rapidly decreased (within a few seconds) by means of a lethal injection of pentobarbital. It was assumed that during this rapid decrease, the vascular resistance would remain constant so that the change in Flow would be only due to the change in this pressure. The measurements demonstrated for both tissues a highly signiÞcant linear (p < 0.01) decrease in Flow as this pressure decreased to zero [6, 77].

7.3.4.3 Scattering Scheme

In conventional LDF in nonocular tissues, where the tissue is directly exposed, the laser beam is delivered through an optical Þber (input Þber), and the scattered light is collected by a second optical Þber (output Þber). Both Þbers, which are in direct contact with the tissue, are separated by a distance r of 250 or 500 mm [6]. Separating the illumination and collection areas provides an optimal compromise in terms of signal-to-noise ratio and depth of the sampled volume [78]. Smaller r yields too shallow depth, and greater r decreases the signal-to-noise ratio by reducing the total scattered light impinging on the photodetector.

In ONH LDF, because of the presence of numerous retinal arterioles or venules close to each other in the disk tissue, such a laser delivery-scattered light detection scheme cannot be implemented, and the detecting Þber is placed on top of the directly illuminated volume [5, 79]. This scheme has the advantage of providing greater scattered light intensity, at the expense of the presence of specular reßection of the incident light and a reduction of the volume sampled by the laser light [6].

7.3.5Reproducibility of LDF

The potential of LDF is mainly realized in the assessment of the changes in blood ßow induced by physiological stimuli or by pathologies which do not alter the optical properties of the tissue. In this type of application, the minimum statistically signiÞcant change that can be determined in

response to a given stimulus for a given population of subjects (sensitivity) depends mainly upon the quality of target Þxation, the stimulus, the site of measurements, the technical experience, and other factors. The reproducibility of LDF for ONH blood ßow has been appraised in humans by Joos et al. [80]. By averaging Þve measurements for each session, an intrasession variation of 18% for Vel and 24% for Flow, and an intersession variation of 12% for Vel and 32% for Flow were reported. Interexaminer variance was small. The measured variability includes components from both technical/measurement error and physiologic variation. Sample size estimates were computed for experimentally induced changes in Flow in single and multiple sessions: to detect a 15% difference in Flow with 80% power by means of a paired test, seven subjects would be needed to evaluate changes within one session, whereas 43 subjects would be needed to detect a change between two sessions. Therefore, LDF was found to be useful in evaluating ONH circulation in humans, particularly when acute perturbation experiments within a single session were assessed. Grunwald et al. [81] found that LDF is practical for assessing differences between patient populations. Comparing measurements in glaucoma subjects and controls and using power calculations based on an independent test with a pooled variance estimate, the statistical power to detect a 20% decrease in the glaucoma patients was 80% or greater for a sample size of 24 glaucoma subjects and 14 control subjects. Variability in terms of coefÞcient of variation (CV) of the ONH blood ßow parameters measured at 3 measurement sites in normal controls (n =13) and glaucoma patients with (10) and without systemic hypertension (12) provided the following values:CV(Vel)=16%,17%,and12%;CV(Vol)=20%, 15%, and 22%; and CV(Flow)=21%, 20% and 13%, respectively.

The reproducibility of ONH Flow responses to ßicker was determined based on consecutive trials performed during sessions of less than 19 min duration [82]. The laser beam was aimed at the temporal rim of the ONH in the right eye.