title: Trypan blue identifies antimetabolite treatment area in trabeculectomy.
abstract: OBJECTIVE
  Colourless solutions of mitomycin C (MMC) and 5-fluorouracil (5-FU) are widely used during trabeculectomy to inhibit postoperative scarring. The poor visibility of these agents on the eye has several drawbacks including the inability to accurately assess the area of treatment. This study examined the utility of using trypan blue dye to colour antimetabolites used during trabeculectomy and the effect of trypan blue on antimetabolite cytotoxicity in vitro.
  
  
  METHODS
  For in vitro experiments, MMC (0.4 mg/ml) and 5-FU (25 mg/ml) were reconstituted with or without trypan blue. A lactate dehydrogenase release assay was used to measure drug induced cell death and viable cell number 7 days after treatment. For clinical assessment, trypan blue 0.1% was added to MMC and 5-FU to final concentrations of between 0.01% and 0.05%. The mixture was applied to Tenon's capsule and sclera via pre-wet or into dry 5x8 mm sponges (MMC and 5-FU) for 3 minutes or by direct subconjunctival injection after completion of surgery (5-FU). Twenty two consecutive patients undergoing trabeculectomy either with or without trypan blue were followed for 2 years postoperatively.
  
  
  RESULTS
  The addition of 0.05% trypan blue to MMC or 5-FU did not alter MMC induced cell death or the number of viable fibroblast in vitro. In vivo, trypan blue clearly delineated the antimetabolite treatment area and facilitated control of excess antimetabolite at the wound margins as well as sponge removal. With direct subconjunctival injection, total staining area varied for a given volume with location of the needle tip. Any leakage from the injection site could be easily seen. No adverse effects attributable to trypan blue were found in 2 years of follow up.
  
  
  CONCLUSIONS
  Trypan blue permits delineation of antimetabolite/tissue interactions without affecting cytoxicity for the assays investigated. Trypan blue can be used to visualise antimetabolite soaked sponges, estimate treatment area, and show areas of unintended tissue contact during trabeculectomy. The addition of trypan blue to antimetabolites has potential benefits in clinical, research, and teaching aspects of ocular surgery and therapy.
authors.affiliation: Centre for Vision Research, Westmead Millennium Institute, University of Sydney, Westmead, NSW 2145, Australia. phealey@glaucoma.net.au
authors.forename: P R
authors.initials: PR
authors.lastname: Healey
authors.forename: J G
authors.initials: JG
authors.lastname: Crowston
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000963:Antimetabolites / Q000032:analysis* / Q000627:therapeutic use; D002478:Cells, Cultured; D004396:Coloring Agents; D005260:Female; D005472:Fluorouracil / Q000032:analysis* / Q000627:therapeutic use; D005500:Follow-Up Studies; D006801:Humans; D008297:Male; D008858:Microscopy, Phase-Contrast; D008875:Middle Aged; D016685:Mitomycin / Q000032:analysis* / Q000627:therapeutic use; D015503:Surgical Sponges; D014130:Trabeculectomy; D014343:Trypan Blue; D014945:Wound Healing
chemical_list: D000963:Antimetabolites; D004396:Coloring Agents; D016685:Mitomycin; D014343:Trypan Blue; D005472:Fluorouracil