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Trypan Blue Staining of Epiretinal Membranes in Proliferative Vitreoretinopathy
Eric J. Feron, MD;
Marc Veckeneer, MD;
Rita Parys-Van Ginderdeuren, MD;
Alfons Van Lommel, PhD;
Gerrit R. J. Melles, MD, PhD;
Peter Stalmans, MD, PhD
Arch Ophthalmol. 2002;120:141-144.
ABSTRACT
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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.
INTRODUCTION
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.
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Characteristics and Outcome of Patients*
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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 1
and 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.
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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).
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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).
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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.
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Figure 3. Electron micrograph of an epiretinal
membrane stained with trypan blue, showing detail of cellular components (nucleus
[asterisk]) and pigment granules (arrows).
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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.
AUTHOR 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{at}chello.nl).
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.
REFERENCES
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1. Girard P, Mimoun G, Karpouzas I, Montefiore G. Clinical risk factors for proliferative vitreoretinopathy after retinal
detachment surgery. Retina. 1994;14:417-424.
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2. Abrams GW, Azen SP, McCuen II BW, Flynn HW Jr, Lai MY, Ryan SJ for 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;115:335-344.
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3. Retina Society Terminology Committee. The classification of retinal detachment with proliferative vitreoretinopathy. Ophthalmology. 1983;90:121-125.
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4. Aaberg TM. Management of anterior and posterior proliferative vitreoretinopathy:
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5. Melles GRJ, de Waard PW, Pameyer JH, Beekhuis H. Trypan blue capsule staining to visualize the capsulorhexis in cataract
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6. Veckeneer M, van Overdam K, Monzer J, et al. Ocular toxicity study of trypan blue applied in the vitreous cavity
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