Role of Chorioretinal Biopsy in Inflammatory Eye Disease CHI-CHAO CHAN, MD,t ALAN G. PALESTINE, MD,I,2 JANET L. DAVIS, MD,1 MARC D. de SMET, MD,1 IAN W. McLEAN, MD,3 MIGUEL BURNIER, MD,1 JOHN H. DROUILHET, MD,4 ROBERT B. NUSSENBLATT, MDI
Abstract: Two patients who had similar clinical presentations of bilateral multiple chorioretinal lesions and needed a correct diagnosis underwent chorioretinal biopsy. The biopsy from one patient demonstrated mainly a B cell infiltrate in choroidal and subretinal nodules, while the biopsy from the second patient showed mainly macrophages in the retina. These findings directed the therapeutic approach taken in each patient. Although chorioretinal biopsy is an invasive procedure with the potential for serious complications, the resultant finding may aid in the diagnosis and guide the subsequent management of certain patients presenting with serious ocular findings of undefined etiology. Ophthalmology 1991; 98: 1281-1286
Chorioretinal biopsy plays a limited but important role in the diagnosis of chorioretinal disorders, which have unusual clinical manifestations and are unresponsive to conventional therapeutic approaches. The pathology and etiology of some ocular diseases, such as those due to infectious organisms or malignancy, may be confined to only the eye. In such cases, examination of the blood or spinal fluid is noncontributory. In addition, there are some ocular diseases that often do not demonstrate pathognomonic clinical features or that have unknown pathology and etiology. Some of these difficult clinical cases can be diagnosed via biopsy of the vitreous. However, there still remains a subtype of sight-threatening chorioretinal disorders in which the diagnostic pathology is primarily confined to the choroid or retina and where only tissue examination of these structures has the potential to provide a correct diagnosis. It is in these cases that a chorioretinal biopsy may be warranted if the clinician finds that conventional therapy has not yielded a positive result and if an alternative diagnosis exists that would indicate a different treatment plan for the patient.
The modem technique of chorioretinal biopsy was pioneered by Peyman et al l •2 and outlined in a series of articles in which procedures were described that had the potential of allowing the retina to remain attached, thereby preserving vision in the eye that underwent biopsy. Many of these eyes survived with retained vision, establishing the relative safety of the procedure for eyes that are at significant risk of visual loss from an unknown pathologic process. 3 Proper patient selection is critical. An example of the usefulness of this procedure is illustrated in the article about a patient with progressive retinitis, which on biopsy demonstrated herpes simplex virus. 4 Another case was the diagnosis of an intraocular large B cell lymphoma in a patient with uveitis who had negative results of a vitreous biopsy and a negative medical evaluation. 5 In this article, we describe chorioretinal biopsies in two patients. Therapeutic decisions were made based on the immunopathologic study, particularly the subset typing of the inflammatory cells obtained from the biopsies.
MATERIALS AND METHODS CHORIORETINAL BIOPSY TECHNIQUE
Originally received: December 10.1990. Revision accepted: February 18. 1991. Laboratory of Immunology. National Eye Institute. Bethesda. Department of Ophthalmology. Georgetown University. Washington. 3 Armed Forces Institute of Pathology. Washington. 4 Straub Clinic. Honolulu. 1
2
Reprint requests to Chi-Chao Chan. MD. Bldg 10. Rm 10N/206. NIH/NEI. Bethesda. MD 20892.
Chorioretinal biopsies were performed using a modification of the technique described by Peyman et al. l Preoperative laser treatment to surround the biopsy site was performed 1 day before the surgery. During the surgery, a supporting ring was not used and only a thin layer of sclera was removed with the specimen. The recti muscles were isolated and a core vitrectomy was performed without removal of the lens before the biopsy was started. This 1281
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vitreous specimen was sent for histopathologic and microbiologic analysis. Removal of the vitreous also improved visualization of the fundus, thereby improving the localization of the biopsy site. The site that underwent biopsy was then marked on the external sclera and markings for a 6 X 6 mm scleral flap were outlined. The infusion line was left in place as a nearly full-thickness scleral dissection was begun 5 to 6 mm posterior to the limbus. After the scleral flap was dissected, two rows of penetrating diathermy were placed along the outside rim of the inner bed. The infusion line was turned off and a long, 75-Beaver blade was used to make the first of two incisions into the eye. The biopsy was grasped firmly once at the edge of the tissue specimen and the piece of remaining sclera with attached retina and choroid was finally separated from the globe using Vannas scissors. The biopsy specimens ranged in size from 2 X 3 mm to 4 X 5 mm, the latter being the currently preferred size. The vitreous in the wound was removed and the scleral flap was used to cover the biopsy site. The sclera was closed with interrupted and then running 9-0 nylon sutures to obtain a watertight closure. Gas-fluid exchange was performed using 15% perfluoropropane. HISTOLOGY AND IMMUNOHISTOLOGY
The chorioretinal biopsy specimen was divided into three parts in a sterile manner under a dissecting microscope for microbiology, tissue culture, routine light histology and immunopathology studies, and electron microscopy.6 Histologic evaluation from both light and electron microscopic examination was done as described previously.7 In brief, the portion ofthe specimen for histology was fixed in 4% glutaraldehyde and half of this portion was embedded in glycol methacrylate, and 3-~m thick sections were cut, processed, and examined by light microscopy. The other halfwas embedded in epoxy resin. Ultrathin sections were stained with uranylacetate and lead citrate for transmission electron microscopy. Immunohistology was performed on another portion of the specimen, which was immediately frozen in optimal cutting temperature compound (Miles Laboratories) after its division. Sections, 6-~m thick, were examined using the ABC peroxidase staining method. The primary antibodies used were lymphocyte cell surface markers CD3 (pan-T lymphocyte), CD4 (helper/inducer T lymphocytes), CD8 (suppressor/cytotoxic T lymphocytes), COlI (NK cell), CD22 (B lymphocyte), markers for macrophages, and MHC class I and class II antigens (BectonDickinson, Mountainview, CA).
CASE REPORTS Case 1. A 53-year-old Japanese-American woman presented in March 1988 with a symptomatic retinal break in her right eye, which was treated with laser photocoagulation. One month later, periorbital pain and iridocyclitis developed. Topical antiinflammatory therapy was not successful in controlling the pain or the inflammation. Multiple chorioretinal lesions were seen in the right eye. At one point, there Was evidence of a nonrhegmatogenous retinal detachment in the right eye. In July, treat-
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ment with 60 mg of oral prednisone was begun. Despite this therapy, the yellow-white chorioretinallesions continued to appear and coalesce. Three weeks later, the patient lost central vision because of progressive juxtafoveal white creamy lesions. In September, a macular hemorrhage developed, with subsequent fibrosis (Fig lA). Prednisone was gradually tapered and visual acuity did not improve. Additional chorioretinal lesions were noted. A systemic work-up disclosed no systemic cause for the uveitis. A mild degree of alopecia developed during the summer of 1988, but results of a spinal tap were normal and no vitiligo or hearing loss was found. In December 1988, infiltrating vitreous cells and small chorioretinallesions, similar to those seen in the right eye, were noted in the left eye. The patient was first examined at the National Eye Institute in January 1989. Her visual acuity was 5/63 in the right eye and 20/20 in the left. Scattered small, white chorioretinal lesions were observed in both eyes, but were more extensive in the right eye (Fig lB). Some of these lesions were atrophic, but others appeared to be less sharply defined and were interpreted as being active sites of inflammation. There was a localized, low-lying serous elevation of the retina inferior and temporal to the right macula in an area overlying a dense grouping of chorioretinal lesions. Results of fluorescein angiography showed hyperfluorescent focal lesions with no evidence of subretinal neovascularization. Results of repeat systemic evaluation including conjuctival biopsy, gallium scan, serologies, and lumbar puncture were unremarkable. Results of serologic examination demonstrated a titer of 1:2560 for herpes simplex virus I, 1:320 for herpes simplex virus II, and 1:640 for herpes zoster virus. Epstein-Barr viral serologies were capsid IgG 1: 160, early diffuse Ag < 1: 10, early restricted Ag 1: 10, nuclear Ag < 1:2, indicating a past infection with this virus. During this evaluation, new chorioretinallesions were noted to appear in the left eye. The patient developed a scotoma on the Amsler grid and her visual acuity decreased to 20/30. The course of the left eye appeared to parallel the course that had been noted in the right eye. Because of the possibility that this might represent a herpes virus infection or uveitis caused by autoimmune etiology such as multifocal choroiditis or VogtKoyanagi-Harada syndrome and vitreous aspiration alone might not be able to provide sufficient information, a chorioretinal biopsy was performed in the right eye in an area where there were several active-appearing lesions. Case 2. A 56-year-old black woman with a 25-year history of insulin-dependent diabetes mellitus noticed sudden onset of photophobia and poor vision in her right eye in May 1988. One week later, she experienced similar symptoms in her left eye. An exudative retinal detachment was found in her right eye. Results of a systemic work-up, including skin tests, angiotensinconverting enzyme, gallium scan, lysozyme, chest x-ray, fungal serologies, fluorescent treponemal antibody, sickle preparation, and orbital computed tomographic scan were normal. She was treated and responded to a high-dose regimen of systemic and topical corticosteroids. Her ocular symptoms resolved by September 1988. The patient remained well until February 1989, when blurred vision developed again in both eyes. At this time, however, a positive tuberculin skin reaction was noted, but repeated sputum and urine cultures were negative. The patient was started on antituberculin treatment, but 9 months later, hepatitis developed, which was induced by isoniazid. She was subsequently treated with other antituberculin combinations. Her vision continued to worsen. The visual acuity dropped from 20/40 in both eyes in February 1990 to 20/60 in the right eye and 20/200 in the left in April 1990. Antituberculous therapy was terminated at that time.
Fig 1. Case I. Results of funduscopic examination of the right eye show a well-defined macular scar (top left, A) and many small yellowish-white subretinallesions in the periphery (top right, B). Fig 2. Center, case 2. Results of funduscopic examination show multiple yellowish-white lesions in the periphery of the left eye. Fig 3. Case I. Results of histopathologic examination show (bottom left, A) loss of retinal ganglion cells, choroidal, and subretinal pigment epithelial infiltrations, which composed with plasma cells, lymphocytes, and histiocytes. Inset. Higher magnification of a infiltrating nodule under the retinal pigment epithelium (hematoxylin-eosin; original magnification X160; inset, X400). Immunohistochemical staining (bottom right, B) shows most infiltrating cells were B lymphocytes (left: CD22, right: CD3; X160).
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Fig 4. Case 2. Results of histopathologic examination show (left, A) inflammatory cells in the retina were stained positively for macrophage marker (left: hematoxylin-eosin, right: ABC staining for macrophages; X250), and (right, B) infiltrating cells in the choroid were mainly histiocytes and CD4 cells (left: macrophages, right: CD4; X250).
The patient was referred to the National Eye Institute for consultation. In May 1990 her visual acuity was 20/63 in the right eye and 20/200 in the left. Multiple, round, yellowish chorioretinallesions located in the peripheral fundi were present in both eyes, however they were more prevalent in the left eye (Fig 2). Some of the lesions appeared to be elevated and whitish in color, while others were confluent. The right macula showed pigmentary mottling and the left macula had a perimacular lesion resembling a neovascular membrane. The fundus was reminiscent of that seen in case 1. Her vitreous reaction was mild at that time. A chorioretinal biopsy of the left eye was performed in June 1990.
RESULTS
terial cultures from the vitreous and the chorioretinal specimen were negative. A diagnosis of multifocal choroiditis with primarily a B lymphocyte infiltrate in the choroid was made. Therapy was begun in February 1989 with 2 mg/kg of oral cyclophosphamide (Cytoxan) combined with 17.5 mg of oral prednisone daily in an attempt to suppress the B celllymphocytic process. There appeared to be an initial stabilization with no further change in the visual field. In addition, many of the active lesions in the left eye appeared to fade. The patient has not been been given cyclophosphamide since summer 1990. Her right eye has retained 20/400 visual acuity with no retinal complications from the biopsy, but a progressive posterior subcapsular cataract developed and was extracted without complication.
CASE 1
CASE 2
Results of the biopsy showed necrosis of the inner retina with loss of ganglion cells, thinning of the nerve fiber layer, and scattered infiltration of plasma cells in the retina. Focal photoreceptor degeneration in the outer retina also was observed. The choroid was infiltrated with many plasma cells, lymphocytes, and had scattered mast cells, eosinophils, and neutrophils (Fig 3A). Small nodules were located beneath the retinal pigment epithelium and were composed of plasma cells, lymphocytes, and histiocytes (Fig 3A, inset). Results of immunohistochemistry showed that these nodules contained plasma cells, B lymphocytes, and macrophages with only an occasional T lymphocyte (Fig 3B). These nodules were similar in appearance to Dalen-Fuchs nodules, but were of B-cell composition, rather than of macrophage and T-cell composition. B lymphocytes were located adjacent to the clumps of plasma cells in the choroid and these B cells were surrounded by T lymphocytes. There were both Band T lymphocytes and macrophages in the vitreous. Virus was not detected by immunoperoxidase or by electron microscopy. Viral, fungal, and bac-
Results of the biopsy showed few inflammatory cells within the retina (Fig 4A). Most of the cells were positive for macrophage markers (Fig 4A). Occasional T lymphocytes, macrophages, plasma cells, and eosinophils were identified in the choroid (Fig 4B). In addition, mainly T lymphocytes and macrophages were present in the vitreous. No organisms were found in the histologic slides. Viral, fungal, and bacterial cultures from the vitreous and from the chorioretinal specimen also were negative. A diagnosis of inflammatory uveitis, most likely sarcoidosis, was made. The patient was treated with oral cyclosporine. She responded to the treatment well and in the 6-month follow-up period, the lesions began to regress and her visual acuity remained 20/40 in the right eye and 20/200 in the left, in which a cataract had developed.
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DISCUSSION The decision to perform a chorioretinal biopsy is the result of a balance between the risks of complications of
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the procedure and the potential gain of knowledge that may help in the treatment of either the eye that undergoes biopsy or the fellow eye. In these two cases, this procedure was beneficial because the histopathologic findings helped direct their management. The cellular compositions of ocular infiltrations showed both T and B cells and macrophages in the vitreous and mainly B lymphocytes and plasma cells in case 1 but only macrophages and T lymphocytes in case 2, despite the similar clinical presentation in both cases. Our knowledge of the pathology of ocular inflammatory disease during the active stages is sparse. In the 1950s, most uveitides were believed to result from syphilis or tuberculosis. 8 A decade later, all uveitis was believed to be mediated by immune complex mechanisms. 9 T lymphocyte-mediated di~eases later came into vogue as the crucial functions of this cell in the immune response were elucidated. to It is clear that some diseases, such as sympathetic ophthalmia, have a very prominent T lymphocyte-mediated inflammation. II Cyclosporine, an immunosuppressive drug that has its primary effect on T lymphocytes, could be considered as the treatment of choice in these diseases. 12 However, different immune responses occurring in other uveitic cases may require alternate specific immunosuppressive agents to be prescribed. Many patients with ocular inflammation do not have a classic clinical presentation of a particular disease and the etiologies of their diseases are unknown. Therefore, choosing a rational therapy can become difficult, especially at a time when vitreous aspiration for cytology may not give the diagnostic information and therapeutic modalities now beginning to target specific aspects of inflammation. Chorioretinal biopsy in selected cases offers a method of obtaining the necessary clinical information to decide whether antimicrobial, antineoplastic, or antiinflammatory therapy is indicated. Case 1 demonstrated a pathologic finding that had not been previously described in multifocal choroiditis. The presence of predominantly B lymphocytes and plasma cells in choroidal and subepithelial nodules without significant numbers of T lymphocytes is unique, as mainly T cell infiltrations are observed in other uveitides such as sympathetic ophthalmia, II Vogt-Koyanagi-Harada syndrome,13 pars planitis,14 and sarcoidosisY Although the underlying etiology could not be identified by the analysis of this tissue, it became clear that cyclosporine, a T lymphocyte-specific drug, should not be prescribed. Since no therapeutic agents specific for B lymphocytes are available at the present, cyclophosphamide, whose effects on B cells are greater than on T cells, 16 was selected for this patient. As a result, she responded well. Case 2 of this report showed a pathology that was confined mainly to the retina despite the impression of "deep lesions" from the clinical examination. The absence of mycobacteria in both culture and the histopathologic findings ruled out the possibility of tuberculosis, which had been originally considered in this case. The predominance of macrophages and T lymphocytes in the lesions, however, suggested an autoimmune disorder. tO ,15 Cyclo-
sporine was prescribed and the patient subsequently showed much improvement. The small amount of tissue that is obtained does lend itself well to histologic analysis and may provide important information on morphology. Culture for various microorganisms requires larger samples, since each organism needs its own specific medium to grow on. Newer techniques of analysis such as the polymerase chain reaction, which can specifically identify unique DNA sequence from infectious agents, will make analysis of the small chorioretinal biopsy specimens even more practical and useful, particularly since several tests (probes) can be applied to the same sample. 17 The biopsy specimen is small and must be handled carefully. The pathologist should be involved from the beginning in planning how to handle it. Once the specimen is removed from the eye, it must be divided immediately into several smaller samples for culture and histopathologic and immunohistochemical studies. One of the major advantages of this technique is the ability to preserve the anatomical relationships between the choroid and retina. This relationship is lost in an endoretinal biopsy or in a needle biopsy. However, if the specimen is not handled properly and the retina is lost, the biopsy may well be of little value. The potential risks of chorioretinal biopsy include retinal detachment, choroidal hemorrhage, infection, proliferative vitreoretinopathy, and cataract. In the two reported patients, cataract, most likely as a result of the intraocular gas, was the only complication. In our opinion, the use of diathermy and not cryopexy is important to prevent vitreoretinal complications. Chorioretinal biopsy is a procedure in which the relative benefits and risks must be carefully reviewed. Ophthalmology is a field of medicine where severe and complicated diseases are often diagnosed without biopsy confirmation. This is usually acceptable if the clinical appearance is sufficiently diagnostic. However, not every clinical ophthalmologic condition has a pathognomonic clinical appearance. The understanding gained from a biopsy can often be generalized to other patients with similar ocular presentations so that pathognomonic aspects of the clinical picture are better defined once the etiology is determined. If the diseases in the differential diagnosis are grave and the corresponding therapeutic approaches significantly different from one another, a histologic and/or culture confirmation by chorioretinal biopsy may provide the information needed for better management of the patient's disease.
REFERENCES 1. Peyman GA, Juarez CP, Raichand M. Full·thickness eye·wall biopsy: long·term results in 9 patients. Br J Ophthalmol1981; 65:723-6. 2. Peyman GA, Raichand M, Schulman J. Diagnosis and therapeutic surgery of the uvea-Part I: Surgical technique. Ophthalmic Surg 1986; 17:822-9.
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3. Peyman GA, Schulman J, Raichand M. Diagnosis and therapeutic surgery of the uvea-Part II: Results. Ophthalmic Surg 1987; 18:310-
6. 4. Grutzmacher RD, Henderson 0, McDonald PJ, Coster OJ. Herpes simplex chorioretinitis in a healthy adult. Am J Ophthalmol1983; 96: 788-96. 5. Constable IJ, Thompson 0, van Bockxmeer F. The value of rational biopsy. Tissue culture of chorioretinal biopsies. Trans Ophthalmol Soc UK 1983; 103:475-9. 6. Nussenblatt RB, Palestine AG. Uveitis: Fundamentals and Clinical Practice. Chicago: Year Book Medical 1989:160-1. 7. Palestine AG, Nussenblatt RB, Chan C-C, et al. Histopathology of the subretinal fibrosis and uveitis syndrome. Ophthalmology 1985; 92: 838-44. 8. Woods AC. Endogenous Inflammations of the Uveal Tract. Baltimore: Williams & Wilkins, 1961. 9. O'Connor GR. Factors related to the initiation and recurrence of uveitis. XL Edward Jackson Memorial Lecture. Am J Ophthalmol1983; 96: 577-99. 10. Nussenblatt RB, Gery I, Ballintine EJ, Wacker WB. Cellular immune
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13.
14.
15.
16. 17.
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responsiveness of uveitis patients to retinal S-antigen. Am J Ophthalmol 1980; 89:173-9. Chan C-C, Nussenblatt RB, Fujikawa LS, et al. Sympathetic ophthalmia: immunopathological findings. Ophthalmology 1986; 93:690-5. Nussenblatt RB, Palestine AG, Chan C-C. Cyclosporin A therapy in the treatment of intraocular inflammatory disease resistant to systemic corticosteroids and cytotoxic agents. Am J Ophthalmol 1983; 96: 275-83. Chan C-C, Palestine AG, Kuwabara T, Nussenblatt RB. Immunopathologic study of Vogt-Koyanagi-Harada syndrome. Am J Ophthalmol 1988; 105:607-11. Wetzig RP, Chan C-C, Nussenblatt RB, et al. Clinical and immunopathological studies of pars planitis in a family. Br J Ophthalmol1988; 72:5-10. Chan C-C, Wetzig RP, Palestine AG, et al. Immunohistopathology of ocular sarcoidosis: report of a case and discussion of immunopathogenesis. Arch Ophthalmol1987; 105:1398-1402. Buckley CE III, Gills JP Jr. Cyclophosphamide therapy of peripheral uveitis. Arch Intem Med 1969; 124:29-35. Stoflet ES, Koeberl DO, Sarkar G, Sommer SS. Genomic amplification with transcript sequencing. Science 1988; 239:491-4.
Abstract: Two patients who had similar clinical presentations of bilateral multiple chorioretinal lesions and needed a correct diagnosis underwent chorioretinal biopsy. The biopsy from one patient demonstrated mainly a B cell infiltrate in choroidal and subretinal nodules, while the biopsy from the second patient showed mainly macrophages in the retina. These findings directed the therapeutic approach taken in each patient. Although chorioretinal biopsy is an invasive procedure with the potential for serious complications, the resultant finding may aid in the diagnosis and guide the subsequent management of certain patients presenting with serious ocular findings of undefined etiology. Ophthalmology 1991; 98: 1281-1286
Chorioretinal biopsy plays a limited but important role in the diagnosis of chorioretinal disorders, which have unusual clinical manifestations and are unresponsive to conventional therapeutic approaches. The pathology and etiology of some ocular diseases, such as those due to infectious organisms or malignancy, may be confined to only the eye. In such cases, examination of the blood or spinal fluid is noncontributory. In addition, there are some ocular diseases that often do not demonstrate pathognomonic clinical features or that have unknown pathology and etiology. Some of these difficult clinical cases can be diagnosed via biopsy of the vitreous. However, there still remains a subtype of sight-threatening chorioretinal disorders in which the diagnostic pathology is primarily confined to the choroid or retina and where only tissue examination of these structures has the potential to provide a correct diagnosis. It is in these cases that a chorioretinal biopsy may be warranted if the clinician finds that conventional therapy has not yielded a positive result and if an alternative diagnosis exists that would indicate a different treatment plan for the patient.
The modem technique of chorioretinal biopsy was pioneered by Peyman et al l •2 and outlined in a series of articles in which procedures were described that had the potential of allowing the retina to remain attached, thereby preserving vision in the eye that underwent biopsy. Many of these eyes survived with retained vision, establishing the relative safety of the procedure for eyes that are at significant risk of visual loss from an unknown pathologic process. 3 Proper patient selection is critical. An example of the usefulness of this procedure is illustrated in the article about a patient with progressive retinitis, which on biopsy demonstrated herpes simplex virus. 4 Another case was the diagnosis of an intraocular large B cell lymphoma in a patient with uveitis who had negative results of a vitreous biopsy and a negative medical evaluation. 5 In this article, we describe chorioretinal biopsies in two patients. Therapeutic decisions were made based on the immunopathologic study, particularly the subset typing of the inflammatory cells obtained from the biopsies.
MATERIALS AND METHODS CHORIORETINAL BIOPSY TECHNIQUE
Originally received: December 10.1990. Revision accepted: February 18. 1991. Laboratory of Immunology. National Eye Institute. Bethesda. Department of Ophthalmology. Georgetown University. Washington. 3 Armed Forces Institute of Pathology. Washington. 4 Straub Clinic. Honolulu. 1
2
Reprint requests to Chi-Chao Chan. MD. Bldg 10. Rm 10N/206. NIH/NEI. Bethesda. MD 20892.
Chorioretinal biopsies were performed using a modification of the technique described by Peyman et al. l Preoperative laser treatment to surround the biopsy site was performed 1 day before the surgery. During the surgery, a supporting ring was not used and only a thin layer of sclera was removed with the specimen. The recti muscles were isolated and a core vitrectomy was performed without removal of the lens before the biopsy was started. This 1281
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vitreous specimen was sent for histopathologic and microbiologic analysis. Removal of the vitreous also improved visualization of the fundus, thereby improving the localization of the biopsy site. The site that underwent biopsy was then marked on the external sclera and markings for a 6 X 6 mm scleral flap were outlined. The infusion line was left in place as a nearly full-thickness scleral dissection was begun 5 to 6 mm posterior to the limbus. After the scleral flap was dissected, two rows of penetrating diathermy were placed along the outside rim of the inner bed. The infusion line was turned off and a long, 75-Beaver blade was used to make the first of two incisions into the eye. The biopsy was grasped firmly once at the edge of the tissue specimen and the piece of remaining sclera with attached retina and choroid was finally separated from the globe using Vannas scissors. The biopsy specimens ranged in size from 2 X 3 mm to 4 X 5 mm, the latter being the currently preferred size. The vitreous in the wound was removed and the scleral flap was used to cover the biopsy site. The sclera was closed with interrupted and then running 9-0 nylon sutures to obtain a watertight closure. Gas-fluid exchange was performed using 15% perfluoropropane. HISTOLOGY AND IMMUNOHISTOLOGY
The chorioretinal biopsy specimen was divided into three parts in a sterile manner under a dissecting microscope for microbiology, tissue culture, routine light histology and immunopathology studies, and electron microscopy.6 Histologic evaluation from both light and electron microscopic examination was done as described previously.7 In brief, the portion ofthe specimen for histology was fixed in 4% glutaraldehyde and half of this portion was embedded in glycol methacrylate, and 3-~m thick sections were cut, processed, and examined by light microscopy. The other halfwas embedded in epoxy resin. Ultrathin sections were stained with uranylacetate and lead citrate for transmission electron microscopy. Immunohistology was performed on another portion of the specimen, which was immediately frozen in optimal cutting temperature compound (Miles Laboratories) after its division. Sections, 6-~m thick, were examined using the ABC peroxidase staining method. The primary antibodies used were lymphocyte cell surface markers CD3 (pan-T lymphocyte), CD4 (helper/inducer T lymphocytes), CD8 (suppressor/cytotoxic T lymphocytes), COlI (NK cell), CD22 (B lymphocyte), markers for macrophages, and MHC class I and class II antigens (BectonDickinson, Mountainview, CA).
CASE REPORTS Case 1. A 53-year-old Japanese-American woman presented in March 1988 with a symptomatic retinal break in her right eye, which was treated with laser photocoagulation. One month later, periorbital pain and iridocyclitis developed. Topical antiinflammatory therapy was not successful in controlling the pain or the inflammation. Multiple chorioretinal lesions were seen in the right eye. At one point, there Was evidence of a nonrhegmatogenous retinal detachment in the right eye. In July, treat-
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ment with 60 mg of oral prednisone was begun. Despite this therapy, the yellow-white chorioretinallesions continued to appear and coalesce. Three weeks later, the patient lost central vision because of progressive juxtafoveal white creamy lesions. In September, a macular hemorrhage developed, with subsequent fibrosis (Fig lA). Prednisone was gradually tapered and visual acuity did not improve. Additional chorioretinal lesions were noted. A systemic work-up disclosed no systemic cause for the uveitis. A mild degree of alopecia developed during the summer of 1988, but results of a spinal tap were normal and no vitiligo or hearing loss was found. In December 1988, infiltrating vitreous cells and small chorioretinallesions, similar to those seen in the right eye, were noted in the left eye. The patient was first examined at the National Eye Institute in January 1989. Her visual acuity was 5/63 in the right eye and 20/20 in the left. Scattered small, white chorioretinal lesions were observed in both eyes, but were more extensive in the right eye (Fig lB). Some of these lesions were atrophic, but others appeared to be less sharply defined and were interpreted as being active sites of inflammation. There was a localized, low-lying serous elevation of the retina inferior and temporal to the right macula in an area overlying a dense grouping of chorioretinal lesions. Results of fluorescein angiography showed hyperfluorescent focal lesions with no evidence of subretinal neovascularization. Results of repeat systemic evaluation including conjuctival biopsy, gallium scan, serologies, and lumbar puncture were unremarkable. Results of serologic examination demonstrated a titer of 1:2560 for herpes simplex virus I, 1:320 for herpes simplex virus II, and 1:640 for herpes zoster virus. Epstein-Barr viral serologies were capsid IgG 1: 160, early diffuse Ag < 1: 10, early restricted Ag 1: 10, nuclear Ag < 1:2, indicating a past infection with this virus. During this evaluation, new chorioretinallesions were noted to appear in the left eye. The patient developed a scotoma on the Amsler grid and her visual acuity decreased to 20/30. The course of the left eye appeared to parallel the course that had been noted in the right eye. Because of the possibility that this might represent a herpes virus infection or uveitis caused by autoimmune etiology such as multifocal choroiditis or VogtKoyanagi-Harada syndrome and vitreous aspiration alone might not be able to provide sufficient information, a chorioretinal biopsy was performed in the right eye in an area where there were several active-appearing lesions. Case 2. A 56-year-old black woman with a 25-year history of insulin-dependent diabetes mellitus noticed sudden onset of photophobia and poor vision in her right eye in May 1988. One week later, she experienced similar symptoms in her left eye. An exudative retinal detachment was found in her right eye. Results of a systemic work-up, including skin tests, angiotensinconverting enzyme, gallium scan, lysozyme, chest x-ray, fungal serologies, fluorescent treponemal antibody, sickle preparation, and orbital computed tomographic scan were normal. She was treated and responded to a high-dose regimen of systemic and topical corticosteroids. Her ocular symptoms resolved by September 1988. The patient remained well until February 1989, when blurred vision developed again in both eyes. At this time, however, a positive tuberculin skin reaction was noted, but repeated sputum and urine cultures were negative. The patient was started on antituberculin treatment, but 9 months later, hepatitis developed, which was induced by isoniazid. She was subsequently treated with other antituberculin combinations. Her vision continued to worsen. The visual acuity dropped from 20/40 in both eyes in February 1990 to 20/60 in the right eye and 20/200 in the left in April 1990. Antituberculous therapy was terminated at that time.
Fig 1. Case I. Results of funduscopic examination of the right eye show a well-defined macular scar (top left, A) and many small yellowish-white subretinallesions in the periphery (top right, B). Fig 2. Center, case 2. Results of funduscopic examination show multiple yellowish-white lesions in the periphery of the left eye. Fig 3. Case I. Results of histopathologic examination show (bottom left, A) loss of retinal ganglion cells, choroidal, and subretinal pigment epithelial infiltrations, which composed with plasma cells, lymphocytes, and histiocytes. Inset. Higher magnification of a infiltrating nodule under the retinal pigment epithelium (hematoxylin-eosin; original magnification X160; inset, X400). Immunohistochemical staining (bottom right, B) shows most infiltrating cells were B lymphocytes (left: CD22, right: CD3; X160).
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Fig 4. Case 2. Results of histopathologic examination show (left, A) inflammatory cells in the retina were stained positively for macrophage marker (left: hematoxylin-eosin, right: ABC staining for macrophages; X250), and (right, B) infiltrating cells in the choroid were mainly histiocytes and CD4 cells (left: macrophages, right: CD4; X250).
The patient was referred to the National Eye Institute for consultation. In May 1990 her visual acuity was 20/63 in the right eye and 20/200 in the left. Multiple, round, yellowish chorioretinallesions located in the peripheral fundi were present in both eyes, however they were more prevalent in the left eye (Fig 2). Some of the lesions appeared to be elevated and whitish in color, while others were confluent. The right macula showed pigmentary mottling and the left macula had a perimacular lesion resembling a neovascular membrane. The fundus was reminiscent of that seen in case 1. Her vitreous reaction was mild at that time. A chorioretinal biopsy of the left eye was performed in June 1990.
RESULTS
terial cultures from the vitreous and the chorioretinal specimen were negative. A diagnosis of multifocal choroiditis with primarily a B lymphocyte infiltrate in the choroid was made. Therapy was begun in February 1989 with 2 mg/kg of oral cyclophosphamide (Cytoxan) combined with 17.5 mg of oral prednisone daily in an attempt to suppress the B celllymphocytic process. There appeared to be an initial stabilization with no further change in the visual field. In addition, many of the active lesions in the left eye appeared to fade. The patient has not been been given cyclophosphamide since summer 1990. Her right eye has retained 20/400 visual acuity with no retinal complications from the biopsy, but a progressive posterior subcapsular cataract developed and was extracted without complication.
CASE 1
CASE 2
Results of the biopsy showed necrosis of the inner retina with loss of ganglion cells, thinning of the nerve fiber layer, and scattered infiltration of plasma cells in the retina. Focal photoreceptor degeneration in the outer retina also was observed. The choroid was infiltrated with many plasma cells, lymphocytes, and had scattered mast cells, eosinophils, and neutrophils (Fig 3A). Small nodules were located beneath the retinal pigment epithelium and were composed of plasma cells, lymphocytes, and histiocytes (Fig 3A, inset). Results of immunohistochemistry showed that these nodules contained plasma cells, B lymphocytes, and macrophages with only an occasional T lymphocyte (Fig 3B). These nodules were similar in appearance to Dalen-Fuchs nodules, but were of B-cell composition, rather than of macrophage and T-cell composition. B lymphocytes were located adjacent to the clumps of plasma cells in the choroid and these B cells were surrounded by T lymphocytes. There were both Band T lymphocytes and macrophages in the vitreous. Virus was not detected by immunoperoxidase or by electron microscopy. Viral, fungal, and bac-
Results of the biopsy showed few inflammatory cells within the retina (Fig 4A). Most of the cells were positive for macrophage markers (Fig 4A). Occasional T lymphocytes, macrophages, plasma cells, and eosinophils were identified in the choroid (Fig 4B). In addition, mainly T lymphocytes and macrophages were present in the vitreous. No organisms were found in the histologic slides. Viral, fungal, and bacterial cultures from the vitreous and from the chorioretinal specimen also were negative. A diagnosis of inflammatory uveitis, most likely sarcoidosis, was made. The patient was treated with oral cyclosporine. She responded to the treatment well and in the 6-month follow-up period, the lesions began to regress and her visual acuity remained 20/40 in the right eye and 20/200 in the left, in which a cataract had developed.
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DISCUSSION The decision to perform a chorioretinal biopsy is the result of a balance between the risks of complications of
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the procedure and the potential gain of knowledge that may help in the treatment of either the eye that undergoes biopsy or the fellow eye. In these two cases, this procedure was beneficial because the histopathologic findings helped direct their management. The cellular compositions of ocular infiltrations showed both T and B cells and macrophages in the vitreous and mainly B lymphocytes and plasma cells in case 1 but only macrophages and T lymphocytes in case 2, despite the similar clinical presentation in both cases. Our knowledge of the pathology of ocular inflammatory disease during the active stages is sparse. In the 1950s, most uveitides were believed to result from syphilis or tuberculosis. 8 A decade later, all uveitis was believed to be mediated by immune complex mechanisms. 9 T lymphocyte-mediated di~eases later came into vogue as the crucial functions of this cell in the immune response were elucidated. to It is clear that some diseases, such as sympathetic ophthalmia, have a very prominent T lymphocyte-mediated inflammation. II Cyclosporine, an immunosuppressive drug that has its primary effect on T lymphocytes, could be considered as the treatment of choice in these diseases. 12 However, different immune responses occurring in other uveitic cases may require alternate specific immunosuppressive agents to be prescribed. Many patients with ocular inflammation do not have a classic clinical presentation of a particular disease and the etiologies of their diseases are unknown. Therefore, choosing a rational therapy can become difficult, especially at a time when vitreous aspiration for cytology may not give the diagnostic information and therapeutic modalities now beginning to target specific aspects of inflammation. Chorioretinal biopsy in selected cases offers a method of obtaining the necessary clinical information to decide whether antimicrobial, antineoplastic, or antiinflammatory therapy is indicated. Case 1 demonstrated a pathologic finding that had not been previously described in multifocal choroiditis. The presence of predominantly B lymphocytes and plasma cells in choroidal and subepithelial nodules without significant numbers of T lymphocytes is unique, as mainly T cell infiltrations are observed in other uveitides such as sympathetic ophthalmia, II Vogt-Koyanagi-Harada syndrome,13 pars planitis,14 and sarcoidosisY Although the underlying etiology could not be identified by the analysis of this tissue, it became clear that cyclosporine, a T lymphocyte-specific drug, should not be prescribed. Since no therapeutic agents specific for B lymphocytes are available at the present, cyclophosphamide, whose effects on B cells are greater than on T cells, 16 was selected for this patient. As a result, she responded well. Case 2 of this report showed a pathology that was confined mainly to the retina despite the impression of "deep lesions" from the clinical examination. The absence of mycobacteria in both culture and the histopathologic findings ruled out the possibility of tuberculosis, which had been originally considered in this case. The predominance of macrophages and T lymphocytes in the lesions, however, suggested an autoimmune disorder. tO ,15 Cyclo-
sporine was prescribed and the patient subsequently showed much improvement. The small amount of tissue that is obtained does lend itself well to histologic analysis and may provide important information on morphology. Culture for various microorganisms requires larger samples, since each organism needs its own specific medium to grow on. Newer techniques of analysis such as the polymerase chain reaction, which can specifically identify unique DNA sequence from infectious agents, will make analysis of the small chorioretinal biopsy specimens even more practical and useful, particularly since several tests (probes) can be applied to the same sample. 17 The biopsy specimen is small and must be handled carefully. The pathologist should be involved from the beginning in planning how to handle it. Once the specimen is removed from the eye, it must be divided immediately into several smaller samples for culture and histopathologic and immunohistochemical studies. One of the major advantages of this technique is the ability to preserve the anatomical relationships between the choroid and retina. This relationship is lost in an endoretinal biopsy or in a needle biopsy. However, if the specimen is not handled properly and the retina is lost, the biopsy may well be of little value. The potential risks of chorioretinal biopsy include retinal detachment, choroidal hemorrhage, infection, proliferative vitreoretinopathy, and cataract. In the two reported patients, cataract, most likely as a result of the intraocular gas, was the only complication. In our opinion, the use of diathermy and not cryopexy is important to prevent vitreoretinal complications. Chorioretinal biopsy is a procedure in which the relative benefits and risks must be carefully reviewed. Ophthalmology is a field of medicine where severe and complicated diseases are often diagnosed without biopsy confirmation. This is usually acceptable if the clinical appearance is sufficiently diagnostic. However, not every clinical ophthalmologic condition has a pathognomonic clinical appearance. The understanding gained from a biopsy can often be generalized to other patients with similar ocular presentations so that pathognomonic aspects of the clinical picture are better defined once the etiology is determined. If the diseases in the differential diagnosis are grave and the corresponding therapeutic approaches significantly different from one another, a histologic and/or culture confirmation by chorioretinal biopsy may provide the information needed for better management of the patient's disease.
REFERENCES 1. Peyman GA, Juarez CP, Raichand M. Full·thickness eye·wall biopsy: long·term results in 9 patients. Br J Ophthalmol1981; 65:723-6. 2. Peyman GA, Raichand M, Schulman J. Diagnosis and therapeutic surgery of the uvea-Part I: Surgical technique. Ophthalmic Surg 1986; 17:822-9.
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3. Peyman GA, Schulman J, Raichand M. Diagnosis and therapeutic surgery of the uvea-Part II: Results. Ophthalmic Surg 1987; 18:310-
6. 4. Grutzmacher RD, Henderson 0, McDonald PJ, Coster OJ. Herpes simplex chorioretinitis in a healthy adult. Am J Ophthalmol1983; 96: 788-96. 5. Constable IJ, Thompson 0, van Bockxmeer F. The value of rational biopsy. Tissue culture of chorioretinal biopsies. Trans Ophthalmol Soc UK 1983; 103:475-9. 6. Nussenblatt RB, Palestine AG. Uveitis: Fundamentals and Clinical Practice. Chicago: Year Book Medical 1989:160-1. 7. Palestine AG, Nussenblatt RB, Chan C-C, et al. Histopathology of the subretinal fibrosis and uveitis syndrome. Ophthalmology 1985; 92: 838-44. 8. Woods AC. Endogenous Inflammations of the Uveal Tract. Baltimore: Williams & Wilkins, 1961. 9. O'Connor GR. Factors related to the initiation and recurrence of uveitis. XL Edward Jackson Memorial Lecture. Am J Ophthalmol1983; 96: 577-99. 10. Nussenblatt RB, Gery I, Ballintine EJ, Wacker WB. Cellular immune
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responsiveness of uveitis patients to retinal S-antigen. Am J Ophthalmol 1980; 89:173-9. Chan C-C, Nussenblatt RB, Fujikawa LS, et al. Sympathetic ophthalmia: immunopathological findings. Ophthalmology 1986; 93:690-5. Nussenblatt RB, Palestine AG, Chan C-C. Cyclosporin A therapy in the treatment of intraocular inflammatory disease resistant to systemic corticosteroids and cytotoxic agents. Am J Ophthalmol 1983; 96: 275-83. Chan C-C, Palestine AG, Kuwabara T, Nussenblatt RB. Immunopathologic study of Vogt-Koyanagi-Harada syndrome. Am J Ophthalmol 1988; 105:607-11. Wetzig RP, Chan C-C, Nussenblatt RB, et al. Clinical and immunopathological studies of pars planitis in a family. Br J Ophthalmol1988; 72:5-10. Chan C-C, Wetzig RP, Palestine AG, et al. Immunohistopathology of ocular sarcoidosis: report of a case and discussion of immunopathogenesis. Arch Ophthalmol1987; 105:1398-1402. Buckley CE III, Gills JP Jr. Cyclophosphamide therapy of peripheral uveitis. Arch Intem Med 1969; 124:29-35. Stoflet ES, Koeberl DO, Sarkar G, Sommer SS. Genomic amplification with transcript sequencing. Science 1988; 239:491-4.
