evaluation of a device using heterochromatic flicker photometry

Macular pigment optical density measurements: evaluation of a device using heterochromatic flicker photometry

R de Kinkelder1,2, R L P van der Veen3, F D Verbaak1,van cleef copy necklace,4, D J Faber1,4, T G van Leeuwen1,5 and T T J M Berendschot3Received 21 June 2010; Revised 17 September 2010; Accepted 17 September 2010; Published online 5 November 2010.

Top of pageAbstractPurpose Accurate assessment of the amount of macular pigment (MPOD) is necessary to investigate the role of carotenoids and their assumed protective functions. High repeatability and reliability are important to monitor patients in studies investigating the influence of diet and supplements on MPOD. We evaluated the Macuscope (Macuvision Europe Ltd., Lapworth, Solihull, UK), a recently introduced device for measuring MPOD using the technique of heterochromatic flicker photometry (HFP). We determined agreement with another HFP device (QuantifEye; MPS 9000 series: Tinsley Precision Instruments Ltd., Croydon, Essex, UK) and a fundus reflectance method.

Methods The right eyes of 23 healthy subjects (mean age 33.9 years) were measured. We determined agreement with QuantifEye and correlation with a fundus reflectance method. Repeatability of QuantifEye was assessed in 20 other healthy subjects (mean age 32.1 years). Repeatability was also compared with measurements by a fundus reflectance method in 10 subjects.

Results We found low agreement between test and retest measurements with Macuscope. The average difference and the limits of agreement were We found high agreement between test and retest measurements of QuantifEye ( and the fundus reflectance method ( MPOD data obtained by Macuscope and QuantifEye showed poor agreement: For Macuscope and the fundus reflectance method, the correlation coefficient was r (P A significant correlation of r (P was found between QuantifEye and the fundus reflectance method. Carotenoids cannot be synthesized by the human body and are only available through diet.15 Hence, the amount of MP depends on the amount of lutein and (meso)zeaxanthin rich foods we ingest, and can be further influenced by the intake of nutritional supplements containing lutein and (meso)zeaxanthin.2, 16, 17

Accurate assessment of the amount of MP, expressed as MP optical density (MPOD), is therefore necessary to investigate the role of carotenoids and their assumed protective functions. High repeatability and reliability are especially important to monitor patients in studies investigating the influence of diet and nutritional (lutein and (meso)zeaxanthin) supplements, or disease processes on MPOD. Methods to determine MPOD should yield reproducible results accompanied by reliable uncertainty estimates, even when operated by non professional or untrained staff. Reproducibility is hampered when the method is applied in older patients, who may suffer from hazy ocular media, compromised physical skills, and early degeneration of the retina.

The most frequently used technique to asses MPOD is heterochromatic flicker photometry (HFP). Different devices using this technique have been validated to produce reproducible and reliable results in healthy research populations of different ages and patients with signs of early AMD.18, 19, 20 HFP setups are relatively inexpensive and easy to use, even for untrained staff. Furthermore, measurements can be performed through an undilated pupil, and are generally not influenced by changes in the ocular media, such as cataracts.

This was performed to evaluate the repeatability and reliability of a recently introduced device using HFP; Macuscope (Macuvision Europe Ltd., Lapworth, Solihull, UK). To this end, we tested the Macuscope against a second device based on HFP (Quantifeye, Tinsley Precision Instruments Ltd., Croydon, Essex, UK) and fundus reflectometry (MP reflectometer (MPR)), two established techniques for measuring MPOD. Special emphasis was put on the agreement between measurements using the Macuscope and the two established devices, and on repeatability between Macuscope measurements within our population. Separate studies showed weak repeatability, however, Macuscope was never tested against other HFP devices or methods.21, 22

Top of pageMaterials and methodsWe investigated the right eyes of 23 healthy subjects without ocular pathology, clear ocular media and a BCVA of at least 1.0. Mean age of the subjects was 34 years. To evaluate the ability of Macuscope to determine the MPOD, we compared the results with another commercially available device that uses psychophysical testing based on HFP, that is, QuantifEye.18, 23 The third method used to determine MPOD was a fundus reflectance technique, used in the MPR.24

MPOD was measured in healthy subjects without any ocular pathology, recruited from the University of Maastricht and the University Eye Clinic Maastricht. Study participants underwent all measurements consecutively on one day following the same protocol, that is, first measurement on the Macuscope, followed by the QuantifEye, followed by the Macuscope (repeatability measurement). The MPR method was the final measurement, because the high intensity of the used light could cause a temporal saturation of the photoreceptors that could influence the result of the other tests. The measurements were scheduled on one day between 0900 and 1500 hours, and were always performed by the same trained operator. When a subject was done with the first Macuscope test, another subject started his Macuscope measurement. Test series did not take longer than 30 per subject to avoid loss of concentration due to fatigue. The test results were only taken into account after a subset of successful pre test measurements,van cleef replica clover necklace, and were performed conform the manual. Repeatability measurements for QuantifEye were done on another day at the Academic Medical Center on 20 healthy subjects. Mean age of these subjects was 32 years. Repeatability data for MPR have already been published by van de Kraats et al,24 and are used in this study. All successful measurements were included for statistical analysis. All research adhered to the tenets of the Declaration of Helsinki. We certify that all applicable institutional and governmental regulations concerning the ethical use of human volunteers were followed during this research.

Heterochromatic flicker photometryBoth the QuantifEye and Macuscope are based on the principle of HFP for measuring MPOD. Flicker is generated by alternating light of two different wavelengths, blue light, which is absorbed by MP, and green light, which is not absorbed by the MP. Because of the pre receptorial location of the MP in the retina, incident light first passes through and is attenuated by the MP (peak absorption at 460 before reaching the photoreceptors. MP has its peak concentration foveally or just parafoveally, and decreases rapidly with eccentricity. Therefore, these setups always use a central (peak MPOD) and peripheral (low or zero MPOD) measurement point, to subtract these values and obtain an individual MPOD value for each different subject. The peripheral measurement is hereby used as a reference point for the central measurement,van cleef necklace knock off wholesale, to correct for the flicker sensitivity of each individual. Using HFP, subjects have to be instructed to fixate a stimulus and indicate when flicker is minimized (Macuscope) or when flicker is first observed (QuantifEye).

The MPOD value is derived by taking the logarithm of the ratio between blue and green luminance measured centrally and peripherally, as is displayed inequation (1):

Here Lbc and Lgc are the luminances of the blue and green light, respectively, during the central measurement and Lbp and Lgp the luminances of blue and green light, respectively, during the peripheral measurements.

MPOD values were determined after training of the subject. For the Macuscope, one or two pre tests were completed depending on how fast the subject comprehended the course of action. For the QuantifEye, the pre test function in the software was used for training of the subject.

MacuscopeMacuscope uses a conventional HFP approach, in which an operator adjusts a (green blue luminance ratio until no or minimal flicker is observed by the subject. Minimal flicker represents a matching of the brightness of both wavelengths. During a measurement, as also described by Hagen et al,21 firstly the subject has to fixate at a disc shaped stimulus of 1.5 in order to measure foveally. A crosshair centred on the stimulus should facilitate central fixation. The presented stimulus alternates between the blue ( 465 and the green ( 530 light, at a fixed frequency of 20 After that, the subject has to fixate one of the crosshairs placed 8 on either side of the central stimulus. Flicker frequency here is changed to 30 and is also fixed. As the crosshair in the centre needs to be in focus, the operator is able to adjust for spherical corrections. The operator then adjusts the luminance of the blue light, and the subject has to verbally indicate when minimal flicker is observed at the centre of the blue disc. The luminescence ratio is then stored. For parafoveal testing of the right eye, the right crosshair is fixated. Before the testing, subjects received verbal instructions on how to perform the test and are given time to make themselves familiar with the task and the machine. When the point of minimal flicker could not be clearly indicated, foveal and parafoveal measurements were repeated. Subjects were reminded of the instructions of the task throughout the test.

QuantifEyeQuantifEye uses a method, in which the user indicates when flicker is first observed. The test consists of two stages. The overall sensitivity of the user to flicker is determined first, and the luminance contrast of the two lights (green and blue) is normalized for that particular subject. Subsequently, during the actual measurement, the subject starts by fixating the central stimulus. The frequency of the blue ( 465 and green ( 530 light of the stimulus is ramped down from above the critical flicker fusion frequency (CFF), for a series of different luminance ratios of the two lights. The subject views the stimulus and presses a button when flicker appears. Each luminance ratio, therefore, generates a certain temporal frequency at which flicker is detected, and directly creates a point on the graph in the software of QuantifEye, as measurements go on. During this sequence, the trained operator can immediately determine if the measurement has been performed well. Each individual will show a luminance ratio for which they are least perceptive, at the lowest detectable flicker frequency. This represents the minimum in the distinct V shaped curve created and easily allows the operator to determine what the luminance ratio is at the minimum flicker frequency. The same sequence is then repeated for eccentric fixation (6 eccentricity). The difference between the minima of determined luminance ratios obtained from central and peripheral viewing determines the MPOD, equation (1). For a detailed description of this technique see van der Veen et al.18

MP reflectometerThe essentials of MPR24 are summarized as follows. The image of the filament of a 30 W halogen lamp is relayed to the pupil plane of the eye. The intensity of the light entering the eye is 1.04 107 Troland. A spot with a diameter of 1 centred on the fovea is illuminated, and the light that reflects from this spot is measured. An image of the retinal spot is focused on an optical fibre that has a mask on its tip to define a diameter spot of 1 at the retinal plane. To keep instrument stray light to a minimum, the detection channel does not overlap with the illumination system. Chin rest and temple pads are used to help maintain head position. MPOD is determined by a full spectral analysis of the reflected light. In brief, the incoming light is assumed to reflect at the inner limiting membrane, at the infoldings in cone outer segments and at the sclera. Using known spectral characteristics of the different absorbers within the eye (lens, MP, melanin, blood), the densities of the pigments and percent reflectance at the interfaces are optimized to fit the measured data at all wavelengths.25, 26 For a detailed discussion of this analysis see Berendschot et al.27, 28

StatisticsThe SPSS statistical software package (Version 15.0.1.1, Release 15.0.1; SPSS Inc., Chicago, IL, USA) was used for data analysis. To evaluate the repeatability, we generated a Bland plot, in which the difference between two measurements in one individual is plotted vs the average of these two measurements,van cleef alhambra knock off necklace, for all individuals. The same analysis was used to evaluate the measurements between the two HFP methods. We also determined relative differences, that is, the differences of two values divided by their mean value. Pearson's correlation tests were used to quantify the linear association of determining MPOD between HFP methods and MPR measurement. MPR determines an MPOD averaged over a 1 field using the reflectance method, whereas with HFP the MPOD is assessed using psycho physical testing. Consequently, as these methods use different analysis methods, a Bland graph is not suitable, and it is more appropriate to use Pearson's correlation tests. Low agreement was found between the test retest measurements done with Macuscope. Using Bland graphs, we determined the average difference between two measurements with Macuscope to be (Figure 1a). The limits of agreement, defined as the average difference plus or minus two times the standard deviation of the differences, were Mean relative difference was 32.2 In this figure two, data points were outside the limits of agreement. We found no reason, however, to exclude these two data points from our analysis. The difference between the first measurement and the second measurement was plotted vs the average of both measurements. Mean of all differences and limits of agreement are displayed. (a) Macuscope. (b) QuantifEye. (c) MPR.

Full figure and legend (99K)

Figure 1 also shows test retest data for QuantifEye and MPR. The limits of agreement for QuantifEye were (Figure 1b).

Full figure and legend (48K)

Figure 3 displays correlation coefficients between Macuscope and MPR measurements, and between QuantifEye and MPR measurements. For first Macuscope measurements and MPR measurements the correlation coefficient was r (P displayed in the upper panel. A significant correlation of r (P was found between the QuantifEye and the MPR, displayed in the lower panel.