Introduction

Recommendations for ISCEV-approved protocols for electroretinography in clinical trials

•1. Preamble This document describes modules, protocols and sequences of modules for full-field (Ganzfeld) electroretinography (ERG) for the special application as a standardized test in clinical trials, in order to improve comparability between recordings from different sites, especially in multicenter trials. It provides globally harmonized recommendations that shall help to exploit the full power of electroretinography by providing a multitude of modules for specific functional targets (e.g. OFF-channel, S-cones etc.), whose function can be modified by specific compounds. The modules can be individually selected and -if properly combined- serve as backbone for a Standard Operating Procedure in a clinical trial that assesses retinal function by ERG. ISCEV-approved protocols do not prevent any scientist responsible for a clinical trial from developing or extending his own ERG- protocol as a basis for a SOP to be applied in a certain study. If approval by ISCEV is desired, the modular structure and the open numbering of the modules provides for additional protocols for modules with specific purposes or applications to particular patient groups or expected drug actions. Specialists in clinical electroretinography are encouraged to complement the list of ISCEV-approved protocols as an ongoing process of further developing the application of electrophysiology in clinical trials on a globally harmonized scale. To put it simply: ISCEV-approved protocols for individual modules are a series of "recipes" that can be selected freely. However, only if adopted by ISCEV t can and -if used- shall they be called "ISCEV-approved protocol No. xx"; ISCEV might add the name of a disease, a drug or a person for easier identification of a particular module/protocol.

2. Purpose

a. Modules The purpose of an ISCEV Module is to specify parameters, settings of stimulation and recording as well as data evaluation procedures for certain purposes, patient populations or compounds in clinical trials, in order to complement the ISCEV standard by addition of modules that allow recording of particular retinal responses (such as OFF and S-Cone responses) or functional alterations ( e.g. by "submicrovolt recordings") that are not part of the standard itself. The list of modules (by Sept.1, 2008), examples of which are shown in fig.1, is as follows

ISCEV Standard modules (*), according to 2008 standard 1: Scotopic 0.01 ERG 5: Photopic 3.0 ERG 2: Scotopic 3.0 ERG 6: Photopic 3.0 flicker 3: Scotopic 3.0 oscillatory potentials 4: Scotopic 10 ERG New modules (being developed by ISCEV) 11: ROD b-wave series 31: Cone b-wave series 41: Focal ERG 12: ROD b-wave V-log(I) 32: S-Cone 13: a-wave analysis 33: ON-OFF response 14: small amplitude ERG 34: Photopic negative response 15: recovery after bleaching 41: Photopic Frequency series

(*) ISCEV Standard for clinical electroretinography (2008 update). Marmor MF, Fulton A, Holder GE, Miyake Y (Chair), Brigell M, Bach M (for the International Society for Clinical Electrophysiology of Vision).

b. Protocols

Protocols are documents that precisely describe each of the modules (presently under development by a small group of ISCEV members appointed for each module at Morgantown meeting). ISCEV has approved a template for the general structure of a protocol which includes: 1. Scope To be defined in all modules 2. Identification 2.1. Number 2.2. Version number 2.3. Date of approval) To be defined in all modules 3. Protocol use(s) 3.1 Patient population To be defined in all modules 4. Technical issues 4.1. Equipment and facilities 4.2. Staff expertise and responsibilities 4.3. Procedure 4.4. Patient Information Include definitions that are not exactly defined in the ISCEV Standard or in the section "general definitions" of this document 5. Response evaluation (with example) To be defined in all modules 6. Reporting and storage Include definitions that are not exactly defined in the ISCEV Standard or in the section "general definitions" of this document 7. Calibration Include definitions that are not exactly defined in the ISCEV Standard or in the section "general definitions" of this document 8. Protocol Specifications To be defined in all modules 9. Relevant References To be defined in all modules

c. Module sequences

After modules have defined, ISCEV will propose sequences of modules to be used in clinical trials' protocols, as shown in fig.1, bottom; the sequences are named A, B, C.... and are designed such as to minimize the effect of preceeding modules on the response of a certain module, with respect to state of adaptation and strength of preceeding light stimulation. Module sequences will be defined in a matrix including all modules defined in the ISCEV standard as well as new modules that are defined by the ISCEV committees.

3. General Definitions

Technical aspects

The correct handling of medical products (MP) according to national, international and European laws and guidelines is the responsibility of a trained and qualified Medical Device Commissioner.

Electronic recording equipment

The band pass of the amplifier and preamplifiers should include at least the range of 0.1 to 1000 Hz and must be adjustable according to the different protocols given in the appendix. The equipment must allow for a sampling frequency of 1 kHz with a effective size of one least significant bit corresponding to no more than 2 µV, corresponding to a 1 µV uncertainty in the 0-250Hz range for a single response and giving a +- 4mV dynamic range with a 12 bit A-to-D converter. The input impedance of the preamplifiers should be at least 100 MΩ. Amplifiers should generally be capable of handling offset potentials that may be produced by the electrodes.

Amplifier Calibration

The gain of modern amplifiers is normally set by reliable components whose values, barring accidents, are unlikely to change appreciably over time. However, it is still desirable for the sensitivity of ERG recording equipment to be routinely and frequently verified. The sensitivity of recording equipment as well as its frequency response is most simply verified by recording square-wave stimuli of appropriate amplitude.

Light sources

The use of LED arrays as light-generating units is highly recommended. The protocols listed in the appendix state distinct settings for colour, flash intensity and flash duration, which are most easily generated by LED arrays. Xenon discharge lamps may be used especially when strong light stimuli are required. Incandescent sources may warrant frequent calibrations and therefore must be avoided.

All stimuli should be presented on a Ganzfeld bowl or dome with diffuser that is visibly white. Background illumination has to be even and steady, both for luminance and spectral distribution.

The light source for white stimuli and background should have a color temperature of 6500 K. For other than Xenon discharge lamps the spectral distribution must be documented.

For colored stimuli and backgrounds the peak wavelength and spectral half-width of the energy distribution must be documented for the used device. Spectral stability over the range of stimuli indicated in the protocols (appendix) must be given.

Standard flash luminance according to ISCEV standard is set here to 3.0 (+/- 10%) photopic cd.s/m² at surface of Ganzfeld bowl and must not be lower than 7.5 scotopic cd.s/m2.

The luminance, duration, pulse form, pulse sequence and pulse color of stimuli should follow exactly the protocols given in the appendix. The variability of the stimulus luminance must be documented.

Specifications of the light stimuli duration

The important requirement is that they should be brief compared to the earliest time after their delivery that a measurement of response amplitude is to be made. It is important that the appropriate mid-energy time be taken as time zero. The maximum permissible duration of the stimulus (except for ON-OFF responses) should be 10 ms and the light flashes should be rectangular in time and follow the specifications given in the protocols. These specifications describe amongst others all relevant flash parameters like flash duration, intensity, color and flash sequence.

Specifications of the recordings

The band pass filter to be used for individual recordings is given in the protocols in the appendix. They may vary between protocols and steps. In general a line filter (notch filter) should be avoided, because they can cause loss of waveform information, since part of the waveform spectrum is affected. Electrode leads should be as short as possible and kept away from any electrical equipment or power lines; twisting the electrode leads together can often reduce noise. With these precautions, electrical noise due to radio frequency equipment will ordinarily be within acceptable limits. Additionally, power transformers produce powerful electromagnetic fields (primarily third harmonic e.g. 150 Hz for 50 Hz and 180 Hz for 60 Hz power sources respectively) that can induce or couple power line interference into the recordings. The closer the patient and the equipment are to these sources, the more interference will be introduced into the recording equipment.

Specifications of the recording sequence

Individual stimuli should not be applied in random order, nor should only selected stimuli of one of the protocols be used. The protocols given in the appendix specify the sequence and the time lag of consecutive stimuli, which have to be performed in that order.

Recorded responses (conditions per eye and per stimulus)

A series of individual sweeps will be recorded for each condition. During recording, sweeps that are obviously heavily disturbed by artefacts may be rejected and another sweep may be taken. Individual sweeps will then be averaged and the whole procedure repeated immediately. Thus the reproducibility of each average will be estimated and if not sufficient, additional averaged results will be recorded until two satisfactorily similar averaged results are obtained for each condition.

If there is a satisfactory reproducibility between the two (or more) averages, the grand average of all averages is calculated immediately. The grand average is the average of individual averages weighted according to the number of sweeps of each average.

The individual sweeps, the two averages as well as the grand average should be displayed and stored in order to be assessable by the central reader.

Data Analysis

Some recordings require offline analysis like Fourier spectrum analysis or Fourier frequency filtering and reconstruction. The respective specifications are given in the protocols in the appendix.

When analyzing data, especially data that underwent Fourier analysis, appropriate statistical means have to be applied to estimate the significance of the extracted signal in relation to underlying noise.

Procedure

Pupil dilatation

Pupils must be fully dilated; this is achieved by instillation of one drop (plus one drop after 1 minute) of mydriatic in both eyes.

Electrode positioning and dark adaptation

DTL fibers are the recommended electrodes for electroretinography in clinical studies, due to patient compliance, single use hygiene and availability of large data bases for age matched normal values. The DTL-fibre needs to be positioned along the lower lid rim in relation to the lower limbus of the cornea. Other electrodes may be used in individual studies if all partners agree and age matched norms are available for all instruments in each center.

Electrode positioning can be performed before or during dark adaptation. If performed during dark adaptation, it must be done under dim red light illumination, and the technical assistant must have experience in performing this procedure at these low illumination conditions.

The reference electrodes should be placed on the zygomatic fossae bilaterally, for right and left eyes respectively, and the ground electrode placed on the central forehead.

Dark adaptation is achieved by positioning the patient comfortably for 20 minutes in a completely dark room with an additional 5 minutes after electrode placement.

The use of topical anesthesia is not necessary but may be applied if the patient cannot tolerate the electrodes. It must be checked that fiber electrodes are always at or just above the rim of the lower lid and do touch the cornea.

Impedance checking

After dark adaptation impedance must be checked, and must be lower than 10 KΩ measured between 10 and 100 Hz (or by use of the ERG machine built-in impedance meter). In case of differences between eyes > than 30% electrodes must be repositioned under dim red light.

Positioning the patient

The patient should be positioned comfortably on the Ganzfeld chin rest. The height of the chin rest, stool and table should be adjusted for patient size. Ensure that the Ganzfeld bowl does not touch the patient or the electrode support.

The patient should be instructed to look towards the fixation light, which is used on its lowest intensity setting, depending on the background intensity. Patient fixation is monitored, if possible, using an infra-red (IR) camera system within the bowl. Dark-adapt the patient for a further 5 minutes.

Control the ERG computer's screen illumination

Curtains should be used to cover the subject from the instrument lights and screens if it is in the same room. If it is unavoidable that the patient and operator cannot be screened off from each other, screen illumination should be reduced to the minimum required for operating comfortably once the operator has dark-adapted. If it is necessary for the operator to leave the room during dark- adaptation, then the patient should be asked to close their eyes whilst this happens, and also whilst the operator re-enters the room. Preferably a black curtain should shield the door. The outside illumination should also be reduced to allow minimum light contamination during re-entry.

While recording

The patient should be told to keep their jaw and face relaxed, and to avoid clenching their teeth. Their eyes should always be open and looking at the fixation light in the Ganzfeld bowl. Gentle blinking can be suggested between flashes.

The technical assistant must inform the patient in cases of major changes of background luminance (i.e. before light adaptation or bleaching) to avoid unexpected abrupt reactions.

After recording

After the ERG protocol is finished: Check if channels 1 and 2 are correctly related to the documented eyes. Measure and document pupil diameter.

Reporting

The amplitudes of the a- and b-waves and their implicit times must be reported in µV and ms respectively for all steps (note that the a-wave is normally not recordable in stimuli with luminance lower than that used for the dark-adapted rod b-wave stimulus of 0.01 cd.s/m²).

a-wave : cursor A must be positioned at the deepest point of the negative deflection; in case of a double peaked a-wave conformation, the first trough will be marked even if not the deepest, if the slope after the first transition point is positive (see diagram, right). This does not apply if there is a shoulder or similar characteristic where there is no trough and the slope continues to be negative (see diagram, left).

b-wave: cursor B must be positioned at the peak of the subsequent positive deflection; if there is no negative deflection (i.e. rod response) the first cursor will be set at the baseline just before the start of the positive deflection. Baseline is defined as the mean of 20 ms pre-trigger time with 50Hz mains and 17 ms for 60 Hz mains (considering 1 kHz sampling frequency).

4. Literature for further reference concerning ERG in Clinical Trials