Use of trypan blue dye during conversionof deep anterior lamellar keratoplasty

to penetrating keratoplastyNamrata Sharma, MD, Vishal Jhanji, MD, Jeewan S. Titiyal, MD, Howard Amiel, MD,

Rasik B. Vajpayee, MS, FRCS (Edin), FRANZCO

We describe a technique that uses trypan blue dye to identify residual recipient corneal stroma andDescemet membrane (DM) during conversion of deep anterior lamellar keratoplasty (DALK) topenetrating keratoplasty (PKP). After the host cornea is dissected, trypan blue dye (0.06%) isused to highlight the remaining host corneal stroma and DM, if any. In 8 DALK procedures thathad to be converted to PKP because of DM perforation, trypan blue staining identified remnantsof DM and parts of the posterior corneal stroma in 7 eyes. Improved visualization of the residualhost tissue enabled complete excision and an overall optimal recipient wound profile.

J Cataract Refract Surg 2008; 34:1242–1245 Q 2008 ASCRS and ESCRS

TECHNIQUE

Deep anterior lamellar keratoplasty (DALK) using thebig-bubble technique is indicated for corneal stromalpathologies that do not involve Descemet membrane(DM) and the corneal endothelium.1–6 The procedureinvolves excising diseased corneal stroma and replac-ing it with healthy donor corneal tissue. The main ad-vantage of the technique is that it does not replace thecorneal endothelium, therefore eliminating the risk forendothelial graft rejection. However, attempts to bareDM are not always successful and can be complicatedby perforation, necessitating conversion to penetratingkeratoplasty (PKP). We observed that in such in-stances, extensive corneal emphysema and opacifica-tion of the corneal stromal lamellae often result inpoor visualization of trephined corneal edges. Insome cases, a shelf of posterior corneal tissue may re-main, compromising the integrity of the wound,

Accepted for publication March 30, 2008.

From the Rajendra Prasad Centre for Ophthalmic Sciences (Shar-ma, Jhanji, Titiyal, Vajpayee), All India Institute of Medical Sciences,New Delhi, India; and the Centre for Eye Research Australia (Amiel,Vajpayee), and the Department of Ophthalmology, University ofMelbourne, Australia.

No author has a financial or proprietary interest in any material ormethod mentioned.

Corresponding author: Rasik B. Vajpayee, MS, FRCS (Edin), Centrefor Eye Research Australia, University of Melbourne, 32, GisborneStreet, East Melbourne, Victoria 3002, Australia. E-mail: rasikv@unimelb.edu.au.

Q 2008 ASCRS and ESCRS

Published by Elsevier Inc.

1242

with the potential to cause significant corneal astigma-tism. Furthermore, it is often difficult to visualize thepartially excised and transparent DM following con-version from DALK to PKP. To overcome these diffi-culties, we used trypan blue dye to stain and identifyremnants of corneal stromal tissue and DM.

TRYPAN BLUE STAINING TECHNIQUE

Trypan blue staining is used after the surgeon has dis-sected the host cornea for conversion of DALK to PKP.The pupil is constricted using acetylcholine chloride(Miochol 2%), and a small bolus of sodium hyaluro-nate 2.3% is placed over the pupil to prevent inadver-tent staining of the crystalline lens capsule (Figure 1,A). Then, 0.1 mL of 0.06% trypan blue (Visiblue) is in-stilled andmechanically ‘‘spread’’ inside the trephinededge of the cornea over the iris (Figure 1, B). After 30seconds, balanced salt solution is used to irrigate andwash out the dye. Any remnants of corneal stromaand/or DM detected after staining with the trypanblue dye are excised (Figure 1, C and D).

Results

The trypan blue staining technique was used in 8cases that were converted from DALK to PKP becauseof inadvertent perforation of DM. Remnants of thehost DM or corneal stromal tissue were detected in 7eyes (87.5%) (Table 1). These included posterior cor-neal stromal remnants, typically forming a shelf ora ledge of tissue along the previously trephined hostcorneal edge. Most remnants of the perforated DM

0886-3350/08/$dsee front matter

doi:10.1016/j.jcrs.2008.03.048

1243TECHNIQUE: USE OF TRYPAN BLUE DYE IN DALK

Figure 1. A: Small bolus of sodium hyaluronate 2.3% placed over the pupil to prevent staining of the crystalline lens capsule. B: Trypan blue0.06% spread inside the trephined edge of the cornea. C: Remnants of stained DM excised inferiorly. D: Remnants of stained DM excisedsuperiorly.

were in the form of small pieces attached to the hostcornea. However, in 2 cases (cases 4 and 5) morethan half of DM was detected after the trypan bluestaining. The remnants were excised and a 0.5 mmoversized graft was secured using 16 interrupted 10-0 nylon sutures.

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

J CATARACT REFRACT SUR

DISCUSSION

Occurrence of a large intraoperative perforation in DMduring DALK requires conversion to PKP after com-plete excision of the host tissue. In many conversioncases, we found that remnants of posterior corneal tis-sue andDMwere left, necessitating another surgical in-tervention. We observed that the use of air in DALKcaused loss of stromal transparency beyond the tre-phined corneal edge. This often prevented adequate vi-sualization of the anterior and posterior edges of thehost cut, and a shelf of posterior stromal tissue was in-advertently left, creating an irregularwoundprofile. Inaddition, because it is a very fine, transparent structure,DM is often hard to identify and excise intraoperativelywhen it is separated from the posterior corneal stroma.

Retention of DM has been known to occur follow-ing corneal transplantation surgery for congenital

G - VOL 34, AUGUST 2008

1244 TECHNIQUE: USE OF TRYPAN BLUE DYE IN DALK

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

Case Indication for DALK Cause of DM Perforation Remnants Detected After Trypan Blue Staining

1 Keratoconus Excessive air injection Remnants of host DM2 Keratoconus Excessive air injection Remnants of host DM, large shelf of stromal

tissue superiorly3 Healed keratitis Instrument trauma Shelf of stromal tissue superiorly4 Keratoconus Instrument trauma Large remnant of DM5 Macular corneal dystrophy Instrument trauma Inferior half of DM6 Keratoconus Excessive air injection None7 Keratoconus Manual dissection Retained DM inferiorly, thin layer of host

stromal tissue8 Keratoconus Excessive air injection Retained DM inferotemporally, large shelf of

stromal tissue

DALK Z deep anterior lamellar keratoplasty; DM Z Descemet membrane; PKP Z penetrating keratoplasty

hereditary endothelial dystrophy and pseudophakicbullous keratopathy.7,8 It has been suggested that se-vere edema and thickening of the recipient corneapredisposes to lamellar splitting and unintentionalpreservation of DM behind the corneal button.9 Di-rect contact of the retained DM with the endotheliumcan compromise graft viability by contact injury orby preventing aqueous humor support.10 However,Loewenstein et al.11 have shown that the cornealgraft remains clear provided contact between thegraft endothelium and retained DM is avoided by al-lowing aqueous flow between them.

Trypan blue dye has a propensity to stain basementmembranes such as the lens capsule and DM and canbe used in intraocular surgery to enhance the visibilityof these structures. Although it is considered to be safe,cases of permanent blue discoloration of hydrogel in-traocular lenses and inadvertent staining of the poste-rior lens capsule have been reported, and possiblelong-term side effects are unknown given its potentialcarcinogenicity in higher concentrations.12 Trypanblue dye is commonly used in cataract surgery to assistin the visualization of the lens capsule,13,14 particularlyfor white cataracts and for staining side-port and cor-neal tunnel incisions for phacoemulsification.15

Trypan blue has also been used in various cornealtransplantation surgeries to identify DM for prepara-tion of donor corneal tissue in DALK and Descemet-stripping automated endothelial keratoplasty16,17

and to stain corneal stromal fibers during dissectionin deep lamellar keratoplasty.18 Roos et al.19 describean innovative technique of staining with trypan blueduring corneal transplantation to judge accurate tissuedepth for optimal suture placement and as a means offacilitating vertical wound alignment. Sinha et al.20 de-scribe descemetorhexis using trypan blue dye for theremoval of inadvertently retained DM following

J CATARACT REFRACT SURG

PKP. The extent of and risk for retained DM is greaterafter DALK than after PKP given that once partiallyseparated from the stiffer corneal stroma, DM ismore compliant and may displace posteriorly againstthe trephined edge, resulting in less effective penetra-tion of DM.

We found that trypan blue was helpful in identify-ing DM remnants as well as the corneal stromalwound edge in 7 of 8 cases (87.5%). Trypan blue stain-ing of corneal stroma was useful for excising the cor-neal shelf close to the trephined corneal edge,providing a near-vertical wound profile at the end ofsurgery.

In the presence of a crystalline lens, staining the an-terior lens capsule or subsequent formation of a visu-ally significant cataract is a theoretical risk. Tominimize this potential risk prior to the use of trypanblue, we induced pupil miosis and used sodium hya-luronate 2.3% to cover the exposed part of the anteriorlens capsule visible through the miosed pupil. We be-lieve that our technique of trypan blue dye stainingduring conversion of DALK to PKP effectively aidsin delineating the edges of the trephined cornealwound and identifying remnants of posterior cornealstroma and DM.

REFERENCES1. Archila EA. Deep lamellar keratoplasty dissection of host tissue

with intrastromal air injection. Cornea 1984–85; 3:217–218

2. Sugita J, Kondo J. Deep lamellar keratoplasty with complete

removal of pathological stroma for vision improvement. Br J

Ophthalmol 1997; 81:184–188

3. Melles GRJ, Lander F, Rietveld FJR, Remeijer L,

Beekhuis WH, Binder PS. A new surgical technique for deep

stromal, anterior lamellar keratoplasty. Br J Ophthalmol 1999;

83:327–333

4. Anwar M, Teichmann KD. Big-bubble technique to bare Desce-

met’s membrane in anterior lamellar keratoplasty. J Cataract Re-

fract Surg 2002; 28:398–403

- VOL 34, AUGUST 2008

1245TECHNIQUE: USE OF TRYPAN BLUE DYE IN DALK

5. Anwar M, Teichmann KD. Deep lamellar keratoplasty; surgical

techniques for anterior lamellar keratoplasty with and without

baring of Descemet’s membrane. Cornea 2002; 21:374–383

6. Vajpayee RB, Tyagi J, Sharma N, Kumar N, Jhanji V, Titiyal JS.

Deep anterior lamellar keratoplasty by big-bubble technique for

treatment corneal stromal opacities. Am J Ophthalmol 2007;

143:954–957

7. Mahmood MA, Teichmann KD, Tomey KF, al-Rashed D. De-

tachment of Descemet’s membrane. J Cataract Refract Surg

1998; 24:827–833

8. Thyagarajan S, Mearza AA, Falcon MG. Inadvertent retention of

Descemet membrane in penetrating keratoplasty. Cornea 2006;

25:748–749

9. Wolter JR, Willey EN. Lamellar splitting of Descemet’s mem-

brane. Am J Ophthalmol 1966; 61:331–334

10. Waring GO III, Bourne WM, Edelhauser HF, Kenyon KR. The

corneal endothelium; normal and pathologic structure and func-

tion. Ophthalmology 1982; 89:531–590

11. Loewenstein A, Geyer O, Lazar M. Intentional retention of De-

scemet’s membrane in keratoplasty for the surgical treatment

of bullous keratopathy. Acta Ophthalmol (Copenh) 1993;

71:280–282

12. Brouzas D, Droutsas D, Charakidas A, Malias I, Georgiadou E,

Apostolopoulos M, Moschos M. Severe toxic effect of methylene

blue 1% on iris epithelium and corneal endothelium. Cornea

2006; 25:470–471

J CATARACT REFRACT SUR

13. Melles GRJ, de Waard PWT, Pameyer JH, Beekhuis WH.

Trypan blue capsule staining to visualize the capsulorhexis in

cataract surgery. J Cataract Refract Surg 1999; 25:7–9

14. Pandey SK, Werner L, Escobar-Gomez M, Werner LP,

Apple DJ. Dye-enhanced cataract surgery. Part 3: poste-

rior capsule staining to learn posterior continuous curvili-

near capsulorhexis. J Cataract Refract Surg 2000;

26:1066–1071

15. Dada T, Sethi HS, Sharma N, Dada VK. Additional uses of try-

pan blue staining during cataract surgery. J Cataract Refract

Surg 2005; 31:1842

16. Wylega1a E, Tarnawska D, Dobrowolski D. Deep lamellar kera-

toplasty for various corneal lesions. Eur J Ophthalmol 2004;

14:467–472

17. Vajpayee RB, Agarwal T, Jhanji V, Sharma N. Modification in

Descemet-stripping automated endothelial keratoplasty; ‘‘hitch

suture’’ technique. Cornea 2006; 25:1060–1062

18. Balestrazzi E, Balestrazzi A, Mosca L, Balestrazzi A. Deep

lamellar keratoplasty with trypan blue intrastromal staining.

J Cataract Refract Surg 2002; 28:929–931

19. Roos JC, Kerr Muir MG. Use of trypan blue for penetrating

keratoplasty. J Cataract Refract Surg 2005; 31:1867–1869

20. Sinha R, Vajpayee RB, Sharma N, Titiyal JS, Tandon R. Trypan

blue assisted descemetorhexis for inadvertently retained De-

scemet’s membranes after penetrating keratoplasty. Br J Oph-

thalmol 2003; 87:654–655

G - VOL 34, AUGUST 2008