Trypan blue staining for capsulorhexis: Ultrastructural effect on lens epithelial cells and capsules Andre ´ Luı ´s F. Portes, MD, Andre ´a C. Almeida, Silvana Allodi, PhD, Ma ´rio L.R. Monteiro, MD, PhD, Na ´dia C.O. Miguel, MSc, PhD PURPOSE: To evaluate the ultrastructural effect of trypan blue 0.1% staining for capsulorhexis on lens epithelial cells (LECs) and capsules. SETTING: Division of Ophthalmology, University of Sa ˜o Paulo, Sa ˜o Paulo, Brazil. METHODS: Before capsulorhexis, patients were randomly assigned to 1 of 2 groups. Trypan blue 0.1% staining was performed in the treatment group. No trypan blue was used in the control group. Samples of capsules with LECs were fixed and analyzed with routine optical microscopy techniques, immunohistochemistry for beclin-1 expression (a marker of autophagy), terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick-end labeling to detect apoptosis, and transmission electron microscopy (TEM). Morphometric analyses were performed, and the 2 sets of data were compared. RESULTS: Each group comprised 15 patients. Cell death by autophagy and apoptosis was observed in the treatment group but not in the control group. The TEM images of subcapsular epithelium cells showed mitochondrial rupture, dilation of the cisterns of the endoplasmic reticulum, increased cy- toplasmic and nuclear electron density, and abnormalities in the nuclear profile of trypan blue– stained cells. Morphometric analysis showed statistically significant differences between the 2 groups in the longest nuclear axes and the ratio between the total nuclear perimeter and the cell area (P Z .03). The difference in capsule thickness between groups was not significant. CONCLUSION: Trypan blue caused LEC death, which supports the hypothesis that staining with try- pan blue 0.1% can help reduce the incidence of posterior capsule opacification after cataract surgery. Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned. J Cataract Refract Surg 2010; 36:582–587 Q 2010 ASCRS and ESCRS Trypan blue dye is used to identify damaged cells and determine tissue viability. 1 It is also widely used to facilitate capsulorhexis in cataract surgery by improv- ing visualization of the anterior lens capsule. 2–5 Although several studies show that trypan blue 0.1% efficiently stains the anterior capsule without being toxic to the anterior segment of the eye, other studies found that ocular structures may be damaged in cases of extended surgery or trypan blue contact with the pos- terior segment. 6 Furthermore, trypan blue modifies the density and viability of lens epithelial cells (LECs). 6,7 Posterior capsule opacification (PCO) is a major cause of decreased vision after cataract surgery and is the most common late complication postoperatively. 8 It is caused by the proliferation and differentiation of remaining LECs and progressively leads to impaired capsule transparency. 9 We recently evaluated the ef- fect of trypan blue on LECs using electron microscopy of 10 anterior capsule specimens obtained during cat- aract surgery. 7 Abnormalities indicating subcellular changes in LECs were found in 5 capsulorhexis sam- ples stained with trypan blue 0.1% but not in the 5 con- trols, suggesting that the use of trypan blue 0.1% can reduce the proliferation of LECs and thereby help pre- vent PCO. 7 We subsequently performed a larger study to further evaluate the effect of staining with trypan blue 0.1% on LECs using routine optical microscopy, terminal deoxynucleotidyl transferase (TdT)–medi- ated dUTP-biotin nick-end labeling (TUNEL) 10 to de- tect apoptosis, immunohistochemistry to detect cell Q 2010 ASCRS and ESCRS 0886-3350/10/$dsee front matter Published by Elsevier Inc. doi:10.1016/j.jcrs.2009.11.005 582 ARTICLE