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We would like to thank Dr Rodrigues and colleagues for bringing up this interesting point of what exactly trypan blue stains.1
In our study, immunohistochemistry was performed to determine the nature of cells involved in the epiretinal membranes (ERM)—not to determine the presence or absence of the ERM. Presence or absence of ERM was determined by examining routinely stained sections (haematoxylin and eosin, periodic acid Schiff) for cytoplasm/nuclei of epiretinal cell elements. All four of the macular hole internal limiting membrane (ILM) specimens were examined in this way.2 Furthermore trypan blue (in low concentrations) stains the anterior lens capsule.3 Since this capsule lacks glia, we do not believe that the evidence supports the contention of the correspondents that the staining of our ILM specimens is due to undetected “glial cell elements of the highly cellular ERM” rather than ILM.
Clinically two features are observed with the use of trypan blue. Firstly, the whole posterior pole that comes into contact with trypan blue is stained a faint blue in all cases. The staining pattern is diffuse and not patchy, suggesting trypan blue staining is indiscriminate of ERM or ILM. Secondly, in cases of macular pucker, the trypan blue stained ERM can be removed separately, leaving intact ILM behind, which can be further stained and removed. In cases of macular hole where a clinical ERM is not present, it appears that only the ILM is stained and peeled. We have harvested these membranes and confirmed that the membranes only consist of ILM and without a secondary ERM.
There is no doubt that trypan blue stains both ERM and ILM. We, however, have no knowledge as to what the structural elements of these membrane that the dye is attached to. We concede that staining of ILM with trypan blue can be variable and sometimes rather faint. Since our publication, Perrier and Sebag have also reported their experience with trypan blue in staining ILM and ERM.4,5 Although histological findings were not given in these studies, clinically the authors found the dye to be useful in both types of membranes. Given the many concerns regarding the use of indocyanine green,6 we believe it is a positive development that an alternative clinically useful dye is available.