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Use of trypan blue dye during conversion of deep anterior lamellar keratoplasty to penetrating keratoplasty

Sharma, Namrata MD; Jhanji, Vishal MD; Titiyal, Jeewan S. MD; Amiel, Howard MD; Vajpayee, Rasik B. MS, FRCS (Edin), FRANZCO

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Journal of Cataract & Refractive Surgery: August 2008 - Volume 34 - Issue 8 - p 1242-1245
doi: 10.1016/j.jcrs.2008.03.048
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Deep anterior lamellar keratoplasty (DALK) using the big-bubble technique is indicated for corneal stromal pathologies that do not involve Descemet membrane (DM) and the corneal endothelium.1–6 The procedure involves excising diseased corneal stroma and replacing it with healthy donor corneal tissue. The main advantage of the technique is that it does not replace the corneal endothelium, therefore eliminating the risk for endothelial graft rejection. However, attempts to bare DM are not always successful and can be complicated by perforation, necessitating conversion to penetrating keratoplasty (PKP). We observed that in such instances, extensive corneal emphysema and opacification of the corneal stromal lamellae often result in poor visualization of trephined corneal edges. In some cases, a shelf of posterior corneal tissue may remain, compromising the integrity of the wound, with the potential to cause significant corneal astigmatism. Furthermore, it is often difficult to visualize the partially excised and transparent DM following conversion from DALK to PKP. To overcome these difficulties, we used trypan blue dye to stain and identify remnants of corneal stromal tissue and DM.


Trypan blue staining is used after the surgeon has dissected the host cornea for conversion of DALK to PKP. The pupil is constricted using acetylcholine chloride (Miochol 2%), and a small bolus of sodium hyaluronate 2.3% is placed over the pupil to prevent inadvertent staining of the crystalline lens capsule (Figure 1, A). Then, 0.1 mL of 0.06% trypan blue (Visiblue) is instilled and mechanically “spread” inside the trephined edge of the cornea over the iris (Figure 1, B). After 30 seconds, balanced salt solution is used to irrigate and wash out the dye. Any remnants of corneal stroma and/or DM detected after staining with the trypan blue dye are excised (Figure 1, C and D).

Figure 1
Figure 1:
A: Small bolus of sodium hyaluronate 2.3% placed over the pupil to prevent staining of the crystalline lens capsule. B: Trypan blue 0.06% spread inside the trephined edge of the cornea. C: Remnants of stained DM excised inferiorly. D: Remnants of stained DM excised superiorly.
Figure 1
Figure 1:
Figure 1
Figure 1:
Figure 1
Figure 1:


The trypan blue staining technique was used in 8 cases that were converted from DALK to PKP because of inadvertent perforation of DM. Remnants of the host DM or corneal stromal tissue were detected in 7 eyes (87.5%) (Table 1). These included posterior corneal stromal remnants, typically forming a shelf or a ledge of tissue along the previously trephined host corneal edge. Most remnants of the perforated DM were in the form of small pieces attached to the host cornea. However, in 2 cases (cases 4 and 5) more than half of DM was detected after the trypan blue staining. The remnants were excised and a 0.5 mm oversized graft was secured using 16 interrupted 10-0 nylon sutures.

Table 1
Table 1:
Cases that were converted from DALK to PKP.

Postoperatively, all patients received 0.3% ciprofloxacin hydrochloride (Ciplox) eyedrops 4 times a day, prednisolone acetate 1% (Predacetate) eyedrops 6 times day, and tropicamide 1% eyedrops twice a day. On the first postoperative day, slitlamp examination revealed a well-apposed graft–host junction in all the cases with a near-vertical host wound profile. No case had remnants of the host DM beyond the trephined corneal edge. At the last follow-up (3 months), no eye demonstrated residual staining of the anterior lens capsule or evidence of cataract formation.


Occurrence of a large intraoperative perforation in DM during DALK requires conversion to PKP after complete excision of the host tissue. In many conversion cases, we found that remnants of posterior corneal tissue and DM were left, necessitating another surgical intervention. We observed that the use of air in DALK caused loss of stromal transparency beyond the trephined corneal edge. This often prevented adequate visualization of the anterior and posterior edges of the host cut, and a shelf of posterior stromal tissue was inadvertently left, creating an irregular wound profile. In addition, because it is a very fine, transparent structure, DM is often hard to identify and excise intraoperatively when it is separated from the posterior corneal stroma.

Retention of DM has been known to occur following corneal transplantation surgery for congenital hereditary endothelial dystrophy and pseudophakic bullous keratopathy.7,8 It has been suggested that severe edema and thickening of the recipient cornea predisposes to lamellar splitting and unintentional preservation of DM behind the corneal button.9 Direct contact of the retained DM with the endothelium can compromise graft viability by contact injury or by preventing aqueous humor support.10 However, Loewenstein et al.11 have shown that the corneal graft remains clear provided contact between the graft endothelium and retained DM is avoided by allowing aqueous flow between them.

Trypan blue dye has a propensity to stain basement membranes such as the lens capsule and DM and can be used in intraocular surgery to enhance the visibility of these structures. Although it is considered to be safe, cases of permanent blue discoloration of hydrogel intraocular lenses and inadvertent staining of the posterior lens capsule have been reported, and possible long-term side effects are unknown given its potential carcinogenicity in higher concentrations.12 Trypan blue dye is commonly used in cataract surgery to assist in the visualization of the lens capsule,13,14 particularly for white cataracts and for staining side-port and corneal tunnel incisions for phacoemulsification.15

Trypan blue has also been used in various corneal transplantation surgeries to identify DM for preparation of donor corneal tissue in DALK and Descemet-stripping automated endothelial keratoplasty16,17 and to stain corneal stromal fibers during dissection in deep lamellar keratoplasty.18 Roos et al.19 describe an innovative technique of staining with trypan blue during corneal transplantation to judge accurate tissue depth for optimal suture placement and as a means of facilitating vertical wound alignment. Sinha et al.20 describe descemetorhexis using trypan blue dye for the removal of inadvertently retained DM following PKP. The extent of and risk for retained DM is greater after DALK than after PKP given that once partially separated from the stiffer corneal stroma, DM is more compliant and may displace posteriorly against the trephined edge, resulting in less effective penetration of DM.

We found that trypan blue was helpful in identifying DM remnants as well as the corneal stromal wound edge in 7 of 8 cases (87.5%). Trypan blue staining of corneal stroma was useful for excising the corneal shelf close to the trephined corneal edge, providing a near-vertical wound profile at the end of surgery.

In the presence of a crystalline lens, staining the anterior lens capsule or subsequent formation of a visually significant cataract is a theoretical risk. To minimize this potential risk prior to the use of trypan blue, we induced pupil miosis and used sodium hyaluronate 2.3% to cover the exposed part of the anterior lens capsule visible through the miosed pupil. We believe that our technique of trypan blue dye staining during conversion of DALK to PKP effectively aids in delineating the edges of the trephined corneal wound and identifying remnants of posterior corneal stroma and DM.


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© 2008 by Lippincott Williams & Wilkins, Inc.