When the visualization of the anterior capsule is compromised due to absent or insufficient red reflex, as in case of mature cataract, dense cortical cataract, dense posterior sub capsular cataract, or eyes with dense asteroid hyalosis, there is a certain risk for anterior capsular tear extending to the periphery, with a subsequent predisposition to posterior capsule rupture, vitreous loss, and posterior dislocation of nucleus.

Staining the anterior capsule with trypan blue (TB) dyes, performed for the first time by Melles et al. in 1999, helps surgeons to complete capsulorhexis in these challenging situations.

A small concentration of TB dye such as 0.05% can provide good anterior capsule staining, and endothelial toxicity is commonly believed as negligible when the TB dye is used in low concentration.

Nevertheless, for ensuring the maximum patient safety, the biocompatibility must always be ascertained, and the determination of cytotoxicity is part of the initial evaluation process according to the ISO standard.

The search for the truly reliable biocompatibility and cytotoxicity test methods has always been one of the main missions of Alchilife. This is the reason why in vitro cytotoxicity test by direct contact method according to ISO 10993-5, which is highly sensitive and able to detect even weak cytotoxicity, has always been the method of choice used in our research laboratories.

Using this approach, the Alchimia R&D department validated quantitatively and qualitatively the cytotoxicity test of vital dyes of the anterior segment according to the ISO 10993-5. By using only 30 µl of the dye applied on ARPE19 and BALB 3T3 cell lines for 24 hours, the absence or the presence of some cytotoxicity in all kinds of TB dyes can be easily and reliably ascertained.

One aspect of the cytotoxicity test methods adopted by our R&D department deserves particular attention, as it allows to “ultra-guarantee” the safety of the TB dyes to be used during anterior segment surgeries: it was chosen to perform the cytotoxicity assay not only on BALB 3T3 murine fibroblast, but also on the human retinal pigment epithelial ARPE19 cell line.

This is our way to ensure that the TB dyes are safe even in a case of inadvertent vitreous staining during cataract surgery – an event that can, although not frequently, occur during anterior segment surgery.


  • Parkash et al. Modified 30 G needle trypan blue staining technique under air for a uniform and consistent anterior capsule staining. Clin Ophthalmol 2017; 11:1651-1659
  • Melles GRI et al. Trypan blue capsule staining of visualize the capsulorhexis in cataract surgery. J Cataract Refract Surg 1999; 25:7-9
  • ISO 10993-5, 2009. Biological evaluation of medical devices – Part 5: Tests for in vitro cytotoxicity. https://www.iso.org/obp/ui/#iso:std:iso:10993:-5:en
  • Coelho RP. Inadvertent vitreous staining by trypan blue during phacoemulsification. Vis Pan-Am 2016; 15:26-27.
  • Gaur A. Inadvertent vitreous staining. J Cataract Refract Surg 2005; 31:649