Diagnostics of paraneoplastic neurological syndromes

Abstract

This document presents the guidelines for onconeural antibody testing 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 paraneoplastic neurological syndromes, indications and limits of onconeural antibody testing, instructions for result interpretation, and an agreed laboratory protocol (Appendix) are reported for the communicative community of neurologists and clinical pathologists.

Appendix

• 1.0 Preanalytical procedures

Refer to the document on “Diagnostics of autoimmune encephalitis associated with antibodies against neuronal surface antigens”

• 2.0 Analytical procedures

  • 2.1 Indirect immunofluorescence. Procedures refer to commercial BIOCHIP Slides by Euroimmun (Lübeck, Germany).

    • 2.1.1 Reagent preparation

      • 2.1.1.1 BIOCHIP Slides with monkey cerebellum are ready for use and sealed in a package that must be opened when room temperature has been reached (18–25 °C) to prevent condensation. Do not touch the biochip.

      • 2.1.1.2 Fluoresceinated goat anti-human Ig antibody (mix thoroughly before use).

      • 2.1.1.3 Mounting medium.

      • 2.1.1.4 Negative and positive control sera.

      • 2.1.1.5 PBS-Tween Wash Solution: dissolve 1 package of “phosphate buffer salt” in 1 l of high-grade distilled water, add 2 mL of Tween 20, and stir for at least 20 min; store in a refrigerator (2–8 °C) for 1 week.

      • 2.1.1.6 Glassy reagent supports: thorough rinses with water and accurate drying are recommended after washing (see the instruction manual).

    • 2.1.2 Sample preparation

      • 2.1.2.1 Vortex the samples after thawing. Dilute serum samples 1:10 in PBS-Tween 20 (11 μL of sample in 100 μL of PBS-Tween 20) and mix thoroughly (vortex). CSF samples are tested undiluted.

      • 2.1.2.2 For titration, go on the 1:10 dilution (1:100, 1:1000, 1:10,000, up to the end-point titration).

    • 2.1.3 Analytical procedure

      • 2.1.3.1 Dispense 25 μL of each diluted serum or undiluted CSF sample on the well of the glassy tray, preventing the formation of air bubbles, and using the polystyrene frame as a reference.

      • 2.1.3.2 Dispense all the samples before starting the incubation.

      • 2.1.3.3 Start the incubation after putting the transfected-cell-equipped biochips onto the glassy tray, checking that each sample is in contact with the biochip, and preventing between-sample cross-contaminations; incubate 30 min at room temperature (RT; 18–25 °C).

      • 2.1.3.4 Washing: Immerse BIOCHIP Slides into 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). Be careful not to completely dry the slides (to prevent faded patterns with or without high background).

      • 2.1.3.5 Dispense 25 μL of conjugated antibody to each well of the clean glassy tray.

      • 2.1.3.6 Remove BIOCHIP Slides from the washing cuvettes, rapidly dry the back and sides of the slides with absorbent paper, and then place then in the appropriate glassy tray areas. Check the contact between biochips and antibody. Incubate for 30 min at RT.

      • 2.1.3.7 Repeat washing (point 2.1.3.4), using fresh PBS-Tween 20.

      • 2.1.3.8 Mount BIOCHIP Slides, placing the cover slides on the polystyrene supports and dispensing a drop of mounting medium (10 μL/well). Check the perfect engagement between biochip slides and cover slides.

    • 2.1.4 Reading and interpretation of results

Use fluorescence microscopes (excitation filter, 450–490 nm; color separator, 510 nm; blocking filter, 515 nm) with 20×-40× magnifications. Slides are interpreted blindly by two expert observers and defined as “Positive” or “Negative”; when the interpretations are discordant, repeat the test.

Describe how fluorescence distributes in the cerebellar tissue at various levels: neuropil, cell layers, myelinated fibers, nuclei, cytoplasm (pictures at www.euroimmun.com). As for the most common reactivities, anti-Yo antibodies stain exclusively the cytoplasm of Purkinje cells, anti-Hu and anti-Ri antibodies neuronal nuclei in the gray matter with granular fluorescence. AINI network of laboratories is available to evaluate and retest dubious samples.

• 2.2 Dot/line blot. Commercial kits are available for confirming the presence of the main onconeural autoantibodies. Recombinant proteins are fixed onto nitrocellulose stripes. Human IgG is the internal control antigen. Neuronal antigens are typically purified by affinity, separated with SDS-PAGE, and immobilized onto nitrocellulose. Autoantibodies are detected with an indirect immunoenzymatic reaction. Procedure: follow the manufacturer’s instructions.

• 2.3 Western blot. In-house Western blot of cerebellum tissue homogenates can be used as an alternative to commercial dot/line blot blots. The rational use of in-house Western blot can be restricted to the analysis of serum samples resulted positive on the immunohistochemistry screening and negative on commercial dot/line blot blots (if ANA negative).

• 3.0 QUALITY CONTROL AND SAMPLE STORAGE

  • 3.1 Indirect immunofluorescence

    • 3.1.1 In every analytical run a positive and a negative control should be used.

    • 3.1.2 If the positive control gives no/dubious staining, and the negative control non-specific or high-background staining, repeat the test.

    • 3.1.3 External quality control schemes should be performed at least yearly (e.g., AINI external quality control schemes).

  • 3.2 Dot/line blot

    • 3.2.1 In every analytical run a positive control should be included.

    • 3.2.2 External quality control schemes should be performed at least yearly (e.g., AINI external quality control schemes).

  • 3.3 Storage, see the document on ‘Cerebrospinal fluid analysis and the determination of oligoclonal bands’

• 4.0 REPORT

  • 4.1 Indirect immunofluorescence. A qualitative result (positive/negative) should be reported, together with a detailed description of the staining localization (see point 2.1.3). Titering is optional, but advisable.

  • 4.2 Dot/line blot. A qualitative result (positive/negative) should be reported, together with the indication of the type of reactivity (e.g., anti-Hu antibodies).

  • 4.3 Reports should contain the following general information:

    • i) Type of method: indirect immunofluorescence, type of tissue and name of the manufacturer.

    • ii) Type of method: dot/line blot, name of the manufacturer.

    • iii) Reference values: report the dilution as cut-off of positivity.

    • iv) Comments: Refer to point 4.1.9 of the document on “Cerebrospinal fluid analysis and the determination of oligoclonal bands.”