To determine through the use of immunohistochemistry the origins of retrocorneal cellular and fibrillar membranes.
Retrospective, clinicopathologic study using surgically removed human corneal tissues.
Clinical records of patients’ ocular diseases and surgical procedures were reviewed. Immunohistochemical staining was performed on 5 enucleated control globes, 32 penetrating keratoplasty specimens, and 6 Descemet stripping endothelial keratoplasty specimens to analyze: (1) the normal corneal epithelium, stroma, and endothelium; and (2) stromal scars, endothelial abnormalities, and retrocorneal membranes. Paraffin sections were stained with hematoxylin and eosin, periodic acid–Schiff, and Masson trichrome methods, and immunohistochemical analyses were performed with commonly available monoclonal and polyclonal antibodies for various cytokeratins (CKs), CD34, α-smooth muscle actin (SMA), and vimentin.
Five subtypes among 28 retrocorneal membranes were characterized. Twelve fibrous (keratocytic) membranes of stromal origin had coarse collagen and immunostained negatively for all CKs, but strongly for vimentin and α-SMA, the last the only marker of diagnostic value. Nine metaplastic endothelium-derived membranes produced delicate collagenous matrices and immunoreacted with CK7, vimentin, and α-SMA. Two epithelial multilaminar or monolaminar membranes reacted with CK cocktail and wide-spectrum CK, mildly with CK7 (not observed in orthotopic surface epithelium), and negatively for α-SMA and vimentin. The final 2 categories were indeterminate or nonimmunoreactive (3 specimens) and mixed (2 specimens).
Immunohistochemistry can diagnose retrocorneal membranes of different provenances reliably in most cases. Clinical correlations established that these membranes develop after serious inflammatory disorders, prolonged wounding or ulcerations, and multiple surgeries (an average of 3.4 per patient).
Retrocorneal (Retro-Descemet) membranes can develop exceptionally from hemorrhage, exudative precipitates with entrapped inflammatory cells that undergo organization or induce endothelial fibrous metaplasia, displaced proliferative iris stromal melanocytes, or adherent proliferating lens epithelial remnants. Most commonly, they arise after surgery or after accidental trauma from 1 of 3 mechanisms: epithelial downgrowth (ingrowth), fibroblastic or stromal (keratocytic) downgrowth, fibrous metaplasia of the corneal endothelium, or a combination thereof. Contemporary textbooks do not cover this subject comprehensively. Immunohistochemical investigations also have not addressed systematically the task of separating these major categories more definitively, nor have they taken up the challenging issue of overlapping or ambiguous histomorphologic features.
Previous immunohistochemical investigations of the normal corneal layers and various pathologic corneal conditions pertinent to this study have established that normal stromal keratocytes express CD34, that retrocorneal fibrous membranes of keratocytic origin are strongly α-smooth muscle actin (α-SMA) positive, and that normal and diseased endothelium can express a variety of cytokeratins (CKs; e.g., in Fuchs dystrophy, posterior polymorphous dystrophy, congenital hereditary endothelial dystrophy, and some cases of the iridocorneal endothelial syndrome). In this study, we amplified these earlier findings by establishing diagnostically useful immunohistochemical profiles for distinguishing among retrocorneal membranes of different sources, with particular attention paid to collagenous membranes.
The regular and consultation diagnostic files of the David G. Cogan Laboratory of Ophthalmic Pathology at the Massachusetts Eye and Ear Infirmary from July 2006 through September 2009 were searched for penetrating keratoplasty (PK) and Descemet stripping endothelial keratoplasty (DSEK) specimens with the diagnoses of endotheliopathy, Fuchs dystrophy, pseudophakic bullous keratopathy, retrocorneal fibrous membrane, retrocorneal epithelial membrane, fibrous metaplasia of corneal endothelium, fibrous downgrowth or ingrowth, and epithelial downgrowth or ingrowth. Microscopic glass slides prepared from paraffin-embedded tissue sections stained with hematoxylin and eosin and periodic acid–Schiff were reviewed critically. Masson trichrome staining also was performed on the specimens exhibiting retrocorneal fibrous membranes.
Twenty-eight cases with retrocorneal membranes, identified in 27 PK and 1 DSEK specimens, were selected for inclusion in this study based on the microscopic findings, proper tissue orientation, and the availability of tissue blocks for additional immunohistochemical investigations. One of these cases has been published elsewhere because of novel therapeutic considerations. Five eyeballs enucleated because of posterior intraocular pathologic features (e.g., choroidal melanoma), but without any anterior segment involvement affecting the cornea, served as controls to determine the baseline immunohistochemical staining characteristics of the normal cellular layers of the cornea. To supplement these findings, individual layers or regions of layers in the 27 PKs that were unaffected by any pathologic abnormalities (present elsewhere in the specimens) also were included in the total numbers of controls (a cross-check of the staining results derived from the globes and the PK specimens evaluated in this manner revealed no differences). Five other PK and 5 DSEK specimens without fibrillar membrane production also were included in this study to evaluate the comparative staining characteristics of agonal (damaged, disturbed, distressed, injured, or marginally viable) endothelial cells in cases of Fuchs endothelial dystrophy or aphakic or pseudophakic endotheliopathy.
All specimens were studied immunohistochemically using the monoclonal and polyclonal antibodies listed in Table 1 . The chromogen diaminobenzidine was used in the immunohistochemical reactions according to methods described elsewhere. These techniques were performed in the Diagnostic Immunopathology Laboratory of the Department of Pathology at the Massachusetts General Hospital, Boston, Massachusetts. After stratification of the membranes into different classes had been accomplished, clinical correlations were sought by reviewing the patients’ clinical and surgical records regarding prior corneal diseases and the number and types of preceding surgeries.
|Antibody||Common Nonocular and Ocular Specificities||Staining Pattern||Source||Dilution|
|CK, a CK 7 b||Large number of simple glandular, lung, breast, complex, and transitional (urothelial) epithelia; mesothelia (but not normal corneal endothelium)||Cytoplasmic||Mouse monoclonal, IgG1/k c||Prediluted|
|CK 20 d||Simple epithelia of intestines and stomach, Merkel cells||Cytoplasmic||Mouse monoclonal, IgG2a/k c||Prediluted|
|CK Cam5.2 e||Basal cells of glandular epithelia, myoepithelia, and simple epithelia||Cytoplasmic||Mouse monoclonal, IgG2a f||1:80|
|CK Cocktail g||Basal cells of glandular epithelia, myoepithelia, simple and complex epithelia, and corneal epithelium||Cytoplasmic||Mouse monoclonal, IgG2a f , and IgG1 h||1:80; 1:160|
|CK wide spectrum i||Either suprabasal or all epidermal layers, all adnexal glandular cells||Cytoplasmic||Polyclonal rabbit j||1:900|
|CD k 34||Capillary endothelium, proliferating vessels, nonvascular spindle cell tumors, embryonic and a subpopulation of dermal fibroblasts, keratocytes||Membranous||Mouse monoclonal, IgG1 c||Prediluted|
|Vimentin||Mesenchymal cells, anterior and posterior iridial pigmented epithelium, pigmented and nonpigmented ciliary epithelium, normal subcapsular lens, and cataractous epithelium (Wedl cells)||Cytoplasmic||Mouse monoclonal, IgG/k c||Prediluted|
|α-Smooth muscle actin||Smooth muscle cells of blood vessels, myofibroblasts, myoepithelial cells||Cytoplasmic||Mouse monoclonal, IgG c||Prediluted|
|EMA||Epithelial and mesothelial cells (indicative of epithelial differentiation as a supplement to cytokeratins)||Cytoplasmic + membranous||Mouse monoclonal, IgG2a/k c||Prediluted|
a Cytokeratins are a class of intermediate cytoplasmic filaments composed of heteropolymers with acidic and basic components encompassing over 25 subtypes, ranging from a low molecular weight of 40 kD to a high of 68 kD.
b CK 7, molecular weight of 54 kD.
c Ventana Medical Systems, Oro Valley, Arizona, USA.
d CK 20, molecular weight of 46 kD.
e CK Cam5.2 recognizes CK 8 (53 kD) and CK 18 (45 kD) exclusively.
f Becton Dickinson, San Jose, California, USA.
g CK cocktail recognizes both high and low molecular weight cytokeratins and is a combination of Cam5.2 and AE1/AE3 cytokeratins with 2 different classes of IgG and dilutions; AE1 recognizes type I acidic cytokeratins (CK 10, CK 13, CK 14, CK 15, CK 16, and CK 19, 56.5 to 40 kD), whereas AE3 recognizes type II basic cytokeratins (CK 1, CK 2, CK 3, CK 4, CK 5, CK 6, CK 7, and CK 8, 68 to 53 kD).
h Signet Laboratories, Dedham, Massachusetts, USA.
i Stains prominently cytokeratins with molecular weights 52, 56, and 58 kD, and less strongly stains cytokeratins with weights of 48, 51, and 60 kD.
j Dako Corporation, Carpinteria, California, USA.
The findings from the immunohistochemical staining of the 3 main cellular corneal tissue layers of normal control specimens as well as of the injured stromas, agonal (distressed) endothelium without an associated membrane, and various categories of membranes encompassed in this study are summarized in Table 2 .
|Tissue||No. of Specimens||CK 7||CK 20||CK Cam5.2||CK Cocktail||CK Wide Spectrum||EMA||CD34||α-SMA||Vimentin|
|Normal components of corneal layers|
|Epithelium||27 a||–||–||–||+++||+++||–||–||–||+ b|
|Stromal keratocytes||32 c||–||–||–||–||–||–||+++||–||+++|
|Pathologic alterations in corneal layers|
|Keratocytes in or near stromal scar||19 e||–||–||–||–||–||–||–||+++||+++|
|Agonal (damaged) endothelium||10 f||+++||–||++||+/++||+||–||–||+/– g||++|
|Retrocorneal membrane origins|
|Fibrous (keratocytic)||12 h||–||–||–||–||–||–||–||+++||++|
|Metaplastic (endothelial)||9 i||+++||–||++||+/++||+/++||–||–||++||++|
|Mixed||2 k||see Results for immunostaining|
a Normal corneal epithelium of 5 enucleated eyeballs and regions with unaffected epithelium and intact Bowman membrane in 22 of 32 pathologic PK procedures.
b Focal staining of the basal epithelial layer in half of the specimens.
c Normal corneas of 5 enucleated eyes and nondiseased stromal regions of 27 PK procedures that were immunoreactive.
d Normal endothelium of 5 enucleated eyeballs.
e PK specimens with focal scars and wounds in which keratocytes manifested strong cytoplasmic α-SMA and vimentin positivity.
f Five PK procedures and 5 DSEK procedures with distressed endothelium but without a membrane.
g Agonal (damaged or marginally viable) endothelium without a membrane may or may not stain for α-SMA (seen in half the specimens).
h Ten purely fibrous and 2 predominantly fibrous membranes covered focally by metaplastic endothelium with appropriate staining characteristics.
i Eight PK procedures and 1 DSEK, the latter obtained from 1 case of toxic anterior segment syndrome.
j Totally nonstaining: probably 2 fibrous and 1 metaplastic, based on trichrome staining characteristics of matrix and Descemet ruptures in the former.
k One vascularized and 1 inflammatory keratocytic–metaplastic–endothelial composite membrane, each example exhibiting 3 cellular elements.
Normal (Undisturbed) Corneal Layers
Five corneas of enucleated control globes and 22 of 32 PK specimens with intact Bowman layer displayed an unperturbed epithelium wherein the suprabasilar cells stained vividly with CK cocktail ( Figure 1 , Top left) and less intensely with wide-spectrum CK. No differences in staining were noted in the 2 groups of specimens. Vimentin immunoreacted positively within some of the basal cells, particularly peripherally, in approximately half of the specimens ( Figure 1 , Top right). CK 7, CK 20, CK Cam5.2, epithelial membrane antigen (EMA), and α-SMA were uniformly negative among the basilar and suprabasilar keratinocytes. CK 20 negativity signaled the absence of Merkel cells.
The stromal fibroblasts (keratocytes) in 5 enucleated eyeballs stained strongly for vimentin ( Figure 1 , Top right) and CD34 ( Figure 1 , Middle left). α-SMA results were negative ( Figure 1 , Middle right). Identical results were obtained for the quiescent keratocytes in 27 PK specimens that were found in nonscarred and nonvascularized stromal regions.
The normal endothelium in the 5 enucleated globes displayed delicate, diaphanous, or pseudovacuolated cytoplasm that stained positively for vimentin ( Figure 1 , Top right inset) and negatively for EMA, CD34, all CKs ( Figure 1 , Top left inset), and α-SMA ( Figure 1 , Middle right top inset).
Pathologic Alterations in Corneal Layers
In 32 PK specimens with retrocorneal membranes, agonal (injured) endothelium, or both, there were 23 with microscopically identifiable stromal scars from a previous infection, injury, or surgery, whereas 9 did not show this finding. The keratocytes in 19 of these scars and wounds, as well as those in the immediately adjacent corneal lamellae, manifested strong cytoplasmic α-SMA and vimentin positivity ( Figure 1 , Middle right bottom inset); the remaining 4 specimens exhibited healed inactive scars in which both α-SMA and CD34 expression were absent (the last wounding events occurred on an average of 2 years before the final keratoplasty). Thirteen of the 19 scarred α-SMA–positive stromas also manifested 2 to 3 lamellae of α-SMA-positive, deep pre-Descemet keratocytes that were not featured in the remaining 6 scarred stromas. α-SMA–positive keratocytes in the scars lost their expression of CD34, which, however, was preserved elsewhere in the stroma away from the scar. No subtype of CK was detected in any of the stromal cells’ cytoplasms. Nine cases with retrocorneal membranes or disturbed endothelium displayed no wounds or scars in the sections examined, and their keratocytes uniformly stained positively for CD34. Among these specimens, there were 3 cases in which the deep stromal keratocytes in an immediate pre-Descemet membrane location expressed α-SMA positivity. The more superficial α-SMA–negative stromal cells immunostained as expected with CD34. The specimens with pre-Descemet α-SMA–positive keratocytes are not included in Table 2 .
Agonal (injured or distressed) endothelium
Five PK and 5 DSEK specimens showed abnormal endothelium in the form of widely spaced nuclei and attenuated cytoplasms ( Figure 1 , bottom left and inset), with or without guttae or diffuse thickening of Descemet membrane in the absence of a conspicuous retrocorneal fibrous membrane. A thread-like deposition of collagen interposed between Descemet membrane and the marginally viable endothelium was identified in 3 specimens. All of these cases revealed strong immunoreactivity for CK 7 and, to a lesser degree, for CK cocktail, CK Cam5.2, and wide-spectrum CK ( Figure 1 , Bottom right and top inset). α-SMA was expressed erratically and weakly in half of the specimens ( Figure 1 , Bottom right, bottom inset), vimentin was moderately and consistently identified, and EMA results were negative. Only 1 PK specimen demonstrated pre-Descemet deep keratocytic α-SMA positivity, which also was positive in a stromal wound located elsewhere.
Twelve thin ( Figure 2 , Top left) or thick ( Figure 2 , Top right) membranes were composed of compact, cellular, eosinophilic, and strongly Masson trichrome-positive wavy collagenous membranes that frequently threw Descemet membranes into undulations or folds. In 10 specimens, an unequivocal interruption in the Descemet membrane was observed, through which histologically continuous stromal keratocytes and associated collagen extended to produce the retrocorneal membrane. All types of CK immunostaining demonstrated negative results ( Figure 2 , Top left, top inset), but α-SMA immunoreactivity was strikingly positive ( Figure 2 , Top left, bottom inset and Figure 2 , Top right inset). CD34 positivity was absent. Three membranes in this category had an interrupted covering of CK 7-positive cells, presumably endothelium-derived cells that overlay strongly α-SMA and CK 7-negative positive intramembranous cells. Seven specimens displayed deep pre-Descemet keratocytic α-SMA positivity, of which 2 did not display a coexistent stromal scar in the sections examined.
Among the 12 patients in this category, 2 had ocular chemical burns, 2 had herpetic keratitis, 2 had multiple retinal detachments, 1 had traumatic open globe repair, 1 had corneal melt, 1 had Fuchs dystrophy with corneal wound dehiscence, 1 had fungal keratitis, 1 had congenital glaucoma, and 1 had bullous keratopathy. A total of 33 ocular surgeries had been performed: 14 PKs, 8 cataract extractions, 6 retinal detachment repairs, 1 corneal wound dehiscence repair, 1 open globe repair, 1 Ahmed valve insertion, 1 secondary intraocular lens (IOL) implantation, and 1 IOL explantation. The average number of surgeries performed per patient was 2.75. One patient with no previous ocular surgery had a corneal alkali burn 2 years earlier that eventually perforated. The other patient with an alkali burn had sustained the injury 23 years earlier and had undergone 2 PKs elsewhere before his referral to our institution.
In 6 thin ( Figure 2 , Middle left) and 3 thick ( Figure 2 , Middle right) of 9 membranes in this category, there was an amorphous or delicately fibrillar, nonrefractile eosinophilic matrix in hematoxylin and eosin–stained sections. It stained light blue with the Masson trichrome and did not cause significant undulations in the Descemet membrane. Faint streaks of periodic acid–Schiff-positive material usually were detected in the matrix; this feature was absent in the fibrous (keratocytic) membranes described above. An attenuated and usually interrupted monolayer of cells covered the membrane on its anterior chamber aspect ( Figure 2 , Middle left), with occasional cells entrapped within the fibrillar membrane itself ( Figure 2 , Middle left inset and Figure 2 , Middle right). Immunostaining of these cells was strongest for CK 7 ( Figure 2 , Bottom left inset and Figure 2 , Bottom right), moderate for CK Cam5.2, and weakest for CK cocktail ( Figure 2 , Bottom right inset) and wide-spectrum CK. Cells embedded in the multilaminar membranes were CK 7 positive ( Figure 2 , Bottom right). α-SMA immunoreactivity and vimentin were moderately positive ( Figure 2 , Bottom left), whereas EMA and CD34 were negative. Three specimens displayed pre-Descemet deep keratocytic α-SMA positivity ( Figure 2 , Bottom left), one of which did not harbor a concomitant stromal scar.
Among the 9 patients in this category, 2 had multiple retinal detachments, 1 had Fuchs corneal dystrophy, 1 had neurotrophic keratitis with anterior uveitis of many years’ duration, 1 had Peter anomaly, 1 had toxic anterior segment syndrome after a cataract extraction, 1 had panuveitis with aphakic bullous keratopathy, 1 had intractable glaucoma, and 1 had repeated inferior subluxations of his intraocular lens implant. No patients in this category experienced ocular trauma, corneal perforation resulting from ulcer or melt, or herpetic keratitis. A total of 31 ocular surgeries had been performed: 8 cataract extractions, 8 PKs, 6 retinal detachment repairs, 4 Ahmed valve insertions and revisions, 3 trabeculectomies, 1 vitrectomy, and 1 multiple IOL dislocation and repositioning. The average number of surgeries performed per patient was 3.4.
Two retrocorneal membranes were composed of multiple ( Figure 3 , Top left) or single ( Figure 3 , Top right) layers of variably eosinophilic cells. These cells were CK cocktail ( Figure 3 , Top left inset and Figure 3 , Top right inset) and wide-spectrum CK positive. Like most normal surface corneal epithelium surmounting the Bowman membrane, they were vimentin negative but CK 7 positive ( Figure 3 , Middle left inset; not detected in normal, nondisplaced corneal epithelium). Most notably, they were totally nonstaining for α-SMA ( Figure 3 , Middle left). Often situated between these cellular membranes and the Descemet membrane was a delicate fibrillar matrical layer with scattered cells that sometimes were moderately CK 7 positive, α-SMA positive ( Figure 3 , Middle left), or both, presumably surviving endothelium capable of elaborating exiguous secretory products. Both epithelial membranes displayed pre-Descemet deep keratocytic α-SMA positivity ( Figure 3 , Middle left), which typically was more prominent in the accompanying stromal scars.