Dr. Namita Dave, D16475, Dr. Rohit Shetty, Dr. Ankush Kawali, Dr.Padmamalini Mahendradas
Introduction
Sarcoidosis isan idiopathic inflammatory disorder affecting multiple tissue and organs, including the eye.1, 2It is usually diagnosed with characteristicclinical and radiological findings along with histological evidence such as widespread non-caseatingepitheloid granulomata.1, 3, 4Ocular involvement of sarcoidosis ranges between 13% – 79% affecting any part of the eye with manifestations such as uveitis, dry eye and conjunctival nodules causing visual impairment.5-7Anterior uveitis is more commonly associated with ocular sarcoidosis in 30% – 70% of sarcoidosis5 and 10% of adult uveitis cases.8, 9 Sarcoidosis associated uveitis can present as either anterior, intermediate, posterior or pan uveitis.10, 11Presently, diagnosis of sarcoidosis presenting with ocular manifestation requires other systemic features3 to confirm diagnosis12.In many patients, sarcoidosis associated uveitis can manifest before exhibiting clinical signs of systemic involvement.The availability ofan international criteria for diagnosing ocular sarcoidosis(International Workshop on Ocular Sarcoidosis – IWOS)13helps in more accuratediagnoses ocular sarcoidosis. However, a considerable number of cases with non-specific signs and negative laboratory investigations makesit difficult to rapidly and accurately diagnose the ocular condition. The laboratory investigations used in the diagnosis of ocular sarcoidosis doesn’t commonly involve ocular tissues and samples. The gold standard for the diagnosis of sarcoidosis is a biopsy of the affected organ or tissue12 which emphasizes on tissue specific investigation for confirmation. However, tissue biopsy is sometimes not preferred due to its invasive nature. Therefore, minimally-invasive strategies are being explored in diagnosis of ocular sarcoidosis. Ocular assessments using imaging modalities such as colour photography, fundus fluorescein angiography, indocyanine green angiography,optical coherence tomography (OCT)and confocal microscopy available to aid in the diagnosis of ocular sarcoidosis many a times does not provide confirmatory evidence for ocular sarcoidosis. Inflammation in sarcoidosis is considered to be due to continued stimulation of CD4+ T helper lymphocytes cells.3, 4 and they comprise the majority of infiltrating cells from the circulation to the sites of disease activity, including the eye.14Unique immunologic findings such as increased CD4+ T helper cell subset and a CD4+/CD8+ ratio in the affected tissue is typical in sarcoidosis.15Bronchoalveolar lavage (BAL) fluid with CD4+/CD8+ ratio greater than 3.5 and high serum levels of angiotensin-converting enzyme were considered as additional confirmatory indicators in diagnosing sarcoidosis, especially in the absence of histopathological confirmation.14, 16, 17Hence, BAL constituents are being investigated to aid in the diagnosis of sarcoidosis uveitis.18More recently, increased CD4+/CD8+ T lymphocyte ratio in the vitreous humour was shown to have diagnostic potential in the confirmation of sarcoidosis associated uveitis.19, 20Inflammation in the anterior chamber inflammation is considered one of themost common sign of sarcoid uveitis.21 Hence, aqueous humour was explored as a sample source to assess angiotensin-converting enzyme (ACE) activity in the confirmation of ocular sarcoidosis in the past.22However, the use of aqueous humor CD4+/CD8+ ratio remains under explored for its potential in the diagnosis of ocular sarcoidosis with only a two case reports published so far indicatingthis capability.23, 24 Hence, in the current study we investigated the potential of CD4+/CD8+ ratios of cellular infiltrates in aqueous humour in the differential diagnosis of patients with sarcoidosis and non-sarcoid uveitis.
Methods
Study cohort
The study approved by the Narayana Nethralaya Institutional Review Board (EC Ref. No.C/2013/04/05) was performed as per guidelines stipulated by the Indian Council for Medical Research (ICMR) and in accordance with the tenets of the Declaration of Helsinki. Subjects were recruited for the study only afterthe purpose of the research and the experimental protocols were explained in detail and informed written consent obtained as per institutional and ethics board guidelines. The subjects for this study were selected from the patients presenting with either anterior uveitis or intermediateand pan uveitis with anterior chamber involvement at Narayana Nethralaya EyeHospital, Bangalore, India. Included in the study were patients with anterior uveitis at presentation as a prerequisite and agreed to undergo anterior chamber paracentesis.Patients with history of intraocular surgery ,diabetes and other active systemic diseaseswere excluded. A group of patients with normal senile cataract surgery were included as the non-uveitis controls. Patients in the study were categorized into 2 groups (sarcoidosis and non-sarcoidosis) according to international criteria for the diagnosis of ocular sarcoidosis established at the International Workshop on Ocular Sarcoidosis (IWOS).13The IWOS criteria have 4 classifications of ocular sarcoidosis based on suggestive clinical signs, appropriate laboratory investigations, and biopsy results into definite,presumed,probable and possible( no need to define)( Briefly, biopsysupported diagnosis with a compatible uveitis is classified as “definite” ocular sarcoidosis; presence of BHL and compatible uveitis but without biopsy is classified as “presumed” ocular sarcoidosis; presence of suggestive intraocular signs and 2 positive investigational test results (negative tuberculin skin test result, elevated angiotensin-converting enzyme, elevated liver enzymes, and chest computed tomography) without BHL and biopsy is classified as “probable” ocular sarcoidosis; negative biopsy, with suggestive intraocular signs and 2 positive investigational test results, is classified as “possible” ocular sarcoidosis. Patients who met the IWOS criteria were categorized as the sarcoidosis group, and other patients were categorized as the non-sarcoidosis group. A total of 61 patients with anterior uveitis, intermediate or pan-uveitis with anterior chamber involvement were recruited in this study in accordance with Standardization of Uveitis Nomenclature (SUN) criteria.25, 26 These subjects were sub-divided into those with sarcoidosis associated uveitis (n=21) and non-sarcoidosis uveitis (n=40) based on IWOS criteria.13 The sarcoidosis associated uveitis subjects were further sub-divided as into Definite, Presumed, Probable and Possibleocular sarcoidosis as per IWOS criteria. Of the 21 patients with ocular sarcoidosis, 3 were diagnosed with “definite” sarcoidosis associated uveitis on the basis of positive histopathologic manifestation by skin biopsy (1 patients) and trans-bronchial lung biopsy (2 patients). Ten patients with bilateral hilar lymphadenopathy (BHL) but without biopsy were categorized as having “presumed” sarcoidosis associated uveitis, and 4 patients without BHL and biopsy were diagnosed with “probable” sarcoidosis associated uveitis on the basis of laboratory investigation. Four patients were diagnosed with “possible” sarcoidosis associated uveitis. The remaining 40 subjects were grouped as non-sarcoidosis uveitis controls.The non-sarcoidosis associated uveitis group included presumed ocular tuberculosis (n=15) on the basis of indirect evidences –Mantoux test,Quantiferon Tb gold and 2 patients with intraocular TB confirmed was diagnosed by nested PCR for IS6110,MPB64 OR BOTH 9 patients were clinically diagnosed as viral uveitis and confirmed with PCR for HSV1(4),HSV2(2) ,VZV(2). autoimmune uveitis –HLAB27+ve associated uveitis, rheumatoid arthritis (n=6) and Idiopathic (n=10). Furthermore, a total 19 non-uveitis controls subjects were also recruited for the study. The peripheral whole blood was obtained from those as part of the routine investigations and those who consented in cases where whole blood was not required as part of routine investigations.
Aqueous humour and peripheral venous whole blood collection
Aqueous humour sample (0.15 ml) was collected by anterior chamber paracentesis using a 30-gauge needle with tuberculin syringe under aseptic precautions and contents stored at 4°C until immunophenotyping of the cells were performed.A part of the sample obtained (0.1 ml) was sent for PCR test for HSV 1&2,VZV, CMV and MTB.The aqueous humor samples (0.5 ml) of non-uveitis control subjects were collected with a 30-gauge needle with tuberculin syringe before cataract surgery commenced. Peripheral venous blood (2 ml) was collected by venipuncture using EDTA coated Vacutainer® (BD, New Jersey, USA) and stored 4°C until further analysis. The cells in the samples were processed for immunophenotyping analysis immediately as described below.
Immunophenotyping of T cell subsets
The cells in the aqueous humour and peripheral whole blood were stained for CD3, CD4 and CD8 using BD Tritest™ (BD Biosciences). Fluorochrome specific staining of CD4 (FITC), CD8 (PE) and CD3 (PerCP) was performed in BD Trucount™ tubes as per manufacturer’s instructions. Briefly, 50 μl of either aqueous humour or whole blood sample was incubated with 20 μl of fluorochrome-labeled antibodies (BD Tritest CD4/CD8/CD3 reagent) and incubated after gentle vortexing for 15 minutes in the dark at room temperature. Red blood cells in the whole blood were lysed following staining by adding 1 x BD FACS lysing solution to the tubes and further incubated after gentle vortexing for an additional 15 minutes in the dark at room temperature. The fluorochrome intensity of the stained cells were analyzed by flow cytometry (BD FACSCalibur) using BD CellQuest Pro software (Version 6). Absolute CD4+ and CD8+ T cell subsets were determined on CD3+ enumerated or gated cells in the sample, i.e., CD3+CD4+ and CD3+CD8+ cells. This was followed by calculating the CD4+/CD8+ ratio in the various samples analyzed
Statistical analysis
The distribution of the data set was determined by Shapiro-Wilk normality test. Mann-Whitney test, Wilcoxon matched-pairs signed rank test and Kruskal-Wallis test were used to analyze data sets that were not normally distributed. ANOVA with Tukey’s multiple comparisons and unpaired t-test were used to analyze normally distributed data. Sensitivity, specificity, accuracy and odds ratio calculation were also performed.
P<0.05 was considered to be statistically significant. Statistical analyses were performed with GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA, USA) and MedCalcⓇ version 12.5 (MedCalc Software bvba, Belgium).
Results
Study cohort characteristics including age, sex and disease sub-groups classifications are shown in Table 1. Cellular infiltrates including T cells (CD3+CD4+ and CD3+CD8+ cells) were not detectable in the aqueous humour of the non-uveitis cataract control subjects. However, T cell infiltrates were observed in the aqueous humour of patients with anterior uveitis, intermediate uveitis with anterior spillover and pan uveitis. The CD4+/CD8+ ratio of cellular infiltrates in the aqueous humour of patients with sarcoidosis associated uveitis (6.3±1.4, 4.7; Mean±SEM, Median) were significantly (P<0.0001) higher than in non-sarcoidosis associated uveitis (1.6±0.1, 1.6; Mean±SEM, Median) subjects as shown in Figure 1a. However, no significant differences in the CD4+/CD8+ ratio of cellular infiltrates in the aqueous humour were observed among the sarcoidosis associated uveitis sub-groups (Figure 1b). Significant differences in the CD4+/CD8+ ratio was not observed among presumed ocular TB, viral uveitis, autoimmune disease associated uveitis and idiopathic uveitis patients, but each group exhibited significantly lower CD4+/CD8+ ratio compared to sarcoid uveitis (Figure 1c).
CD4+/CD8+ T lymphocyte ratio in whole blood of patients with sarcoid uveitis (1.8±0.2, 1.9; Mean±SEM, Median) and non-sarcoidosis associated uveitis (1.5±0.1, 1.5; Mean±SEM, Median) were not significantly different (Figure 2a), unlike that observed with the ratio in aqueous humour. Aqueous humour CD4+/CD8+ ratio (7.1±1.8, 4.8; Mean±SEM, Median) was significantly (P=0.001) higher than whole blood ratio (1.8±0.2, 1.9; Mean±SEM, Median) in sarcoid uveitis (Figure 2a). Similar difference was not observed between aqueous humour (1.6±0.2, 1.5; Mean±SEM, Median) and whole blood (1.5±0.1, 1.5; Mean±SEM, Median) CD4+/CD8+ ratio in non-sarcoidosisassociated uveitis (Figure 2a). The fold increase in CD4+/CD8+ ratio in aqueous humour over corresponding whole blood ratio in sarcoidosisassociated uveitis (4.6±0.9, 4.0; Mean±SEM, Median) was significantly higher than in non-sarcoidosis associated uveitis subjects as shown in Figure 2b.
Aqueous humour CD4+/CD8+ ratio >3.5 was observed in 14/21 patients categorized under sarcoidosis associated uveitis and in 2/40 patients with non-sarcoidosis associated uveitis. Sarcoidosisassociated uveitis was observed to be associated with aqueous humour CD4+/CD8+ ratio >3.5 as indicated by an odds ratio of 38 (95% confidence interval 7.0-205.2, P<0.0001) (Figure 3). The sensitivity, specificity and accuracy of the aqueous humour CD4+/CD8+ ratio with a cut-off value of 3.5 were 66.7%, 95.0% and 83.6% respectively for the confirmation of sarcoid uveitis.
Discussion
Ocular manifestations of sarcoidosiscan mimic many eye diseases, including uveitis associated with ocular tuberculosis, viral infections and autoimmune diseases, masquerade syndrome, chronic Vogt–Koyanagi–Harada and Blau syndrome.2 Differential diagnosis of ocular sarcoidosis from other eye disease is primarily determined based on clinical manifestations. In the absence of known aetiology or other confirmatory findings the diagnosis of sarcoidosis is largely by exclusion of other diseases.2 CD4+/CD8+ Tlymphocyte ratio of >3.5 in bronchoalveolar lavage (BAL) fluid and increased angiotensin-converting enzyme (ACE) levels are used confirmatory tests for sarcoidosis along with other clinical and radiological findings.27 Hence, CD4+/CD8+ ratio in broncho-alveolar lavage (BAL)18 and combined serum angiotensin-converting enzyme with gallium scan are suggested for its use in diagnosing ocular sarcoidosis.28It is important to note that serum ACE levels may not be elevated in patients with subclinical disease. Therefore, determination of CD4+/CD8+ ratio in aqueous humour or vitreous humour can be beneficial in identification of early disease in ocular sarcoidosis. In the current flow cytometry based study, we observed an increase in the CD4+/CD8+ ratios of cellular infiltrates in aqueous humour obtained by anterior chamber paracentesis in patients with sarcoidosis associated uveitis compared to patients with non-sarcoidosis associated uveitis (Figure 1). Hence, CD4+/CD8+ T lymphocytes ratio in the aqueous humour can be used as a biomarker to differentiate between sarcoidosis and non-sarcoidosis uveitis.This observation would be of particular relevance since the diagnosis of ocular sarcoidosis based on systemic findings proves difficult when ocular involvement is the first sign of sarcoidosis. This strategy will be of use in the absence of non-caseating epithelioid cell granuloma in the ocular tissues and/or when biopsies are accompanied with the risk of complications. Findings from our current observation, increased aqueous humor CD4+/CD8+ ratioin sarcoid uveitis is similar to two case reports on ocular sarcoidosis where they observed aqueous humor CD4+/CD8+ ratio of 29.6 and 9.5, respectively.23, 24the CD4+/CD8+ ratio in aqueous humor may be useful in patients with active ocular inflammation, and itmay be especially useful in patients with ocular sarcoidosis. Kojima K et al reported elevated (>3.5) CD4+/CD8+ ratio elevated (>3.5) in the vitreous humour of patients with sarcoidosis associated uveitis.20They further validated this observation by reporting that CD4/CD8 ratio in the vitreous lavage fluid and bronchoalveolar lavage fluid have similar diagnostic value in confirmation of sarcoidosis.19Anterior chamberparacentesishas the advantage of being quick and relatively straightforward to perform and can be carried out in an outpatient setting. The measurement of CD4+/CD8+ values is relatively straightforward and has long been clinically approved diagnostic methods to monitor inflammation.29Thus, it allows us to confirm the focal lymphocytosis observed in ocular sarcoidosis, avoiding pars plana vitrectomy. Vitrectomy carries the risk of retinal detachments and is hence not favored clinically.Peripheral blood CD4+/CD8+ ratio ranges between 1.5 – 2.5 in normal healthy population.30-33Alteredperipheral blood CD4+/CD8+ ratio are observed in infections34Kojima K et al reported increased peripheral blood CD4+/CD8+ ratio in patients with sarcoidosis comparted to non-sarcoidosis controls.20We did not observe any difference in the peripheral blood CD4+/CD8+ ratio between sarcoidosis and non-sarcoidosis subjects in the current study. This could be due to presence of ocular symptoms, irrespective of systemic signs being the primary inclusion criteria in our study cohort.However, CD4+/CD8+ ratio was found to higher in the aqueous humour (current study) and vitreous humour20 compared to peripheral blood in both the studies.The distinction between ocular and peripheral lymphocytes subset population can be used effectively in thediagnosis of ocular sarcoidosis. In our study, the odds ratio of 38 (Figure 3) and the specificity of 95% (Figure 3) suggests that the CD4+/CD8+ ratio could be of significant relevance as a biomarker for confirming clinical ocular sarcoidosis.Presumed Ocular sarcoidosis and Definite ocular sarcoidosis are already proven and does not require confirmation
The dilemma in diagnosis of ocular sarcoidosis exists mainly in probable and possible subgroups where the systemic search for sarcoidosis is negative hence the possible role of CD4/CD8 ratio as a biomarker in the identification seems promising .
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