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Immunoprotein Deposition in the Cornea
Immunoprotein Deposition in the Cornea OJ
KATHLEEN H. MILLER, MD,* W. RICHARD GREEN, MD,* WALTERJ. STARK, MD,* HAWEY A. WELLS, MD;t GEOFFREY MENDELSOHN, MD,* HARRY KANHOFER, MD*
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Abstract: A 63-year-old woman had bilateral, multi-level corneal deposits distributed as fine , discrete crystals and in dense, deep geographic patches. She had a long history of sero-positive rheumatoid arthritis . Autopsy revealed an unsuspected Iymphoproliferative disorder and immune-complex disease. Histologic examination of the eyes revealed eosinophilic, PAS-positive , noncollagenous deposits in the cornea at all levels and also in the ciliary processes, pars plana, and choroid. Stains for gold, amyloid, and acid mucopolysaccharides were negative. Immunoperoxidase stains were positive for IgG most strongly, and also for IgA, kappa and lambda light chains. Transmission electron microscopy showed needle-like electron-dense extracellular particles which we presume are immunoglobulins. [Key words: benign monoclonal gammopathy, Bietti's crystalline dystrophy, chrysiasis, corneal crystalline deposits, cystinosis , immunoperoxidase stains, immunoprotein, multiple myeloma, Schnyder's crystalline dystrophy.] Ophthalmology 87:944-950, 1980
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Corneal crystalline deposits are known to be a relatively rare manifestation of hypergammaglobulinemicstates, often long preceding the discovery of systemic disease. Corneal crystals were described in individual patients more than five years prior to the diagnosis of multiple myeloma,I-4 benign monoclonal gammopathy,5 and an uncharacterized hyperproteinemia. 6 In one case of multiple myeloma the crystals' appeared during treatment with urethane and decreased after cessation of this therapy.7 The crystalline deposits are usually present in the
From the Eye Pathology Laboratory of the Wilmer Institute and Department of Pathology, The Johns Hopkins Medical Institutions,* and the Department of Pathology,t and Department of Ophthalmolog y. :j: Titusville Hospital, Titusville, PA. Supported in part by Research Grant 1 R01 EY 01684-03 from the National Eye Institute, US Public Health Service. Reprint requests to W. R. Green, MD, Eye Pathology Laboratory , The Johns Hopkins Hospital, 600 North Wolfe Street, Baltimore, MD 21205.
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stroma, although at varying levels (anterior,3 posterior,5,s or diffuse1,2,9,10); but epithelial involvement has also been reported. 4 ,11 Conjunctival involvement is occasionally seen,3,6,9,12 and similar material has been noted in plasma cells in the bone marrow. 3.9.12 In the majority of cases, this crystalline material seems to have infiltrated normal corneal structures but Pinkerton and Robertson 12 have observed an infiltrate of plasma cells with cytoplasmic inclusions apparently responsible for the physical findings. . Until recently, this curious material was poorly characterized, but Klintworth et al4 and Rodrigues et al5 have both identified histologically and electron microscopically these deposits in two patients with immunoglobulin disorders. Our case of bilateral multi-level crystalline corneal deposits was studied in similar manner, with histochemical stains, immunoperoxidase and electron microscopy. Our findings appear to support the view that these corneal opacities are protein and, more specifically, immunoglobulin, in nature.
0161-6420/80/0900/0944/$00.85
©
American Academy of Ophthalmoiogy
CASE REPORT A 63-year-old woman had a history of sero-positive rheumatoid arthritis for 19 years. She was referred to the Wilmer Institute for ophthalmologic evaluation in September 1976. Prior to that time she had been treated with various medications, including indomethacin (Indocin), prednisone, thyroxine, chlorothiazide (Diuril), and vitamin C. She had been on chloroquine for her arthritis and also had received gold injections for one year. Ocular symptoms of keratoconjunctivitis sicca were controlled with topical artificial tears. On examination visual acuity was 20/20 minus 3 in the right eye and 20/25 minus 3 in the left eye. Slitlamp examination revealed very mild changes of keratitis sicca without significant staining with fluorescein. The corneas showed two types of opacities: fine refractile bodies in the epithelium (Fig 1) and large, white, lobulated, discrete opacities within the stroma at all levels (Fig 2). In the right eye, these opacities were most obvious superiorly, the middle stromal layers were most often affected, and in one area the corneal stroma just anterior to Descemet's membrane was involved. Superotemporally, superficial stroma and epithelium were affected, with light staining over this area. In the left eye, most of the opacities were present in a ring configuration in the mid periphery. In one area there was almost a full-thickness, 1 x 1 mm area of deposition. On retroillumination the more superficial lesions had a granular appearance. The lenses were without opacities and the remainder of the ocular findings were normal. At that time it was thought that the fine corneal-epithelial crystals might be gold deposits from therapy, but the stromal opacities could not be further characterized clinically. The patient's clinical findings did not change from the time of consultation in 1976 to her death in July 1978. Postmortem examination performed at the patient's local hospital disclosed an unsuspected lymphoproliferative disorder and immune-complex disease. There were lymphocytic infiltrates in the skin with telangiectasis, renal and pulmonary lesions, and virtual replacement of the liver and spleen. Terminal events included the Budd-Chiari syndrome and multiple pulmonary emboli. Further studies documented infiltrates of immune complexes containing only kappa and lambda chains in the cyto-
plasm of mature-looking lymphocytes and in the basement membranes of many vessels.
MATERIALS AND METHODS The eyes were obtained postmortem and fixed in buffered 10% formaldehyde. The right eye, with the majority of the corneal opacities located superiorly, was opened vertically. The left eye was opened horizontally, bisecting the cornea, with opaque lesions present in both halves. Portions of each cornea containing these lesions were prepared for light microscopy and transmission electron microscopy. Tissue submitted for light microscopy was progressively dehydrated in alcohol, cleared in xylol, embedded in paraffin, and stained differentially with the following stains: hematoxylineosin for glycogen; periodic-acid - Schiff for collagen; Van der Grift, Masson's trichrome for amyloid; Congo red for acid mucopoly· saccharides; Alcian blue for reticulin (Manuel); and for gold (p-dimethylaminobenzylidene rhodamine). Additional tissue sections were processed by the unlabeled antibody immunoperoxidase technique for immunoglobulins (IgG, IgA, kappa and lambda light chains). Controls included substituting normal rabbit serum IgM for the immunoglobulin antisera. Specimens for transmission electron microscopy were fixed in 1% glutaraldehyde and 4% formaldehyde solution, secondarily fixed in 2.5% glutaraldehyde for two hours, washed with buffer, and post-fixed in 2% phosphatebuffered osmium tetroxide for two hours. Further processing included standard dehydration and embedding in Araldite/Epon mixture (Mollenhauer).
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RESULTS GROSS POSTMORTEM EXAMINATION
The right eye was firm and measured 25 x 25 x 25 mm. The cornea had numerous deep
Fig 1. Left, appearance of subepithelial glistening deposits. Fig 2. Right, large geographic crystalline patches involving the full thickness of the cornea of right eye.
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opacities, especially superiorly and fewer inferiorly. The opacities had discrete margins with surrounding clear cornea. No other gross abnormalities were noted. The left eye was firm and measured 24 x 25 x 25 mm. Deep discrete corneal opacities were present in a ring configuration in the midperiphery. There was an uninvolved limbal clear-zone and the central cornea was also relatively clear. HISTOLOGIC EXAMINATION Q)
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In the right eye, unusual deposits were seen to involve the middle and posterior (Fig 3) corneal stroma and one area between the corneal epithelium and Bowman's membrane (Fig 4). The most prominent deposition of this material was seen in an area of overall corneal thickening and involved the posterior stroma adjacent to Descemet's membrane in the superior midperiphery. These deposits were consistently eosinophilic and PAS-positive. They stained red with Masson's trichrome and Van der Grift stains for collagen. Stains for amyloid and gold were negative, as were Alcian blue stains for acid mucopolysaccharide. A small amount of similar material was seen in the pars plicata including ciliary processes, pars plana, and choroid. One deposit in the ciliary body had a surrounding area of granulomatous inflammatory cell infiltration (Fig 5). In the left eye the deposits were also present in the subepithelial area, Bowman's membrane, and the superficial one-third of the corneal stroma, mainly nasally (Fig 6). The posterior stroma ofthe left eye was not involved histologically. The immunoperoxidase staining technique for immunoglobulins disclosed a positive staining reaction of this unusual material was most marked with anti-IgG (Fig 7) and to a lesser degree with antisera against IgA, kappa and lambda light chains. There was no reaction with normal rabbit serum and IgM. ELECTRON MICROSCOPY
Transmission electron microscopy disclosed large aggregates of needle-like electron-dense osmiophilic material within the corneal stroma in an extracellular location (Fig 8). In some areas the deposits seemed to incorporate stromal collagen fibrils, while in other areas there was dissolution of the surrounding collagen. The deposits appeared to be continuous with parallel and sometimes pleated filaments with a diameter of approximately 170 A (17 nm) (Fig 9). A questionable periodicity of 84 A (8.4 nm) was evident in other areas.
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DISCUSSION The differential diagnosis of crystals in the cornea may present a difficult problem to the clinician. Crystals may be seen in a variety of conditions including cystinosis, Bietti's crystalline retinal and corneal dystrophy, Schnyder's crystalline dystrophy, gout, chronic kidney disease and uremia, lipid keratopathy, chrysiasis, mUltiple myeloma, and monoclonal gammopathies. In infantile nephropathic cystinosis, which is a congenital, autosomal recessive, renal tubular defect with an associated metabolic defect in cystine metabolism, tinsel-like, polychromatic crystals representing intracellular accumulation of free cystine are seen in the cornea, conjunctiva, and uveal tissue within the first years of life. Deposits of L-cystine may also be found in the extraocular muscles, retina, retinal pigment epithelium, sclera, and in cells of the reticuloendothelial system throughout the body. The cornea generally is cloudy and shows dense needle-like, glistening crystals, concentrated peripherally more than centrally and often involving the whole thickness of the cornea at the periphery. Central deposits usually involve the anterior cornea, sparing the deeper one-third of the stroma. The characteristic uniform, needle-shaped crystals may occasionally be seen at Bowman's and Descemet's membranes. Peripheral pigmentary retinopathy is also a feature. Pigmentary changes in the macular area may also be observed. These children have severe vitamin-D-resistant rickets, dwarfism, the effects of chronic renal tubular disease (Fanconi syndrome), and they often die before reaching puberty. The adolescent and adult forms of this disease (cystinuria) are more mild, with typical corneal crystals, normal ocular fundi and kidneys, and are without functional impairmenL 13 - 15 Bietti's crystalline corneal and retinal dystrophy is an entity, seen initially in individuals around age 30, which is slowly progressive and tends to develop in family groups. The retinal findings are the most striking, with yellowish glittering crystalline areas of tapetoretinal degeneration, advancing to choroidal sclerosis in late cases. The corneal findings are more subtle. With careful examination one may find tiny, yellowish-white sparkling crystals near the limbus. The crystals are polygonal or needle-like, are mainly located in the anterior stroma with a scattering in the posterior layers, and react negatively to tests for fat and protein. 16 Welchl7 reported lipid deposition in fibrocytes and epithelium in a corneal and conjunctival biopsy of a patient with Bietti's dystrophy. Electron
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Fig 3. First row left, large deposits in the posterior corneal stroma. Descemet's membrane and endothelium are normal (Masson's trichrome, original magnification x 4(0). Fig 4. First row right, subepithelial corneal deposit with surrounding normal epithelium, Bowman's membrane, and stroma (Van der Grift, original magnification x 100). Fig 5. Second row left, deposit in the ciliary body with surrounding granulomatous inflammatory-cell infiltrate (Van der Grift, original magnification x 400). Fig 6. Second row right, superficial stromal deposit seen in the left eye (Van der Grift, original magnification x 100). Fig 7. Third row left, deep stromal substance stains positive for polyvalent IgG. Descemet's membrane and endothelium are normal (immunoperoxidase, IgG 1:40; original magnification x 100). Fig. 8. Third row right. transmission electron micrograph showing extracellular accumulation of large aggregates of a needle-like material in the corneal stroma (original magnification x 3950). Fig. 9. Fourth row right, some deposits appear to be continuous with filaments that have a diameter of approximately 170 A (original magnification x49,000).
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microscopic study of a corneal biopsy specimen from a patient with this syndrome disclosed extracellular deposits of material similar to the immunoglobin described in this case report. Extensive studies, however, failed to show any evidence of a hyperproteinemic state. IS Schnyder's crystalline corneal dystrophy is a slowly progressive, autosomal dominant disorder that becomes manifest in early life. Crystals are composed of cholesterol and are deposited as a symmetrical, grayish discoid central or paracentral corneal opacity in the anterior stroma, including Bowman's membrane. The epithelium is unaffected. Occasionally the opacity is ringshaped with a clear central area. With the biomicroscope, fine needle-like crystals are seen which are either dull white or scintillating with variegated red and green colors. Normal corneal stroma surrounds the crystals. The eye generally is not irritated, no corneal vascularization is seen, and the effect on a patient's vision is often slight. 19- 21 Crystals seen in gout are composed of uric acid and may take various forms: a band keratopathy in the corneal epithelium; multicolored, needle-like crystals scattered diffusely; or cornea urica, a band keratopathy of urate crystals in the cornea in the absence of hyperuricemia. The band keratopathy exhibits golden-yellow crystals in the epithelium and subepithelial region. There is no staining of the epithelium with dyes, and the crystals may be seen to be typically birefringent when obtained by scraping. Urates may also accumulate in the sclera, tarsus, lens, and conjunctiva. Findings of hyperuricemia, typical arthritis, tophi, and urate crystals in affected joint fluid aid in diagnosis; but in cases in which these symptoms and signs are not really evident, a difficult diagnosis may be made on the basis of the ocular findings. 22 - 24 In kidney disease, the corneal pathology is in the form of a band keratopathy of calcium salts. Hydroxyapatite crystals are found in the vicinity of Bowman's membrane. In a majority of uremic patients (up to 80%), a grossly visible, chalk-white limbal opacity in the palpebral fissure may be seen, continuous with the sclera without intervening clear cornea. These asymptomatic deposits of hydroxyapatite are in epithelial and subepithelial locations. Curiously, these patients do not have elevated serum calcium but do have hyperphosphatemia. These limbal opacities do not change with hemodialysis .25-26 Secondary lipid keratopathy (also known as cholesterol keratopathy) is usually a result of local corneal disease (eg, chemical bums, longstanding herpes simplex or zoster, or interstitial keratitis) and is almost always associated with
corneal vascularization. This is seen as a dense yellow-white infiltrate. In some cases, there is no disease or history of trauma; and multicolored, needle-like crystals thought to be cholesterol have been documented. This may be considered "primary" lipid keratopathy,27 Chrysiasis, or gold deposition in tissues, may be seen following elemental gold therapy. Crystals are found anywhere in connective tissue and the reticuloendothelial system. In the cornea there are minute, irregular brownish depositions at various depths in the stroma, more concentrated posteriorly and in Descemet's membrane. These may have a golden metallic gleam in the light of the biomicroscope. 2s In gammopathies (benign monoclonal gammopathy and mUltiple myeloma, specifically), corneal crystals may be seen in variable patterns and locations. Klintworth et al4 reported two cases. The first of these showed epithelial crystals in a patient with mUltiple myeloma and elevated serum IgG with kappa light chains. Histologic and immunoperoxidase study of the corneal button from this patient yielded results identical to our case. On electron microscopic examination they identified intra- and extracellular, electron-dense deposits with contours of varying irregUlarity. Some crystals, however, were quite regular hexagons. Internal periodicity was measurable in a few areas at approximately 10 to 11 nm. Rodrigues et al5 described a case in which crystals were limited to the posterior stroma in the mid periphery of the cornea, with a clear limbal zone. The patient was later discovered to have Bence-Jones-positive monoclonal gammopathy; and, again, a corneal button revealed histological and immunoperoxidase characteristics identical to our case. Extracellular electron-dense deposits were present on electron microscopy, and in this case also had a 10 to 11 nm internal periodicity. It becomes obvious in reviewing the first case of Klintworth et al4 and the case presented by Rodrigues et al,s with the added evidence of our report, that the specific location of the corneal crystals does not seem to be a differential factor in their identification. The crystalline corneal deposits in gammopathies appear clinically as white, yellow, gray, or polychromatic opacities which may be variably iridescent. They may be deposited as discrete, diffuse, fine crystals or in an irregular, more dense geographic distribution (both of these forms were seen in our case). Location is also variable, with the majority of cases reported as having stromal involvement either centrally, peripherally, or in the midperiphery with clear central and limbal zones. In reviewing the literature, one finds that corneal crystals in patients with multiple
myeloma have been documented since 1934,1 but advances in identifying these crystals have only recently emerged. Aronson and Shaw3 concluded from a conjunctival biopsy specimen in a patient with multiple myeloma that the crystals were probably cholesterol or lipid in nature. Laibson and Damian09 studied corneal, conjunctival, and bone marrow crystals with X-ray and electron diffraction techniques and concluded that the crystals were similar to cholesterol stearate. The corneas in our case and the corneal buttons in the cases presented by Klintworth et al4 and Rodrigues et aP show no evidence of cholesterol or lipid. The protein nature of these deposits has long been postulated, based on the patient's underlying disease, l but similar corneal deposits were first characterized as protein by Francois and RabaeyB in 1961. They used benzidine followed by staining with Schiff reagent which resulted in a positive reaction for protein. The mechanism by which this crystalline substance now presumed to be immunoglobulin of the IgG type is deposited in the corneas of these patients is unknown. Because the deposits were in the epithelium in the first case reported by Klintworth et al4 it was postulated that the tears were the source. Rodrigues et al5 state that the posterior location of the protein deposits in their case makes the tears an unlikely vehicle, and tends to implicate the limbal vessels as the source of the immunoglobulin. In our patient, these protein deposits were present at all levels of the cornea (more posteriorly in the right eye and more anteriorly in the left), and additionally, slight scattered deposition was evident in the ciliary processes, pars plana, and choroid. To date, this is the only case in which these proteins have been characterized in ocular tissues other than the cornea and cOrUunctiva, but this is probably because most of the case reports had only cOrUunctival biopsy material and corneal buttons to analyze. In the normal eye, IgG is the predominant immunoglobulin in the cornea, although IgA is also present in significant quantities. 29 AIlansmith et apo report that the serum IgG:IgA ratio is 5: 1 and the corneal IgG:IgA ratio is 10: 1, and they suggest that the small molecular weight of IgG (140,000) in relation to IgA (170,000) and IgM (900,000) may allow IgG to diffuse into the corneal stroma more readily. If so, then the deposition of IgG in the corneas of patients with hypergammaglobulinemia of the IgG type may simply be a concentration phenomenon, although this becomes unlikely as the sole mechanism because the crystals are seen in only a minority of these patients. In conclusion, the corneal opacities in the cases discussed herein had variable clinical pre-
sentations. Thus, discovery of them should prompt the clinician to investigate further, keeping in mind the differential diagnosis of corneal crystals. Obtaining serum and urine electrophoretic and immunoelectrophoretic studies and bone marrow analysis for mUltiple myeloma or another immunoglobulin disorder may often be indicated. If the present patient had been studied further prior to her death, her previously unsuspected lymphoproliferative disorder and immunologic disease, which was discovered at autopsy, would no doubt have come to light at an earlier date.
REFERENCES 1. Meesmann A. Uber eine eigenartige Hornhautdegeneration? (Ablagerung des Bence-Jonesschen EiweisskOrpers in der Hornhaut.) Ber Dtsch Ophthalmol Ges 1934; 50:311-15. 2. Borki E. Uber Hornhautveranderungen bei einem Fall von multiplem Myelom (Plasmocytom). Ophthalmologica 1958; 135:565-72. 3. Aronson SB, Shaw R. Corneal crystals in multiple myeloma. Arch Ophthalmol 1959; 61 :541 -46. 4. Klintworth GK, Bredehoeft SJ, Reed JW. Analysis of corneal crystalline deposits in multiple myeloma. Am J Ophthalmol1978; 86:303-13. 5. Rodrigues MM, Krachmer JH, Miller SO, Newsome DA. Posterior corneal crystalline deposits in benign monoclonal gammopathy. A clinicopathologic case report. Arch Ophthalmol 1979; 97:124-28. 6. Blobner F. Kristallinische Degeneration der Bindehaut und Hornhaut. Klin Monatsbl Augenheilk d 1938; 100:588-93. 7. Markoff N. Ueber Kristallbilbung in der Hornhaut bei. Urethantherapie des Myeloms. Schweiz Med Wochenschr 1948; 78:987 -88. 8. Francois J, Rabaey M. Corneal dystrophy and paraproteinemia. Am J Ophthalmol 1961; 52:895-901. 9. Laibson PR, Damiano W. X-ray and electron diffraction of ocular and bone marrow crystals in paraproteinemia. Science 1969; 163:581-83. 10. Palazon Godinez A. Depot de cristaux dans la cornee. Am Hosp Jose y Adela 1933; 4:235-37, Cited by Burki (1958). 11. Palm E. A case of crystal deposits in the cornea. Precipitation of a spontaneously crystallizing plasma globulin. Acta Ophthalmol 1947; 25:165-74. 12. Pinkerton RMH, Robertson OM. Corneal and conjunctival changes in dysproteinemia. Invest Ophthalmol 1969; 8:357-64. 13. Francois J, Hanssens M, Coppieters R, Evens L. Cystinosis. A clinical and histopathologic study. Am J Ophthalmol 1972; 73:643-50. 14. Kraus E, Lutz P. Ocular cystine deposits in an adult. Arch Ophthalmol 1971; 85:690-94. 15. Sanderson PO, Kuwabara T, Stark WJ, Wong VG, Collins EM. Cystinosis: a clinical, histopathologic, and ultrastructural study. Arch Ophthalmol 1974; 91:270-74. 16. Bagolini B, loli-Spada G. Bietti's tapetoretinal degen-
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eration with marginal corneal dystrophy. Am J Ophthalmol 1968; 65:53-60. Welch, RB. Bietti's tapetoretinal degeneration with marginal corneal dystrophy: crystalline retinopathy. Trans Am Ophthalmol Soc 1977; 75:164-79. Green WR, Klien M. Unpublished data. Gillespie FD, Covelli B. Crystalline corneal dystrophy. Report of a case. Am J Ophthalmol 1963; 56:465-67. Luxenberg M. Hereditary crystalline dystrophy of the cornea. Am J Ophthalmol1967 ; 63:507-11 . Duke-Elder S, ed. System of Ophthalmology, vol. VIII, part II. Diseases of the Outer Eye: Cornea and Sclera. St. Louis : CV Mosby, 1965; 945-46. Fishman RS, Sunderman FW. Band keratopathy in gout. Arch Ophthalmol 1966; 75:367 -69. Siansky HH, Kuwabara T. Intranuclear urate crystals in corneal epithelium. Arch Ophthalmol 1968; 80:338-44.
24. McWilliams JR. Ocular findings in gout . Am J Ophthalmol 1952; 35:1778-83. 25. Harris LS, Cohn K, Toyofuku H, et al. Conjunctival and corneal calcific deposits in uremic patients . Am J Ophthalmol1971; 72:130-33. 26. Caldeira JAF, Sabbaga E, lanhez LE. Conjunctival and corneal changes in renal failure. Influence of renal transplantation . Br J Ophthalmol 1970; 54 :399-404. 27. Davidson A. Primary lipid dystrophy of the cornea. Arch Ophthalmol 1947; 37:433-43. 28. Roberts WH, Wolter JR. Ocular chrysiasis. Arch Ophthalmol 1956; 56:48 -52. 29. Rahi AHS, Garner A. Immunopathology of the eye. Philadelphia: JB Lippincott, 1976; 86-7 . 30. Allansmith MR, Whitney CR, McClellan BH, Newman LP. Immunoglobulins in the human eye . Location, type, and amount. Arch Ophthalmol 1973; 89:36-45.
KATHLEEN H. MILLER, MD,* W. RICHARD GREEN, MD,* WALTERJ. STARK, MD,* HAWEY A. WELLS, MD;t GEOFFREY MENDELSOHN, MD,* HARRY KANHOFER, MD*
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Abstract: A 63-year-old woman had bilateral, multi-level corneal deposits distributed as fine , discrete crystals and in dense, deep geographic patches. She had a long history of sero-positive rheumatoid arthritis . Autopsy revealed an unsuspected Iymphoproliferative disorder and immune-complex disease. Histologic examination of the eyes revealed eosinophilic, PAS-positive , noncollagenous deposits in the cornea at all levels and also in the ciliary processes, pars plana, and choroid. Stains for gold, amyloid, and acid mucopolysaccharides were negative. Immunoperoxidase stains were positive for IgG most strongly, and also for IgA, kappa and lambda light chains. Transmission electron microscopy showed needle-like electron-dense extracellular particles which we presume are immunoglobulins. [Key words: benign monoclonal gammopathy, Bietti's crystalline dystrophy, chrysiasis, corneal crystalline deposits, cystinosis , immunoperoxidase stains, immunoprotein, multiple myeloma, Schnyder's crystalline dystrophy.] Ophthalmology 87:944-950, 1980
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Corneal crystalline deposits are known to be a relatively rare manifestation of hypergammaglobulinemicstates, often long preceding the discovery of systemic disease. Corneal crystals were described in individual patients more than five years prior to the diagnosis of multiple myeloma,I-4 benign monoclonal gammopathy,5 and an uncharacterized hyperproteinemia. 6 In one case of multiple myeloma the crystals' appeared during treatment with urethane and decreased after cessation of this therapy.7 The crystalline deposits are usually present in the
From the Eye Pathology Laboratory of the Wilmer Institute and Department of Pathology, The Johns Hopkins Medical Institutions,* and the Department of Pathology,t and Department of Ophthalmolog y. :j: Titusville Hospital, Titusville, PA. Supported in part by Research Grant 1 R01 EY 01684-03 from the National Eye Institute, US Public Health Service. Reprint requests to W. R. Green, MD, Eye Pathology Laboratory , The Johns Hopkins Hospital, 600 North Wolfe Street, Baltimore, MD 21205.
944
stroma, although at varying levels (anterior,3 posterior,5,s or diffuse1,2,9,10); but epithelial involvement has also been reported. 4 ,11 Conjunctival involvement is occasionally seen,3,6,9,12 and similar material has been noted in plasma cells in the bone marrow. 3.9.12 In the majority of cases, this crystalline material seems to have infiltrated normal corneal structures but Pinkerton and Robertson 12 have observed an infiltrate of plasma cells with cytoplasmic inclusions apparently responsible for the physical findings. . Until recently, this curious material was poorly characterized, but Klintworth et al4 and Rodrigues et al5 have both identified histologically and electron microscopically these deposits in two patients with immunoglobulin disorders. Our case of bilateral multi-level crystalline corneal deposits was studied in similar manner, with histochemical stains, immunoperoxidase and electron microscopy. Our findings appear to support the view that these corneal opacities are protein and, more specifically, immunoglobulin, in nature.
0161-6420/80/0900/0944/$00.85
©
American Academy of Ophthalmoiogy
CASE REPORT A 63-year-old woman had a history of sero-positive rheumatoid arthritis for 19 years. She was referred to the Wilmer Institute for ophthalmologic evaluation in September 1976. Prior to that time she had been treated with various medications, including indomethacin (Indocin), prednisone, thyroxine, chlorothiazide (Diuril), and vitamin C. She had been on chloroquine for her arthritis and also had received gold injections for one year. Ocular symptoms of keratoconjunctivitis sicca were controlled with topical artificial tears. On examination visual acuity was 20/20 minus 3 in the right eye and 20/25 minus 3 in the left eye. Slitlamp examination revealed very mild changes of keratitis sicca without significant staining with fluorescein. The corneas showed two types of opacities: fine refractile bodies in the epithelium (Fig 1) and large, white, lobulated, discrete opacities within the stroma at all levels (Fig 2). In the right eye, these opacities were most obvious superiorly, the middle stromal layers were most often affected, and in one area the corneal stroma just anterior to Descemet's membrane was involved. Superotemporally, superficial stroma and epithelium were affected, with light staining over this area. In the left eye, most of the opacities were present in a ring configuration in the mid periphery. In one area there was almost a full-thickness, 1 x 1 mm area of deposition. On retroillumination the more superficial lesions had a granular appearance. The lenses were without opacities and the remainder of the ocular findings were normal. At that time it was thought that the fine corneal-epithelial crystals might be gold deposits from therapy, but the stromal opacities could not be further characterized clinically. The patient's clinical findings did not change from the time of consultation in 1976 to her death in July 1978. Postmortem examination performed at the patient's local hospital disclosed an unsuspected lymphoproliferative disorder and immune-complex disease. There were lymphocytic infiltrates in the skin with telangiectasis, renal and pulmonary lesions, and virtual replacement of the liver and spleen. Terminal events included the Budd-Chiari syndrome and multiple pulmonary emboli. Further studies documented infiltrates of immune complexes containing only kappa and lambda chains in the cyto-
plasm of mature-looking lymphocytes and in the basement membranes of many vessels.
MATERIALS AND METHODS The eyes were obtained postmortem and fixed in buffered 10% formaldehyde. The right eye, with the majority of the corneal opacities located superiorly, was opened vertically. The left eye was opened horizontally, bisecting the cornea, with opaque lesions present in both halves. Portions of each cornea containing these lesions were prepared for light microscopy and transmission electron microscopy. Tissue submitted for light microscopy was progressively dehydrated in alcohol, cleared in xylol, embedded in paraffin, and stained differentially with the following stains: hematoxylineosin for glycogen; periodic-acid - Schiff for collagen; Van der Grift, Masson's trichrome for amyloid; Congo red for acid mucopoly· saccharides; Alcian blue for reticulin (Manuel); and for gold (p-dimethylaminobenzylidene rhodamine). Additional tissue sections were processed by the unlabeled antibody immunoperoxidase technique for immunoglobulins (IgG, IgA, kappa and lambda light chains). Controls included substituting normal rabbit serum IgM for the immunoglobulin antisera. Specimens for transmission electron microscopy were fixed in 1% glutaraldehyde and 4% formaldehyde solution, secondarily fixed in 2.5% glutaraldehyde for two hours, washed with buffer, and post-fixed in 2% phosphatebuffered osmium tetroxide for two hours. Further processing included standard dehydration and embedding in Araldite/Epon mixture (Mollenhauer).
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RESULTS GROSS POSTMORTEM EXAMINATION
The right eye was firm and measured 25 x 25 x 25 mm. The cornea had numerous deep
Fig 1. Left, appearance of subepithelial glistening deposits. Fig 2. Right, large geographic crystalline patches involving the full thickness of the cornea of right eye.
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opacities, especially superiorly and fewer inferiorly. The opacities had discrete margins with surrounding clear cornea. No other gross abnormalities were noted. The left eye was firm and measured 24 x 25 x 25 mm. Deep discrete corneal opacities were present in a ring configuration in the midperiphery. There was an uninvolved limbal clear-zone and the central cornea was also relatively clear. HISTOLOGIC EXAMINATION Q)
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In the right eye, unusual deposits were seen to involve the middle and posterior (Fig 3) corneal stroma and one area between the corneal epithelium and Bowman's membrane (Fig 4). The most prominent deposition of this material was seen in an area of overall corneal thickening and involved the posterior stroma adjacent to Descemet's membrane in the superior midperiphery. These deposits were consistently eosinophilic and PAS-positive. They stained red with Masson's trichrome and Van der Grift stains for collagen. Stains for amyloid and gold were negative, as were Alcian blue stains for acid mucopolysaccharide. A small amount of similar material was seen in the pars plicata including ciliary processes, pars plana, and choroid. One deposit in the ciliary body had a surrounding area of granulomatous inflammatory cell infiltration (Fig 5). In the left eye the deposits were also present in the subepithelial area, Bowman's membrane, and the superficial one-third of the corneal stroma, mainly nasally (Fig 6). The posterior stroma ofthe left eye was not involved histologically. The immunoperoxidase staining technique for immunoglobulins disclosed a positive staining reaction of this unusual material was most marked with anti-IgG (Fig 7) and to a lesser degree with antisera against IgA, kappa and lambda light chains. There was no reaction with normal rabbit serum and IgM. ELECTRON MICROSCOPY
Transmission electron microscopy disclosed large aggregates of needle-like electron-dense osmiophilic material within the corneal stroma in an extracellular location (Fig 8). In some areas the deposits seemed to incorporate stromal collagen fibrils, while in other areas there was dissolution of the surrounding collagen. The deposits appeared to be continuous with parallel and sometimes pleated filaments with a diameter of approximately 170 A (17 nm) (Fig 9). A questionable periodicity of 84 A (8.4 nm) was evident in other areas.
946
DISCUSSION The differential diagnosis of crystals in the cornea may present a difficult problem to the clinician. Crystals may be seen in a variety of conditions including cystinosis, Bietti's crystalline retinal and corneal dystrophy, Schnyder's crystalline dystrophy, gout, chronic kidney disease and uremia, lipid keratopathy, chrysiasis, mUltiple myeloma, and monoclonal gammopathies. In infantile nephropathic cystinosis, which is a congenital, autosomal recessive, renal tubular defect with an associated metabolic defect in cystine metabolism, tinsel-like, polychromatic crystals representing intracellular accumulation of free cystine are seen in the cornea, conjunctiva, and uveal tissue within the first years of life. Deposits of L-cystine may also be found in the extraocular muscles, retina, retinal pigment epithelium, sclera, and in cells of the reticuloendothelial system throughout the body. The cornea generally is cloudy and shows dense needle-like, glistening crystals, concentrated peripherally more than centrally and often involving the whole thickness of the cornea at the periphery. Central deposits usually involve the anterior cornea, sparing the deeper one-third of the stroma. The characteristic uniform, needle-shaped crystals may occasionally be seen at Bowman's and Descemet's membranes. Peripheral pigmentary retinopathy is also a feature. Pigmentary changes in the macular area may also be observed. These children have severe vitamin-D-resistant rickets, dwarfism, the effects of chronic renal tubular disease (Fanconi syndrome), and they often die before reaching puberty. The adolescent and adult forms of this disease (cystinuria) are more mild, with typical corneal crystals, normal ocular fundi and kidneys, and are without functional impairmenL 13 - 15 Bietti's crystalline corneal and retinal dystrophy is an entity, seen initially in individuals around age 30, which is slowly progressive and tends to develop in family groups. The retinal findings are the most striking, with yellowish glittering crystalline areas of tapetoretinal degeneration, advancing to choroidal sclerosis in late cases. The corneal findings are more subtle. With careful examination one may find tiny, yellowish-white sparkling crystals near the limbus. The crystals are polygonal or needle-like, are mainly located in the anterior stroma with a scattering in the posterior layers, and react negatively to tests for fat and protein. 16 Welchl7 reported lipid deposition in fibrocytes and epithelium in a corneal and conjunctival biopsy of a patient with Bietti's dystrophy. Electron
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Fig 3. First row left, large deposits in the posterior corneal stroma. Descemet's membrane and endothelium are normal (Masson's trichrome, original magnification x 4(0). Fig 4. First row right, subepithelial corneal deposit with surrounding normal epithelium, Bowman's membrane, and stroma (Van der Grift, original magnification x 100). Fig 5. Second row left, deposit in the ciliary body with surrounding granulomatous inflammatory-cell infiltrate (Van der Grift, original magnification x 400). Fig 6. Second row right, superficial stromal deposit seen in the left eye (Van der Grift, original magnification x 100). Fig 7. Third row left, deep stromal substance stains positive for polyvalent IgG. Descemet's membrane and endothelium are normal (immunoperoxidase, IgG 1:40; original magnification x 100). Fig. 8. Third row right. transmission electron micrograph showing extracellular accumulation of large aggregates of a needle-like material in the corneal stroma (original magnification x 3950). Fig. 9. Fourth row right, some deposits appear to be continuous with filaments that have a diameter of approximately 170 A (original magnification x49,000).
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microscopic study of a corneal biopsy specimen from a patient with this syndrome disclosed extracellular deposits of material similar to the immunoglobin described in this case report. Extensive studies, however, failed to show any evidence of a hyperproteinemic state. IS Schnyder's crystalline corneal dystrophy is a slowly progressive, autosomal dominant disorder that becomes manifest in early life. Crystals are composed of cholesterol and are deposited as a symmetrical, grayish discoid central or paracentral corneal opacity in the anterior stroma, including Bowman's membrane. The epithelium is unaffected. Occasionally the opacity is ringshaped with a clear central area. With the biomicroscope, fine needle-like crystals are seen which are either dull white or scintillating with variegated red and green colors. Normal corneal stroma surrounds the crystals. The eye generally is not irritated, no corneal vascularization is seen, and the effect on a patient's vision is often slight. 19- 21 Crystals seen in gout are composed of uric acid and may take various forms: a band keratopathy in the corneal epithelium; multicolored, needle-like crystals scattered diffusely; or cornea urica, a band keratopathy of urate crystals in the cornea in the absence of hyperuricemia. The band keratopathy exhibits golden-yellow crystals in the epithelium and subepithelial region. There is no staining of the epithelium with dyes, and the crystals may be seen to be typically birefringent when obtained by scraping. Urates may also accumulate in the sclera, tarsus, lens, and conjunctiva. Findings of hyperuricemia, typical arthritis, tophi, and urate crystals in affected joint fluid aid in diagnosis; but in cases in which these symptoms and signs are not really evident, a difficult diagnosis may be made on the basis of the ocular findings. 22 - 24 In kidney disease, the corneal pathology is in the form of a band keratopathy of calcium salts. Hydroxyapatite crystals are found in the vicinity of Bowman's membrane. In a majority of uremic patients (up to 80%), a grossly visible, chalk-white limbal opacity in the palpebral fissure may be seen, continuous with the sclera without intervening clear cornea. These asymptomatic deposits of hydroxyapatite are in epithelial and subepithelial locations. Curiously, these patients do not have elevated serum calcium but do have hyperphosphatemia. These limbal opacities do not change with hemodialysis .25-26 Secondary lipid keratopathy (also known as cholesterol keratopathy) is usually a result of local corneal disease (eg, chemical bums, longstanding herpes simplex or zoster, or interstitial keratitis) and is almost always associated with
corneal vascularization. This is seen as a dense yellow-white infiltrate. In some cases, there is no disease or history of trauma; and multicolored, needle-like crystals thought to be cholesterol have been documented. This may be considered "primary" lipid keratopathy,27 Chrysiasis, or gold deposition in tissues, may be seen following elemental gold therapy. Crystals are found anywhere in connective tissue and the reticuloendothelial system. In the cornea there are minute, irregular brownish depositions at various depths in the stroma, more concentrated posteriorly and in Descemet's membrane. These may have a golden metallic gleam in the light of the biomicroscope. 2s In gammopathies (benign monoclonal gammopathy and mUltiple myeloma, specifically), corneal crystals may be seen in variable patterns and locations. Klintworth et al4 reported two cases. The first of these showed epithelial crystals in a patient with mUltiple myeloma and elevated serum IgG with kappa light chains. Histologic and immunoperoxidase study of the corneal button from this patient yielded results identical to our case. On electron microscopic examination they identified intra- and extracellular, electron-dense deposits with contours of varying irregUlarity. Some crystals, however, were quite regular hexagons. Internal periodicity was measurable in a few areas at approximately 10 to 11 nm. Rodrigues et al5 described a case in which crystals were limited to the posterior stroma in the mid periphery of the cornea, with a clear limbal zone. The patient was later discovered to have Bence-Jones-positive monoclonal gammopathy; and, again, a corneal button revealed histological and immunoperoxidase characteristics identical to our case. Extracellular electron-dense deposits were present on electron microscopy, and in this case also had a 10 to 11 nm internal periodicity. It becomes obvious in reviewing the first case of Klintworth et al4 and the case presented by Rodrigues et al,s with the added evidence of our report, that the specific location of the corneal crystals does not seem to be a differential factor in their identification. The crystalline corneal deposits in gammopathies appear clinically as white, yellow, gray, or polychromatic opacities which may be variably iridescent. They may be deposited as discrete, diffuse, fine crystals or in an irregular, more dense geographic distribution (both of these forms were seen in our case). Location is also variable, with the majority of cases reported as having stromal involvement either centrally, peripherally, or in the midperiphery with clear central and limbal zones. In reviewing the literature, one finds that corneal crystals in patients with multiple
myeloma have been documented since 1934,1 but advances in identifying these crystals have only recently emerged. Aronson and Shaw3 concluded from a conjunctival biopsy specimen in a patient with multiple myeloma that the crystals were probably cholesterol or lipid in nature. Laibson and Damian09 studied corneal, conjunctival, and bone marrow crystals with X-ray and electron diffraction techniques and concluded that the crystals were similar to cholesterol stearate. The corneas in our case and the corneal buttons in the cases presented by Klintworth et al4 and Rodrigues et aP show no evidence of cholesterol or lipid. The protein nature of these deposits has long been postulated, based on the patient's underlying disease, l but similar corneal deposits were first characterized as protein by Francois and RabaeyB in 1961. They used benzidine followed by staining with Schiff reagent which resulted in a positive reaction for protein. The mechanism by which this crystalline substance now presumed to be immunoglobulin of the IgG type is deposited in the corneas of these patients is unknown. Because the deposits were in the epithelium in the first case reported by Klintworth et al4 it was postulated that the tears were the source. Rodrigues et al5 state that the posterior location of the protein deposits in their case makes the tears an unlikely vehicle, and tends to implicate the limbal vessels as the source of the immunoglobulin. In our patient, these protein deposits were present at all levels of the cornea (more posteriorly in the right eye and more anteriorly in the left), and additionally, slight scattered deposition was evident in the ciliary processes, pars plana, and choroid. To date, this is the only case in which these proteins have been characterized in ocular tissues other than the cornea and cOrUunctiva, but this is probably because most of the case reports had only cOrUunctival biopsy material and corneal buttons to analyze. In the normal eye, IgG is the predominant immunoglobulin in the cornea, although IgA is also present in significant quantities. 29 AIlansmith et apo report that the serum IgG:IgA ratio is 5: 1 and the corneal IgG:IgA ratio is 10: 1, and they suggest that the small molecular weight of IgG (140,000) in relation to IgA (170,000) and IgM (900,000) may allow IgG to diffuse into the corneal stroma more readily. If so, then the deposition of IgG in the corneas of patients with hypergammaglobulinemia of the IgG type may simply be a concentration phenomenon, although this becomes unlikely as the sole mechanism because the crystals are seen in only a minority of these patients. In conclusion, the corneal opacities in the cases discussed herein had variable clinical pre-
sentations. Thus, discovery of them should prompt the clinician to investigate further, keeping in mind the differential diagnosis of corneal crystals. Obtaining serum and urine electrophoretic and immunoelectrophoretic studies and bone marrow analysis for mUltiple myeloma or another immunoglobulin disorder may often be indicated. If the present patient had been studied further prior to her death, her previously unsuspected lymphoproliferative disorder and immunologic disease, which was discovered at autopsy, would no doubt have come to light at an earlier date.
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eration with marginal corneal dystrophy. Am J Ophthalmol 1968; 65:53-60. Welch, RB. Bietti's tapetoretinal degeneration with marginal corneal dystrophy: crystalline retinopathy. Trans Am Ophthalmol Soc 1977; 75:164-79. Green WR, Klien M. Unpublished data. Gillespie FD, Covelli B. Crystalline corneal dystrophy. Report of a case. Am J Ophthalmol 1963; 56:465-67. Luxenberg M. Hereditary crystalline dystrophy of the cornea. Am J Ophthalmol1967 ; 63:507-11 . Duke-Elder S, ed. System of Ophthalmology, vol. VIII, part II. Diseases of the Outer Eye: Cornea and Sclera. St. Louis : CV Mosby, 1965; 945-46. Fishman RS, Sunderman FW. Band keratopathy in gout. Arch Ophthalmol 1966; 75:367 -69. Siansky HH, Kuwabara T. Intranuclear urate crystals in corneal epithelium. Arch Ophthalmol 1968; 80:338-44.
24. McWilliams JR. Ocular findings in gout . Am J Ophthalmol 1952; 35:1778-83. 25. Harris LS, Cohn K, Toyofuku H, et al. Conjunctival and corneal calcific deposits in uremic patients . Am J Ophthalmol1971; 72:130-33. 26. Caldeira JAF, Sabbaga E, lanhez LE. Conjunctival and corneal changes in renal failure. Influence of renal transplantation . Br J Ophthalmol 1970; 54 :399-404. 27. Davidson A. Primary lipid dystrophy of the cornea. Arch Ophthalmol 1947; 37:433-43. 28. Roberts WH, Wolter JR. Ocular chrysiasis. Arch Ophthalmol 1956; 56:48 -52. 29. Rahi AHS, Garner A. Immunopathology of the eye. Philadelphia: JB Lippincott, 1976; 86-7 . 30. Allansmith MR, Whitney CR, McClellan BH, Newman LP. Immunoglobulins in the human eye . Location, type, and amount. Arch Ophthalmol 1973; 89:36-45.