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Corneal stroma demarcation line after standard and high-intensity collagen crosslinking determined with anterior segment optical coherence tomography

Kymionis, George D. MD, PhD*; Tsoulnaras, Konstantinos I. MD; Grentzelos, Michael A. MD; Plaka, Argyro D. MD, MSc; Mikropoulos, Dimitrios G. MD, PhD; Liakopoulos, Dimitrios A. MD; Tsakalis, Nikolaos G. MD; Pallikaris, Ioannis G. MD, PhD

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Journal of Cataract & Refractive Surgery: May 2014 - Volume 40 - Issue 5 - p 736-740
doi: 10.1016/j.jcrs.2013.10.029
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Abstract

Keratoconus is a noninflammatory corneal disorder characterized by progressive thinning, irregular steepening of the cornea, and apical scarring causing gradual loss of visual acuity.1 Corneal collagen crosslinking (CXL) is a minimally invasive surgical treatment used to strengthen the tissue of the ectatic cornea and arrest keratoconus progression.2,3 The CXL procedure involves 30 minutes of ultraviolet-A (UVA) irradiation at an intended irradiance of 3.0 mW/cm2 with a total surface dose of 5.4 J/cm2 (Dresden protocol2).

According to the photochemical law of reciprocity (Bunsen-Roscoe law), the same photochemical effect is achieved with reduced illumination time and correspondingly increased irradiation intensity, meaning that a 10-minute illumination at 9.0 mW/cm2 should provide the same effect as a 30-minute illumination at 3.0 mW/cm2. Several new commercially available CXL devices offer high UVA irradiation intensity with relevant time settings.

After CXL, a corneal stroma demarcation line is detectable on slitlamp examination at a depth of approximately 300 μm as early as 2 weeks after treatment.4 A corneal stroma demarcation line after CXL can also be detected using confocal microscopy and anterior segment optical coherence tomography (AS-OCT), possibly representing the effectiveness of the CXL treatment.5–8

The purpose of this study was to evaluate and compare the depth of the corneal stroma demarcation line using AS-OCT after CXL for 2 treatment protocols using a new high-intensity UVA irradiation device.

Patients and methods

This prospective comparative interventional case series comprised patients with progressive keratoconus. Institutional review board approval was obtained. All patients were appropriately informed before their participation in the study and gave written informed consent in accordance with institutional guidelines according to the Declaration of Helsinki.

The clinical diagnosis of keratoconus was based mainly on corneal topography data (iTrace, Tracey Technologies) and clinical signs, such as Fleischer rings, Vogt striae, stromal thinning, and conical protrusion. Inclusion criteria were progressive keratoconus, age older than 18 years, corneal thickness greater than 400 mm, no other corneal or anterior segment pathologic signs, and no pregnancy or lactation. Keratoconus was graded with the Amsler-Krumeich classification9; only patients with grades I and II were included in the study.

In all cases, the CXL treatment was performed with a new high-intensity UVA illuminator (CCL-365, Peschke Meditrade GmbH). The selection of the CXL illumination treatment protocol was random. Patients were treated for 30 minutes at an intended irradiance of 3.0 mW/cm2 according to the Dresden protocol2 (Group 1) or for 10 minutes in an intended irradiance of 9.0 mW/cm2 (Group 2).

Data obtained from the patient records included age, sex, preoperative ultrasound corneal pachymetry (Corneo-Gage Plus, Sonogage, Inc.), preoperative keratometry (K) readings, and AS-OCT scans (Visante OCT 3.0, Carl Zeiss Meditec AG) 1 month postoperatively.

Surgical Technique

The same surgeon (G.D.K.) performed all procedures under sterile conditions. After topical anesthesia of proxymetacaine hydrochloride 0.5% eyedrops (Alcaine) was administered, the corneal epithelium within an 8.0 to 9.0 mm diameter was mechanically removed with a rotating brush. After epithelial removal, riboflavin (0.1% solution of 10 mg riboflavin-5-phosphate in 10 mL dextran-T-500 20% solution, Medio-Cross, Peschke Meditrade GmbH) was instilled on the center of the cornea every 3 minutes for approximately 30 minutes. Ultraviolet-A irradiation was applied using the high-intensity UVA illuminator. Before treatment, the intended irradiance was calibrated using a UVA light meter (YK-35UV, Lutron Electronic Enterprise Co. Ltd.), which is supplied with the UVA device. Irradiance was performed for 30 minutes at an intended irradiance of 3.0 mW/cm2 and for 10 minutes at an intended irradiance of 9.0 mW/cm2, corresponding to a total surface dose of 5.4 J/cm2 in all cases. During UVA irradiation, riboflavin solution was applied every 3 minutes to maintain corneal saturation with riboflavin. At the end of the procedure, a silicone–hydrogel bandage contact lens with a 14.0 mm diameter, 8.6 base curvature, and oxygen permeability of 140 barrers (lotrafilcon B [Air Optix], Alcon Laboratories Inc.) was applied until full reepithelialization.

Postoperative medication included nepafenac suspension 0.1% (Nevanac) 3 times a day for the first 2 postoperative days and chloramphenicol–dexamethasone drops (Dispersadron) 4 times daily until the removal of the bandage contact lens. After removal of the contact lens, patients received fluorometholone 0.1% drops (FML), which were tapered over 2 weeks. Patients were encouraged to use artificial tears at least 6 times a day for 3 months.

Anterior Segment Optical Coherence Tomography

All scans were performed under the same light conditions 1 month postoperatively. Patients were asked to fixate on the optical target in the system. The image was captured when the corneal reflex, a vertical white line along the center of the cornea, was visible. The high-resolution corneal scan was used to produce an enhanced image of the cornea on the horizontal meridian (0 to 180 degrees). The stromal demarcation line was identified in the enhanced corneal image, and the demarcation line depth was measured using the flap tool provided by the manufacturer. The demarcation line depth was measured centrally by 2 independent observers (K.I.T., M.A.G.). The visibility of the demarcation line was scored to obtain accuracy of the measurements (0 = line not visible; 1 = line visible, but measurement not very accurate; 2 = line clearly visible). Only measurements with a score of 2 were included in the study.

Statistical Analysis

All data were collected in an Excel spreadsheet (Microsoft Corp.). Statistical analysis of the results was performed using Stata software (version 12.0, Statacorp LP). Normality of the distribution of all measurements was confirmed using the Shapiro-Wilk test, which is more appropriate for small sample sizes (<50 participants) than the Kolmogorov-Smirnov test. The t test for unpaired data was used; a P value less than 0.05 was considered statistically significant. Continuous variables are presented as the mean ± standard deviation (minimum, maximum). The agreement between the 2 observers was assessed using the Bland Altman method. The 95% limits of agreement (LoA) were computed for the 30-minute and 10-minute irradiation protocols. The mean of the 2 corneal stroma demarcation line depth measurements in each group was used in the statistical analysis.

Results

The study comprised 16 patients (21 eyes). Of the 9 men and 7 women, 7 (9 eyes) were in Group 1 and 9 (12 eyes) were in Group 2. The 2 groups were similar in age, preoperative corneal pachymetry, and in steep and flat K readings (Table 1). No intraoperative or postoperative complications occurred.

Table 1
Table 1:
Age, preoperative ultrasound corneal pachymetry, and keratometry readings by group.

The corneal stroma demarcation line was identified easily (ie, scored as clearly visible) on AS-OCT in all eyes by both observers. All measurements were reliable and used in the statistical analysis. The LoA between the 2 observers were −22.854 to 21.076 (Group 1) and −17.755 to 22.588 (Group 2). The Pitman test of the difference in variance was statistically insignificant in both groups.

The mean depth of the corneal stroma demarcation line was 350.78 ± 49.34 μm (range 256.5 to 410 μm) in Group 1 and 288.46 ± 42.37 μm (range 238.5 to 353.5 μm) in Group 2. There was a statistically significant difference in the corneal stroma demarcation line depth between the 2 groups (P=.0058, t test for unpaired data). Figures 1 and 2 show examples of high-resolution AS-OCT scans of the corneal stroma demarcation line from each of the 2 groups.

Figure 1
Figure 1:
High-resolution AS-OCT scan of the corneal stroma demarcation line 1 month after CXL in Group 1; the central corneal demarcation line depth was 404 μm.
Figure 2
Figure 2:
High-resolution AS-OCT scan of the corneal stromal demarcation line 1 month after CXL in Group 2; the central corneal demarcation line depth was 240 μm.

Discussion

Corneal CXL with riboflavin and UVA irradiation is a minimally invasive surgical treatment that increases the mechanical stability of the ectatic cornea and halts keratoconus progression.2,3 The CXL procedure involves 30 minutes of UVA irradiation at an intended irradiance of 3.0 mW/cm2 with total surface dose of 5.4 J/cm2 (standard Dresden protocol2).

According to the photochemical law of reciprocity (Bunsen-Roscoe law), the biological effect of irradiation on a tissue should stay similar when the total energy dose is maintained; the same photochemical effect is achieved with reduced illumination time and correspondingly increased irradiation intensity, meaning that 10 minutes of illumination at 9.0 mW/cm2 should provide the same corneal stroma demarcation line (same effect) as 30 minutes of illumination at 3.0 mW/cm2 (total surface dose of 5.4 J/cm2 in both cases). Several new commercially available CXL devices offer high-UVA irradiation intensity with relevant time settings.

Seiler and Hafezi4 first reported identification of a corneal stroma demarcation line at a depth of approximately 300 μm that was visible as early as 2 weeks after CXL. The corneal stroma demarcation line after CXL can be detected using confocal microscopy and AS-OCT and may represent the effectiveness of the CXL treatment.5–8

In this study, we used AS-OCT to compare the depth of the corneal stroma demarcation line after CXL for 2 illumination times (10 minutes versus 30 minutes) with a new high-intensity UVA irradiation device 1 month postoperatively. The mean corneal stroma demarcation line depth was significantly deeper in Group 1 (350.78 ± 49.34 μm) than in Group 2 (288.46 ± 42.37 μm). Considering that the corneal stroma demarcation line after CXL may represent the effectiveness of the CXL treatment, CXL could be more effective after a 30-minute procedure at an intended irradiance of 3.0 mW/cm2 (Group 1) than after a 10-minute procedure at an intended irradiance of 9.0 mW/cm2 (Group 2) using the CCL-365 UVA irradiation device. On the other hand, a deeper corneal stroma demarcation line may increase the possibility of corneal damage and endothelial cell toxicity.

Until now, the ideal corneal stroma demarcation line depth in terms of the effectiveness and safety after CXL treatment has not been determined. A shallower corneal stroma demarcation line could be less effective but is probably safer than a deeper line. Therefore, the 10-minute CXL procedure could be a possible treatment option in eyes with a thin cornea that are excluded from CXL treatment based on the Dresden protocol. Furthermore, if this high-intensity treatment protocol is less effective than the standard 30-minute protocol, additional adjustments could be made to the energy and treatment time settings to obtain equal results.

As stated (photochemical law of reciprocity; Bunsen-Roscoe law), 10-minute irradiation at 9.0 mW/cm2 should provide the same effect as 30-minute irradiation at 3.0 mW/cm2. Thus, the corneal stroma demarcation line should have been at the same depth in both groups in our study. Nevertheless, our results indicate that the corneal stroma demarcation line depth differed significantly between the 2 groups, suggesting that the law of reciprocity is not directly applicable to living cornea tissue and other possible unknown factors may also play a vital role.

In conclusion, our study found that the corneal stroma demarcation line depth was deeper after a 30-minute CXL treatment according to the Dresden protocol than after a 10-minute CXL procedure with high-intensity UVA irradiation. To our knowledge this is the first clinical study evaluating corneal stroma demarcation line depth with 2 UVA illumination treatment protocols during CXL.

What Was Known

  • Standard CXL involves 30 minutes of UVA irradiation at an intended irradiance of 3.0 mW/cm2 with a total surface dose of 5.4 J/cm2. According to the photochemical law of reciprocity (Bunsen-Roscoe law), a 10-minute illumination at 9.0 mW/cm2 should provide the same effect as a 30-minute illumination at 3.0 mW/cm2.
  • A corneal stroma demarcation line is detectable 1 month after CXL on AS-OCT and confocal microscopy.

What This Paper Adds

  • The depth of the corneal stroma demarcation line was significantly deeper after a 30-minute CXL treatment at an intended UVA irradiance of 3.0 mW/cm2 than after a 10-minute CXL procedure at an intended UVA irradiance of 9.0 mW/cm2.

References

1. Rabinowitz YS. Keratoconus. Surv Ophthalmol. 42, 1998, p. 297-319, Available at: http://www.keratoconus.com/resources/Major+Review-Keratoconus.pdf. Accessed December 9, 2013.
2. Wollensak G, Spoerl E, Seiler T. Riboflavin/ultraviolet-A-induced collagen crosslinking for the treatment of keratoconus. Am J Ophthalmol. 135, 2003, p. 620-627, Available at: http://grmc.ca/assets/files/collagen_crosslinking_2003_wollensak.pdf. Accessed December 9, 2013.
3. Kymionis GD, Grentzelos MA, Kounis GA, Diakonis VF, Limnopoulou AN, Panagopoulou SI. Combined transepithelial phototherapeutic keratectomy and corneal collagen cross-linking for progressive keratoconus. Ophthalmology. 2012;119:1777-1784.
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8. Kymionis GD, Grentzelos MA, Plaka AD, Stojanovic N, Tsoulnaras KI, Mikropoulos DG, Rallis KI, Kankariya VP. Evaluation of the corneal collagen cross-linking demarcation line profile using anterior segment optical coherence tomography. Cornea. 2013;32:907-910.
9. Krumeich JH, Daniel J. Lebend-Epikeratophakie und Tiefe Lamelläre Keratoplastik zur Stadiengerechten chirurgischen Behandlung des Keratokonus (KK) I-III [Live-epikeratophakia and deep lamellar keratoplasty for stage-related treatment of keratoconus]. Klin Monatsbl Augenheilkd. 1997;211:94-100.
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