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Cite this article: Pescosolido N, Fazio S, Rusciano D (2014) Reliability of the Implicit Time of Flicker Erg B-Wave as an Objective Diagnostic Tool for Reti-nopathies. JSM Biotechnol Bioeng 2(2): 1037.
*Corresponding authorDario Rusciano, Sooft italia SpA, Scientific Department, Via Salvatore Quasimodo 136, 00144 Roma, Italy, E-mail:
Reliability of the Implicit Time of Flicker Erg B-Wave as an Objective Diagnostic Tool for RetinopathiesNicola Pescosolido1, Stefano Fazio2 and Dario Rusciano3*1Department of Cardiovascular, Sapienza University of Rome, Italy 2Department of Sense Organs, Sapienza University of Rome, Italy3Scientific Department, Sapienza University of Rome, Italy
Abstract
Flicker ERG represents an objective method to evaluate photoreceptors functional state. Aim of the present work was to assess which of its two parameters – implicit time or amplitude – could better discriminate between normal and pathologic subjects affected by different types of retinopathies.
Two groups of patients were enrolled. The first group included 34 eyes of 17 patients with no eye pathologies; the second group included 23 eyes of 12 patients with different retinopathies. The Retimax Advanced Plus® was used to generate a flash, the intensity of which was calibrated according to the ISCEV standard. Both the implicit time and the amplitude of the flicker ERG B wave were recorded, and related to the pathological state of the retina.
The average value of the amplitude was significantly decreased in retinopathic patients (29.42 ± 12.05 mV) with respect to normal patients (53.57 ± 19.24 mV), however with a high variability and a wide zone of overlapping between normal and pathologic patients. Implicit time values had a much lower variability, with no overlapping between the two groups of patients: average normal values were 31.70 ± 1.40 ms for healthy patients and 39.48 ± 2.13 ms for retinopathic patients.
Our results indicate that the implicit time value of the flicker ERG B-wave can be a quantifiable objective measurement of photoreceptor damage in different types of retinal diseases. Therefore, it can be a valuable alternative for their diagnosis and follow up, which nowadays is still mainly based on morphologic criteria.
International Society for Clinical Electrophysiology of Vision; OCT: Optical Coherence Tomography; VEP: Visual Evoked Potentials; OP: Oscillatory Potentials; µV: microvolts; ms: milliseconds
INTRODUCTIONThe neuro-sensitive retina is devoted to the reception and
transmission of light stimuli, and contains several neuronal cell types arranged in multiple layers [1]:
· The photoreceptors (cones and rods), embedded in the pigmented epithelium, constitute the external layer of the retina, and have the function of converting light energy into electrical signals;
· The retinal ganglion cells make up the most internal layer of the retina, and their axons form the optic nerve that conveys light signals to the lateral geniculate nucleus and finally to the visual cortex in the brain;
· The interneuron’s (bipolar, horizontal and amacrine cells), form the intermediate retinal layers, and form the connections between photoreceptors and RGC.
An electro retino gram (ERG) shows the mass electrical response to photopic stimulation of all the neuronal components of the retina. However, the photopic response of individual classes of neurons can also be recorded separately according to the standard protocols proposed by the International Society for Clinical Electrophysiology of Vision (ISCEV) (flash strength in cd/m2) (Figure 1A and ref. 2):
· Dark-adapted 0.01 ERG (rod response);
· Dark-adapted 3.0 ERG (maximal or standard combined rod–cone response);
The evaluation of the flicker ERG response is based on two
Special Issue on
The Biology of Retinal Photoreceptor Cells
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retinal diseases: 6 atherosclerotic retinopathy, 2 diabetic retinopathy, 2 hypertensive retinopathy, 1 myopic retinopathy and 1 age related macular degeneration (see Table 2 for details). The retinopathic condition was unequivocally assessed by ocular fundus examination with an ophthalmoscope after pupil dilatation with atropine, and by OCT analysis.
The bioelectric activity of the retina was recorded by the flicker ERG accessory of the Retimax Advanced Plus instrument (CSO, Florence, Italy). The intensity of the flash source of the instrument was calibrated according to the light adapted 3.0 flicker ERG protocol of the ISCEV standard. The instrument used also includes a special software application that allows the evaluation of the retina response to the flicker stimulation of the patient. Such a response is acquired through HK loop electrodes (CSO, Florence, Italy) positioned inside the inferior eye fornix, after a drop of local anesthetic. The flicker ERG test was carried out on patients under conditions of mesopic ambient light. The amplitude and implicit time of the flicker ERG generated B wave were the two parameters considered in this study. Moreover, two pathologic patients were randomly selected to undergo a test-retest variability assay: they were subjected, 3 times, on consecutive days to flicker ERG, to evaluate the stability and reproducibility of each parameter recorded.
A non-parametric statistics (Mann-Whitney) with 99% of confidence limits was applied to evaluate the different response between normal and pathologic subjects.
RESULTSFigure 2 illustrates the flicker response of normal, non-
Figure 1 A: Full-field electroretinograms (ERGs) recorded according to the International Society for Clinical Electrophysiology of Vision (ISCEV) standards protocol. The rod, combined rod-cone, cone, oscillatory potentials, and 30-Hz flicker full-field ERGs are shown. Ordinate values are expressed in µVolts, and abscissa values in ms. B: 30 Hz flicker ERG: a repetitive high-intensity flash (30/sec) produces this all-cone response. Amplitude (µV) and implicit time (ms) are indicated.
parameters: the amplitude of the B wave, and its implicit time (from flash to response peak: time to peak, Figure 1B) [3]. Both parameters are altered in all those pathologies that involve the photoreceptors: a decrease in amplitude and an increase of the implicit time are usually observed [4].
Experimental ocular electrophysiology studies after different types of pharmacological treatments have shown that the flicker ERG response mainly depends on post-receptorial components. Injections of DL-2-amino-4-phosphonobutyric acid and cis-2,3-piperidinedicarboxylic acid given into the vitreous of primates to block the activity of ON- and OFF-bipolar cells, respectively, reduced the peak-to-peak flicker amplitudes by 80% from control amplitudes, independently from the shape of the flicker stimulus given, at frequencies above 30 Hz [5]. More recently, Verma and Planta [6] have shown that also in humans the postreceptoral components dominate the photopic flicker ERG at 15, 30 and 60 Hz.
However, all these studies only considered the amplitude of the flicker wave. In this study we set up to analyze and compare the behavior of both the amplitude and the implicit time of the flicker response in a group of non-pathologic subjects, and in a group of patients affected by different retinopathies.
PATIENTS AND METHODSFifty-seven eyes of 29 patients were enrolled in this pilot
study, subdivided into two groups: the first, the control group, included 34 eyes of 17 patients with no eye disease (Table 1); the second group included 23 eyes of 12 patients with different
AGE IMPLICIT TIME(ms)
AMPLITUDE(µv)
RE LE RE LE
32 33.07 33.07 47.59 50.47
40 29.83 29.83 58.93 58.04
46 32.1 33.07 40.16 35.39
48 33.39 33.39 33.27 30.51
50 28.86 29.83 77.38 86.86
55 32.42 32.42 67.13 55.71
55 30.48 30.15 66.89 59.01
56 31.45 31.45 45 33.6
57 33.39 34.04 29.45 26.5
63 30.48 33.07 74.94 48.64
66 31.77 31.45 91.02 69.41
67 30.8 30.15 61.41 63.84
68 30.8 30.8 65.43 61.65
68 33.39 33.07 34.71 31.35
70 32.42 32.42 74.27 78.16
72 32.1 33.07 54.02 53.99
84 30.15 29.83 53.35 43.89
Mean 58.65 31.58 31.83 57.35 52.18
SD 13.01 1.37 1.47 17.40 17.27
Table 1: Light Adated 3.0 flicker ERG (30Hz) values recorded for non pathologic patients.
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RETINOPATHY AGE IMPLICIT TIME(ms)
AMPLITUDE(µV)
RE LE RE LE
RI 68 34.04 39.88 56.54 14.64
RI 72 40.53 39.23 31.97 30.7
RD 80 39.23 38.26 49.48 32.86
RD 73 38.26 41.5 27.2 33.73
RA 63 41.5 33.72 22.96 42.23
RA 87 40.2 38.58 30.09 29.27
RA 67 39.23 38.26 15.32 15.53
RA 80 40.53 39.88 21.8 16.23
RA 50 41.82 40.2 39.58 47.31
RA 82 40.85 41.5 39.19 17.43
AMD 94 39.23 21.99
M 70 42.47 39.23 13.98 26.24
MEAN 74 39.88 39.12 31.65 27.38
SD 11.71 2.29 2.01 13.53 10.71
Table 2: Light adapted 3.0 flicker ERG (30Hz) values recorded for retinopathic patients.
HR: Hypertensive Retinopathy; DR: Diabetic Retinopathy; AR: Atherosclerotic Retinopathy; AMD: Age related Macular Degradation; MR: Myopic Retinopathy
Figure 2 Box plot analyses of 30 Hz flicker ERG implicit time (A) and amplitude (B) of normal and retinopathic patients. RE (right eye); LE (left eye). ** p < 0.001
pathologic subjects, and retinopathic patients. Retinopathy results in highly significant (p < 0.001) variations of both the implicit time (Figure 2A) and the amplitude (Figure 2B). It is also evident that the implicit time is less variable than the amplitude, and the increase that is observed with the occurrence of pathologic alterations of the retina is highly consistent, and leaves almost no overlap between normal and pathologic values: the range of variation observed for normal subjects stays between 29.83 and 34.04 ms, while for retinopathic patients it is between 34.04 and 42.47 ms. On the other hand, the range of amplitude variation in normal subjects stays between 26.50 and 91.02 µV, and in retinopathic patients between 13.98 and 56.54 µV, with a large enough area of values overlap that does not allow a complete discrimination between normal and pathologic. This is also reflected by the higher standard deviation observed for amplitude values (around 30% of the mean) than for implicit time values (around 5% of the mean) (see Tables 1 and 2).
Figure 3(A and B) shows a different interpolation of these data, in which implicit time and amplitude of each eye are plotted against each other, with the implicit time on the abscissa. All the points appear to be more or less aligned along vertical lines, which theoretically cross the abscissa close to 31.7 ms for normal subjects (circles), and 39.5 ms for retinopathic patients (squares). The span of the values along the vertical axis is much higher, indicating again a higher variability of the amplitude recording than the implicit time value.
Another interpolation of the data is shown in Figure 3 (C and D), where the value (either implicit time or amplitude) of each eye is plotted against the value of the control lateral eye. It is evident that in normal patients (circles) the recorded values are similar in both eyes, with a high correlation coefficient for the regression lines(r >0.7). However, in retinopathic eyes (squares), any correlation between the two eyes is lost, and each eye shows values independently from the fellow eye (r ≤ 0.05).
Finally, two patients, one affected by atherosclerotic retinopathy, the other by hypertensive retinopathy, were subjected to 3 repeated measurements on consecutive days to evaluate the test-retest variability of the flicker ERG assay. Table 3 and Figure 4 show the results of this experiment. It can be easily seen that while the implicit time values remain the same throughout all measurements (the SD remains well below 2% of the mean), the amplitude shows bigger intrinsic variations (SD between 7 and 20% of the mean), that appears to be intrinsic to the measurement, and cannot be related to a real change of the pathologic situation.
DISCUSSIONWe have presented in this paper novel data from a pilot study,
showing that the implicit time of the photopic 30 Hz flicker ERG is a stable and reliable parameter for the diagnosis and, probably, the follow up of different types of retinopathies.
The methodology of flicker ERG has been known for many years to be able to evaluate photoreceptor function in the retina [7-9]. However, up until now the prevalent analyses of retinal damage have been mainly based on imaging methods such as OCT or fluorangiography [10-12], and classical electrophysiological
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Figure 3 A, B: Amplitude and implicit time values plotted against each other for right and left eye. Circles represent values of normal patients, squares values of retinopathic patients.C, D: Implicit time (C) and amplitude (D) of each eye plotted against the same values of the controlateral eye in normal (circles) and retinopathic (squares) patients. Regression lines with correlation values are reported.
PATIENT 1: atherosclerotic retinopathy
IMPLICIT TIME (ms) AMPLITUDE (µV)
RE LE RE LE
41.82 40.2 39.58 47.31
41.18 38.91 41.31 63.55
41.44 39.16 45.64 57.36
MEAN 41.48 39.42 42.18 56.07
SD 0.32 0.68 3.12 8.20
PATIENT 2: hypertensive retinopathy
IMPLICIT TIME (ms) AMPLITUDE (µV)
RE LE RE LE
34.04 39.88 56.54 14.64
34.04 39.88 40.1 10.54
34.69 40.2 39.61 11.2
MEAN 34.26 39.99 45.42 12.13
SD 0.38 0.18 9.64 2.20
Table 3: Test-retest variability of light adapted 3.0 flicker ERG (30Hz) values recorded for two different Retinopathic patients.
analyses such as flash ERG [13], pattern ERG [14], and focal/multifocal stimulation [15-18]. The electro-oculogram and visual evoked potentials have also been used to support the ERG analysis in the characterization of the functional response of the retina [14]. Oscillatory potentials have also been employed for diagnostic
and prognostic purposes [19-20], and Tahara et al. in 1993 [21] described a correlation between the implicit time of the flicker ERG and the OP in diabetic retinopathy. Such OP appear to derive from a feedback response of the slower depolarizing bipolar cells to the light trigger [22], where as the B wave latency time might
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pathologic subjects such correlation is generally lost, consistent with a differential progression in each eye.
In conclusion, 30 Hz flicker ERG has found its main applications far in the generic evaluation of the retinal damage of diabetic retinopathy [21]. We have now extended these observations to other types of retinopathies, such as hypertensive or atherosclerotic retinopathy, or even age related macular degeneration (Table 2), showing the higher reliability of the latency time over the amplitude of the B wave in different types of analyses. We therefore propose the B wave latency time of the photopic flicker ERG as a valid parameter for the diagnosis, and probably also the follow up, of different types of retinopathies.
ACKNOWLEDGEMENTSThe authors wish to thank Dr Antony Bridge wood for critical
proofreading of the manuscript.
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Figure 4 Repeated measurements of implicit time (■) and amplitude (▲) in a patient affected by atherosclerotic retinopathy (A) and a patient affected by hypertensive retinopathy (B) (see table 3).
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This whole system naturally depends on a correct metabolism supported by the blood supply, which is normally deficient in all types of retinopathies. Therefore, since in our experience we have noted little correlation between OP and 30 Hz flicker ERG response (not shown: subject of a forthcoming paper), we tend to believe that it could be the chorio capillaris damage that may influence in a significant way the latency time of the photopic flicker ERG, and in a lesser way its amplitude.
Our data, although obtained in a reduced number of patients, are consistent with this picture, and show that the latency time could be indeed a reliable indicator of the pathologic state of the photoreceptor layer of the retina. The amplitude, by itself, appears as a more variable parameter, but coupled with the latency time it can reinforce the diagnostic value of the photopic 30 Hz flicker ERG.
Finally, it is interesting to note that the 30 Hz flicker ERG clearly indicates that retinopathies progress independently in the two eyes. In fact, while in normal subjects the implicit time and amplitude values in the two eyes are related to each other, in
Pescosolido N, Fazio S, Rusciano D (2014) Reliability of the Implicit Time of Flicker Erg B-Wave as an Objective Diagnostic Tool for Retinopathies. JSM Biotechnol Bioeng 2(2): 1037.
Cite this article
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