The diagnostic accuracy of photopic negative responses evoked by broadband and chromatic stimuli in a clinically heterogeneous population

Routine electrophysiological assessment of optic nerve and macular retinal ganglion cell (RGC) function often involves cortical visual evoked potential (VEP) and pattern electroretinogram (PERG) methods, performed according to well-established standardised protocols [1‐3]. There is increasing interest in use of the photopic negative response (PhNR) to assess global [4‐7] and focal [8‐11] RGC function. The PhNR can be evoked with many different flash strength and wavelength combinations including white flashes on a white background (WW PhNR) and red flashes on blue background (RB PhNR). The International Society for Clinical Electrophysiology of Vision (ISCEV) extended protocol for the PhNR recommends the use of RB stimuli as studies have reported that this yields a larger amplitude response than broadband (WW) stimuli [12‐14], with a few exceptions [15‐17]. Diagnostic accuracy relates to the ability of a test to discriminate between health and a target condition [18], and previous studies have compared the diagnostic accuracy of different PhNR stimuli using a case–control methodology [14‐16, 19‐22], with most focussed on the diagnostic and prognostic potential in glaucoma [23‐28]. There is a lack of published studies that compare the relative diagnostic accuracy of RB and WW PhNR stimuli without prior knowledge of the diagnosis in a heterogeneous clinical population.

This study investigates the diagnostic accuracy of WW and RB PhNRs, compared with a test battery of clinical tests routinely used in the diagnosis of retinal ganglion cell disease (our target condition). The aim was to test whether WW PhNR stimuli, used routinely to record the ISCEV standard light-adapted full-field (LA 3) ERG [29], are a suitable alternative to RB stimuli for the detection of retinal ganglion cell dysfunction.

This was a prospective, paired diagnostic accuracy study conducted at Moorfields Eye Hospital, London, UK. Ethics committee approval was granted by UK National Research Ethics Committee Wales 6 (reference: 20/WA/0300). All eligible adult (18 +) patients attending the Department of Electrophysiology within the recruitment window were identified through the triage of outpatient referrals and invited to participate in the study.

Each participant underwent examinations and electrophysiology according to routine clinical management with an additional PhNR protocol added at the end of electrophysiological testing. Participants were excluded from the study if they met any of the following criteria:

This study examines the diagnostic accuracy of PhNRs in the largest patient cohort to date, by comparison with multi-modal assessments of optic nerve structure and standardised electrophysiological tests of function. Uniquely, the diagnostic accuracy of PhNRs evoked by chromatic and broadband stimuli is compared in a heterogeneous rather than a case-controlled clinical population, providing a robust estimation of sensitivity and specificity more applicable to the general patient population. The potential of using a widely available ISCEV standard full-field ERG protocol to assess retinal ganglion cell function is examined.

This study showed that PhNR amplitudes are larger when elicited by the ISCEV-recommended RB [12] rather than ISCEV standard (LA 3 ERG) WW [29] stimuli, consistent with several previous comparisons of chromatic and broadband stimuli [14, 19]. It has been suggested that a chromatic stimulus may preferentially stimulate a single subtype of cone, reducing the amount of spectral antagonism in the receptive fields of the RGCs [18]. It is noted, however, that PhNR amplitudes are only minimally influenced by the chromaticities of the flash and background when stimuli are expressed in photopic photometric terms [15, 36] and photopically matched, with one report that RB are larger than WW PhNRs only at higher flash strengths (3 phot cd·s·m⁻²). In the present study, the statistically significant amplitude difference between PhNRs seen in those without RGC pathology was not apparent in patients with RGC dysfunction. The possibility that dysfunction disproportionately attenuates chromatic-evoked responses and that this may be related to disease type and severity warrants further investigation.

The study shows that the RB PhNR has a higher sensitivity than the WW PhNR for the detection of RGC dysfunction (62% vs 53%) and that this difference is statistically significant. The confidence intervals of the difference between the sensitivities (95% CI − 17% to − 1%) further support this finding as the range does not encompass zero [37]. These findings suggest that the RB PhNR stimulus is better able to detect RGC pathology than the WW stimulus in a heterogeneous clinical population. Estimations of specificity did not significantly differ for the two stimuli (78% and 80% for RB and WW), suggesting that both methods can identify unaffected individuals to a similar degree. The findings suggest that the RB PhNR is more likely to detect RGC disease in affected individuals, but as the specificities of the PhNR stimuli are equivalent, a positive finding in the WW PhNR is just as likely to be a true positive as a positive result from the RB PhNR. This is an important finding in the context of clinical practice, as RB PhNR protocols are not fully standardised and are less widely available than WW PhNRs, as the latter form part of the ISCEV standard full-field LA 3 ERG, used routinely to assess retinal function.

In our cohort, the PhNR:b-wave ratio lowered the sensitivity of the test when compared with the PhNR amplitude measure. Perhaps not surprisingly, specificity increased as the ratio takes into account ERG amplitude variability and the possibility of ERG b-wave attenuation, e.g. due to retinal (non-RGC) pathology. In support of this, the specificity of both measurements was equivalent when cases with an abnormal LA 3 ERG a- and b-wave were excluded from the comparison, highlighting the importance of considering retinal function and light-adapted ERG a- and b-waves when interpreting the PhNR clinically.

There were no significant differences in the ROC AUCs of WW and RB PhNRs measured with the amplitude or ratio methods, consistent with some previous studies on patients with glaucoma [10, 38]. However, differences have also been reported; Cvenkel et al., [23] found that the PhNR amplitude provided significantly larger AUCs than the ratio for both suspect and early glaucoma, and Preiser et al. [39] found that the PhNR:b-wave ratio yielded higher AUC values than amplitude measures in pre-perimetric but not manifest glaucoma. These conflicting reports may relate to different methods and individual differences, and it may be prudent to consider both measurements (of the same waveform) for diagnostic or monitoring purposes.

In this study, the estimated area under the ROC curves for RB and WW PhNRs suggests only a modest level of diagnostic accuracy, as defined by ROC reporting guidelines [33]. A contributory factor may be the heterogeneity and diversity of the clinical patients examined, with different disorders and at different stages of disease severity. The overall diagnostic accuracies of RB and WW PhNRs estimated by AUCs were equivalent (0.74 vs 0.73 for RB and WW), a finding in contrast with some prior reports. Sustar et al. [14] reported AUC values of 0.97 and 0.74 for RB and WW stimuli, respectively, and Banerjee et al. [21] also reported higher RB AUC values of 0.90 compared with 0.76 for WW stimuli. Hara et al. [16] calculated AUCs for a range of photopically matched RB and WW stimuli and found that the 3.0 cd·s·m⁻² RB stimulus provided the best diagnostic accuracy overall (AUC = 0.94); the best WW PhNR stimulus was obtained with a 2.0 cd·s·m⁻² flash (AUC = 0.88). In these studies, examinations of pathology were restricted to patients with glaucoma, which may account for some of the divergence with our findings; nonetheless, the AUC evidence suggests that RB stimuli may have better overall diagnostic accuracy than WW stimuli for the detection of glaucoma.

Our heterogeneous group of patients with RGC dysfunction included seven with mitochondrial optic neuropathies (Leber hereditary optic neuropathy (LHON) and autosomal dominant optic atrophy (DOA)). These disorders primarily affect the papillomacular bundle, with relative sparing of peripheral RGC axons [40], particularly in the early stages of the disease process. As the full-field PhNR is a global measure of RGC function it is likely to be less sensitive to focal/central RGC dysfunction than the PERG or the focal PhNR. Majander et al. [41] reported that the majority of full-field PhNR responses from a cohort of patients with LHON were normal or near the lower limit of normal. Likewise, Miyata et al. [42] found only mildly abnormal/borderline reductions in the full-field PhNR in patients with DOA. Tamada et al. [43] investigated the ability of focal and full-field PhNRs to detect optic nerve atrophy and found that focal PhNRs were more sensitive to damage in eyes with a central visual field defect. The inclusion of patients with both central and diffuse RGC damage in the present study is likely another reason that the estimations of sensitivity and specificity are lower than previous reports.

There are some limitations to this study. In our preliminary experiments, a stimulus–response series was used to determine the optimal RB PhNR stimuli to compare against the broadband PhNR. An aim was to assess the diagnostic accuracy of the ISCEV LA 3.0 ERG PhNR component, which may be of lower diagnostic accuracy compared with optimised WW stimuli. Our aim to compare the LA 3 WW PhNR against the RB PhNR from the ISCEV extended protocol also meant that there was a slight difference between the amplifier bandwidths of the two protocols. The high-pass filter of the WW PhNR was 0.31 Hz, while the high pass of the RB PhNR was 0.13 Hz. As the PhNR is a relatively slow frequency response, this difference may have contributed to the smaller PhNR amplitude that we reported for the WW PhNR. Another limitation is that there is no single gold standard test of retinal ganglion cell dysfunction and therefore multiple tests combined into a test battery were needed to establish the diagnostic status of the participants. Not all participants performed all of the reference tests; this means that not all participants were compared to an identical reference standard, raising the possibility of some verification bias [44]. The group comparison showed that sensitivity to RGC dysfunction was higher for PhNR amplitude than for the PhNR:b-wave ratio, but it is acknowledged that the ratio is likely to be informative in cases of dual pathology, e.g. to judge the severity of optic nerve/RGC dysfunction in the presence of retinopathy (manifest as ERG b-wave and ‘downstream’ PhNR reduction). Strengths of the study include that it was appropriately powered and based on an ethnically diverse and relatively large number of patients, from a consecutive sample referred to an electrophysiology department rather than a case–control population. This enabled a robust estimation of sensitivity and specificity generalisable to the general population.

The PhNR yields moderate levels of diagnostic accuracy for the detection of retinal ganglion cell dysfunction in a heterogeneous clinical cohort. PhNRs evoked by red flashes on a blue background are more sensitive to dysfunction than white-on-white stimuli, but there is no significant difference between the relative specificities of the two PhNR methods. The study highlights the value and potential convenience of using the WW stimulus, already used widely for routine ERG assessment of retinal function.