Effects of the storage conditions on the stability of natural and synthetic cannabis in biological matrices for forensic toxicology analysis: An update from the literature

Optical coherence tomography angiography (OCTA) is a noninvasive imaging technique that is capable of visualizing retinal vasculature down to the capillary level without the use of injected dye. OCTA uses low-coherence interferometry to measure changes in backscattered signal intensity or amplitude in order to differentiate areas of blood flow from areas of static tissue. To correct for patient movement during scanning, bulk tissue changes in the axial direction are eliminated, ensuring that all detected changes are due to red blood cell movement.^[1] This form of OCT requires a very high sampling density in order to achieve the resolution needed to detect the tiny capillaries found in the retina. Recent advancements in OCT acquisition speed have made it possible the required sampling density to obtain a high enough resolution for OCTA.^[1][2] This has allowed OCTA to become widely used clinically to diagnose a variety of ophthalmological diseases, such as, age related macular degeneration (AMD), diabetic retinopathy, artery and vein occlusions, and glaucoma. ^[1]

Prior to the emergence of OCTA, the most common angiographic techniques were fluorescein (FA) or indocyanine green angiography (ICGA), which both involve the use of an injectable dye. Intravenous dye injection is time consuming and can have adverse side effects. Furthermore, the edges of the capillaries can become blurred due to dye leakage and imaging of the retina can only be 2D when using this method.^[3] With OCTA, dye injection is not needed making the imaging process faster and more comfortable while at the same time improving the quality of the image.

Initial efforts to measure blood flow using OCT utilized the Doppler effect.^[4][5] By comparing the phase of successive A-mode scans, the velocity of blood flow can be determined via the Doppler equation. This was deemed Optical Doppler Tomography; the development of spectral domain OCT (SD-OCT) and swept-source OCT (SS-OCT) greatly improved scan times since this phase information was readily accessible. Still, Doppler techniques were fundamentally limited by bulk eye motion artefacts, especially as longer scan times became important for increasing sensitivity.^[6]

In the mid 2000s systems began compensating for bulk eye motion, which significantly reduced motion artefacts. Systems also began to measure the variance and power of the Doppler phase between successive A-mode and B-mode scans; later it was shown that successive B-mode scans must be corrected for motion and the phase variance data must be thresholded to remove bulk eye motion distortion.^[7][8][9]

By 2012, split spectrum amplitude decorrelation was shown to be effective at increasing SNR and decreasing motion artefacts.^[10] Commercial OCT-A devices also emerged around this time, beginning with the OptoVue AngioVue in 2014 (SD-OCT) and the Topcon Atlantis/Triton soon after (SS-OCT).^[11]

OCTA functions by comparing sequential B-scans of the same cross-section of the retina. Many cross-sections are scanned over time to get a 3D view of the retina over time. Low-coherence interferometry is used to perform each B-scan. The device shines an infrared laser onto the retina and measures the interference patterns of the light that is backscattered from the tissue. ^[12] Areas of change between each sequential B-scan can then be distinguished as sites of blood flow. The B-scans are then added together to get a 3D image of the retina. The resulting image provides structural and functional information of the retinal layers and the vasculature found at each layer, as shown in Figure 1 from Gao et al.^[3] Interpretation of the OCT scans and segmentation of the retinal layers rely on software analysis of the collected data. However, once the data processed, OCTA is capable of providing images of the retinal and choroidal vasculature which, as seen in Figure 1, can be split into five layers: the superficial plexus, the deep plexus, the outer retina, the choriocapillaris, and the deeper choroid.

An algorithm developed by Jia et al.^[13], is used to determine blood flow in the retina. The split-spectrum amplitude decorrelation angiography (SSADA) algorithm calculates the decorrelation in the reflected light that is detected by the OCT device.

The blood vessels are where the most decorrelation occurs allowing them to be visualized, while static tissue has low decorrelation values. ^[15] The equation takes into account fluctuations of the received signal amplitude or intensity over time. Greater fluctuations receive a greater decorrelation value and indicate more movement.

A significant challenge when trying to image the eye is patient movement and saccadic movement of the eye. Movement introduces a lot of noise into the signal making tiny vessels impossible to distinguish. One approach to decreasing the influence of movement on signal detection is to shorten the scanning time. A short scan time prevents too much patient movement during signal acquisition. With the development of Fourier-domain OCT, spectral-domain OCT, and swept source signal acquisition time was greatly improved making OCTA possible.^[3] OCTA scan time is now around three seconds, however, saccadic eye movement still causes a low signal-to-noise ratio. This is where SSADA proves to be very advantageous as it is able to greatly improve SNR by averaging the decorrelation across the number of B-scans, making the microvasculature of the retina visible. ^[16]

Diabetic retinopathy is a leading cause of blindness globally and typically does not manifest until long after the diagnosis of diabetes. OCT-A is a simple, fast, and noninvasive manner of diagnosing early onset diabetic retinopathy, as patients with this disease have demonstrated choriocapillaris abnormalities and retinal microvascular abnormalities (including microaneurysms, enlarged foveal avascular zone and capillary dilation).^[1][17] Macular degeneration refers to age-related loss of vision. It has been shown that the onset of macular degeneration results in choriocapillaris flow impairment, even outside areas of the eye experiencing geographic atrophy.^[1][18] OCT-A can also detect relevant features indicative of venous occlusions.^[19] In theory, this may also aid in the early diagnosis of arterial occlusion, but no clinical studies have been performed so far.^[20] OCT-A has been successful in showing the optic nerve head perfusion and optic disc perfusion in patients with glaucoma.^[21][22] This allows it to be useful for monitoring glaucomatous eyes over time.

Despite the ease of use, OCTA is limited by several factors. Limited penetration depth; can be overcome by increasing wavelength to above 1000 nm, which is absorbed less than shorter wavelengths, such as 800 nm. This workaround is employed in SS-OCTA, but is offset by worse axial resolution.^[23]

OCTA also necessitates a tradeoff among scanning time and size of scanned region and scan resolution. Longer scanning time can increase motion artefacts due to conscious saccades and unconscious microsaccades.

OCTA offers an extremely small field of view for diagnostic-quality images: a 3x3mm (9mm2) region of interest is commonly used, with the ability to scan 6x6mm (36mm2) or 12x12mm (144mm2) areas at the expense of image quality. For comparison, typical fundus images have a surface area of around 400mm2.^[24]

Other methods of angiography have existed long before the development of OCTA. The current gold standards of angiography, fluorescein angiography (FA) and indocyanine green angiography (ICGA), both require dye to be injected.^[25][26]

OCTA does not need dye, but this method takes a long time to capture an image and is susceptible to motion artefacts. The dyes used in FA and ICGA can cause nausea, vomiting, and general discomfort, and only have an effective lifetime on the order of a few minutes.^[27] In addition, it has been shown that only a 3x3mm OCTA scan is comparable in diagnostic quality to FA or OCTA.

From a physics perspective, both dye-based methods utilize the phenomenon of fluorescence. For FA, this corresponds to an excitation wavelength of blue (around 470nm) and an emission wavelength near yellow (520nm).^[28] For IGCA, the newer method, the excitation wavelength is between 750 and 800nm while emission occurs above 800nm.^[29] These longer wavelengths are closer to the isobestic point of hemoglobin and oxyhemoglobin, which corresponds to very low absorption and a high amount of emitted light reaching the detector. On the other hand, OCTA only detects the emitted light that is reflected from the anatomical structures of interest, namely blood vessels. The use of near-infrared light by OCTA confers the benefits of the penetration depth of ICGA, while the lack of dye removes the adverse patient reaction. However, a longer wavelength corresponds to a worse axial resolution, which implies that the absolute resolution of FA is greater than that of ICGA or OCTA.

OCTA has been a novel use of OCT to measure and detect blood flow in the eye. Thus, any advancements to OCT technology itself will be translated into improved performance for OCTA. General advancements to improve OCT image quality include increasing the number of B scans acquired per second and achieving the ability to image deeper in tissue.

WIth increasing image quality, automated image segmentation and anatomical feature detection will be feasible. This will assist in interpretation of the OCTA images and increase their diagnostic quality.

Because OCTA detects the presence of blood vessels, it could be applied to any surface of the body to observe the underlying vasculature. This means that OCTA may have clinical application in diagnosing superficial cancers, which are characterized by neovascularization. For example, OCTA may theoretically be used to estimate the depth of melanoma on the surface of the skin by probing how deep abnormal blood vessels are found below the skin. This process is already being applied to melanomas on and within the eye.^[30]