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Conjunctival Biopsy in Sarcoidosis
Conjunctival Biopsy in Sarcoidosis Zeynel A. Karcioglu, M.D., and Russell Brear, M.D.
In a prospective study of systemic sarcoidosis, 28 patients underwent 55 conjunctival biopsies. The incidences of positive granulomatous lesions in proven and suspected sarcoidosis cases were 71.4% (ten of 14 patients) and 28.5% (four of 14 patients), respectively. Thus, random, bilateral conjunctival biopsies should be performed as soon as possible in all cases of presumed sarcoidosis. THE VALUE OF CONJUNCTIVAL biopsy in the diagnosis of sarcoidosis was emphasized by Walsh l in 1939. He wrote that "when the lids or external structures of the eye are involved ... the diagnosis may be readily ascertained by biopsy of the tumor." Crick" later stressed that "examination of the conjunctiva and biopsy of any follicles had been a routine part of the investigation ... " Although the common occurrence of the disease in conjunctiva and the value of a conjunctival biopsy in diagnosis are widely accepted, certain aspects of the biopsy procedure are still controversial. Some investigators suggest searching for typical conjunctival lesions to biopsy.v' whereas others propose a random biopsy of normalappearing conjunctiva in every patient with a presumptive clinical diagnosis of sarcoidosis."! Our prospective study was designed to determine the value of random, bilateral conjunctival biopsy in the diagnosis of sarcoidosis.
Subjects and Methods A total of 55 conjunctival biopsies were performed on 28 patients. All but one patient underwent bilateral biopsies. The only unilateral biopsy included in Accepted for publication Oct. 22, 1984. From the O'Brien Ocular Pathology Laboratory, Department of Ophthalmology, Tulane Medical Center, New Orleans, Louisiana. Read in part before the Association for Research in Vision and Ophthalmology, Sarasota, Florida, May 2, 1984. Reprint requests to Zeynel A. Karcioglu, M.D., O'Brien Ocular Pathology Laboratory, 1430 Tulane Ave., New Orleans, LA 70112.
68
our study was positive. The study included 20 women and eight men between 20 and 57 years old (mean age, 36 years). The subjects were divided into two groups containing 14 patients each. Group 1 included 14 patients with histologically confirmed sarcoidosis (skin, lymph node, or liver biopsy). Group 2 included 14 other individuals suspected of having sarcoidosis. These patients had pulmonary or ocular manifestations, or both, typical or strongly suggestive of sarcoidosis at the time of conjunctival biopsy and had been referred here as a part of the diagnostic examination. None had histologically confirmed sarcoidosis at the time of consultation but the diagnosis was subsequently confirmed in each case. Each patient underwent a complete ophthalmologic examination, and four patients in Group 1 and three patients in Group 2 had ocular findings consistent with sarcoidosis. Nonspecific follicular reaction of upper and lower tarsal conjunctiva was observed in ten of 14 proven cases and one of 14 suspected cases. None of the patients had the typical solitary, yellow "millet-seed" nodule in the biopsy site or elsewhere in the conjunctiva. The follicles were not particularly selected for the biopsy, but if they happened to be in the inferior fornix they were included in the specimen. With the patient under topical anesthesia (a few drops of proparacaine hydrochloride), the inferior eyelid was retracted and the inferior forniceal conjunctiva infiltrated with 1% lidocaine; a piece of bulging conjunctiva was then excised with the Wescott scissors. Pressure was then applied to the eyes for hemostasis, antibiotic ointment was instilled into the inferior fornix, and the patient was allowed to leave the clinic. No complications or morbidity occurred. The conjunctival tissue was fixed in 10% formalin and embedded in paraffin blocks. A minimum of 60 4-jJ.msections from six different levels were prepared from each biopsy block and stained with hematoxylin and eosin. We considered the biopsy specimen positive for sarcoidosis when discrete, hard (noncaseating) granulomas were observed.
©AMERICAN JOURNAL OF OPHTHALMOLOGY 99:68-73, JANUARY, 1985
Conjunctival Biopsy in Sarcoidosis
Vol. 99, No.1
The biopsy specimens and granulomatous lesions were measured with a calibrated eyepiece micrometer. To exclude false-positive results, all sections were examined for foreign bodies with polarizing light. None were found. Additionally, Ziehl-Neelsen and Gomori's silver methenamine stains failed to disclose any causative organisms within the granulomas in the positive specimens. All specimens were examined by the same pathologist who had no knowledge of the patients' clinical histories and findings.
Results We obtained a total of 55 biopsy specimens from the 28 patients with proven (14 patients and 28 specimens in Group 1) and suspected (14 patients and 27 specimens in Group 2) sarcoidosis and examined approximately 3,300 (60 cuts from 55 specimens) tissue sections. The average biopsy specimen measured 5.35 x 1.1 mm. The average diameter of the granuloma was 0.39 mm (range, 0.05 to 0.6 mm) (Table). In Group 1, 14 of 28 specimens (50%) were positive. In some cases only one of the two bilateral specimens was positive, giving an incidence of positive granulomatous lesions of 71.4%, that is, ten of 14 patients in this category had at least one positive biopsy specimen. In Group 2, four of 27 specimens (14.8%) were positive. One patient had positive bilateral specimens and in two patients one of two specimens was positive. In the fourth patient only one side underwent biopsy and the specimen was positive. Thus, in
TABLE
SUMMARY OF BIOPSY DATA BIOPSY DATA
Positive specimens Group 1 Group 2 Patientswith positive specimens Group 1 Group 2 Average biopsy specimen Size Diameter Positivespecimens In initial section In deeper sections
NO.
14 of 28 4 of 27 10 of 14 4 of 14
5.35 x 1.1 mm 0.39 mm 10 18
69
Group 2 four of 14 patients (28.5%) had positive specimens. In all four patients the conjunctival lesion was the initial histopathologic confirmation of the disease. The histopathologic appearance of the granulomatous lesions consisted of aggregates of epithelioid histiocytes, giant cells, and varying numbers of lymphocytes and plasma cells at the periphery of the granuloma. In many slides, inflammatory cells were present between the granuloma and the overlying epithelium (Fig. 1). When the granuloma was located immediately beneath the epithelium, the inflammatory infiltrate was continuous within the overlying epithelial layer (Fig. 2). In some instances, mild hyperplasia of the epithelium was observed. Most granulomas, however, were small and located within the substantia propria with a band of fibroconnective tissue between the lesion and the overlying epithelium (Figs. 3 and 4). In the latter case, the overlying epithelium was slightly elevated without atrophy, hyperplasia, keratinization, or loss of goblet cells. The epithelial changes were only observed in specimens in which the granulomas were large and adjacent to the overlying epithelium. Also, haphazard aggregates of lymphocytes and well-formed follicles are identified in the tissue material. Changes in the overlying epithelium of correspondingly sized follicles were more severe than the effects of granulomas. These abnormalities consisted primarily of alternating zones of epithelial atrophy and hyperplasia and inflammatory cell infiltrates within the epithelium.
Discussion The reported incidences of ocular involvement in sarcoidosis vary from 15% to 75% depending on the technique of the study. 3,8-11 The predilection of different components of the visual apparatus for this granulomatous disease is also variable. Orbital involvement, for example, excluding the lacrimal gland is rare in sarcoidosis. Conjunctiva, anterior uveal structures, and lacrimal gland, however, are often involved. Granulomatous conjunctivitis, according to some, may be the most frequent form of ocular sarcoidosis.v':" There has been a great deal of confusion concerning conjunctival sarcoidosis. Many clinical investigators underestimate the diagnostic value of conjunctival involvement, primarily because of the absence of symptoms in this area and because of the nonspecific and inconsistent nature of the follicular reaction. Although some believe that conjunctival biopsy has a role in the diagnostic examination, the guidelines for patient selection and biopsy procedure are not firmly established.
70
AMERICAN JOURNAL OF OPHTHALMOLOGY
January, 1985
Fig. 1 (Karcioglu and Brear). Chronic inflammatory cells between the granuloma (G) and the overlying conjunctival epithelium (arrows) (hematoxylin and eosin, x40).
Crick, Clifford, and Smellie" described a series of 139 patients, in 59 of whom histologic confirmation was lacking when the conjunctival biopsy was done. Of these 59 specimens, 36 contained conjunctival follicles and only four (11%) were proven to have
granulomas. In other words, 11 % of the purposeful conjunctival biopsy specimens from patients thought to have sarcoidosis were positive. In the remaining 80 patients sarcoidosis was histologically confirmed by other means. Forty-three specimens from these 80 patients contained conjunctival follicles and 16 (37%)
Fig. 2 (Karcioglu and Brear). Chronic inflammatory infiltrate involving the epithelial layer overlying the granuloma (G) (hematoxylin and eosin, x 40).
Vol. 99, No.1
Conjunctival Biopsy in Sarcoidosis
71
Fig. 3 (Karcioglu and Brear). Small granulomatous lesion with giant cells (G). Few inflammatory cells are present between the lesion and the normalappearing conjunctival epithelium (hematoxylin and eosin, x 100).
had granulomatous lesions. All the biopsies were unilatera!. Merrit and associates!' described a small but interesting group of 16 black patients. Biopsy specimens from the inferior fornix demonstrated granulomatous inflammation consistent with sarcoidosis in 12
(75%) of the patients. None of the patients had undergone previous tissue analysis so the entire group contained suspected sarcoidosis cases. All specimens were taken unilaterally from the lower fornix and digital pressure through the lower eyelid "usually revealed solid yellowish-grey nodules."!'
Fig. 4 (Karcioglu and Brear). Small subepithelial granulomas (G) delineated with bands of fibrous tissue between the lesion and the normal-appearing conjunctival epithelium (hematoxylin and eosin, x x40).
72
AMERICAN JOURNAL OF OPHTHALMOLOGY
Although the exact number of patients with these nodules was not given, the impression is that these were present in most cases. Although this study was based on a small group, 75% positivity among sarcoidosis suspects without previous tissue diagnosis is quite impressive. Further, this high yield of positive granulomatous inflammation was obtained with unilateral biopsies, which are usually less informative than the bilateral biopsies. In another biopsy study by Nichols and associates,? 55 of 146 patients had sarcoidosis proven by biopsy specimens from sites other than conjunctiva. This series included only random bilateral conjunctival specimens from the lower fornix. Thirty of 55 patients (55%) showed granulomatous lesions in the conjunctival specimens. The remaining 91 patients were suspected of having sarcoid but general tests for sarcoidosis-including negative nonocular biopsy specimens and the subsequent follow-up-have demonstrated that their systemic problems were not related to sarcoidosis. All of the specimens from the later group were negative for sarcoid granulomas. Although it is considered superfluous to do a conjunctival biopsy in patients with biopsy-proven sarcoidosis, this study provides two important pieces of information: (1) Random conjunctival biopsy (four patients with typical millet-seed conjunctival nodules were excluded from their series) is a valuable adjunct in diagnosis of sarcoidosis. In their series, every patient underwent bilateral biopsy and 75 to 100 sections from each site were examined for granulomatous lesions. Nichols and associates" suggested that the bilateral biopsy technique and the exhaustive sectioning of the material can explain the increased yield. (2) This study also provided an important negative finding in 91 patients suspected of having sarcoid but later proved to have another type of systemic disease. In a report by Karma," 17% of 218 conjunctival biopsy specimens contained granulomas. The study included both biopsy-proven and suspected sarcoidosis cases, and therefore the number of positive specimens in each group cannot be determined. Also, the study does not help us to compare the random vs purposeful techniques because only the nodular areas of the conjunctiva were sampled. According to Karma, the granulomas were identified in approximately 25% of the specimens taken from "ordinary nodules" and in 41 % of the specimens taken from nodules suspected of being sarcoid lesions. All the biopsies were apparently unilateral. Thus, the data are confusing and the differences in study designs make the results difficult to compare. The relative values of the random and the purposeful biopsy techniques cannot be properly evaluated with the information now available.
January, 1985
Our study was specifically designed to address the question of the usefulness of random bilateral conjunctival biopsy by comparing proven sarcoid cases with suspected cases. We excluded patients with conjunctival findings other than nonspecific follicular change and made a particular effort to standardize the biopsy site in the central portion of the inferior fornix in all cases. In many cases, the biopsy site was not even close to follicular reaction of the tarsal conjunctiva. In our Group 1, 50% of the bilateral biopsy specimens were positive, a figure in agreement with those of Nichols and associates? whose study was also based on random bilateral conjunctival biopsies. Our positive yield in patients with proven sarcoidosis was higher than many of the previously reported series in which special attention was paid to the biomicroscopic appearance of conjunctival changes. Elliott" emphasized that a repeat random biopsy of conjunctiva in patients with otherwise biopsyproven cases is superfluous. Although we agree with this concept, we used this method in our study to prove that the yield of positive specimens from random bilateral conjunctival biopsies is as high as, if not higher than, the yield from purposeful biopsies of clinical conjunctival lesions. We firmly believe that bilateral sampling and the methods of tissue processing are closely related to the yield of positive specimens. In our Group 1, granulomas were identified in one of the specimens 71 % of the time, indicating that bilateral biopsy technique definitely increases the yield. Further, we fully agree with Nichols and associates," that "the examination of multiple sections of each biopsy is essential to obtain a high yield." In our study, only ten of 18 positive specimens showed positive granulomatous lesions on examination of the initial histologic sections. Multiple deeper cuts into the paraffin block, however, enabled us to demonstrate additional granulomas. The histopathologic and quantitative analysis of the biopsy material disclosed several interesting features. Most of the granulomatous lesions were small, with the smallest being 0.05 mm in diameter, and without much reaction in adjacent tissues. Particular attention was focused on the segment of conjunctival epithelium overlying the granulomatous lesion because these changes affect the accuracy of the biornicroscopic examination and determine the value of clinical documentation of the lesions before biopsy. After examining approximately 3,300 sections, we concluded that the biomicroscopic identification of lesions smaller than 0.25 mm in diameter (eight in our series) would be extremely difficult since these minute subepithelial granulomas do not affect the overlying epithelium in terms of contour and histologic appearance. The overlying epithelium of these
Vol. 99, No.1
Conjunctival Biopsy in Sarcoidosis
lesions was absolutely normal or showed minimal changes. The quantitative analysis of the granulomas and the histopathologic examination of our biopsy material produced strong evidence in favor of random conjunctival biopsy. Because many of these granulomatous lesions are extremely small and rather inactive, the slit-lamp examination should not be considered an effective way to pick up these minute subepithelial foci. We have seen cases in which the diagnosis of sarcoidosis was histologically documented with systemic biopsy procedures, and the patients were referred for ophthalmology consultation within a few weeks after the biopsy. In some of these patients, conjunctival specimens obtained from normalappearing lower fornix were proven to be positive for granulomatous inflammation. If the conjunctival biopsy specimen had been obtained before systemic biopsy, the patients could have been spared higherrisk invasive procedures. Therefore, we not only agree with Elliott's" statement that "blind biopsy of the conjunctiva in all patients with clinically presumptive sarcoidosis should be performed," but stress that it should be done very early, before any other invasive procedure. We also believe that the biopsy should be done randomly even if the conjunctiva appears to be normal. Our study and other reports clearly indicate that sarcoid granulomas may be too small to be detected even by slit-lamp examination. Conversely, if an obviously suspect lesion is present, that particular lesion should be removed. In our experience more prominent nodules of conjunctiva usually turn out to be large follicles, ectopic lacrimal gland tissue, foreign-body fibrosis, and the like rather than sarcoid granulomas. This has also been observed by others (R. C. Eagle, [r., oral communication, May 1984). Unfortunately, we could not compare the specimens from suspect nodular lesions with those obtained from random biopsies because we excluded any patient who had conjunctival changes other than a nonspecific follicular reaction. The obvious advantages of the conjunctival biopsy
73
are its extremely small morbidity and low cost. According to our data, approximately 25% of patients with suspected sarcoid can be histologically proven to have the disease with random conjunctival biopsies and spared higher-risk invasive techniques such as trimsbronchial biopsy, mediastinoscopy, and liver biopsy.
References 1. Walsh, F. B.: Ocular importance of sarcoid. Its relation to uveoparotid fever. Arch. Ophthalmo!. 21:421, 1939. 2. Crick, R. P.: Diagnostic conjunctival biopsy in sarcoidosis. Trans. Ophthalmo!. Soc. U.K. 76:403, 1956. 3. Crick, R. P., Clifford, H., and Smellie, H.: The eyes in sarcoidosis. Br. J. Ophthalmo!. 45:461, 1961. 4. Karma, A.: Conjunctival biopsy in sarcoidosis. In Henkind, P. (ed.): Proceedings of the 24th International Congress of Ophthalmology. San Francisco, J. B. Lippincott, 1982, p. 846. 5. Bornstein, J. S., Frank, M. 1., and Radner, D. B.: Conjunctival biopsy in the diagnosis of sarcoidosis. N. Eng!. J. M~d. 267:60, 1961. 6. Khan, F., Wessely, Z., Chazin, 9. R., and Seriff, N. S.: Conjunctival biopsy in sarcoidosis. A simple safe and specific diagnostic procedure. Ann. Ophthalmo!. 9:671, 1976. 7. Nichols, C. W., Eagle, R. C., Yanoff, M., and Manocal, N. G.: Conjunctival biopsy as an aid in the evaluation of the patient with sarcoidosis. Ophthalmology 87:287, 1980. 8. Langcope, W. T., and Freiman, D. G.: Study of sarcoidosis based on combined investigation of 160 cases including 30 autopsies. Medicine 31:1, 1952. 9. Henkind, P.: Sarcoidosis. An expanding ophthalmic horizon. J. R. Soc. Med. 75:153, 1982. 10. Weinreb, R. N., and Tessler, H.: Laboratory diagnosis of ophthalmic sarcoidosis. Surv. Ophthalmo!. 28:653, 1984. 11. Merrit, J. c., Lipper, S. L, Peiffer, R. L., and Hale, L. M.: Conjunctival biopsy in sarcoidosis. J. Nat!., Med. Assoc. 72:347, 1980. 12. Elliott, J. H., in discussion, Nichols, C. W., Eagle, R. c., Jr., Yanoff, M., arid Menocal, N. G.: Conjunctival biopsy as an aid in the evaluation of the patient with suspected sarcoidosis. Ophthalmology 87:287, 1980.
In a prospective study of systemic sarcoidosis, 28 patients underwent 55 conjunctival biopsies. The incidences of positive granulomatous lesions in proven and suspected sarcoidosis cases were 71.4% (ten of 14 patients) and 28.5% (four of 14 patients), respectively. Thus, random, bilateral conjunctival biopsies should be performed as soon as possible in all cases of presumed sarcoidosis. THE VALUE OF CONJUNCTIVAL biopsy in the diagnosis of sarcoidosis was emphasized by Walsh l in 1939. He wrote that "when the lids or external structures of the eye are involved ... the diagnosis may be readily ascertained by biopsy of the tumor." Crick" later stressed that "examination of the conjunctiva and biopsy of any follicles had been a routine part of the investigation ... " Although the common occurrence of the disease in conjunctiva and the value of a conjunctival biopsy in diagnosis are widely accepted, certain aspects of the biopsy procedure are still controversial. Some investigators suggest searching for typical conjunctival lesions to biopsy.v' whereas others propose a random biopsy of normalappearing conjunctiva in every patient with a presumptive clinical diagnosis of sarcoidosis."! Our prospective study was designed to determine the value of random, bilateral conjunctival biopsy in the diagnosis of sarcoidosis.
Subjects and Methods A total of 55 conjunctival biopsies were performed on 28 patients. All but one patient underwent bilateral biopsies. The only unilateral biopsy included in Accepted for publication Oct. 22, 1984. From the O'Brien Ocular Pathology Laboratory, Department of Ophthalmology, Tulane Medical Center, New Orleans, Louisiana. Read in part before the Association for Research in Vision and Ophthalmology, Sarasota, Florida, May 2, 1984. Reprint requests to Zeynel A. Karcioglu, M.D., O'Brien Ocular Pathology Laboratory, 1430 Tulane Ave., New Orleans, LA 70112.
68
our study was positive. The study included 20 women and eight men between 20 and 57 years old (mean age, 36 years). The subjects were divided into two groups containing 14 patients each. Group 1 included 14 patients with histologically confirmed sarcoidosis (skin, lymph node, or liver biopsy). Group 2 included 14 other individuals suspected of having sarcoidosis. These patients had pulmonary or ocular manifestations, or both, typical or strongly suggestive of sarcoidosis at the time of conjunctival biopsy and had been referred here as a part of the diagnostic examination. None had histologically confirmed sarcoidosis at the time of consultation but the diagnosis was subsequently confirmed in each case. Each patient underwent a complete ophthalmologic examination, and four patients in Group 1 and three patients in Group 2 had ocular findings consistent with sarcoidosis. Nonspecific follicular reaction of upper and lower tarsal conjunctiva was observed in ten of 14 proven cases and one of 14 suspected cases. None of the patients had the typical solitary, yellow "millet-seed" nodule in the biopsy site or elsewhere in the conjunctiva. The follicles were not particularly selected for the biopsy, but if they happened to be in the inferior fornix they were included in the specimen. With the patient under topical anesthesia (a few drops of proparacaine hydrochloride), the inferior eyelid was retracted and the inferior forniceal conjunctiva infiltrated with 1% lidocaine; a piece of bulging conjunctiva was then excised with the Wescott scissors. Pressure was then applied to the eyes for hemostasis, antibiotic ointment was instilled into the inferior fornix, and the patient was allowed to leave the clinic. No complications or morbidity occurred. The conjunctival tissue was fixed in 10% formalin and embedded in paraffin blocks. A minimum of 60 4-jJ.msections from six different levels were prepared from each biopsy block and stained with hematoxylin and eosin. We considered the biopsy specimen positive for sarcoidosis when discrete, hard (noncaseating) granulomas were observed.
©AMERICAN JOURNAL OF OPHTHALMOLOGY 99:68-73, JANUARY, 1985
Conjunctival Biopsy in Sarcoidosis
Vol. 99, No.1
The biopsy specimens and granulomatous lesions were measured with a calibrated eyepiece micrometer. To exclude false-positive results, all sections were examined for foreign bodies with polarizing light. None were found. Additionally, Ziehl-Neelsen and Gomori's silver methenamine stains failed to disclose any causative organisms within the granulomas in the positive specimens. All specimens were examined by the same pathologist who had no knowledge of the patients' clinical histories and findings.
Results We obtained a total of 55 biopsy specimens from the 28 patients with proven (14 patients and 28 specimens in Group 1) and suspected (14 patients and 27 specimens in Group 2) sarcoidosis and examined approximately 3,300 (60 cuts from 55 specimens) tissue sections. The average biopsy specimen measured 5.35 x 1.1 mm. The average diameter of the granuloma was 0.39 mm (range, 0.05 to 0.6 mm) (Table). In Group 1, 14 of 28 specimens (50%) were positive. In some cases only one of the two bilateral specimens was positive, giving an incidence of positive granulomatous lesions of 71.4%, that is, ten of 14 patients in this category had at least one positive biopsy specimen. In Group 2, four of 27 specimens (14.8%) were positive. One patient had positive bilateral specimens and in two patients one of two specimens was positive. In the fourth patient only one side underwent biopsy and the specimen was positive. Thus, in
TABLE
SUMMARY OF BIOPSY DATA BIOPSY DATA
Positive specimens Group 1 Group 2 Patientswith positive specimens Group 1 Group 2 Average biopsy specimen Size Diameter Positivespecimens In initial section In deeper sections
NO.
14 of 28 4 of 27 10 of 14 4 of 14
5.35 x 1.1 mm 0.39 mm 10 18
69
Group 2 four of 14 patients (28.5%) had positive specimens. In all four patients the conjunctival lesion was the initial histopathologic confirmation of the disease. The histopathologic appearance of the granulomatous lesions consisted of aggregates of epithelioid histiocytes, giant cells, and varying numbers of lymphocytes and plasma cells at the periphery of the granuloma. In many slides, inflammatory cells were present between the granuloma and the overlying epithelium (Fig. 1). When the granuloma was located immediately beneath the epithelium, the inflammatory infiltrate was continuous within the overlying epithelial layer (Fig. 2). In some instances, mild hyperplasia of the epithelium was observed. Most granulomas, however, were small and located within the substantia propria with a band of fibroconnective tissue between the lesion and the overlying epithelium (Figs. 3 and 4). In the latter case, the overlying epithelium was slightly elevated without atrophy, hyperplasia, keratinization, or loss of goblet cells. The epithelial changes were only observed in specimens in which the granulomas were large and adjacent to the overlying epithelium. Also, haphazard aggregates of lymphocytes and well-formed follicles are identified in the tissue material. Changes in the overlying epithelium of correspondingly sized follicles were more severe than the effects of granulomas. These abnormalities consisted primarily of alternating zones of epithelial atrophy and hyperplasia and inflammatory cell infiltrates within the epithelium.
Discussion The reported incidences of ocular involvement in sarcoidosis vary from 15% to 75% depending on the technique of the study. 3,8-11 The predilection of different components of the visual apparatus for this granulomatous disease is also variable. Orbital involvement, for example, excluding the lacrimal gland is rare in sarcoidosis. Conjunctiva, anterior uveal structures, and lacrimal gland, however, are often involved. Granulomatous conjunctivitis, according to some, may be the most frequent form of ocular sarcoidosis.v':" There has been a great deal of confusion concerning conjunctival sarcoidosis. Many clinical investigators underestimate the diagnostic value of conjunctival involvement, primarily because of the absence of symptoms in this area and because of the nonspecific and inconsistent nature of the follicular reaction. Although some believe that conjunctival biopsy has a role in the diagnostic examination, the guidelines for patient selection and biopsy procedure are not firmly established.
70
AMERICAN JOURNAL OF OPHTHALMOLOGY
January, 1985
Fig. 1 (Karcioglu and Brear). Chronic inflammatory cells between the granuloma (G) and the overlying conjunctival epithelium (arrows) (hematoxylin and eosin, x40).
Crick, Clifford, and Smellie" described a series of 139 patients, in 59 of whom histologic confirmation was lacking when the conjunctival biopsy was done. Of these 59 specimens, 36 contained conjunctival follicles and only four (11%) were proven to have
granulomas. In other words, 11 % of the purposeful conjunctival biopsy specimens from patients thought to have sarcoidosis were positive. In the remaining 80 patients sarcoidosis was histologically confirmed by other means. Forty-three specimens from these 80 patients contained conjunctival follicles and 16 (37%)
Fig. 2 (Karcioglu and Brear). Chronic inflammatory infiltrate involving the epithelial layer overlying the granuloma (G) (hematoxylin and eosin, x 40).
Vol. 99, No.1
Conjunctival Biopsy in Sarcoidosis
71
Fig. 3 (Karcioglu and Brear). Small granulomatous lesion with giant cells (G). Few inflammatory cells are present between the lesion and the normalappearing conjunctival epithelium (hematoxylin and eosin, x 100).
had granulomatous lesions. All the biopsies were unilatera!. Merrit and associates!' described a small but interesting group of 16 black patients. Biopsy specimens from the inferior fornix demonstrated granulomatous inflammation consistent with sarcoidosis in 12
(75%) of the patients. None of the patients had undergone previous tissue analysis so the entire group contained suspected sarcoidosis cases. All specimens were taken unilaterally from the lower fornix and digital pressure through the lower eyelid "usually revealed solid yellowish-grey nodules."!'
Fig. 4 (Karcioglu and Brear). Small subepithelial granulomas (G) delineated with bands of fibrous tissue between the lesion and the normal-appearing conjunctival epithelium (hematoxylin and eosin, x x40).
72
AMERICAN JOURNAL OF OPHTHALMOLOGY
Although the exact number of patients with these nodules was not given, the impression is that these were present in most cases. Although this study was based on a small group, 75% positivity among sarcoidosis suspects without previous tissue diagnosis is quite impressive. Further, this high yield of positive granulomatous inflammation was obtained with unilateral biopsies, which are usually less informative than the bilateral biopsies. In another biopsy study by Nichols and associates,? 55 of 146 patients had sarcoidosis proven by biopsy specimens from sites other than conjunctiva. This series included only random bilateral conjunctival specimens from the lower fornix. Thirty of 55 patients (55%) showed granulomatous lesions in the conjunctival specimens. The remaining 91 patients were suspected of having sarcoid but general tests for sarcoidosis-including negative nonocular biopsy specimens and the subsequent follow-up-have demonstrated that their systemic problems were not related to sarcoidosis. All of the specimens from the later group were negative for sarcoid granulomas. Although it is considered superfluous to do a conjunctival biopsy in patients with biopsy-proven sarcoidosis, this study provides two important pieces of information: (1) Random conjunctival biopsy (four patients with typical millet-seed conjunctival nodules were excluded from their series) is a valuable adjunct in diagnosis of sarcoidosis. In their series, every patient underwent bilateral biopsy and 75 to 100 sections from each site were examined for granulomatous lesions. Nichols and associates" suggested that the bilateral biopsy technique and the exhaustive sectioning of the material can explain the increased yield. (2) This study also provided an important negative finding in 91 patients suspected of having sarcoid but later proved to have another type of systemic disease. In a report by Karma," 17% of 218 conjunctival biopsy specimens contained granulomas. The study included both biopsy-proven and suspected sarcoidosis cases, and therefore the number of positive specimens in each group cannot be determined. Also, the study does not help us to compare the random vs purposeful techniques because only the nodular areas of the conjunctiva were sampled. According to Karma, the granulomas were identified in approximately 25% of the specimens taken from "ordinary nodules" and in 41 % of the specimens taken from nodules suspected of being sarcoid lesions. All the biopsies were apparently unilateral. Thus, the data are confusing and the differences in study designs make the results difficult to compare. The relative values of the random and the purposeful biopsy techniques cannot be properly evaluated with the information now available.
January, 1985
Our study was specifically designed to address the question of the usefulness of random bilateral conjunctival biopsy by comparing proven sarcoid cases with suspected cases. We excluded patients with conjunctival findings other than nonspecific follicular change and made a particular effort to standardize the biopsy site in the central portion of the inferior fornix in all cases. In many cases, the biopsy site was not even close to follicular reaction of the tarsal conjunctiva. In our Group 1, 50% of the bilateral biopsy specimens were positive, a figure in agreement with those of Nichols and associates? whose study was also based on random bilateral conjunctival biopsies. Our positive yield in patients with proven sarcoidosis was higher than many of the previously reported series in which special attention was paid to the biomicroscopic appearance of conjunctival changes. Elliott" emphasized that a repeat random biopsy of conjunctiva in patients with otherwise biopsyproven cases is superfluous. Although we agree with this concept, we used this method in our study to prove that the yield of positive specimens from random bilateral conjunctival biopsies is as high as, if not higher than, the yield from purposeful biopsies of clinical conjunctival lesions. We firmly believe that bilateral sampling and the methods of tissue processing are closely related to the yield of positive specimens. In our Group 1, granulomas were identified in one of the specimens 71 % of the time, indicating that bilateral biopsy technique definitely increases the yield. Further, we fully agree with Nichols and associates," that "the examination of multiple sections of each biopsy is essential to obtain a high yield." In our study, only ten of 18 positive specimens showed positive granulomatous lesions on examination of the initial histologic sections. Multiple deeper cuts into the paraffin block, however, enabled us to demonstrate additional granulomas. The histopathologic and quantitative analysis of the biopsy material disclosed several interesting features. Most of the granulomatous lesions were small, with the smallest being 0.05 mm in diameter, and without much reaction in adjacent tissues. Particular attention was focused on the segment of conjunctival epithelium overlying the granulomatous lesion because these changes affect the accuracy of the biornicroscopic examination and determine the value of clinical documentation of the lesions before biopsy. After examining approximately 3,300 sections, we concluded that the biomicroscopic identification of lesions smaller than 0.25 mm in diameter (eight in our series) would be extremely difficult since these minute subepithelial granulomas do not affect the overlying epithelium in terms of contour and histologic appearance. The overlying epithelium of these
Vol. 99, No.1
Conjunctival Biopsy in Sarcoidosis
lesions was absolutely normal or showed minimal changes. The quantitative analysis of the granulomas and the histopathologic examination of our biopsy material produced strong evidence in favor of random conjunctival biopsy. Because many of these granulomatous lesions are extremely small and rather inactive, the slit-lamp examination should not be considered an effective way to pick up these minute subepithelial foci. We have seen cases in which the diagnosis of sarcoidosis was histologically documented with systemic biopsy procedures, and the patients were referred for ophthalmology consultation within a few weeks after the biopsy. In some of these patients, conjunctival specimens obtained from normalappearing lower fornix were proven to be positive for granulomatous inflammation. If the conjunctival biopsy specimen had been obtained before systemic biopsy, the patients could have been spared higherrisk invasive procedures. Therefore, we not only agree with Elliott's" statement that "blind biopsy of the conjunctiva in all patients with clinically presumptive sarcoidosis should be performed," but stress that it should be done very early, before any other invasive procedure. We also believe that the biopsy should be done randomly even if the conjunctiva appears to be normal. Our study and other reports clearly indicate that sarcoid granulomas may be too small to be detected even by slit-lamp examination. Conversely, if an obviously suspect lesion is present, that particular lesion should be removed. In our experience more prominent nodules of conjunctiva usually turn out to be large follicles, ectopic lacrimal gland tissue, foreign-body fibrosis, and the like rather than sarcoid granulomas. This has also been observed by others (R. C. Eagle, [r., oral communication, May 1984). Unfortunately, we could not compare the specimens from suspect nodular lesions with those obtained from random biopsies because we excluded any patient who had conjunctival changes other than a nonspecific follicular reaction. The obvious advantages of the conjunctival biopsy
73
are its extremely small morbidity and low cost. According to our data, approximately 25% of patients with suspected sarcoid can be histologically proven to have the disease with random conjunctival biopsies and spared higher-risk invasive techniques such as trimsbronchial biopsy, mediastinoscopy, and liver biopsy.
References 1. Walsh, F. B.: Ocular importance of sarcoid. Its relation to uveoparotid fever. Arch. Ophthalmo!. 21:421, 1939. 2. Crick, R. P.: Diagnostic conjunctival biopsy in sarcoidosis. Trans. Ophthalmo!. Soc. U.K. 76:403, 1956. 3. Crick, R. P., Clifford, H., and Smellie, H.: The eyes in sarcoidosis. Br. J. Ophthalmo!. 45:461, 1961. 4. Karma, A.: Conjunctival biopsy in sarcoidosis. In Henkind, P. (ed.): Proceedings of the 24th International Congress of Ophthalmology. San Francisco, J. B. Lippincott, 1982, p. 846. 5. Bornstein, J. S., Frank, M. 1., and Radner, D. B.: Conjunctival biopsy in the diagnosis of sarcoidosis. N. Eng!. J. M~d. 267:60, 1961. 6. Khan, F., Wessely, Z., Chazin, 9. R., and Seriff, N. S.: Conjunctival biopsy in sarcoidosis. A simple safe and specific diagnostic procedure. Ann. Ophthalmo!. 9:671, 1976. 7. Nichols, C. W., Eagle, R. C., Yanoff, M., and Manocal, N. G.: Conjunctival biopsy as an aid in the evaluation of the patient with sarcoidosis. Ophthalmology 87:287, 1980. 8. Langcope, W. T., and Freiman, D. G.: Study of sarcoidosis based on combined investigation of 160 cases including 30 autopsies. Medicine 31:1, 1952. 9. Henkind, P.: Sarcoidosis. An expanding ophthalmic horizon. J. R. Soc. Med. 75:153, 1982. 10. Weinreb, R. N., and Tessler, H.: Laboratory diagnosis of ophthalmic sarcoidosis. Surv. Ophthalmo!. 28:653, 1984. 11. Merrit, J. c., Lipper, S. L, Peiffer, R. L., and Hale, L. M.: Conjunctival biopsy in sarcoidosis. J. Nat!., Med. Assoc. 72:347, 1980. 12. Elliott, J. H., in discussion, Nichols, C. W., Eagle, R. c., Jr., Yanoff, M., arid Menocal, N. G.: Conjunctival biopsy as an aid in the evaluation of the patient with suspected sarcoidosis. Ophthalmology 87:287, 1980.