Accepted Manuscript Intraoperative Corneal Thickness Measurement by Optical Coherence Tomography in Keratoconic Patients Undergoing Corneal Collagen Cross-Linking Cosimo Mazzotta, Stefano Caragiuli PII:

S0002-9394(14)00125-1

DOI:

10.1016/j.ajo.2014.02.042

Reference:

AJOPHT 8847

To appear in:

American Journal of Ophthalmology

Received Date: 18 December 2013 Revised Date:

18 February 2014

Accepted Date: 19 February 2014

Please cite this article as: Mazzotta C, Caragiuli S, Intraoperative Corneal Thickness Measurement by Optical Coherence Tomography in Keratoconic Patients Undergoing Corneal Collagen Cross-Linking, American Journal of Ophthalmology (2014), doi: 10.1016/j.ajo.2014.02.042. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Title Intraoperative Corneal Thickness Measurement by Optical Coherence Tomography in Keratoconic Patients Undergoing Corneal Collagen Cross-Linking Short title

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Cosimo Mazzotta and 1Stefano Caragiuli Department of Ophthalmology, Siena University, Siena, Italy

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Intraoperative OCT Pachymetry in Corneal Cross-Linking

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Department of Ophthalmology Unità Operativa Complessa di Oculistica, Policlinico Santa Maria delle Scotte, Siena

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On behalf of all Authors Cosimo Mazzotta MD, PhD

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Reprint requests to: Cosimo Mazzotta MD, PhD U.O.C Oculistica Policlinico Santa Maria delle Scotte, Viale Bracci 8, 53100 Siena, Italy Tel. + 39 0577 356618 Fax + 39 0577 356618 Email: [email protected]

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ACCEPTED MANUSCRIPT Abstract Purpose: To assess intra-operative variation of corneal thinnest point in keratoconic patients undergoing riboflavin/UV-A (Ultraviolet type A) cross-linking treatment using noncontact time domain optical pachymetry. Design: Prospective, non-comparative, Interventional Study.

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Methods: 10 patients underwent epithelium-off riboflavin/Ultraviolet type A corneal crosslinking at Siena University Hospital; corneal thickness was measured by using the Visante™ Optical Coherence Tomography (Zeiss, Jena, Germany) at the following times: preoperatively with epithelium-on, after removal of an 9-mm diameter disc of epithelium, immediately after instillation of two drops of riboflavin 0.1%, dextran T 500 20% solution, after repeated instillation of riboflavin 0.1%, dextran T 500 20% solution every 2.5 minutes, at 10, 20 and 30 minutes of soaking time and at the end of the treatment after 30 minutes of UV-A exposure with the riboflavin biofilm in situ and finally after washing the riboflavin biofilm.

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Results: The most significant decrease in thinnest point measurement was detected meanly in the first 10 minutes of corneal soaking. In this interval, the minimum recommended in various studies, a mean reduction of - 79.28 µm (- 17.61% of initial thinnest point value after removal of the epithelium) was recorded. No adverse events were recorded.

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Conclusions: the study demonstrates a statistically significant intraoperative reduction in corneal thinnest point value during epithelium-off cross-linking procedure using standard riboflavin 0.1%-dextran 20% solutions. Intra-operative optical pachymetry evaluation during cross-linking should be recommended before starting UV-A irradiation. and if a thickness under 350 µm is detected, the stroma should be re-expanded with hypotonic or dextran-free riboflavin solutions.

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Key Words: Optical pachymetry, OCT pachymetry, Intraoperative Optical pachymetry, Cross-linking, Keratoconus

ACCEPTED MANUSCRIPT Introduction

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Keratoconus is a degenerative non-inflammatory ectatic disease of the cornea characterized by biomechanical instability of stromal collagen leading to a reduction in corneal thickness, a variation in posterior and anterior corneal curvature and a loss of visual acuity due to irregular astigmatism with or without a reduction in corneal transparency.1 Riboflavin-Ultraviolet A (UV-A)-induced corneal collagen crosslinking is increasingly proposed as a therapeutic approach to progressive keratoconus and for progressive ectasia of the cornea in general. The aim of the method is to slow down or block progress, avoiding or delaying recourse to keratoplasty. The surgical technique of standard crosslinking involves removal of the epithelium to favour penetration of riboflavin into the stroma and the anterior chamber, ensuring sufficient efficacy while protecting the endothelium and posterior eye structures. A controversial aspect of the technique is how long the stroma should be imbibed with the solution (usually riboflavin 0.1% and dextran T 500 20%) in order to ensure a sufficient intra-stromal concentration of vitamin B2 and its presence in the anterior chamber to maximize the efficacy of treatment and shielding. Since the literature does not suggest univocal imbibition times, the crosslinking procedure is still not fully standardized. Indeed, certain authors continue to recommend and use an imbibition time of 5 minutes after removal of the epithelium, as originally proposed by Wollensak, Seiler et al.2 in 2003 (first international report), repeated by Caporossi, Mazzotta et al.3 in 2006 (second international report). Subsequently, Spoerl, Seiler et al. 4 proposed an imbibition time of 30 minutes, while the Siena School5 proposed a standard time of 10-15 minutes. Actually, no significant differences in clinical results of crosslinking in relation to imbibition time have ever been demonstrated, making the time beyond the first necessary 10 minutes still an open question of subjective experience. However, a study of our group6 on intrastromal concentration of riboflavin, measured by high precision liquid chromatography (HPLC), demonstrated that 10 minutes of imbibition are more than enough to ensure a sufficient concentration of riboflavin in the stoma. This aspect is not secondary, since Kymionis et al.7 demonstrated by intraoperative ultrasound (US) pachymetry that during impregnation of the stroma with standard 0.1% riboflavin solution containing 20% dextran of molecular weight (MW) 500,000 Da, a significant intraoperative reduction in stromal thickness occurs, exposing the endothelium and eye structures behind it to radiation damage. However, ultrasound pachymetry has certain limits because it is a non-repeatable contact method in identifying the thinnest point of the keratoconic cornea.8,9 The present prospective observational clinical study aimed to assess intraoperative variation in corneal thickness during crosslinking treatment by time domain no-contact optical pachymetry mapping, involving readings at regular significant intervals during corneal soaking time and at the end of treatment (after 30 minutes of UV-A irradiation). An in vivo intraoperative pachymetric map seeks to determine when the best time is to interrupt imbibition and safely begin UV-A exposure. Patients and methods Study design was an open prospective clinical study. Ten patients (8 males and 2 females) with progressive keratoconus undergoing riboflavin-UV-A-induced corneal collagen crosslinking (CXL) with removal of epithelium (ten eyes) were enrolled in the study after the approval of the Ethics Committee of Siena 2

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University Hospital. Inclusion criteria were: age between 16 and 28 years (average age of 21.5 years); Krumeich’s stage I or II progressive keratoconus; documented clinical, topographic and pachymetric deterioration; minimum corneal thickness greater than 400 µm measured by optical pachymetry at the thinnest point; absence of stromal opacities, concomitant infections, autoimmune diseases and severe dry eye. Pachymetric readings before, during and after the operation were made with a Visante OCT (time domain Optical Coherence Tomography) (Zeiss, Jena, Germany) non-contact optical system inside the operatory room. Statistical analysis was performed using a Wilcoxon test. All analyses were done using SPSS v16.0. A p value ≤ 0.05 was considered to be statistically significant. Main outcome measures

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Pachymetric measurements of corneal thickness were made at the following times: preoperative (epithelium-on), outside the operating room before the operation; epitheliumoff, immediately after removal of an 8-mm diameter disc of epithelium with a blunt metal spatula and instillation of a drop of oxybuprocaine to anaesthetize the naked stroma; immediately after instillation of two drops of sterile riboflavin 0.1%, dextran T 500 20% solution (Ricrolin Sooft, Montegiorgio AP, Italy), to assess any effects of the solution on corneal thickness (reflection of light, meniscus thickness); at 10 minutes, after repeated instillation of solution every 2.5 minutes; at 20 minutes, after repeated instillation of solution every 2.5 minutes; at 30 minutes, after repeated instillation of solution every 2.5 minutes; at the end of treatment, after 30 minutes of exposure to UVA and instillation of solution every 2.5 minutes, with riboflavin biofilm in situ; at the end of treatment, after washing the riboflavin biofilm with a balanced saline solution.

Results

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In all patients, CXL was performed according to the standard protocol (Dresden modified): exposure to the UV-A source (Caporossi, Baiocchi, Mazzotta, Vega X-Linker™; C.S.O., Florence, Italy) in 6 five-minute steps (total 30 minutes), irradiating an area of 8 mm in diameter (power 3 mW/cm2); instillation of a solution containing 0.1% riboflavin and 20% dextran every 2.5 minutes; corneal washing with sterile balanced saline solution and antibiotic medication with levofloxacin and cyclopentolate eye drops, followed by application of a therapeutic soft corneal contact lens (Shalcon Professional™). Patients were given topical antibiotic therapy (ofloxacin eye drops), Non-steroidal anti-inflammatory drug (diclofenac eye drops) and artificial tears (sodium hyaluronate 0.2%) to be applied four times a day until removal of the contact lens on day 4. Then a topical steroid was prescribed (fluorometholone 0.2% drops four times a day for 4 weeks, subsequently modified to zero over 4-6 weeks). Artificial tears were maintained at 1 drop three times per day for 3 months after removal of the corneal lens.

The results for the average thinnest point provided by the Visante OCT are reported below and summarized in Table 1. Average thickness measured with the epithelium in situ was 473.29 µm ± 16.98 (range 437-526 µm). After epithelial removal, average thickness was 450.29 µm ± 15.71 (range 413-492 µm) p=0.06, equal to 95.14% of the initial thickness, with an average decrease of 23 µm ± 8.86 (4.86% of the initial thickness). Average thickness immediately after the instillation of two drops of Ricrolin™ solution (riboflavin 0.1% and dextran T 500 20%) and 3

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one drop of oxibuprocaine chlorhydrate, was 438.71 µm ± 19.02 (range 402-504 µm), p=0.10 with a not statistically significant change of -11.57 µm ± 11.54 (2.57% of average thickness after epithelial removal), Figure 1. Average thicknesses measured after 10, 20 and 30 minutes of imbibition with Ricrolin,™ instilled every 2.5 minutes, were: after 10 minutes of imbibition: 371.00 µm ± 19.07 (range 335-440), p=0.031; after 20 minutes of imbibition: 335.71 µm ± 19.07 (range 305-414), p=0.031; after 30 minutes of imbibition: 305.86 µm ± 23.29 (range 280-410), p=0.039; with respective decrements of: - 79.28 µm ± 9.25 after 10 minutes (-17.61%); 114.57 µm ± 11.83 after 20 minutes (-25.44%); - 144.43 µm ± 18.85 after 30 minutes (32.07%), Figure 2. All measurements refer to stromal thickness after removal of the epithelium (epi-off). Average pachymetric measurements recorded after treatment, after a total of 30 min. exposure to UV A and instillation of riboflavin 0.1%, dextran T 500 20% solution every 2.5 min. were as follows: 346.71 µm ± 24.12 (range 306-451 µm) p=0.031with riboflavin biofilm in situ; 359.71 µm ± 10.03 (range 332-390 µm) p=0.031 after washing with BSS and antibiotic medication; with respective average reductions in corneal thickness with respect to initial thickness after epithelial removal of: - 103.57 µm ± 19.69 (-23.00%) p=0.031, 90.57 µm ± 16.75 (-20.11%) p=0.031. The results are showed in Figure 3. Discussion

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Non-contact optical pachymetry performed during the standard crosslinking procedure, effected with 0.1% riboflavin - 20% dextran solution, confirms a significant reduction in intraoperative corneal thickness during pre-radiation soaking time and at the end of treatment, i.e. at the end of UV-A irradiation. The study demonstrates that even if high molecular weight (MW) dextran used in the solution makes it iso-osmolar with respect to the stroma (380-400 mosm/l), it is hyper-oncotic. The most significant decrease in thickness was detected in the first 10 minutes of corneal soaking. In this interval, the minimum recommended in various studies, an average reduction of - 79.28 µm (17.61% loss of initial corneal thickness after removal of the epithelium) was recorded. A small percentage reduction in thickness associated with reflection of light induced by the biofilm of riboflavin, estimated at about 2% (based on instantaneous measurement after the first riboflavin drops), did not significantly affect the basic measurement, which means that the reduction in corneal (stromal) thickness after 10 minutes of imbibition can be estimated with certainty at around 15.6% of the initial thickness, excluding epithelium. The decrease in thickness proceeded in the next 10 minutes (to 20 min) and the subsequent 10 minutes (to 30 min) of imbibition, reaching a final value (calculated at the end of the operation) that was about 20% less than the initial value. Our results confirm those obtained by US pachymetry7 providing an important insight: intraoperative pachymetry during crosslinking treatment should be recommended before irradiation. Eventually, the stroma should be re-expanded with hypotonic riboflavin 0.1% solution (dextran free) to preserve the endothelium and posterior eye structures if intraoperative optical thickness measurement is found under 350 µm at the thinnest point. Some endothelial problems encountered by other authors10,11 in their patient series could have been due to excessively long soaking times. The good clinical results and absence of complications reported in the long-term studies from Dresden and Siena,5,12 where imbibition times not exceeding 10-15 minutes were used, suggests that longer imbibition 4

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times (over 15-20 minutes) are unnecessary (HPLC studies6 have established the optimal intrastromal concentrations of riboflavin), if not dangerous (due to excessive dehydration of the stroma). Another fundamental finding emerging from the present study was that the optimal imbibition time may not be the same in all cases, apart from the minimum imbibition time of 10 minutes demonstrated by the original studies to be necessary and sufficient. These studies did not encounter any complications due to low corneal impregnation; indeed, the first cases2,3 treated with only 5 minutes of imbibition are all well. Anyway soaking more than 15-20 minutes should be considered excessive and not useful. The main cause of intra-operative reduction in corneal thickness can be attributed to stromal dehydration and inter-lamellar compaction during imbibition, caused by the colloidosmotic gradient, the so called oncotic pressure13 between the corneal stroma (hypoosmolar) and the corneal surface (biofilm of riboflavin), related to the relative hyperosmolarity of the solution containing 0.1% riboflavin and 20% dextran having a MW of 500,000 Da. This consideration, arising from observations confirmed by this study and intraoperative US pachymetry,7 demonstrates that even if high MW dextran used in the solution makes it iso-osmolar with respect to the stroma (380-400 mosm/l), it is hyperoncotic. A reduction of dextran MW or an alternative Riboflavin diffusion medium, such as the hydroxyl-propyl-methyl-cellulose (HPMC) instead of dextran, could avoid the corneal thinning caused by corneal dehydration reported in literature with dextran solutions. HPMC has long been established for ophthalmic safety and optimal tolerability.14,15,16 As reported in literature by Spoerl et al17 and by Kamaev et al.,18 riboflavin and fluorescein have similar polarity and molecular weight (376 g/mol) with similar diffusion coefficients of 6 x 10-7 cm2/s at 35°C. Since the hydroxyl-propyl methylcellulo se significantly increases the penetration of topical fluorescein as compared to other commonly used ophthalmic vehicles,7 it is theoretically reliable that HPMC could be a good vehicle for riboflavin molecules allowing its faster diffusion into the corneal stroma reducing the problem of intraoperative loss of thickness, with its associated risks. Another consideration is the underestimation of corneal thickness estimated by the Visante™ OCT compared with Us pachymetry 14.74+/-10.84.19,20 At the end of treatment, after UV-A exposure, we recorded a slight recovery of thickness, probably due to oedema caused by the treatment, and demonstrated by postoperative confocal microscopy,20,21 as well as to lower light reflection. However, the clinical and technical significance of the fundamental fact of thickness loss during imbibition remains. Washing of the residual biofilm of riboflavin on the anterior surface of the cornea largely but not completely eliminates the reflective action of vitamin B2 on the partially coherent light of the optical measurement system, reducing the artifact of underestimation of stromal thickness. However, the reduction in pachymetric thickness at the end of the operation was significant and averaged about 90 µm, which was 20% less than corneal thickness measured immediately after removal of the epithelium and before the start of imbibition. In the next 10 minutes, dehydration continues to reduce stromal thickness by about 30 µm for every 10 minutes of imbibition (the relative decrements from 10th to 20th minute and from 20th to 30th minute were 7% and 6%, respectively). I recommend a guideline for calculating the minimum corneal thickness to avoid the risk of endothelial burns during UVA irradiation. Since the depth of treatment penetration demonstrated ex vivo and in vivo human corneas averages 300 µm without epithelium, since calculated average epithelial thickness is 50 µm or even as low as 20 µm in keratoconus as demonstrated by Reinstein et al.,23 and since without epithelium we obtain a stromal depth (apoptosis of keratocytes) comprised between 250 and 350 µm21 to which we add a safety margin of ± 20 µm due to 5

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variability in corneal thickness measurements, at least 400 µm ± 20 µm is the recommended intraoperative stromal thickness. The most significant decrease in thickness was detected in the first 10 minutes of corneal imbibition. In this interval, the minimum recommended in various studies, an average reduction of - 79.28 µm (17.61% loss of initial corneal thickness after removal of the epithelium) was recorded. Optical pachymetry should be performed: 1) intra-operatively once the epithelium has been removed; 2) after 10 minutes of imbibition; and 3) before beginning UV-A irradiation, possibly using a noncontact optical system to identify the average thinnest point, or otherwise centrally by ultrasound in a sterile manner, after marking to ensure that thickness is always measured in more or less the same point. If stromal thickness is more than 350 µm, UV-A treatment can begin. Imbibition can be prolonged to a maximum of 15 minutes (considering a 6% decrement about -30 µm for every 10 minutes after the first 10 min) and stromal thickness should always be measured again before starting UV irradiation. Imbibition for more than 10 minutes should be considered risky, especially when performed on corneas with borderline thicknesses of 400 µm with epithelium at the time of enrolment in the CXL treatment protocol. It is important to remember that in certain cases, it can be useful to exploit intraoperative oedema by using a 0.1% riboflavin solution without dextran (corneal expansion); this can still be done if stromal thickness falls below 350 µm during riboflavin soaking time. Different solutions with low dextran molecular weight or alternative diffusion medium for Riboflavin should be used avoiding intraoperative corneal thickness reduction and relative risk for endothelium. In conclusion, riboflavin 0.1% plus dextran 20% solution, commonly used during epithelium-off crosslinking treatment, induces an intraoperative reduction of mean central corneal thickness that was demonstrated by contact ultrasound (US) pachymetry. Present study confirms the previous data of a statistically significant intraoperative reduction of the thinnest point value of the corneal during epithelium-off cross-linking by using 0.1% - 20% dextran solution. This demonstration was conducted by non-contact optical pachymetry map allowing a precise and repeatable measurement of the thinnest point of the corneal. The most significant decrease in thickness was detected in the first 10 minutes of corneal soaking addressing the concept that prolonged soaking, over a minimum of 10 minutes, is not always recommended. Due to corneal dehydration, the thinnest point of the cornea should always be re-measured before starting UV-A irradiation during epithelium-off crosslinking and also if thickness falls below 350 µm corneal expansion with dextran free 0.1% riboflavin solution should be attempted. Hypooncotic dextran free solutions may be a good solution to avoid early postoperative complications of cornea dehydration such as deep permanent haze and endothelial burn.

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ACCEPTED MANUSCRIPT Acknowledgement section 1) Funding/Support: None 2) Financial disclosures: None

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3) Contribution of Authors: Conception And Design (CM); Analysis And Interpretation (CM); Writing The Article (CM); Critical Revision Of The Article (CM, SC); Final Approval Of The Article (CM, SC); Data Collection (CM, SC); Literature Search (CM, SC).

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4) Other acknowledgments: None

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ACCEPTED MANUSCRIPT References

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1. Rabinowitz YS. Keratoconus. Surv Ophthalmol. 1998 Jan-Feb;42(4):297-319. 2. Wollensak G, Spoerl E, Seiler T. Riboflavin/ultraviolet-a-induced collagen crosslinking for the treatment of keratoconus. Am J Ophthalmol. 2003 May;135(5):620-7. 3. Caporossi A, Baiocchi S, Mazzotta C, Traversi C, Caporossi T. Parasurgical therapy for keratoconus by riboflavin-ultraviolet type A rays induced cross-linking of corneal collagen: preliminary refractive results in an Italian study. J Cataract Refract Surg. 2006 May;32(5):837-45. 4. Spoerl E, Hoyer A, Pillunat LE, Raiskup F. Corneal cross-linking and safety issues. Open Ophthalmol J. 2011 Feb 11;5:14-6. 5. Caporossi A, Mazzotta C, Baiocchi S, Caporossi T. Long-term results of riboflavin ultraviolet a corneal collagen cross-linking for keratoconus in Italy: the Siena eye cross study. Am J Ophthalmol. 2010 Apr;149(4):585-93. 6. Baiocchi S, Mazzotta C, Cerretani D, Caporossi T, Caporossi A. Corneal crosslinking: riboflavin concentration in corneal stroma exposed with and without epithelium. J Cataract Refract Surg. 2009 May;35(5):893-9. 7. Kymionis GD, Kounis GA, Portaliou DM, Grentzelos MA, Karavitaki AE, Coskunseven E, Jankov MR, Pallikaris IG. Intraoperative pachymetric measurements during corneal collagen cross-linking with riboflavin and ultraviolet A irradiation. Ophthalmology. 2009 Dec;116(12):2336-9. 8. Chow VW, Biswas S, Yu M, Wong VW, Jhanji V. Intraoperative pachymetry using spectral-domain optical coherence tomography during accelerated corneal collagen crosslinking. Biomed Res Int. 2013;2013:848363. 9. Nemeth G, Tsorbatzoglou A, Kertesz K, Vajas A, Berta A, Módis L Jr: Comparison of central corneal thickness measurements with a new optical device and a standard ultrasonic pachymeter. J Cataract Refract Surg 2006, 32:460-463. 10. Gokhale NS. Corneal endothelial damage after collagen cross-linking treatment. Cornea. 2011 Dec;30(12):1495-8. 11. Faschinger C, Kleinert R, Wedrich A. Corneal melting in both eyes after simultaneous corneal cross-linking in a patient with keratoconus and Down syndrome. Ophthalmologe. 2010 Oct;107(10):951-2, 954-5. 12. Raiskup-Wolf F, Hoyer A, Spoerl E, Pillunat LE. Collagen crosslinking with riboflavin and ultraviolet-A light in keratoconus: long-term results. J Cataract Refract Surg. 2008 May;34(5):796-801. 13. Vetter JM, Brueckner S, Tubic-Grozdanis M, Vossmerbäumer U, Pfeiffer N, Kurz S. Modulation of central corneal thickness by various riboflavin eyedrop compositions in porcine corneas. J Cataract Refract Surg. 2012 Mar;38(3):525-32. 14. Fechner PU. Methylcellulose, a viscous cushioning material in ophthalmic surgery. Trans Ophthalmol Soc UK 1983; 103: 259–262. 15. Glaser DB, Matsuda M, Edelhauser HF. A comparison of the efficacy and toxicity of and intraocular pressure response to viscous solutions in the anterior chamber. Arch Ophthalmol 1986; 104: 1819–1824. 23. 16. Waltman SR, Patrowicz TC. Effects of hydroxypropyl methylcellulose and polyvinyl alcohol on intraocular penetration of topical fluorescein in man. Invest Ophthalmol. 1970 Dec;9(12):966-70. 17. Kamaev P, Friedman MD, Sherr E, Muller D. Photochemical kinetics of corneal cross-linking with riboflavin. Invest Ophthalmol Vis Sci. 2012 Apr 30;53(4):2360-7. 8

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18. Spoerl E, Mrochen M, Sliney D, Trokel S, Seiler T. Safety of UVA-riboflavin crosslinking of the cornea. Cornea. 2007 May;26(4):385-9. 19. Cheng AC, Rao SK, Lau S, Leung CK, Lam DS. Central corneal thickness measurements by ultrasound, Orbscan II, and Visante OCT after LASIK for myopia. J Refract Surg. 2008 Apr;24(4):361-5. 20. Li EY, Mohamed S, Leung CK, Rao SK, Cheng AC, Cheung CY, Lam DS. Agreement among 3 methods to measure corneal thickness: ultrasound pachymetry, Orbscan II, and Visante anterior segment optical coherence tomography. Ophthalmology. 2007 Oct;114(10):1842-7. 21. Mazzotta C, Balestrazzi A, Traversi C, Baiocchi S, Caporossi T, Tommasi C, Caporossi A. Treatment of progressive keratoconus by riboflavin-UVA-induced cross-linking of corneal collagen: ultrastructural analysis by Heidelberg Retinal Tomograph II in vivo confocal microscopy in humans. Cornea. 2007 May;26(4):3907. 22. Mazzotta C, Caporossi T, Denaro R, Bovone C, Sparano C, Paradiso A, Baiocchi S, Caporossi A. Morphological and functional correlations in riboflavin UV A corneal collagen cross-linking for keratoconus. Acta Ophthalmol. 2012 May;90(3):259-65. 23. Reinstein DZ, Gobbe M, Archer TJ, Silverman RH, Coleman DJ. Epithelial, stromal, and total corneal thickness in keratoconus: three-dimensional display with artemis very-high frequency digital ultrasound. J Refract Surg. 2010 Apr;26(4):259-71.

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ACCEPTED MANUSCRIPT Figure captions

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Figure 1. Intraoperative Optical Coherence Tomography pachymetry during Riboflavin Ultraviolet A corneal collagen crosslinking. Left image corneal OCT scan (left) shows the riboflavin biofilm in situ (yellow arrows) in the de-epithelialized area of 9 mm (red arrow). Average corneal thickness measured immediately after the instillation of two drops of riboflavin 0.1% plus dextran T 500 20% and one drop of oxibuprocaine chlorhydrate demonstrated a not-statistically significant change by a mean value of -11.57 µm ± 11.54 (2.57% of mean corneal thickness after epithelial removal); this instantaneous measurement was carried out to verify the underestimation of corneal thickness due to light source reflection provided by riboflavin itself. Right image (right) shows the patient during the Intraoperative OCT pachymetry evaluation inside the operatory room.

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Figure 2. Intraoperative Optical Coherence Tomography pachymetry during Riboflavin Ultraviolet A corneal collagen crosslinking. Example of OCT sequence in a patient of our series showing pre and intraoperative non-contact optical global pachymetry map (GPM) provided by the Visante™ OCT. Top left scan shows a preoperative minimum corneal thickness of 472 µm; top intermediate scan shows corneal thinnest point immediately after epithelial removal at 457 µm; top right scan demonstrated after 10 minutes of Riboflavin 0.1% plus Dextran 20% 500.000 Dalton MW, a thinnest point of 372 µm with 85 µm difference in stromal thickness (- 19%); bottom left scan after 20 minutes of corneal soaking demonstrated a minimum corneal thickness of 339 µm with a difference of 118 µm (- 26%); bottom intermediate scan performed after 30 minutes of corneal soaking demonstrated a minimum corneal thickness of 283 µm with a difference of 174 µm (38%); bottom right scan shows the final pachymetry map at the end of UV A irradiation with a minimum corneal thickness of 332 µm with a difference of 125 µm (- 27%); adding 12 µm related to light source reflection the final thickness was 344 µm (- 24 % of the initial stromal thickness). The data suggests that intraoperative corneal thinnest point under 350 µm requires a corneal expansion with hypotonic dextran-free solutions before starting UV A irradiation or at least to avoid soaking for more than 10-15 minutes.

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Figure 3. Intraoperative Optical Coherence Tomography pachymetry during Riboflavin Ultraviolet A corneal collagen crosslinking. The graphic shows that the most significant decrease in thickness was detected in the first 10 minutes of corneal soaking. In this interval a mean reduction of - 79.28 µm was reached. A small percentage reduction in thickness estimated at about 2% was associated with reflection of light induced by the biofilm of riboflavin. The decrease in stromal thickness proceeded in the next 10 minutes (to 20 min.) and the subsequent 10 minutes (to 30 min.) of imbibition. The reduction of stromal thickness at the end of the treatment averaged about 90 µm, which was 20% less than corneal thickness measured immediately after removal of the epithelium and before the start of corneal soaking.

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ACCEPTED MANUSCRIPT Table 1. Pre, intra and postoperative optical pachymetry measurements in Riboflavin UV A corneal collagen cross-linking.

Mean corneal thickness ∆ thickness ± SD

%

Preop Epi. ON

473.29 ± 16.98

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Epi. OFF

450.29 ± 15.71 p=0.06

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Immed. after Ricrolin™ 438.71 ± 19.02 p=0.10

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TP ± SD (µm)

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-11.57 ± 11.54

10 min Imbib. Ricrolin™ 371.00 ± 19.07 p=0.031 -79.28 ± 9.25

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20 min Imbib. Ricrolin™ 335.71 ± 19.00 p=0.031 -114.57 ± 11.83 30 min Imbib. Ricrolin™ 305.86 ± 23.29 p=0.039 -144.43 ± 18.85 30

min

UVA,

biofilm in situ After

30

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without biofilm

UVA,

-17.61

-25.44 -32.07

346.71 ± 24.12 p=0.031 -103.57 ± 19.69

-23.00

359.71 ± 10.03 p=0.031 -90.57 ± 16.75

-20.11

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After

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Preop: pre-operative; Epi. ON: epithelium on; UVA: Ultraviolet type A.

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