Specular microscopy is a non-invasive diagnostic modality to image the corneal endothelium. It allows detailed in vivo analysis of corneal endothelium in healthy and diseased eyes. Specular microscopy helps in the diagnosis and management of several endothelial pathologies. Common endothelial pathologies such as Fuch's endothelial dystrophy, congenital hereditary endothelial dystrophy, iridocorneal endothelial syndrome, viral endothelitis, drugs, trauma, and uveitis may alter the endothelial cell morphology and functional status and may result in corneal edema and vision loss. Even overuse of contact lenses and intraocular surgery may damage the endothelium. The technique can be contact, non-contact, automated, manual, or both. A specular microscope has computer-assisted morphometry, which analyses endothelial cells' size, shape, number, and density. It is a valuable tool in clinical practice to examine the health of corneal endothelium before planning surgery in compromised endothelial cases and explain the prognosis to the patients. This activity is focused on indications, contraindications, equipment used, technique, complications, and clinical significance of specular microscopy in detail.
Describe the indications of specular microscopy.
Review the contraindications of specular microscopy.
Summarize the technique of specular microscopy.
Outline the complications of specular microscopy.
The corneal endothelium is made up of a monolayer of hexagonal cells. It is imperative to assess the health of the corneal endothelium normally and when the cornea is diseased.
Specular microscopy is a non-invasive modality to document the healthy and diseased endothelium photographically. Specular microscopy is also crucial in assessing the preoperative endothelial health before high-risk surgeries, comparing various techniques, the impact of lasers during refractive surgery, and the assessment of donor cornea before transplantation.
The various endothelial pathologies where specular microscopy plays an important role are Fuch's endothelial dystrophy, corneal dystrophies, posterior polymorphous dystrophy, pseudophakic bullous keratopathy, congenital hereditary endothelial dystrophy, viral endothelitis, ICE syndrome, trauma, uveitis and pharmacological disruption of the endothelium.
Various specular microscopes are available for documenting endothelial cell details at various magnifications and calibrations. Approximately 75 years ago, Vogt tried to obtain the endothelial cell morphology by using the reflected light of a slit lamp. However, limited magnification and rapid eye movements precluded a clear image. David Maurice first described specular microscopy in 1968.
In 1975 Laing first used the specular microscope for clinical use. Baurne et al. in 1976 used the specular microscope at 200X for rapid endothelial examination and photography. In corneal edema, the specular reflection is masked and hampers visualization of the corneal endothelium. Eye banks also need to assess the donor corneal endothelial status.
Anatomy and Physiology
Anatomy of Corneal Endothelial Cells
Before embarking on specular microscopy, it is imperative to be thorough with the anatomy and physiology of corneal endothelial cells. This helps in the better and correct interpretation of specular microscopy results. The corneal endothelial layer is a monolayer of hexagonal cells. The hexagonality allows for a more efficient geometric shape and even distribution of membrane surface tension and gives the advantage of a larger surface area than its perimeter.
These cells lie dormant in the G1 phase of the cell cycle, and there is no evidence of mitotic activity under normal conditions. When there is endothelial injury, the healing is by the process of cellular elongation and spread to create a uniform cellular layer over the inner surface of the cornea. This results in increased surface area and reduced endothelial cell count. When there is variation in cell areas of individual cells, it is called polymegathism or coefficient of variation. A perfect cornea should have 100% hexagonal cells. But a normal cornea is expected to have 60% hexagonality. Under stress or insult to the endothelium, the cell count and hexagonality reduce.
The endothelial cell's morphological analysis is characterized by polymegathism (coefficient of variation, CV), pleomorphism (% of hexagonal cells), cellular density (cells/ mm), and cell area +/-S.D. (μm). The cell density is calculated by the formula 10/ average cell area. Polymegathism is denoted by the coefficient of variation and is calculated by the formula CV= SD / mean cell area, where CV is the coefficient of variation and SD denotes the standard deviation of the cell area.
The endothelial cell density reduces with aging at an average of 0.6% per year. This has two phases, a rapid phase and a slow phase. The endothelial cell density is approximately 6000 cells/ mm in the first month after birth and slowly declines to 3500 cells/mm by five years of age, 3000 cells/mm at the age of 15 to 20 years, and 2500 cells/mm by the age of 50 years.
This is a normal aging process in the cornea with a change in cellular dimensions and senescence of endothelial cells. The endothelial cell density is also affected and declines with racial, environmental, and geographical variations. The endothelial cells lack regenerative capacity; hence any traumatic injury to the endothelial cells is repaired by the surviving adjacent cells. The most sensitive index of corneal endothelial dysfunction is a CV and its percentage of hexagonality for wound healing.
Physiology of Corneal Endothelial Cells
The monolayer of endothelial cells contains approximately 350,000 to 500,000 cells on the posterior surface of the cornea. The major function of the endothelium is the formation of the Descemet membrane by secretion of the collagen matrix. The pumping activity of the endothelium helps maintain the cornea's health and transparency.
The cornea is avascular and provides a physiological barrier to external stimuli. The nutrient supply and diffusion of glucose and other solutes in the aqueous humour occur through the corneal endothelium. The aqueous influx into the stroma from the anterior chamber is due to a change in intraocular pressure, which facilitates diffusion.
The endothelium plays a significant role in maintaining hydration of the cornea to prevent edema. The endothelium acts as a physiological and anatomical barrier to maintain corneal hydration, which makes it semipermeable.
Moreover, on the lateral aspect of the cell membrane, tight junctions between the endothelial cells help control the endothelial permeability and the amount of fluid influx in the corneal stroma.
The endothelium also has an active NaKATPase pump and carbonic anhydrase pump that maintain stromal deturgescence by fluid efflux from the stroma by a dynamic transport mechanism. The carbonic anhydrase pump also helps transport bicarbonate ions across the cell membrane, allowing the aqueous flow in the anterior chamber.
Specular microscopy gives a peculiar and distinctive endothelial picture in various corneal endotheliopathies, which are discussed below. The corneal endotheliopathies are divided into primary and secondary. Primary endotheliopathies are not secondary to any ocular or systemic pathology. In secondary endotheliopathy, the endothelial damage is secondary to an underlying ocular or systemic insult.
The most common primary corneal endotheliopathy is corneal guttate. They are seen as a part of the normal aging process. These are also evidenced in some pathologies like Fuch's endothelial dystrophies, secondary to inflammation and trauma. Guttae are present in the central cornea and have minimal to no effect on visual acuity. Up to 70% of the population above the age of 40 years can have corneal guttae. They are best visualized by the specular reflection technique and are observed as dark areas resembling a hole in the endothelial mosaic. The dark areas are formed because the apex of the gutta lies outside the specular plane of focus. The guttae have five stages of development which are observed on specular microscopy.
Stage 1 Guttae - Nodular, small, dark structure in the endothelial cell center
Stage 2 Guttae - Nodule are of the same size as endothelial cells, surrounding cells stretched
Stage 3 Guttae - Very large, multiple guttae may be seen, and one nodule has many endothelial cells; the adjacent cells lose their boundaries
Stage 4 Guttae - The guttae coalesce, and endothelial cells in between appear abnormal
Stage 5 Guttae - There is a continued coalescence of guttae, and the endothelial mosaic becomes challenging to visualize
Fuchs Endothelial Corneal Dystrophy
FECD is characterized by the presence of guttae, which are focal excrescences of DM in the central cornea. The guttae may or may not affect visual acuity, and there is no direct correlation between guttae and loss of visual acuity. Guttae morphology can vary from fine, coarse, and confluent to nonconfluent, and their number can vary from few to numerous.
These appear as drop-out areas on the endothelial image. In the specular images with few discrete and nonconfluent guttae, the endothelial cells between the dropout areas can still be made to make a precise diagnosis. However, when there are numerous guttae, the endothelial cells cannot be imaged, and the specular images are not readable to make a correct diagnosis. The specular image should always be correlated clinically, and the limitations of the imaging in FECD should be well understood.
The functionality of endothelium should be associated primarily with the patient's signs and symptoms, such as early morning blurring of vision, subepithelial haze, and central versus peripheral pachymetry changes. The changes in the FECD endothelium start from the center and progresses towards the periphery.
One method of assessment of endothelium is to perform Descematorhexis without endothelial keratoplasty. Specular microscopy should be obtained in different gazes to have a preoperative workup of the patient and helps in decision making. The peripheral corneal endothelial cell's health will help in the assessment of patients that are considered for DWEK. The peripheral endothelial count should be above 1000 cells/ mm. When FECD and cataract coexist, the decision for endothelial keratoplasty is based on patient symptoms, pachymetry changes, and specular count.
Good specular count with minimal guttae can be managed with cataract surgery with a good viscoelastic cover of the endothelium. While subjecting the patient to cataract surgery, guarded prognosis and chances of corneal decompensation should be explained to the patient.
Sometimes, the guttae can be misleading and mimic many conditions such as pigments in anterior uveitis, pseudoguttae or secondary guttae as in ocular inflammation, infection or endothelial swelling, Hassal Henle bodies, which are DM excrescences in the peripheral cornea. Pseudo guttae are transient and reverse when the pathology reverses. Hence the specular finding should always be correlated with slit lamp imaging.
Posterior Polymorphous Endothelial Dystrophy
This condition is characterized by the presence of vesicles, band-shaped structures, and placoid mosaics on the endothelium. The pathology can be bilateral or unilateral. In many patients, the endothelial changes are subtle and can be easily overlooked.
Posterior polymorphous endothelial dystrophy can be mistaken for keratoconus due to steep cornea, and unilateral cases may result in amblyopia. Specular microscopy picks up these cases. The endothelial cell density is reduced in these cases compared to age-matched eyes, and endothelial cells are missing in the areas of vesicles and bands.
Congenital Hereditary Endothelial Dystrophy
Stromal haze in CHED acts as a barrier in specular imaging and precludes a clear image of the endothelium. Hence it is not possible to obtain a clear image in these cases.
Iridocorneal Endothelial Syndrome
ICE syndrome comprises Chandler syndrome, Progressive iris atrophy, and Cogan Reese syndrome. Specular microscopy gives distinct morphological changes in these cases. There are two grading systems for describing the endothelial changes- Hirst's grading system and Sherrard's grading system. Specular microscopy in these cases reveals- rounded cell borders, increased areas of blackouts, and a dark and light reversal pattern of normal endothelium.
Senile Endothelial Degeneration
With advanced age, there is an abnormal reduction in endothelial cell density and an increase in pleomorphism, polymegathism, and proliferation in guttae.
Endothelial Changes after Surgery
A variable degree of endothelial cell loss has been reported after intraocular surgeries such as cataract surgery and phakic IOLs. As a result, specular count reveals a lower count and larger mean cell area in the pseudophakic eye versus the age-matched group. There is also a good amount of endothelial cell loss after keratoplasty, and it appears rapidly after the initial years of penetrating keratoplasty.
Approximately 4 to 10% of endothelial cell loss occurs after routine cataract surgery. The cell loss postoperatively is called iatrogenic endotheliopathy. This results in corneal decompensation and stromal edema. The various risk factors for iatrogenic endotheliopathy are ocular surgery, diabetes mellitus, uncontrolled glaucoma, uveitis, polymegathism, pleomorphism, and corneal guttae in FECD.
In a study by Armitage et al., there was biexponential cell loss after keratoplasty. In another study, endothelial cell loss was 7.8% per year between 3 to 5 years post keratoplasty and 4.2 per year between 5 to 10 years. The mean five-year endothelial cell loss after endothelial keratoplasty has been reported to be 47 to 48%.
Contact Lens Endotheliopathy
Regular contact lens wear has been reported to cause acute and chronic corneal endothelium changes as contact lens wear duration increases and the rate of polymegathism increases. Wearing contact lenses for a longer duration reduces oxygen permeability, which results in hypoxia. Chronic hypoxia causes stromal edema and later decompensation. Discontinuing contact lenses doesn't result in acute reversal of endothelial morphological changes.
However, the changes reverse over an extended period or if an alternate contact lens is used with higher oxygen permeability. Contact lens-induced endotheliopathy results in corneal edema, foreign body sensation, photophobia, reduced visual acuity, and corneal exhaustion syndrome.
Pseudoexfoliation Induced Endotheliopathy
Pseudoexfoliation is known to cause endothelial cell alteration. This result in reduced cell density and guttae.
Endotheliopathy after Glaucoma
Uncontrolled IOP may reduce endothelial cell density and morphological changes in the endothelium. Another school of thought is physiological alternation in the glaucomatous eyes, reduced aqueous outflow, or decreased oxygen concentration of the aqueous.
Endotheliopathy after Ocular Inflammation (Uveitis, Endothelitis)
Uveitis can result in reduced endothelial cell loss and cell function. In uveitis, immune-reactive proteins are released in the anterior chamber, leading to endothelial cell loss. The inflammatory cell can penetrate the junction between endothelial cells during acute inflammation. This results in cells dislodging and free float in the aqueous. In acute anterior uveitis, the specular microscopy reveals dark dropout areas located at the endothelial cell intersections, invading the large white blood cells.
Endotheliopathy after Forceps Injury
Forceps trauma can lead to DM rupture and result in corneal edema at birth. The tear heals, and corneal edema is resolved with the migration of endothelial cells to cover the defect. The endothelial cell density is lower in the eyes compared to the normal.
Endotheliopathy in Corneal Dystrophy
In macular corneal dystrophy (MCD), there is the deposition of GAGs in the stroma and corneal endothelium. In MCD, guttae similar to FECD are observed. Endothelial morphological changes and reduced cell density can occur in these cases after cataract surgery. Hence a proper preoperative workup is necessary.
Patients who are on antidiabetic drugs and chronic kidney disease who are on hemodialysis can have endothelial abnormalities. These patients undergoing cataract surgery should be thoroughly evaluated to avoid the risk of corneal decompensation. Certain medications can lead to endothelial cell loss and result in bilateral corneal edema. Antiparkinsonism drugs, certain toxins like madar juice (calotropis), and plant medication can result in drug-induced epitheliopathy.
Role of Specular Microscopy in Eye Banks
Assessing the donor corneal endothelial status before subjecting the tissue to keratoplasty is a regular and standard practice. The microscopes available in the eye banks help magnify endothelial cells' vies. An ex vivo wide filed specular microscope with dual imaging characteristics is employed to assess a larger area of the endothelial surface. The donor tissue is warmed to 25 C before the examination, ranging from 1 to 2 hours to obtain a clear endothelial image.
The endothelial count is an imperative assessment parameter to decide donor suitability for optical penetrating or lamellar keratoplasty. The cut-off taken for OPK is 2000 cells/mm, and for lamellar transplant, it is 2200 to 2500 cells/ mm, which varies based on the eye bank and surgeon preference. A donor count of less than 2000 is employed for either therapeutic or tectonic purposes or research and education.
The contraindications of specular microscopy can be
In this microscope, a contact lens with a coupling fluid is used. This has a similar index of refraction to that cornea, which eliminates the corneal specular reflection. In this method, the corneal thickness also includes the contact lens thickness.
The reflection from the contact lens surface replaces that of the corneal surface. The contact apparatus provides good resolution as well as magnification. Due to the contact procedure, there is a risk of infection if sterile precautions are not taken, and the majority time, the patient feels uncomfortable. Manipulation with this technique can induce artifacts and especially in diseased corneas.
Non-Contact Specular Microscope
In a non-contact specular microscope, the reflections from the anterior surface are eliminated by increasing the angle of incidence. By modifying the angle of incidence, the anterior review is moved to the side, and there is less specular reflection. This technique being non-contact is more tolerated and accepted by the patient, and there is minor infection. Hence a broader view is obtained.
Wide Field Specular Microscope
This is a modification of the previous version with the addition of a scanning mirror. A field of 800 um is obtained with no loss of contrast. This technique allows a continuous view of an 800 um diameter area because of the mirror's higher rate of oscillation. The wide field provides for a 10 to 15 times larger picture, with higher resolution, and image quality is less affected by eye movements. A wide field provides a more accurate cell count, the topography is easily evaluated, and the relocation of a particular portion of endothelium is easy.
The paramedical technicians have working knowledge and are well-trained in handling specular microscopy is needed for perfect patient management. The optometrists help in assessing the visual as well as the refractive status of the patient. The ophthalmic corneal surgeon, well trained in evaluating specular count, helps delineate the endothelial pathology and provides the targeted treatment. The mid-level ophthalmic personnel also assist in patient counseling and follow-up.
The procedure is first fully explained to the patient to relieve any anxiety and make the patient comfortable. The cornea is systemically scanned, and all quadrants should be scanned centrally, superior, inferior, temporal, and nasal. The specular microscope is a light-reflecting microscope.
The slit beam of light is projected onto the cornea and the light reflected from the tissue interface helps form the image. The difference between the refractive index of aqueous humor and endothelial cells gives rise to specular or mirror-like reflection on the posterior surface.
The reflected light is approximately 0.02% of the incident beam of light. If a broad beam of light is used, the reflected beam of light from the stroma and epithelium obscures a clear view of the endothelium. Liang described four distinct zones on specular microscopy using a slit beam of evaluation.
Zone 1- Epithelium/ Coupling fluid of lens
Zone 2- Corneal stroma
Zone 3- Endothelium
Zone 4- Aqueous humor
The area between zone 3 and 4 is dark and is called a dark boundary.
The area between zone 3 and zone 2 is dark and is called a light boundary.
There are various ways to get quantitative information on the endothelium, such as the corner, comparison, frame, center to center, and flex center. Errors can also occur while evaluating the corneal endothelium.
In the frame method, cell density can be assessed by counting the number of cells within a frame.
In the comparison method, cell density is assessed by visually comparing the image of a known set with a hexagonal pattern of various cell densities.
In the corner method, the intersecting sides of the endothelial image frame are located.
Fixed Frame Method
This method can induce many errors when many border cells are present. This error can be eliminated by the variable frame counting method and is preferable to the fixed-frame method.
This means tracing the individual cell areas and other parameters.
Digested Cell Analysis
The cells can be digested after tracing outlines using a digested tablet. The analysis can be done by using a photograph- a negative image on a television screen or a videotaped recording.
The computerized analysis help to assess the cell density, frequency, size, and shape.
Subjective decision-making can induce errors in the center-to-center method and cell border. The accuracy of endothelial cell morphology depends on the clarity and quality of the endothelial scan obtained. The specular scans are based on the specular reflex. Hence any barrier in the endothelial monolayer's optical pathway will affect the scan quality. The other factors that hamper the scan quality are tear film abnormalities, epithelial haze, stromal scar, guttae, and DM damage. Based on the quality of the endothelial scan, it can be graded as good, fair, poor, or impossible.
Automated Specular Microscopy
In this method, automatic analysis of individual cell layers is done which are well delineated. If the cell boundaries are not visible, then the manual method should be used to perform a specular count. The interobserver subjective variability can be around 0 to 6% for the excellent quality of endothelial images and 6-11% for acceptable quality.
Specular microscopy is challenging to ascertain in patients with small micromovements and nystagmus patients. It gives information only about the morphological status and not the functional level of the endothelium. It is a vital tool for assessing the endothelial status in primary and secondary corneal endotheliopathies.
Specular microscopy has revolutionized the management of FECD and cataract patients and is also an important tool for assessing the endothelial status on serial follow-ups. Understanding and working knowledge of specular microscopy are highly imperative for all ophthalmologists to guide the management and prognosis of all patients.
Enhancing Healthcare Team Outcomes
It is essential for the ophthalmologist, optometrists, specular trained technicians, and nursing staff to work in tandem for better management of patients with endothelial pathologies. These patients can gain good visual acuity if managed perfectly.
Nursing, Allied Health, and Interprofessional Team Interventions
The nursing and allied health staff working as an interprofessional team help perform the specular scan and guide the patients regarding the need for further intervention and follow-up.
Nursing, Allied Health, and Interprofessional Team Monitoring
The nursing, allied health staff, and interprofessional team help assess and evaluate specular microscopy of these patients with regular follow-up. The skilled staff can understand the progression or stability of endotheliopathy by assessing the specular scans and also help in counseling these patients.
(Click Image to Enlarge)
Specular image of a patient with Fuch's Endothelial Corneal Dystrophy depicting reduced endothelial cell count, coefficient of variation and reduced hexagonality
Contributed by Bharat Gurnani, MD
(Click Image to Enlarge)
Specular count of the patient depicting large areas of drop outs suggestive of gutta but with adequate endothelial cell count in a patient with FECD
Contributed by Bharat Gurnani, MD
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