Incubate for 30 min at room temperature, in the dark.

• viii) Repeat the washing as in point iv, using fresh PBS-Tween-20.

• ix) Mount the slides with the biochip, placing the cover slides on the polystyrene support and place a drop (10 μL per well) of mounting liquid (glycerol/PBS) on each slide. Take out a slide at a time and dry the back and four sides with absorbent paper. The biochip slides must be delicately placed on the cover and slot in perfectly.

• 2.2 Observation and interpretation of results. Use fluorescence microscopes (excitation filter, 450–490 nm; color separator, 510 nm; blocking filter, 515 nm), with ×20 and ×40 magnifications. Results are interpreted blindly by two independent observers and defined as “positive” or “negative”; when interpretations differ, repeat the test. When interpreting results, keep in mind the general principle that the distribution of fluorescence across all the cells present on the biochip indicates a false positive since, by definition, transfection is always incomplete; therefore, fluorescence is distributed on only one part of the transfected cells in positive samples. Typically, the surface reactivity draws the outline of the cells, which is generally irregular, and extends along the cellular processes. Cell bodies are generally poorly fluorescent. Broadly homogeneous reactivity is likely non-specific and generated by apoptotic cells. Using biochips of non-transfected cells as controls help to rule out false positive results (in addition to using control samples from well-known healthy seronegative subjects). The AINI laboratory network is willing to evaluate doubtful results.

• 3.0 Quality control and sample storage

• 3.1 CBA

• 3.1.1 A positive internal control and a negative internal control should be included in each analytical session.

• 3.1.2 If the positive control does not produce the expected result, or if the negative control unexpectedly shows positive fluorescence, the entire analytical session must be repeated.

• 3.1.3 An external quality control scheme should be planned at least annually (e.g., AINI external quality control schemes).

• 3.2 Storage, see the document on cerebrospinal fluid analysis and the determination of oligoclonal bands.

• 4.0 Report

• 4.1 The following information should be reported:

• i) Type of sample (serum, CSF), with indication of the dilution.

• ii) Commercial CBA (first level): type of the test, and manufacturer (Euroimmun, Lübeck, Germany); in-house tests (second level): type of test (specify that the test is performed for research purpose).

• iii) Presence or absence of the detected NS-Ab, with the indication of the sample dilution (serum; CSF), and titer (if performed).

• iv) Comments: refer to the document on cerebrospinal fluid analysis and the determination of oligoclonal bands.