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A Flow Cytometric Analysis of Vitreous Inflammatory Cells in Patients with Proliferative Diabetic Retinopathy

A Flow Cytometric Analysis of Vitreous Inflammatory Cells in Patients with Proliferative Diabetic Retinopathy
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  Hindawi Publishing CorporationBioMed Research InternationalVolume 󰀲󰀰󰀱󰀳, Article ID 󰀲󰀵󰀱󰀵󰀲󰀸, 󰀷 pages󰀱󰀰.󰀱󰀱󰀵󰀵/󰀲󰀰󰀱󰀳/󰀲󰀵󰀱󰀵󰀲󰀸 Clinical Study   A Flow Cytometric Analysis of Vitreous Inflammatory Cells inPatients with Proliferative Diabetic Retinopathy  MojcaUrban I i I , 1  VeronikaKlobovesPrevodnik, 2 DanielPetrovi I , 3 andMojcaGlobo I nikPetrovi I 1 󰀱 University Medical Centre Ljubljana, Eye Hospital, Grabloviˇceva 󰀶, 󰀱󰀰󰀰󰀰 Ljubljana, Slovenia 󰀲 Institute of Oncology, Zaloˇska 󰀲, 󰀱󰀰󰀰󰀰 Ljubljana, Slovenia 󰀳 Institute of Histology and Embryology, Medical Faculty, University Ljubljana, Korytkova 󰀲, 󰀱󰀱󰀰󰀵 Ljubljana, Slovenia Correspondence should be addressed to Mojca Urbanˇciˇc; mojca.urbancic@kclj.siReceived 󰀵 August 󰀲󰀰󰀱󰀳; Accepted 󰀸 September 󰀲󰀰󰀱󰀳Academic Editor: Goran PetrovskiCopyright © 󰀲󰀰󰀱󰀳 Mojca Urbanˇciˇc et al.isisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,which permits unrestricted use, distribution, and reproduction in any medium, provided the srcinal work is properly cited.e purpose of this study was to investigate inflammatory cells in vitreous from patients with proliferative diabetic retinopathy (PDR) using flow cytometric analysis. Twenty-eight patients with PDR requiring vitrectomy because of macular traction ortractional retinal detachment were enrolled in the study (  = 28 ), and 󰀶 patients with macular hole (MH) formed the controlgroup. Samples of vitreous and peripheral venous blood were obtained at the beginning of vitrectomy. T lymphocytes were foundin vitreous from patients with PDR, and CD/CD󰀸 ratio was higher in vitreous (median .󰀳) compared to blood (median 󰀱.󰀹;  = 0.003 ). No B lymphocytes were detected in vitreous. e percentage of histiocytes/macrophages was significantly higher in vitreous(median󰀶󰀲.󰀱)incomparisonwithblood(median󰀵.󰀵;  < 0.0001 ).Nolymphocytesweredetectedinvitreousofthecontrolgroup. ere were more T lymphocytes in vitreous from patients with active PDR. No association between cells in the vitreous and visual acuity improvement a󰀀er surgery was found. In conclusion, T lymphocytes are found in vitreous from patients with PDR and reflect the activity of PDR but do not seem to predict visual prognosis. Higher CD/CD󰀸 ratio in vitreous compared to bloodfrom patients with PDR is consistent with local inflammatory response in PDR. 1. Introduction Diabetic retinopathy (DR) is a late microvascular complica-tion of diabetes mellitus and a leading cause of blindness intheworkingagepopulation.TypicalriskfactorsofDRincludehyperglycemia,hypertension,andhyperlipidemia.esefac-tors have been shown to induce retinal inflammation by a variety of mechanisms [󰀱]. ere is now general acceptance that DR is a low-grade chronic inflammation [󰀲]. Inflammation is a nonspecific response to injury. Many molecular mediators and functional changes with immunecell and resident macrophage activation are involved in theinflammatory response. In acute inflammation, inflamma-tory cells contribute to tissue repair. However, leukocytes’prolonged secretion of inflammatory mediators and toxicoxygen radicals in persisting, chronic inflammation can leadto tissue damage [󰀳, ]. Leukocytesareinvolvedinendothelialcelldamage,capil-lary occlusions, and blood-retinal barrier breakdown in DR.Leukocyte adhesion to the endothelial wall and leukostasisis an early event in the development of DR  [󰀵]. Leukocyte adhesion molecules are upregulated in the vessels of thediabetic retina and choroid, and consequently inflammatory cells accumulate in the chorioretinal tissues [󰀳]. Studies on diabetic rats revealed increased leukocyte adhesion associ-atedwithvasculardamageandincreasedvascularpermeabil-ity [󰀶–󰀸].Increasednumbersofaccumulatedleukocyteshave been shown to correlate with the retinal capillary damagein spontaneously diabetic monkeys [󰀹]. Accumulations of  polymorphonuclear leukocytes have been observed in thelumen of human microaneurysms [󰀱󰀰]. Leukocytes enhance the formation of new vessels by releasing angiogenic factors and increasing the activity of matrix metallopeptidase [󰀱]. T lymphocytes have been found  󰀲 BioMed Research Internationalin fibrovascular membranes of patients with PDR  [, 󰀱󰀱] and correlated well with the severity of retinopathy and visualprognosis [󰀳, 󰀱󰀱]. Monocytes/macrophages were found in the neovascular tu󰀀s [󰀱󰀲, 󰀱󰀳]. It is well known that inflammatory mediators areincreased not only in the retina but also in the vitreous. e vitreous actively participates in the etiopathogenesis of DR by means of accumulating inflammatory molecules. A lot of data regarding pathogenesis of DR was obtained indirectly by studying inflammatory molecules in vitreous samples of patients undergoing vitrectomy  [󰀱]. Less is known about inflammatory cells in the vitreous, especially about the roleof specific subsets of these cells in the pathogenesis of DR.Vitreous is composed mainly of collagen fibers, hyalu-ronic acid, and hyalocytes. Hyalocytes belong to the mono-cyte/macrophage lineage and have characteristics of tissuemacrophages. By acting as modulators of the intraocularimmune system and intraocular inflammation, they play asignificant role in maintaining the vitreous transparent andavascular. ey have been found to be present in diabeticmacular edema and PDR  [󰀱󰀵]. Normally, there are no leuko- cytes in the vitreous as this is an immune-privileged site [󰀱󰀶,󰀱󰀷].However,whentheblood-retinalbarrierisdisrupted,likein DR, leukocytes gain access to the vitreous. T lymphocyteshave been found in most of the vitreous samples from PDR patients and they were not present in the vitreous samples of nondiabetic patients. Moreover, differences in percentages of T lymphocytes between vitreous and peripheral blood werereported by Cant´on and coworkers [󰀱󰀸].In our study, we used flow cytometry to investigate in-flammatory cells in the vitreous of diabetic patients. Ourpurpose was to observe pattern changes in lymphocyte sub-sets (CD󰀳, CD, CD󰀸, and CD󰀱󰀹) and macrophage (CD󰀱)in the vitreous of patients with PDR in comparison withperipheral blood and in comparison with the vitreous of nondiabetic patients. Additionally, we have searched for thepossible association of pattern changes in leukocyte subsetswith the activity of DR and visual acuity improvement a󰀀er vitrectomy. 2. Patients and Methods 󰀲󰀸 patients with PDR requiring vitrectomy because of mac-ular traction/tractional retinal detachment were consideredfor the study (󰀱 men and 󰀱 women; age: 󰀶󰀳.  ±  󰀱󰀲.󰀹years). Only patients with no vitreous haemorrhage or with vitreous haemorrhage of more than three months durationwere enrolled. Exclusion criteria were vitreous haemorrhageof less than three months, recent retinal photocoagulation(less than six months), previous vitrectomy, and glycatedhaemoglobin of more than 󰀱󰀰 percent. Six patients requiring vitrectomy because of macular hole (MH) were enrolled inthe study as the control group (󰀳 men and 󰀳 women; age:󰀷󰀲.󰀵 ± 󰀷.󰀸). Patientswere notincludedin thestudy if they hadany other ocular disease or known systemic inflammatory orhematological disease. Informed consent was obtained fromall patients.All patients had complete ophthalmological evaluationbefore surgery with best corrected visual acuity (BCVA)determination (Early Treatment Diabetic Retinopathy Study (ETDRS)), slit lamp examination, fundus examination,intraocular pressure measurement, gonioscopy, and opticalcoherence tomography (OCT). Based on ophthalmologicalevaluation (preoperative and intraoperative), activity of thedisease was noted. e activity of the disease was defined asbeingactiveretinopathywhentherewereperfusedcapillariesin neovascular membranes or inactive, quiescent retinopathy when there were nonperfused capillaries in fibrotic mem-branes [󰀱󰀹]. Data regarding the patient’s general condition anddiabetescontrolwereobtainedfromthepatientandfromthe patient’s general practitioner or diabetologist. Arterialhypertension was defined by a systolic blood pressure of 󰀱󰀰mmHg or higher and/or diastolic blood pressure of 󰀸󰀵mmHg or higher or defined as the condition treated withantihypertensivemedications.Hyperlipidemiawasdefinedastotal cholesterol higher than 󰀵mmol/L and/or triglycerideshigher than 󰀲mmol/L or defined as condition treated withhypolipemic medications. Patients were followed a󰀀er thesurgery and BCVA a󰀀er 󰀶 months was compared to preoper-ative BCVA. Visual acuity improvement was defined as gainof more than 󰀵 ETDRS letters. For statistical analysis ETDRS visual acuity was converted to logarithm of the MinimumAngle of Resolution (logMAR).Samples of undiluted vitreous and samples of peripheral venous blood were obtained at the beginning of vitrectomy.Undiluted vitreous samples were obtained by aspiration intoa syringe attached to a vitreouscutter before the infusionlinewith balanced salt solution was opened. 󰀰.󰀵mL of undiluted vitreous sample was then put immediately into 󰀱mL of cell media (.󰀵% bovine serum albumin and 󰀰.󰀵% EDTAin phosphate buffer solution with 󰀵󰀰IE/mL of penicillin).For microscopic examination with light microscope twocytospins were prepared (Giemsa and Papanicolaou stainingmethod)andsamples witherythrocytespresentwereconsid-ered as blood contaminated. Based on this vitreous samplecontamination, patients with PDR were further divided intotwogroups:vitreoussamplesof󰀱󰀲patientswereconsideredasnoncontaminatedwithblood(󰀳menand󰀹women;age:󰀶󰀷.󰀳 ± 󰀱.󰀹); blood contaminated vitreous samples of 󰀱󰀶 patientsformed the other group (󰀱󰀱 men and 󰀵 women; age: 󰀶󰀰.  ± 󰀱󰀰.󰀷).Flow cytometric analysis (FCA) for markers CD󰀵(leukocyte common antigen), CD󰀱 (monocyte/macrophagemarker), CD󰀳 (T lymphocyte marker), CD󰀱󰀹 (B lymphocytemarker), kappa (kappa light chain B cell marker), lambda(lambda light chain B lymphocyte marker), CD (T helperlymphocyte marker), and CD󰀸 (T cytotoxic lymphocytemarker) was done from vitreous and peripheral venousblood samples. Samples of undiluted vitreous were preparedaccording to the modified protocol adopted for cytologicalsamples at the Institute of Oncology, Ljubljana, Slovenia[󰀲󰀰]. Antibodies by BD Biosciences were applied (Table 󰀱). Peripheral venous blood samples were prepared accordingto red blood cell lysis protocol for peripheral blood. 󰀲.󰀵mLof TRIS ammonium chloride solution for erythrocyte lysiswas first added to 󰀱󰀰󰀰mL of peripheral venous blood. A󰀀er󰀱󰀰-minute incubation at room temperature in dark place,samples were centrifuged for 󰀵 minutes at 󰀱󰀵󰀰󰀰 turns per  BioMed Research International 󰀳 T󰁡󰁢󰁬󰁥 󰀱: Antibodies for 󰀸-colour immunophenotyping of vitreous and peripheral venous blood samples.Test tube FITC PE PerCP-Cy󰀵.󰀵 APC PE-Cy󰀷 APC-Cy󰀷mAbs mAbs mAbs mAbs mAbs mAbs󰀱 Kappa ∗ + CD ∗ Lambda ∗ + CD󰀸 ∗ CD󰀵 ∗ CD󰀱 ∗∗ CD󰀱󰀹 ∗∗ CD󰀳 ∗∗ Legend: mAbs: monoclonal antibodies, FITC: fluorescein isothiocyanate, PE: phycoerythrin, PerCP-Cy󰀵.󰀵: peridinin chlorophyll protein-Cy󰀵.󰀵, APC:allophycocyanin, PE-Cy󰀷: phycoerythrin-cyanin 󰀷, APC-Cy󰀷: allophycocyanin-cyanin 󰀷;  ∗ 󰀵  L monoclonal antibody was added;  ∗∗ 󰀳  L monoclonal antibody was added. T󰁡󰁢󰁬󰁥 󰀲: Data of patients.PDR (  = 28 ) MH (  = 6 )Age (years) 󰀶󰀳.  ±  󰀱.󰀹 󰀷󰀲.󰀵  ±  󰀷.󰀸󰀱Gender 󰀱 men (󰀵󰀰%), 󰀱 women (󰀵󰀰%) 󰀳 men (󰀵󰀰%), 󰀳 women (󰀵󰀰%)Duration of diabetes (years) 󰀱󰀵.󰀸  ±  󰀸.󰀸 󰀰HbA󰀱c (%) 󰀷.󰀸  ±  󰀱.󰀰 󰀰BMI (kg/m 󰀲 ) 󰀳󰀰.󰀰  ±  .󰀹 󰀲󰀵.󰀰  ±  󰀵.󰀲Incidence of arterial hypertension 󰀲󰀶 (󰀹󰀲.󰀸%)  (󰀶󰀶.󰀷%)Incidence of hyperlipidemia 󰀱 (󰀵󰀰%) 󰀲 (󰀳󰀳.󰀳%)Incidence of insulin therapy 󰀲󰀰 (󰀷󰀱.%) 󰀰 Legend: PDR: patients with proliferative diabetic retinopathy; MH: patients with macular hole; HbA󰀱c: glycated haemoglobin; BMI: body mass index. minute. Supernatant was discarded and washed with 󰀲.󰀵mLof buffer (Cell Wash, BD Biosciences). Antibodies (BDBiosciences, Table 󰀱) were added and samples then incubatedindarkatroomtemperaturefor󰀲󰀰minutes.A󰀀er󰀲󰀰minutessuperfluous antibodies were washed away with 󰀲.󰀵mL of buffer and 󰀳󰀰󰀰  L of buffer (Cell Wash, BD Biosciences) wasadded. FCA was done with flow cytometer FACSCanto II(BectonDickinson,SanJose, CA).Measurementresultswereanalyzed with FACSDiva programme.Measurement data in our study did not meet the nor-mality assumption, so median and range between minimumand maximum variable were used for the description of data.e comparison of related samples was done with Wilcoxonsignedranktest.MannWhitney   testwasusedforassessingdifferences between independent groups. A    value of lessthan 󰀰.󰀰󰀵 was considered statistically significant.estudywasapprovedbytheNationalEthicalCommit-tee(NationalEthicalCommitteenumber󰀱󰀱󰀸/󰀱󰀲/󰀲󰀰󰀱󰀱)andwasperformed in compliance with the Helsinki declaration. 3. Results ClinicaldataofpatientsenrolledinthestudyarepresentedinTable 󰀲.Vitreous samples were first evaluated by light micro-scope. e samples with erythrocytes present under micro-scopic examination were considered as blood contaminated.According to these findings, patients with PDR were dividedinto two groups: blood noncontaminatedgroup (nPDR) (  =12 ) and blood-contaminated group (cPDR) (  = 16 ). In thenPDR group 󰀰–󰀱󰀰 lymphocytes and 󰀱–󰀳󰀰 hystiocytes werefound on microscopic examination. In the control group(MH group) no erythrocytes were found; therefore thesesamples were considered uncontaminated with blood. Nolymphocytes and only 󰀰–󰀲 hystiocytes were found in thisgroup.FCA measurement results of vitreous samples andperipheral venous blood samples from patients with PDR arepresented in Table 󰀳. Since no kappa and lambda chains weredetected in vitreous samples, they are not presented in thetables.epairedcomparisonofvitreousandperipheralvenousblood samples from patients with PDR (  = 28 ) showedsignificant differences in the number of CD󰀵+ cells and inthe percentages of CD󰀱+, CD󰀱󰀹+, and CD󰀸+ cells between vitreous and blood samples (  < 0.0001 ). Ratio betweenCD󰀱+ cells and lymphocytes was higher in vitreous. Per-centages of lymphocytes, CD󰀳+, and CD+ cells were notsignificantly different, but CD󰀳/CD󰀱󰀹 ratio and CD/CD󰀸ratio were significantly higher in vitreous compared to blood(Table 󰀳).e comparison of blood noncontaminated and bloodcontaminated vitreous samples is presented in Table . erewere more leukocytes (CD󰀵+ cells) in blood-contaminatedsamples and further immunophenotyping revealed that thiswas mainly due to CD󰀳+ cells. CD/CD󰀸 ratio was notsignificantly different between both groups.Onlybloodnoncontaminatedvitreoussampleswereusedfor further comparison between patients with PDR andnondiabeticpatientswithMHandforsearchingforapossibleassociation between cell pattern in the vitreous and theactivity of the disease and visual gain a󰀀er surgery.ecomparisonofbloodnoncontaminatedvitreoussam-ples of patients with PDR (nPDR group) and patients withMH (MH group) is presented in Table 󰀵. Significantly moreCD󰀵+ cells were found in vitreous samples of patients withPDR with highly variable percentage of lymphocytes present.ere were no lymphocytes detected in the vitreous samplesof the control MH group, except in two patients wherethe percentage of lymphocytes was less than 󰀳 and furtherimmunophenotypingdidnotshowCD󰀳,CD󰀱󰀹,CD,orCD󰀸positive cells. e percentages of CD󰀱+ cells were similar   BioMed Research International T󰁡󰁢󰁬󰁥󰀳:PairedcomparisonofvitreousandbloodsamplesinpatientswithPDR(median,minimum–maximum;Wilcoxonsignedranktest).Patients with PDR (  = 28 )Vitreous Blood  P   valueCD󰀵+ (number) 󰀶󰀸.󰀰 (󰀱󰀸󰀱.󰀰–󰀹󰀲󰀶.󰀰) 󰀱󰀰󰀶󰀸󰀶󰀹.󰀵 (󰀸󰀵󰀵󰀳󰀹.󰀰–󰀲󰀶󰀵󰀲󰀲󰀷.󰀰)  󰀰.󰀰󰀰󰀰󰀱 Ly (%) 󰀹.󰀹 (󰀰.–󰀸󰀹.󰀶) 󰀲󰀶.󰀸 (󰀸.󰀵–󰀸󰀲.󰀱) 󰀰.󰀱󰀵󰀸CD󰀱+ (%) 󰀶󰀲.󰀱 (󰀵.󰀳–󰀸󰀷.󰀷) 󰀵.󰀵 (󰀱.󰀲–󰀱󰀲.󰀰)  󰀰.󰀰󰀰󰀰󰀱 CD󰀱󰀹+ (%) 󰀰 (󰀰–󰀹.󰀷) 󰀸.󰀹 (󰀲.󰀵–󰀸󰀳.󰀰)  󰀰.󰀰󰀰󰀰󰀱 CD󰀳+ (%) 󰀸󰀲. (󰀰–󰀱󰀰󰀰.󰀰) 󰀷󰀱.󰀰 (󰀱󰀲.󰀶–󰀹󰀵.󰀶) 󰀰.󰀳󰀶󰀲CD󰀳/CD󰀱󰀹 󰀸󰀳.󰀵 (󰀱.󰀱–󰀱󰀰󰀰.󰀰) 󰀸.󰀲 (󰀳.󰀱–󰀲󰀷.󰀰)  󰀰.󰀰󰀰󰀰󰀱 CD+ (%) 󰀷󰀵.󰀹 (󰀰–󰀱󰀰󰀰.󰀰) 󰀶󰀲. (󰀲.󰀷–󰀷󰀸.) 󰀰.󰀵󰀲CD󰀸+ (%) 󰀱.󰀰 (󰀰–󰀶󰀶.󰀷) 󰀳󰀳.󰀱 (󰀱󰀶.󰀹–󰀶󰀸.󰀲)  󰀰.󰀰󰀰󰀰󰀱 CD/CD󰀸 .󰀳 (󰀰.󰀵–󰀱󰀰󰀰.󰀰) 󰀱.󰀹 (󰀰.–󰀲󰀳.󰀰)  󰀰.󰀰󰀰󰀳 Legend: PDR: proliferative diabetic retinopathy; CD󰀵+: leukocytes; Ly: lymphocytes; CD󰀱+: macrophages; CD󰀱󰀹+: B lymphocytes; CD󰀳+: T lymphocytes;CD+: T helper lymphocytes; CD󰀸+: T cytotoxic lymphocytes. T󰁡󰁢󰁬󰁥:Comparisonofbloodnon-contaminated(nPDR)andcon-taminated (cPDR) vitreous samples of patients with PDR (median,minimum–maximum; Mann Whitney     test).VitreousnPDR (  = 12 ) cPDR (  = 16 )  P   valueCD󰀵 (  ) 󰀰󰀱.󰀰 (󰀱󰀸󰀱.󰀰–󰀱󰀶.󰀰) 󰀹󰀳󰀸.󰀰 (󰀲󰀷󰀳.󰀰–󰀹󰀲󰀶.󰀰)  󰀰.󰀰󰀲󰀹 Ly (%) 󰀳.󰀳 (󰀰.–󰀵󰀵.󰀵) 󰀱.󰀷 (󰀰.󰀶–󰀸󰀹.󰀶)  󰀰.󰀰󰀴󰀸 CD󰀱all (%) 󰀶.󰀱 (󰀳.󰀰–󰀷󰀹.󰀳) 󰀶󰀰.󰀵 (󰀵.󰀳–󰀸󰀷.󰀷) 󰀰.󰀵󰀶CD󰀱󰀹 (%) 󰀰 (󰀰-󰀰) 󰀰 (󰀰–󰀹.󰀷) 󰀰.󰀲󰀱󰀱CD󰀳 (%) 󰀵󰀷.󰀵 (󰀰–󰀱󰀰󰀰.󰀰) 󰀸󰀸 (󰀰–󰀱󰀰󰀰.󰀰)  󰀰.󰀰󰀱󰀲 CD󰀳/CD󰀱󰀹 󰀸.󰀰 (󰀷.󰀷–󰀱󰀰󰀰.󰀰) 󰀸󰀶.󰀹 (󰀱.󰀱–󰀱󰀰󰀰.󰀰) 󰀰.󰀷󰀲󰀳CD (%) 󰀶.󰀰 (󰀰–󰀱󰀰󰀰.󰀰) 󰀷󰀷.󰀵 (󰀰–󰀱󰀰󰀰.󰀰) 󰀰.󰀰󰀶󰀵CD󰀸 (%) 󰀰 (󰀰–󰀲󰀶.󰀰) 󰀱󰀵.󰀹 (󰀰–󰀶󰀶.󰀷) 󰀰.󰀰󰀹󰀸CD/CD󰀸 󰀳.󰀹 (󰀲.󰀲–󰀱󰀰󰀰.󰀰) .󰀶 (󰀰.󰀵–󰀱󰀰󰀰.󰀰) 󰀰.󰀷󰀲 Legend: nPDR: PDR patients with blood non-contaminated vitreous; cPDR:PDR patients with blood contaminated vitreous; CD󰀵+: leukocytes; Ly:lymphocytes; CD󰀱+: macrophages; CD󰀱󰀹+: B lymphocytes; CD󰀳+: T lym-phocytes; CD+: T helper lymphocytes; CD󰀸+: T cytotoxic lymphocytes. in both groups. Peripheral venous blood samples were alsocompared and there were no significant differences in thenumber of CD󰀵+ cells and in the percentages of observedcells between the two groups.Active neovascularization was observed in 󰀱󰀷 patientswith PDR (󰀶 noncontaminated with blood and 󰀱󰀱 bloodcontaminated);󰀱󰀱patientshadquiescentPDR(󰀶noncontam-inated and 󰀵 blood contaminated). When comparing bloodnoncontaminated vitreous samples from patients with activeretinopathy and from patients with quiescent retinopathy (presented in (Table 󰀶)), there were more CD󰀵+ cells andthe percentages of CD󰀳+, CD+, and CD󰀸+ cells weresignificantly higher in vitreous samples from patients withactive PDR. e percentages of lymphocytes were very low in samples from patients with quiescent PDR and furtherimmunophenotyping was negative for CD󰀱󰀹+ cells in allsamples and negative for CD󰀳+ cells in five samples. In thesample from patient with quiescent PDR where CD󰀳+ cellswere detected, only CD+ cells were detected with furtherimmunophenotyping.Visual acuity improved 󰀶 months a󰀀er surgery in 󰀲󰀳(󰀸󰀲%) patients with PDR (󰀸 patients with blood noncon-taminated vitreous samples and 󰀱󰀵 patients with blood con-taminated vitreous samples), remained the same in 󰀳 (󰀱󰀱%)patients (󰀲 patients with noncontaminated vitreous samplesand 󰀱 contaminated), and worsened in 󰀲 (󰀷%) patients (bothnoncontaminated). e average BCVA before surgery was󰀱.󰀲  ±  󰀰.󰀶󰀲 logMAR and 󰀶 months a󰀀er surgery 󰀰.󰀷󰀵  ±  󰀰.󰀵󰀵logMAR. Considering only patients with blood noncontam-inated vitreous samples, preoperative average BCVA was󰀱.󰀰󰀲  ±  󰀰.󰀶󰀸 and postoperative BCVA six months later was󰀰.󰀸󰀶  ±  󰀰.󰀵󰀷. No association was found between cells in the vitreous and visual acuity improvement a󰀀er surgery. 4. Discussion In our study, differences in the percentages of lymphocytesand macrophages in the vitreous between patients withPDR and either peripheral blood of patients with PDR or vitreous from nondiabetic patients using flow cytometry were observed. T lymphocytes were found in most vitreoussamples from patients with PDR, and CD/CD󰀸 ratio washigher in vitreous samples in comparison with peripheralblood. To our knowledge, this is the first report of higherCD/CD󰀸 ratio in vitreous in patients with PDR indicatingthe importance of local inflammation. In our study, higherpercentages of T lymphocytes were associated with activePDR. Similar findings, that is, higher percentages of Tlymphocytes (CD+ and CD󰀲󰀸+ cells) in the vitreous incomparison with peripheral blood in patients with PDR,were reported by Cant´on and coworkers [󰀱󰀸]. In their study, however,allthepatientswithPDRreportedhadthequiescentform of PDR [󰀱󰀸]. CD/CD󰀸 ratio was approximately twotimes higher in vitreous (.󰀳) compared to blood (󰀱.󰀹) inour study. is suggests that inflammatory cells in vitreousare under the influence of local environment. Consideringdiabetic retinopathy as low-grade chronic inflammation,theseresultsareinconcordancewithsomestudiesreportedinuveiticpatients.Tlymphocytesareknowntoparticipateinalltypes of uveitis [󰀲󰀱, 󰀲󰀲]. Moreover, intraocular inflammation was reported to be mediated by activated CD+ T cells  BioMed Research International 󰀵 T󰁡󰁢󰁬󰁥 󰀵: Comparison of samples from the nPDR group and the MH group (median, minimum–maximum; Mann Whitney     test).Vitreous BloodnPDR (  = 12 ) MH (  = 6 )  P   value nPDR (  = 12 ) MH (  = 6 )  P   valueCD󰀵 (number) 󰀰󰀱.󰀰 (󰀱󰀸󰀱.󰀰–󰀱󰀶.󰀰) 󰀱󰀳󰀲.󰀰 (󰀸󰀰.󰀰–󰀲󰀹.󰀰)  󰀰.󰀰󰀰󰀵  󰀱󰀰󰀷󰀰󰀷󰀹.󰀵 (󰀱󰀰󰀰󰀹󰀱󰀱.󰀰–󰀱󰀶󰀸󰀸󰀱󰀲.󰀰) 󰀱󰀰󰀹󰀸󰀶󰀲.󰀵 (󰀱󰀰󰀰󰀶󰀱󰀸.󰀰–󰀱󰀲󰀷󰀶󰀵󰀹.󰀰) 󰀰.󰀶Ly (%) 󰀳.󰀳 (󰀰.–󰀵󰀵.󰀵) 󰀱.󰀰 (󰀰–󰀲.󰀶)  󰀰.󰀰󰀴󰀴  󰀳󰀰.󰀲 (󰀱󰀰.–󰀸󰀲.󰀱) 󰀲󰀳.󰀰 (󰀱.󰀳–󰀶󰀳.󰀹) 󰀰.󰀵󰀱󰀲CD󰀱all (%) 󰀶.󰀱 (󰀳.󰀰–󰀷󰀹.󰀳) 󰀵󰀲.󰀷󰀵 (󰀱󰀷.󰀹–󰀸󰀳.󰀰) 󰀰.󰀲󰀲󰀳 󰀶.󰀰 (󰀲.󰀲–󰀱󰀲.󰀰) 󰀸.󰀰 (󰀵.󰀰–󰀲󰀱.󰀰) 󰀰.󰀱CD󰀱󰀹 (%) 󰀰 (󰀰-󰀰) 󰀰 (󰀰-󰀰) 󰀰.󰀸 󰀸. (󰀳.󰀰–󰀸󰀳.󰀰) 󰀸.󰀵 (.󰀹–󰀱󰀹.) 󰀰.󰀹󰀲󰀵CD󰀳 (%) 󰀵󰀷.󰀵 (󰀰–󰀱󰀰󰀰.󰀰) 󰀰 (󰀰-󰀰)  󰀰.󰀰󰀲󰀵  󰀵󰀹.󰀹 (󰀱󰀲.󰀶–󰀹󰀵.󰀶) 󰀶󰀱.󰀱 (󰀳󰀶.󰀶–󰀶󰀷.󰀰) 󰀰.󰀵󰀳CD󰀳/CD󰀱󰀹 󰀸.󰀰 (󰀷.󰀷–󰀱󰀰󰀰.󰀰) 󰀷.󰀵 (󰀳.󰀱–󰀲󰀷.󰀰) .󰀶 (󰀲.󰀸–󰀱󰀳.󰀳) 󰀰.󰀵CD (%) 󰀶.󰀰 (󰀰–󰀱󰀰󰀰.󰀰) 󰀰 (󰀰-󰀰)  󰀰.󰀰󰀲󰀵  󰀶󰀱.󰀳 (󰀲.󰀷–󰀷󰀸.) 󰀵󰀸.󰀸 (󰀳󰀲.󰀰–󰀷󰀵.󰀳) 󰀰.󰀸󰀵󰀱CD󰀸 (%) 󰀰 (󰀰–󰀲󰀶.󰀰) 󰀰 (󰀰-󰀰) 󰀰.󰀰󰀷󰀵 󰀳󰀲.󰀳 (󰀱󰀶.󰀹–󰀶󰀸.󰀲) 󰀳󰀳. (󰀲󰀱.–󰀵󰀹.) 󰀱.󰀰CD/CD󰀸 󰀳.󰀹 (󰀲.󰀲–󰀱󰀰󰀰.󰀰) 󰀲.󰀰󰀵 (󰀰.–󰀸.󰀲) 󰀱.󰀸 (󰀱.󰀳–󰀳.󰀵) 󰀰.󰀹󰀶󰀳 Legend: nPDR: PDR patients with blood non-contaminated vitreous; MH: patients with macular hole; CD󰀵+: leukocytes; Ly: lymphocytes; CD󰀱+:macrophages; CD󰀱󰀹+: B lymphocytes; CD󰀳+: T lymphocytes; CD+: T helper lymphocytes; CD󰀸+: T cytotoxic lymphocytes. T󰁡󰁢󰁬󰁥 󰀶: Comparison of vitreous samples from nPDR group withactive and quiescent PDR (median, minimum–maximum; MannWhitney     test).nPDR (  = 12 )Active PDR (  = 6 ) Quiescent PDR (  = 6 )  P   valueCD󰀵 ( n ) 󰀶󰀶󰀶.󰀰 (󰀳󰀲.󰀰–󰀱󰀶.󰀰) 󰀲󰀰.󰀵 (󰀱󰀸󰀱.󰀰–󰀶󰀰󰀵.󰀰) 󰀰.󰀰󰀵󰀵Ly (%) 󰀱󰀷.󰀰 (󰀲.󰀰–󰀵󰀵.󰀵) 󰀱.󰀷 (󰀰.–󰀹.󰀷)  󰀰.󰀰󰀲󰀵 CD󰀱all (%) 󰀸.󰀸 (󰀳.󰀰–󰀷󰀷.󰀰) 󰀶󰀶.󰀲 (󰀵󰀳.–󰀷󰀹.󰀳) 󰀰.󰀲CD󰀱󰀹 (%) 󰀰 (󰀰–󰀱.󰀰) 󰀰 (󰀰-󰀰) 󰀰.󰀳󰀱󰀷CD󰀳 (%) 󰀸󰀲.󰀵 (󰀶󰀸.󰀰–󰀱󰀰󰀰.󰀰) 󰀰 (󰀰–󰀷.)  󰀰.󰀰󰀰󰀳 CD󰀳/CD󰀱󰀹 󰀸󰀸.󰀵 (󰀶󰀸.󰀰–󰀱󰀰󰀰.󰀰)CD (%) 󰀷󰀳.󰀹 (󰀵󰀷.󰀰–󰀱󰀰󰀰.󰀰) 󰀰 (󰀰–󰀱󰀰󰀰.󰀰)  󰀰.󰀰󰀳󰀷 CD󰀸 (%) 󰀲󰀰.󰀰 (󰀰–󰀲󰀶.󰀰) 󰀰 (󰀰-󰀰)  󰀰.󰀰󰀰󰀷 CD/CD󰀸 󰀳.󰀸󰀵 (󰀲.󰀲–󰀱󰀰󰀰.󰀰) Legend: nPDR: PDR patients with blood non-contaminated vitreous;CD󰀵+: leukocytes; Ly: lymphocytes; CD󰀱+: macrophages; CD󰀱󰀹+: Blymphocytes; CD󰀳+: T lymphocytes; CD+: T helper lymphocytes; CD󰀸+: Tcytotoxic lymphocytes. [󰀲󰀱, 󰀲󰀲]. CD/CD󰀸 ratio higher than .󰀰 has been reported to have positive predictive value of 󰀷󰀰% in patients havingnoninfectious uveitis [󰀲󰀳, 󰀲]. High CD/CD󰀸 ratio (higher than 󰀳.󰀵) in the vitreous of patients with ocular sarcoidosishas also been found to be of high diagnostic value withsensitivity and specificity of 󰀱󰀰󰀰% and 󰀹󰀶.󰀳%, respectively [󰀲󰀵].ere were significantly more CD󰀵+ cells (leukocytes)inthevitreousofpatientswithPDR(nPDRgroup)comparedtonondiabeticpatientswithMH.erewerenolymphocytesdetectedinthevitreousofnondiabeticpatients.Lymphocyteswere detected in all vitreous samples from patients withPDR, although there were also five samples with small per-centages of detected lymphocytes, and further phenotypingwas negative. Nevertheless, these results are in concordancewith the concept that blood-retinal barrier breakdown isnecessary for inflammatory cells to gain access into the vitreous, since leukocytes have an active role in blood-retinalbarrier breakdown during inflammation [󰀵].Great variations in the percentages of lymphocytes in vitreous from patients with PDR (nPDR) (󰀰. to 󰀵󰀵.󰀵) inour study seem to be in association with the activity of PDR. T lymphocytes were detected in all samples with activePDR and only in one sample with quiescent PDR. Ourfindings are in contrast with the findings of Cant´on andcoworkers who reported T lymphocytes in 󰀵󰀵% of patientswith no vitreous haemorrhage, but all patients in whom Tlymphocytes were detected had quiescent PDR [󰀱󰀸]. Cant´on and coworkers assumed that T lymphocytes infiltrating the vitreous cavity had a protective role in the outcome of PDR since patients in whom T lymphocytes were detected in the vitreoushadquiescentdiseaseandbetteroutcomeintermsof early bleeding a󰀀er vitrectomy [󰀱󰀸]. Based on our results, wewere not able to confirm Cant´on’s conclusions. In our study,T lymphocytes were associated with active PDR. None of our patients had early postoperative bleeding. No associationbetween cells in the vitreous and visual acuity improvementa󰀀er surgery was found in our study to support the idea of the protective role of T lymphocytes. Visual acuity dependson many factors, with one of them being the preservation of photoreceptors in macula, which was not evaluated in thisstudy but could be in relation with local inflammation in theretina itself. It has been shown in the study of Kase et al.[󰀱󰀱] that high infiltration of fibrovascular membranes withT lymphocytes in patients with PDR is associated with poor visual prognosis. In our opinion, T lymphocytes in vitreouscavity might reflect the activity of PDR but could not be usedas a predictor of visual prognosis.We did not observe significant difference in the cellpatterninbloodsamplesfrompatientswithPDR(nPDR)andnondiabetic patients. Moreover, we did not find statistically significantdifferencesineitherCD󰀳/CD󰀱󰀹ratioorCD/CD󰀸ratio in patients with PDR. Our findings are consistentwith some previous reports [󰀲󰀶]. Additional diagnostics isnecessary to detect systemic inflammation in type 󰀲 diabetesmellitus.Higher percentages of CD󰀱+ cells (macrophage/histio-cyte) in vitreous in comparison to blood were observed inpatientswithPDR. esepercentages weresimilarregardlessof blood contamination. Macrophages are known to play animportant role in the pathogenesis of proliferative vitreo-retinal disorders and they have been found in proliferativemembranes in patients with PDR [󰀲󰀷]. Increased number
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