Cross-linking ultrathin corneas, the sub400 protocol

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The landmark paper that describes how the sub400 protocol is used to perform CXL in thin corneas with keratoconus has now been published in the American Journal of Ophthalmology (1).

To understand how much of an advance that the sub400 individualised corneal cross-linking (CXL) approach is, it is worth reviewing why CXL cannot be performed in very thin corneas (such as those with advanced keratoconus and a corneal thickness less than 400 µm) that are likely to require corneal transplant surgery), and the techniques (or “hacks”) surgeons used to perform to allow cross-linking to be performed in these corneas.

Traditional cross-linking of thin corneas

CXL involves the irradiation of the cornea with ultraviolet (UV) light (2). Before this, the corneal stroma is saturated with riboflavin. Riboflavin absorbs the UV light, which causes oxidative reactions that covalently bond together molecules in the stroma, strengthening it, and making the cornea more resistant to further weakening. The riboflavin is consumed during the process, from the top down. At the base of the cornea, however, there is a layer of cells that are important to keeping the cornea clear and healthy: corneal endothelial cells. These cells can be damaged when UV irradiation exceeds a certain level. In order to avoid doing so, and leaving a good safety margin, traditional CXL protocols (2) avoid crosslinking the bottom 70 µm of the stroma when delivering the standard total UV dose of 5.4 J/cm² delivered by 30 minutes of UV irradiation at an intensity of 3 mW/cm².

Surgeons took two main approaches to get around the 400 µm minimal thickness limit. First, was to swell the cornea with hypoosmolar riboflavin to a thickness greater than 400 µm (3). The other approach tried is to artificially thicken the cornea with a riboflavin-soaked contact lens (4). However, both approaches are less than ideal. The first approach can result in unpredictable swelling effects, and variability in the cross-linking effect; the latter approach hinders the entry of an essential component of the cross-linking photochemical reaction into the stroma: oxygen, and this results in a poorer cross-linking effect in terms of corneal strengthening produced.

CXL in thin corneas: traditional protocols vs sub400 protocol

Don’t manipulate the cornea; manipulate the light

But rather than trying to manipulate the thickness of the cornea to maintain a 70 µm safety margin above the corneal endothelial cell layer, why not moderate the amount of energy delivered to the cornea, based on the thickness of each patient’s cornea, to give the same effect? To do so is as simple as adjusting the amount of time of irradiation. A treatment algorithm taking exactly this approach: customising irradiation time to each patients’ individual corneal thickness was developed (5), and has now been validated in the clinic: the sub400 protocol (1).

CXL in thin corneas: the sub400 protocol

The sub400 approach

The sub400 approach has a number of advantages over previous approaches: it removes the variability introduced by hypoosmolar CXL, and permits oxygen to diffuse into the cornea unhindered (unlike contact lens-assisted CXL). Further, these legacy approaches were only feasible in corneas that were at least ~330 µm. The sub400 approach has been used to successfully treat corneas as thin as ~200 µm and still maintain the 70 µm safety margin above the corneal endothelium.

Click on the paper preview image to read the manuscript:

Prof. Hafezi, Medical Director of the ELZA Institute recorded a short video explainer of the sub400 protocol:


  1. Hafezi F, Kling S, Gilardoni F, et al. Individualized corneal cross-linking with riboflavin and uv-a in ultrathin corneas: the sub400 protocol. Am J Ophthalmol. 2021;224:133-142.
  2. Wollensak G, et al. Riboflavin/ultraviolet-A–induced collagen crosslinking for the treatment of keratoconus. Am J Ophthalmol 2003; 135:620-627.
  3. Hafezi F, et al. Collagen crosslinking with ultraviolet-A and hypoosmolar riboflavin solution in thin corneas. J Cataract Refract Surg 2009; 35:621-624.
  4. Jacob S, et al. Contact lens-assisted collagen cross-link-ing (CACXL): A new technique for cross-linking thin corneas. J Refract Surg 2014; 30:366-72.
  5. Kling S, Richoz O, Hammer A, et al. Increased Biomechanical Efficacy of Corneal Cross-linking in Thin Corneas Due to Higher Oxygen Availability. J Refract Surg. 2015;31(12):840-6.

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