Ten Tenets on Corneal Collagen Crosslinking

Dr. Amanda Mohanan
Dr. Sanjeev P Srinivas
Dr. Srinivas K Rao
Published Online: April 1st, 2021 | Read Time: 18 minutes, 55 seconds

Corneal collagen crosslinking (CXL) is a minimally invasive procedure that is currently the gold standard treatment to arrest the progression of corneal ectatic conditions. It promotes the induction of crosslinks in stromal collagen of the cornea, thereby strengthening and stiffening the cornea in ectatic conditions.


It was observed that corneas in diabetic patients were stiffer due to the natural crosslinking occurring from glycosylation and hence had a lower incidence of keratoconus. Based on this observation, various experiments were conducted to crosslink the cornea manually using chemical agents and sugar aldehydes. Following multiple trials, it was found that this could be safely done by making use of the photochemical reaction induced by the absorption of UV light by riboflavin.1 The first in vivo study was done by Wollensak et al., and they reported in 2003 that CXL was effective in stopping the progression of moderate to advanced progressive keratoconus, in 23 eyes for four years. Since then, various studies have validated these results.2


The main components of CXL are a photosensitizer, an effective and safe light source, and the resulting photochemical reaction.


A photosensitizer is a molecule that absorbs light energy and produces a chemical change in another molecule. In CXL, Riboflavin is used as the photosensitizer. It is safe systemically and can be adequately absorbed by the corneal stroma after topical application. It has an absorption peak at 370 nm.3


As the absorption peak of riboflavin was noted to be 370 nm, UV-A light was found to be ideal for CXL, while at the same time protecting the other ocular structures. The total fluence required was found to be 5.4J/cm2. The Bunsen Roscoe law of reciprocity states that the photochemical effect should be similar if the total fluence remains constant. Based on this, various protocols have been devised with different combinations of the intensity and duration of UV-A exposure.4 However, it has been noted that CXL fails to be effective once the energy intensity exceeds 45mW/cm2.


Once exposed to UV-A light, the riboflavin generates reactive oxygen species, which induce the formation of covalent bonds both between collagen molecules and between collagen molecules and proteoglycans.5


Recent studies indicate that the presence of oxygen is essential for effective CXL.5


The standard treatment protocol, called the Dresden protocol2, was formulated by Wollensak et al for corneas with minimal thickness of 400µm, and is as follows:

  • Instill topical anesthetic drops in the eye
  • Debride the central 7-9mm of corneal epithelium
  • Instill 0.1% riboflavin 5-phosphate drops in 20% dextran solution every 5 minutes for 30 minutes
  • Exposure to UVA (370nm, 3mw/cm2) for 30 minutes while continuing instilling the above drops every 5 minutes.
  • At the end of the procedure, apply topical antibiotics and soft BCL with good oxygen permeability.



Epithelium-off method

As the corneal epithelium offers a barrier to the diffusion of riboflavin to the stroma, the epithelium is manually debrided to enable better penetration. The epithelium-off method is the standard method and remains the most effective.4

Epithelium-on method / Trans-epithelial method

Various techniques have been tried to avoid epithelium debridement. These include the use of pharmacological agents to loosen the intraepithelial junctions, the creation of intrastromal pockets for direct introduction of riboflavin, and iontophoresis. Even though debridement induced complications like postoperative pain and corneal haze are avoided, studies thus far have demonstrated lower effectiveness of CXL in this method.5


Hypo-osmolar riboflavin is used in thin corneas with a thickness between 400 and 320 µm when Dresden protocol is precluded.6



Several protocols have been tried to reduce the treatment time by increasing the intensity of UV exposure. Studies have shown that a middle path with an irradiation dose of 10mW/cm2 for 9 minutes has a better therapeutic and safety profile than higher irradiation doses for shorter periods.5


Traditionally, CXL is performed in the supine position in the operation room. There are a few recent reports on the nuances of crosslinking performed at the slit lamp by Hafezi et al. 7


Crosslinking effect and stiffening are seen more in the anterior cornea, as riboflavin concentration reduces with increasing depth. IOP measurements are not affected significantly post CXL. The debrided epithelium is replaced in 3-4 days. Limbal stem cells are not damaged by CXL, as riboflavin is kept away by the remaining peripheral epithelium. CXL causes apoptosis of keratocytes in the anterior stroma, and in the following weeks, new keratocytes are found migrating from the periphery to the center. As the stroma heals, collagen compaction, and a hyperdense extracellular matrix are seen, No endothelial damage is caused by CXL, when the appropriate technique is used. However, if performed in very thin corneas, if excessive energy is used or if the delivery distance is shortened, endothelial damage can occur. The subepithelial basal nerve plexus is obliterated by this procedure, but it starts to regenerate after seven days. Hence, CXL alters normal corneal structure and cellularity at least for 36 months.6



There is no dispute regarding the fact that CXL is a boon to keratoconus patients as it helps arrest the progress of a progressively debilitating condition. Post CXL, as the cornea becomes stiff, most eyes have stable keratometry values, with some even showing flattening of the curvature. Visual acuity may remain stable or improve.

However, controversies exist as to when to perform CXL. Given the natural history of the disease, it is prudent to perform CXL in children with keratoconus as it is known that the earlier the disease starts, the worse the prognosis. However, documenting progression in adults is the logical requirement irrespective of the method used to do the same, considering the prevalence of forme fruste keratoconus, keratoconus suspects, and subclinical keratoconus, which may not progress. confusion persists due to the different definitions of what constitutes progression.3,4

For further visual rehabilitation along with stabilization of keratoconus, different approaches to combining CXL with refractive surgery, CXL Plus, have been described. Customized CXL depending on the stage of keratoconus and refractive error, CXL along with photorefractive keratectomy and CXL with Intracorneal stromal rings or phakic IOL are some of the techniques.4,8


Being a rare ectatic disorder usually involving the inferior peripheral cornea, CXL has been attempted in these eyes by decentering the focus of irradiation to involve the pathological site. Reports suggest improvement in visual acuity, keratometry, and astigmatism parameters. Even though long-term stability is yet to be studied, in the absence of serious complications, CXL does buy time to postpone further tectonic surgical interventions.3,9


CXL for post-Lasik ectasia has been found to stabilize or improve visual acuity and keratometric parameters. The ‘Athens protocol’ described by Kanellopoulos et al. combines CXL with PRK for managing post Lasik ectasia. Lasik Xtra is a new procedure for Lasik followed by modified CXL to prevent post-Lasik ectasia.3,8,9


PhotoActivated Chromophore for infectious Keratitis – corneal collagen crosslinking

Strengthening of the cornea by CXL and the microbicidal activity of UV irradiation has been utilized successfully in the management of keratitis with stromal melt. As the consistency of results is yet to be demonstrated, currently CXL is considered only in the cases resistant to standard antimicrobial therapy, although the results are variable.8


Studies have shown that CXL causes a reduction in corneal edema and thickness with improvement in visual acuity in patients with bullous keratopathy due to different causes. However, these changes last for only about six months and due to this transient effect, CXL may only have a palliative role, if at all, for now, .9



Postoperative pain is extremely common due to the epithelial debridement and can be managed similar to post PRK pain with a BCL, and topical and systemic analgesics. Pain reduces with each day as the epithelium regenerates.9,10


Both infective keratitis and sterile infiltrates are reported in the literature following CXL. The predisposing factors and management are like usual keratitis. It has also been postulated that the procedure may incite herpetic keratitis reactivation. Diffuse lamellar keratitis has been reported in patients undergoing CXL for post-Lasik ectasia.9.10


Transient corneal haze is commonly noted after CXL due to edema and keratocyte loss. Unlike the subepithelial haze seen post-PRK, post-CXL haze is at the stromal level and responds to topical steroids. However, advanced keratoconus cases have a higher risk of persistent corneal haze which can negatively impact vision quality.10


Epithelial healing problems are noted in around 3-8% cases. As no apparent risk factors, apart from thin corneas are identified, it is pertinent that patients are followed up meticulously till epithelial healing is complete.10


Rates of progression reported vary between 1% and 7.6% according to different protocols. It can be concluded that eccentric cones and thinner corneas have a poorer prognosis and it might be beneficial to customize the CXL protocol in these patients.10



Keratoconus diagnosed in children is usually associated with an unfavorable prognosis and an increased need for a corneal transplant. Hence, in this age group, CXL is advised immediately without the need for documenting progression, as the disease tends to be more aggressive.

The Siena Paediatrics CXL study was conducted on 152 keratoconus patients between 10 and 18 years of age. It demonstrated rapid functional improvement and better long-term stability irrespective of initial corneal thickness in 80% of patients. As expected, patients with thicker corneas did better than the patients with thinner corneas.4,9


According to the Dresden protocol, for safe crosslinking of the cornea, the minimal corneal thickness required is 400 µm. However, as many cases of keratoconus have thin corneas, different modifications have been tried to cross-link these eyes safely.

In corneas with a minimum thickness of 350 µm, hypo-osmolar riboflavin solution has been used to cause corneal swelling. The CXL effect is not compromised as the anterior stroma remains the same while it is the posterior corneal stroma that swells considerably, in turn also protecting the endothelium.

Prior to the advent of hypo-osmolar riboflavin, other methods like reducing the UV irradiation intensity and not debriding the central epithelium were tried; however, CXL effectiveness was compromised.

Using higher concentration riboflavin of 0.2% has been tried to increase the UV absorption in the anterior stroma and hence protect the endothelium.6


Pregnancy is associated with hormonal changes that can negatively impact corneal biomechanics. Hence it is advisable to monitor pregnant patients with keratoconus or recent refractive surgery closely. CXL is avoided during pregnancy, keeping in mind the possibility of complications that may require systemic therapy or additional procedures. If needed, CXL may be done after delivery. Despite CXL being done, these patients need to be informed that the ectasia may progress during subsequent pregnancy due to the hormonal changes. For the same reason, it would be prudent for these patients to avoid hormonal contraception methods.9



It has been postulated that pulsed delivery of UVA radiation, would permit better oxygen diffusion into the stroma. As oxygen has an essential role in the photochemical reaction, the presence of more oxygen would translate to greater effect. Further studies are required to determine the ideal pulsing approach.8


UV-A fluence delivered in all cases irrespective of the protocol has remained constant at 5.4J/cm2. Hafezi and Kling have put forward the concept of adapted fluence where the energy delivered is customized for the cornea. While this may increase the safety profile of CXL especially in thin corneas, it remains to be validated.8


Lasik Xtra is a procedure where CXL is combined with Lasik. After raising the flap and performing laser ablation, higher concentration riboflavin (0.25%) is applied to the stromal bed for about 90 seconds. After this, the interface is washed, and flap replaced. Then half fluence high-intensity UV irradiation is performed. Results so far have shown promise, with better refractive stability and reduced incidence of post Lasik regression and ectasia.8


High‑Fluence CXL is being tried in patients with low myopia to induce subsequent flattening and refractive correction.8


Progressive myopia is accompanied by scleral thinning and elongation. In vitro studies are being conducted to crosslink the sclera and hence stall the progression.8


Photochemical CXL with other agents like Rose Bengal dye and derivatives of photosynthetic agents like chlorophyll is being studied. Using a contact lens to increase the corneal thickness in thin corneas, during crosslinking has been suggested and termed CACXL – contact lens assisted cross linking. Purely chemical CXL, using molecules like Genipin and β-nitro alcohols, is being investigated.3


  1. Spoerl E, Huhle M, Seiler T: Induction of crosslinks in corneal tissue. Exp Eye Res 1998; 66: 97–103.
  2. Wollensak G, Spoerl E, Seiler T: Riboflavin/ ultraviolet-A-induced collagen crosslinking for the treatment of keratoconus. Am J Ophthalmol 2003; 135: 620–627.
  3. Sorkin N, Varssano D. Corneal collagen crosslinking: a systematic review.Ophthalmologica. 2014;232(1):10-27.
  4. Randleman, J.B., Khandelwal, S.S., Hafezi, F. Corneal crosslinking. Surv Ophthalmol 2105; 60, 509–523.
  5. Subasinghe, S.K., Ogbuehi, K.C., Dias, G.J. Current perspectives on corneal collagen crosslinking (CXL). Graefes Arch. Clin. Exp. Ophthalmol. 2018; 256, 1363–1384.
  6. Raiskup, F., Spoerl, E. Corneal crosslinking with riboflavin and ultraviolet A. I. Principles. Ocul Surf 2013; 11, 65–74.
  7. Salmon B, Richoz O, Tabibian D, Kling S, Wuarin R, Hafezi F. CXL at the Slit Lamp: No Clinically Relevant Changes in Corneal Riboflavin Distribution During Upright UV Irradiation.J Refract Surg. 2017;33(4):281.
  8. Sachdev, G.S., Sachdev, M. Recent advances in corneal collagen crosslinking. Indian Journal of Ophthalmology 2017; 65, 787.
  9. Raiskup, F., Spoerl, E. Corneal crosslinking with riboflavin and ultraviolet A. Part II. Clinical indications and results. Ocul Surf 2013;11, 93–108.
  10. Evangelista, C.B., Hatch, K.M. Corneal Collagen Cross-Linking Complications. Semin Ophthalmol 2018; 33, 29–35.

Dr. Amanda Mohanan
Darshan Eye Care
Dr Amanda Mohanan is currently pursuing uvea fellowship at Sankara Nethralaya, Chennai. She has completed her MS from Amala Institute of Medical Sciences, Thrissur in 2014. Subsequently she has undergone fellowship in cataract surgery from Ahalia Eye Institute, Palakkad, medical retina from Giridhar Eye Institute, Kochi and cornea and ocular surface from Darshan Eye Care, Chennai. She was awarded FICO in 2016 and MRCS Ed in 2019. She also holds an MBA in health care services and PG diploma in Medicolegal studies.
Dr. Sanjeev P Srinivas
Cornea Fellow, Disha Eye Hospital, Barrackpore
Dr. Sanjeev P Srinivas (DNB ophthalmology) completed his residency training from Aravind Eye Hospital and Post Graduate Institute, Salem, Tamilnadu, India. He is currently undergoing his specialty training in cornea and anterior segment surgery at Disha Eye Hospital, Barrackpore, West Bengal.
Dr. Srinivas K Rao
Darshan Eye Care & Surgical Centre, Chennai
Dr. Srinivas K. Rao DO, DNB, FRCSEd has worked as a Senior Consultant at Sankara Nethralaya for 15 years before joining as Professor in the Chinese University of Hong Kong from September 2004 till August 2006. Since October 2006 he is Director of Darshan Eye Care and Darshan Surgical Centre, Chennai. He is also Visiting Professor, Sri Ramachandra Medical University, Cornea Head at Sankara Hospital, Pammal, and Sunetra Eye Care, Kolkata, and Ophthalmic Consultant to Madras Medical Mission, Chennai. He is Visiting Professor, Chinese University of Hong Kong, and Honorary Professor at the Shantou Medical University in China. He has published extensively in these areas and is much sought after as a speaker and teacher. He has authored multiple books in the areas of Cataract, refractive surgery, and Corneal diseases. He is actively involved in efforts to promote knowledge in the management of Corneal Diseases, and Mentors Fellows in his CORNEA Fellowship program. He has produced a Module called HOST (Handbook of Ocular Surface Treatment) which will serve as a comprehensive resource for the diagnosis and management of problems of the Ocular Surface. He is also currently the President of the Cornea Society of India.
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