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Trypan blue staining of antiproliferative agents for trabeculectomy surgery and bleb needling
  1. W Franks
  1. Correspondence to: Wendy Franks Glaucoma Service, Moorfields Eye Hospital, City Road, London EC1V 2PD, UK;

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Staining could be a useful tool in allowing comparison of the effects of different antiproliferative agents

Healey and Crowston, in the September issue of BJO,1 describe a novel and ingenious study using trypan blue to stain antiproliferative agents used during trabeculectomy surgery and trabeculectomy bleb needling. Trypan blue is commercially available as Vision Blue (Dorc, Zuidland, Netherlands). It is widely used in cataract surgery to stain the anterior capsule, and in vitreoretinal surgery to improve the visibility of preretinal membranes and is therefore readily available in most eye theatres.

They describe a clinical safety and efficacy trial backed up by laboratory based studies of colouring mitomycin C or 5-fluorouracil with the vital dye trypan blue 0.1%. Addition of trypan blue in vitro had no effect on cell death rates in controls or on mitomycin C and 5-fluorouracil treated cells. In vivo, there was no difference in outcome in a series of eyes undergoing trabeculectomy surgery with trypan blue stained antiproliferative agent compared to controls. This technique has the potential to be useful both in research to compare outcomes of surgery as well as in clinical practice by enhancing safety in the use of antiproliferative agents and improving surgical technique.

Trabeculectomy is the surgical procedure of choice in most countries for treatment of chronic open angle glaucoma. Since the first description in 1968 by Cairns2 the operation has survived challenges from procedures such as laser trabeculoplasty,3 holmium laser sclerostomy,4 artificial drainage devices5–7 and, more recently, deep sclerectomy8,9 and viscocanalostomy.10,11 Some of the latter remain as lesser weapons in the glaucoma surgery armamentarium but none has stood the test of time to take trabeculectomy’s foremost place in the surgical management of glaucoma.

Trabeculectomy is the most effective treatment for glaucoma for reducing intraocular pressure and preventing visual field loss,12–15 but relatively few cases come to surgery because of fear of complications. Eye drops are the first line treatment of chronic open angle glaucoma and the number of different types available leads to a myriad of possible combinations. The popularity and variety of medical treatments were greatly increased by the introduction of prostaglandin agonist eye drops in the mid 1990s and have been associated with a halving of trabeculectomy cases,16,17 although the number performed at Moorfields Eye Hospital now appears to have stabilised (fig 1).

Figure 1

 Number of trabeculectomy operations at Moorfields Eye Hospital 1997–2004.

The realisation that patients with progressive visual field loss need lower target pressures,18 and that this can be difficult to achieve even with multiple eye drops, means that trabeculectomy surgery will continue to be an important treatment to prevent blinding disease. In countries where the cost of drops is prohibitively expensive and the supply uncertain, trabeculectomy is the only feasible sight saving treatment.

The most important refinement to trabeculectomy surgery has been the use of antiproliferative agents to reduce postoperative subconjunctival fibrosis, prevent bleb failure, and achieve better intraocular pressure control.19,20 Evidence is accumulating that at lower intraocular pressures visual field progression is slowed or even arrested.21,22 In the pre-antiproliferative era an intraocular pressure of 21 mm Hg or less was considered a successful outcome, whereas now intraocular pressures of 12 mm Hg are being sought and achieved with surgery using the more potent antiproliferative agents. Over the past few years nearly all cases of trabeculectomy at Moorfields Eye Hospital have been with mitomycin C (fig 2).

Figure 2

 Trabeculectomy and antiproliferative agents 1997–2004. 5-FU, 5-fluorouracil, MMC, mitomycin C.

It was soon recognised that the use of antiproliferative agents was associated with cystic bleb formation.23 Cystic blebs lead to an uncomfortable eye, late wound leaks, hypotony, and an increased risk of bleb related infections with potentially devastating consequences.24,25 If a small treatment area is used subconjunctival fibrosis occurs at the margins of the trabeculectomy bleb, confining drainage of aqueous humour to the treated area. The pressure of aqueous humour in this confined subconjunctival space causes thinning of the overlying conjunctiva over time. This led to the idea that cystic bleb formation would be less likely if peroperative treatments with antiproliferative agents were applied over a wide area of the upper fornix. This results in a greater area for aqueous humour drainage and reduced local tissue pressure. This necessitated a change in surgical technique from limbal to fornix based conjunctival flaps.26,27 The downside to this change has been early bleb leaks because the antiproliferative agent inhibits healing of the anterior edge of the conjunctival flap. Surgeons learning trabeculectomy surgery are doing fewer cases at a time when the technique has become more demanding. By staining the antiproliferative agent with trypan blue it is clear, firstly, if an adequate area has been treated and, secondly, any areas of inadvertent treatment are highlighted. If the conjunctival edge is contaminated this will be visible and give an indication if additional sutures are needed to prevent wound leaks.

Healey and Crowston found that by using sponges pre-soaked with dyed antiproliferative agent the treatment area was larger than when the antiproliferative agent was injected into dry pre-placed sponges. This means that surgeons will need to be careful to do a thorough dissection of the subconjunctival space before inserting sponges as squeezing in wet sponges may cause leakage. In particular, extra care will need to be taken to prevent inadvertent treatment of the conjunctival edge by the dampened sponges and this will be easier to see if the antiproliferative agent is stained with trypan blue.

Healey and Crowston also describe the use of dyed 5-fluorouracil when needling failing trabeculectomy blebs. Trypan blue staining can show both the extent of the treatment area as the antiproliferative agent is injected into the subconjunctival space and, very importantly, can make penetration of the antiproliferative agent into the anterior chamber via the sclerostomy visible. It will make leakage back through the injection site into the tear film more obvious and demonstrate whether there is a risk of 5-fluorouracil induced keratopathy.

Comparing surgical outcomes between centres in trials of new surgical techniques in trabeculectomy surgery is problematic. Intraocular pressure lowering and visual field stability are insufficient alone to assess success. The comfort and appearance of the trabeculectomy bleb are also important outcomes for the patient. Clarke et al have published a useful guide to bleb appearances to aid researchers in quantifying morphological outcomes of trabeculectomy surgical technique.28

The surgeon chooses the concentration of mitomycin C but the area treated is variable. Dyeing antiproliferative agents with trypan blue may aid in standardising treatment areas. This makes comparing outcomes of treating series of patients difficult as the area and therefore the dosage of antiproliferative agent are not standardised. Staining could be a useful tool in the future in developing protocols allowing comparison of the effects of treating standardised areas of different antiproliferative agents.

Trabeculectomy is likely to remain the most commonly performed surgical operation for glaucoma for many years to come. However, concern remains about potential complications. Healey and Crowston’s work will be of interest to clinicians as it promises to make surgery more predictable and to help in teaching safe surgical technique. It also promises to be of use in developing research protocols.

Some caution is necessary however. Trypan blue is not yet licensed for this application and, as the authors point out, further in vitro studies of fibroblast contraction may be needed before such approval is given.

Note in Proof

Staining could be a useful tool in allowing comparison of the effects of different antiproliferative agents


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