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Figure 1.
Intraoperative view of a poorly delineated epiretinal membrane in proliferative vitreoretinopathy before (A) and after (B) trypan blue staining (digitally processed images).

Intraoperative view of a poorly delineated epiretinal membrane in proliferative vitreoretinopathy before (A) and after (B) trypan blue staining (digitally processed images).

Figure 2.
Margin of this previously unsuspected epiretinal membrane clearly visualized after staining with trypan blue, which facilitated safe and complete dissection from the retinal surface (digitally processed image).

Margin of this previously unsuspected epiretinal membrane clearly visualized after staining with trypan blue, which facilitated safe and complete dissection from the retinal surface (digitally processed image).

Figure 3.
Electron micrograph of an epiretinal membrane stained with trypan blue, showing detail of cellular components (nucleus[asterisk]) and pigment granules (arrows).

Electron micrograph of an epiretinal membrane stained with trypan blue, showing detail of cellular components (nucleus[asterisk]) and pigment granules (arrows).

Characteristics and Outcome of Patients*
Characteristics and Outcome of Patients*
1.
Girard  PMimoun  GKarpouzas  IMontefiore  G Clinical risk factors for proliferative vitreoretinopathy after retinal detachment surgery. Retina. 1994;14417- 424Article
2.
Abrams  GWAzen  SPMcCuen  BW  IIFlynn  HW  JrLai  MYRyan  SJfor the Silicone Study Group, Vitrectomy with silicone oil or long-acting gas in eyes with severe proliferative vitreoretinopathy: results of additional and long-term follow-up: Silicone Study report 11. Arch Ophthalmol. 1997;115335- 344Article
3.
Retina Society Terminology Committee, The classification of retinal detachment with proliferative vitreoretinopathy. Ophthalmology. 1983;90121- 125Article
4.
Aaberg  TM Management of anterior and posterior proliferative vitreoretinopathy: XLV Edward Jackson Memorial Lecture. Am J Ophthalmol. 1988;106519- 532Article
5.
Melles  GRJde Waard  PWPameyer  JHBeekhuis  H Trypan blue capsule staining to visualize the capsulorhexis in cataract surgery. J Cataract Refract Surg. 1999;257- 9Article
6.
Veckeneer  Mvan Overdam  KMonzer  J  et al.  Ocular toxicity study of trypan blue applied in the vitreous cavity of rabbit eyes. Graefes Arch Clin Exp Ophthalmol. 2001;239698- 704Article
7.
Gandorfer  AMessmer  EMUlbig  MWKampik  A Indocyanine green selectively stains the internal limiting membrane. Am J Ophthalmol. 2001;131387- 388Article
Clinical Sciences
February 2002

Trypan Blue Staining of Epiretinal Membranes in Proliferative Vitreoretinopathy

Author Affiliations

From the Rotterdam Eye Hospital, Rotterdam, the Netherlands (Drs Feron and Veckeneer); Departments of Ophthalmology (Drs Parys-Van Ginderdeuren and Stalmans) and Pathology (Dr Van Lommel), Universitair Ziekenhuis Leuven, Leuven, Belgium; and Netherlands Institute for Innovative Ocular Surgery, Rotterdam(Dr Melles). The Netherlands Institute for Innovative Ocular Surgery has proprietary and financial interest in a commercially available 0.06% solution of trypan blue for intraocular use (VisionBlue; Dutch Ophthalmic Research Center, Zuidland, the Netherlands), which is not yet available in the United States.

Arch Ophthalmol. 2002;120(2):141-144. doi:10.1001/archopht.120.2.141
Abstract

Objective  To determine whether trypan blue staining facilitates epiretinal membrane(ERM) removal in proliferative vitreoretinopathy.

Methods  In 10 patients undergoing vitrectomy for proliferative vitreoretinopathy, ERM peeling was performed without staining the tissue, until no additional ERMs were clearly visible. Then, after a fluid-air exchange, 0.06% trypan blue solution was applied onto the retinal surface. After 1 minute, all excess dye was removed and, after an air-fluid exchange, ERM peeling was completed. Excised ERM specimens were analyzed by transmission electron microscopy.

Main Outcome Measures  For each patient, the efficacy of trypan blue staining of ERMs during surgery was scored.

Results  In all patients, intraoperative staining of ERMs with trypan blue was found to be a useful adjunct, since the dye consistently improved direct visualization and delineation of ERMs and facilitated ERM removal. A clear contrast was created between the stained ERMs and the nonstaining, underlying retina. Electron microscopy showed that only ERM tissue was removed. No adverse reactions related to the use of the dye were observed up to 3 months after surgery.

Conclusions  Trypan blue may be an important new tool in the surgical management of proliferative vitreoretinopathy, since it may allow a more complete and safer ERM removal.

PROLIFERATIVE vitreoretinopathy (PVR) is characterized by the proliferation and contraction of nonvascular epiretinal membranes (ERMs) at the retinal-vitreous interface after rhegmatogenous retinal detachment. It is the most common cause of failure of rhegmatogenous retinal detachment surgery, with recurrent retinal detachment occurring in 5% to 10% of eyes.1

Although long-acting gases and silicone oil as tamponading agents have improved the outcome of PVR surgery, incomplete removal or ERMs during the first surgery may be a major cause of recurrent PVR with redetachment of the retina. Better visibility and delineation of the ERMs during PVR surgery may result in a more complete ERM removal and a higher rate of long-term retinal reattachment.2

The purpose of our study was to evaluate whether trypan blue staining facilitates visualization and delineation of ERMs, allowing a more complete ERM removal in PVR surgery.

PATIENTS AND METHODS

The study was approved by an institutional review board of the University of Leuven, Leuven, Belgium. Ten patients were enrolled (Table 1). All patients had advanced PVR (stage C3 and more) after rhegmatogenous retinal detachment or complicated posterior segment surgery, and all agreed to the study by informed consent.

In each patient, ERM peeling was performed, until no residual membranes could be clearly observed under wide-angle viewing conditions (Zeiss operating microscope with EIBOS wide-angle viewing system; Zeiss; Weesp, the Netherlands). A complete fluid-air exchange was then performed, and 0.5 mL of 0.06% trypan blue in a sodium phosphate buffer (VisionBlue; Dutch Ophthalmic Research Center, Zuidland, the Netherlands) was injected in the vitreous cavity close to the retinal surface through a blunt-tipped needle. The eye was rotated in different directions to disperse the dye over the peripheral retinal surface. After 1 minute, all excess dye was carefully removed with a back-flush needle and air was exchanged with fluid.

Removed ERM membranes were immediately fixed in 2.5% glutaraldehyde–0.1M phosphate buffer. After postfixation in 1% osmium tetroxide–0.1M phosphate buffer, the specimens were processed for routine transmission electron microscopy.

All patients were examined at days 1, 2, 3, 10, and 21 after surgery, and then at 2- to 4-week intervals.

RESULTS

After "complete" ERM removal, the injection of the dye resulted in light blue staining of previously poorly visible or invisible residual PVR membranes in all patients. Stained ERMs were well delineated and showed a clear contrast with the nonstaining retina (Figure 1and Figure 2). In all patients, the best-corrected visual acuity returned to preoperative values within 10 days after surgery and showed slow improvement up to 3 months after surgery. No residual staining of posterior segment tissues was observed at the first day after surgery, and no adverse reactions related to the dye were detected up to 3 months after surgery.

With transmission electron microscopy, excised tissues were found to be ERMs containing fibrous astrocytes and cells derived from the retinal pigment epithelium (Figure 3). No internal limiting membrane or other retinal tissue elements could be detected in our specimens. Trypan blue staining particles could not be visualized with light or electron microscopy.

COMMENT

The completeness of removal of tractional membranes is one of the most important prognostic factors influencing the outcome of PVR surgery.4 However, ERMs are often poorly visible because of their transparency, and a mild sheen or atypical wrinkling of the underlying retina may be the only indirect clue of their presence. When ERMs are visible, their actual extent may be much greater than that expected from their ophthalmoscopic aspect.

Recently, trypan blue staining of the anterior lens capsule was introduced to facilitate the capsulorrhexis during phacoemulsification procedures in the absence of a red fundus reflex.5 To our knowledge, no adverse effects have been reported after the intraocular use of the dye. We therefore speculated that trypan blue could have a use in posterior segment surgery.

Before our clinical study was conducted, the biocompatibility of 0.06% trypan blue was evaluated in vitro by an independent laboratory (BioScan, Laboratory for Medical Device Evaluation, Bilthoven, the Netherlands): cytotoxicity, extract, 24-hour end-point dilution tests were conducted according to the International Standardization Organization (ISO) 10993 and European Norm (EN) 30993 (H. W. B. Jansen, PhD, unpublished data, 2000). Retinal tissue changes after long-term exposure to trypan blue were also evaluated in an in vivo rabbit model. In that study, no tissue changes were detected with light and electron microscopy after continuous exposure of 0.06% trypan blue to the retina for 1 month, whereas high concentrations of the dye were associated with tissue changes in the inferior retinal quadrant.6

In the present study, trypan blue was found to create a useful contrast between the ERM and the nonstaining retina, thereby clearly delineating the extent of the ERM. This enabled a more complete removal of the ERMs, since ERMs that were unsuspected before injection of the dye were clearly visualized. Because the margins of the membranes were better delineated, the risk of inadvertent damage to the retina was also minimized.

Trypan blue was particularly useful in visualizing ERMs in long-standing and/or recurrent PVR. In contrast, in cases of early PVR with a majority of fresh, immature membranes, the density of trypan blue staining of the ERMs was found to be highly variable. Trypan blue also proved to be a helpful tool to assess whether the retinal surface was completely free of membranes at the time of silicone oil removal. During this procedure, trypan blue was applied to the retina after aspiration of the oil. The absence of staining of the retina at this stage supported our decision that silicone oil removal was safe to perform.

Recently, we also detected that trypan blue and indocyanine green may have complementary staining properties at the vitreoretinal interface in PVR: although trypan blue shows high affinity for mature ERMs, indocyanine green binds more selectively to the internal limiting membrane7 but may also stain some epiretinal PVR membranes (data not shown). A double staining technique with trypan blue and indocyanine green proved useful in patients with idiopathic premacular fibrosis (P.S., unpublished data, 2001).

In conclusion, trypan blue staining of ERMs was found to be a useful adjunct in the surgical management of PVR, since it allows a more complete and safer removal of ERMs. Long-term clinical studies are needed to determine whether this novel technique will ultimately result in a better anatomic and functional outcome of PVR surgery.

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Article Information

Accepted for publication October 5, 2001.

Corresponding author and reprints: Eric J. Feron, MD, Rotterdam Eye Hospital, Schiedamsevest 180, 3011 BH Rotterdam, the Netherlands (e-mail: ericferon@chello.nl).

References
1.
Girard  PMimoun  GKarpouzas  IMontefiore  G Clinical risk factors for proliferative vitreoretinopathy after retinal detachment surgery. Retina. 1994;14417- 424Article
2.
Abrams  GWAzen  SPMcCuen  BW  IIFlynn  HW  JrLai  MYRyan  SJfor the Silicone Study Group, Vitrectomy with silicone oil or long-acting gas in eyes with severe proliferative vitreoretinopathy: results of additional and long-term follow-up: Silicone Study report 11. Arch Ophthalmol. 1997;115335- 344Article
3.
Retina Society Terminology Committee, The classification of retinal detachment with proliferative vitreoretinopathy. Ophthalmology. 1983;90121- 125Article
4.
Aaberg  TM Management of anterior and posterior proliferative vitreoretinopathy: XLV Edward Jackson Memorial Lecture. Am J Ophthalmol. 1988;106519- 532Article
5.
Melles  GRJde Waard  PWPameyer  JHBeekhuis  H Trypan blue capsule staining to visualize the capsulorhexis in cataract surgery. J Cataract Refract Surg. 1999;257- 9Article
6.
Veckeneer  Mvan Overdam  KMonzer  J  et al.  Ocular toxicity study of trypan blue applied in the vitreous cavity of rabbit eyes. Graefes Arch Clin Exp Ophthalmol. 2001;239698- 704Article
7.
Gandorfer  AMessmer  EMUlbig  MWKampik  A Indocyanine green selectively stains the internal limiting membrane. Am J Ophthalmol. 2001;131387- 388Article
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