Invasive Diagnostic Procedures Böke WRF, Manthey KF, Nussenblatt RB (eds): Intermediate Uveitis. Dei Ophthalmol. Basel, Karger, 1992, vol 23, pp 120-132
Diagnostic Vitrectomy in Intermediate Uveitis Janet L. Davis°, Chi-Chao Chanb, Robert B. Nussenblatt' of Ophthalmology, University of Miami, Bascom Palmer Eye Institute, Miami, Fla.; hNational Eye Institute, Laboratory of Immunology, Bethesda, Md., USA
The intraocular cells and snowbanks of intermediate uveitis are logical targets for biopsy and study. They are the visible evidence of the disease, correlate with its activity and may in fact be the effectors of damage to the eye [ 1 ]. The vitreous potentially contains a variety of clues as to the etiology of intermediate uveitis and of other vitreoretinal diseases in which peripheral neovascularization, cystoid macular edema and retinal vascular leakage play a part. Diagnostic vitrectomy has become a more common surgical procedure. It is ordinarily performed to confirm the fairly restricted list of diagnoses that can be made by the cytological or histological examination of small numbers of cells or tissue fragments. The usefulness of vitrectomy specimens in the diagnosis of intraocular lymphoma is proven [2-4]. The clinical indications for diagnostic vitrectomy are typically limited to cases where either neoplasia or bacterial or fungal infection is suspected. Cytological and histological diagnoses of a wider variety of diseases have been established using the technique, however, including lens and blood-induced glaucoma, metastatic tumors and amyloidosis [2]. In uveitis, a precise diagnosis is usually not confirmed by examination of vitrectomy specimens as chronic inflammatory cells are seen without other specific diagnostic features. Conventional pathological examinations have not been useful in discriminating between the various types of uveitis. Research techniques practiced on intraocular specimens removed incidentally in the course of other indicated surgery, such as lensectomy and vitrectomy for uveitis-associated cataract or retinal detachment repair [5-7], have expanded the number of laboratory investigations to which vitreous fluid can be subjected. As laboratory techniques are improved and useful diagnostic information is pro-
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 2:06:32 AM
a Department
Diagnostic Vitrectomy in Intermediate Uveitís
121
duced, the indications for diagnostic vitrectomy in intermediate uveitis may increase.
Diagnostic vitrectomy is generally performed by surgeons with specialized training in vitreoretinal surgery. In order to obtain adequate specimens with good visualization and control over the intraocular pressure, most surgeons prefer the standard three-port pars plana approach developed for more complicated vitreous procedures [8]. The exact sequence of surgery depends on other procedures planned at the time of vitrectomy and the objectives of the biopsy. Paracentesis of the anterior chamber may be performed as a first step in phakic eyes or pseudophakic eyes with posterior chamber lenses and intact posterior capsules. This should be performed before other fluids are infused into the eye if a pure aqueous humor specimen is desired. The anterior chamber should be reformed with balanced saline solution since there is a risk that even transient hypotony will produce miosis. About 200 µl of fluid can be obtained by careful aspiration into a 30gauge needle. Pure specimens of vitreous need to be removed before lensectomy by using the vitreous cutter through one of the superior sclerotomies while either (1) infusing air via an air pump into the preplaced pars plana cannula or (2) removing vitreous until the eye softens to the maximal safe extent. Simultaneous scleral depression can help control hypotony [9]. Air infusion is probably the safest procedure in eyes which are intended to remain phakic, but requires a high-minus contact lens and endoillumination to maintain a good view. In addition, the cannula tip must be visible in the vitreous cavity. Usually no more than 0.5-1.0 ml of pure vitreous can be removed from the eye if air is not simultaneously infused. If lensectomy is planned, this is usually performed before vitrectomy, using a fragmentation needle through the pars plans. Secondary infusion through a 20- to 23-gauge bent needle should be used until the medium is sufficiently clear to visualize the cannula tip. Lensectomy can compromise the quality of the vitreous specimen in two ways. First, infusion fluid must enter the eye, either through the posterior infusion cannula or through the secondary infusion line in the lens capsule. This will necessarily dilute the vitreous specimen and is undesirable if quantitative determinations are planned.
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 2:06:32 AM
Techniques of Diagnostic Vitrectomy
122
The second problem created by lens material is that it markedly interferes with the quality of cytological and histological preparations. This problem can be lessened by emptying the collection chamber of the vitrectomy instrument before proceeding with vitrectomy. There will usually still be enough residual material clinging to the sides of the container to affect the quality of the vitreous specimen. An alternative is to have an assistant manually aspirate the lens material into a separate syringe attached to the aspiration line. The syringe is connected by a three-way stopcock or by flexible tubing to the rigid aspiration port on the vitrectomy handpiece. The initial lensectomy specimen is discarded and the aspiration line flushed before performing the vitrectomy. If an absolutely pure vitreous specimen is not needed for quantitative determinations, it may still be useful to partition the vitreous specimen in two parts. The first portion of the vitrectomy always contains a less dilute specimen because it is easier to keep the tip of the vitrectomy instrument embedded in vitreous early in the case. It is a useful practice to aspirate the first 10 ml of vitreous mixed with infusate into a separate syringe. This will roughly correspond to the central core vitreous diluted with infused saline and provides a cell suspension of the appropriate density for preparation of slides by cytocentrifugation. After the central formed vitreous is removed, peripheral vitreous skirt can be removed. This results in a more dilute specimen which can be conveniently collected using automatic suction into the machine cassette. A low cutting rate (200 cuts/ min) has been recommended to ensure optimal specimen quality [9]. As complete a vitrectomy as possible without endangering the retina or lens is generally performed. Removing the central formed vitreous from a phakic eye of a uveitis patient does not much increase the complication rate beyond that associated with removal of only a small amount of fluid with the vitreous cutter. Furthermore, standard pars plana vitrectomy is a more controlled method than simple needle aspiration of the vitreous cavity with its inherent risks of creating vitreoretinal traction and retinal detachment. Uveitis patients may benefit from diagnostic vitrectomy in that the clearing of the central visual axis of opaque vitreous may allow better visual acuity. Reroyal of formed vitreous may also decrease the risk of tractional or rhegmatogenous retinal detachment. Subtotal vitrectomy is acceptable when removal of the residual vitreous skirt is not possible because of pupillary miosis or a compromised view of the periphery. In general, however, attempts should be made to remove as much of the vitreous as possible, including attempts to disinsert the posterior cortical vitreous from the retinal surface with a membrane pick or a soft-tipped extrusion needle [10].
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 2:06:32 AM
Davis/Chan/Nussenblatt
Diagnostic Vitrectomy in Intermediate Uveitis
1 23
In young intermediate uveitis patients, collapse of the globe due to massive loss of liquid vitreous from the eye may occur when the initial sclerotomy is made. The vitreous may be difficult to evaluate accurately preoperatively as to its degree of liquefaction. If pure vitreous specimens are necessary, an initial attempt can be made to aspirate through the pars plana with a 25-gauge needle on a tuberculin syringe before opening the 20-gauge sclerotomy for the infusion cannula.
Risks of Diagnostic Vitrectomy Pars plana vitrectomy ranks as a major ocular surgical procedure and should not be undertaken unless the information gained is likely to lead to a change in therapy or diagnosis for the patient or result in improved vision. Phakic patients run the immediate risk of damage to the crystalline lens during the surgical procedure or accelerated cataract formation after the surgery. Reported rates of cataract formation 1 year after vitrectomy range from 15% of patients [1] to 47% [12]. Conventional cataract extraction procedures are generally successful if cataracts do form [13]. Retinal complications of vitrectomy, including retinal detachment, occur in about 2% of patients in whom the retina is attached prior to surgery and in whom membrane peeling is not performed [14]. Uveitis patients may be at increased risk for this complication because inflammatory changes in the vitreous may produce abnormal vitreoretinal adhesions [15]. Both improved and persistent cystoid macular edema following vitrectomy for uveitis have been reported [16, 17]. Vitreous hemorrhage has been reported as a complication of vitrectomy surgery in intermediate uveitis patients with active neovascularization [17]. Endophthalmitis following vitrectomy has been reported but is rare [ 18, 19].
Cell Culture For techniques requiring live cells such as lymphocyte proliferative studies, the vitreal cells must be separated by centrifugation and transferred to a supportive growth medium as rapidly as possible. The length of survival of live cells in vitreous alone is unknown. There has been an impression that cells retrieved from vitreous specimens were of marginal viability. This may be due to the presence of many dead and degenerating cells in vivo rather
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 2:06:32 AM
Specimen Handling and Brief Methodology
Davis/Chan/Nussenblatt
124
than death after removal from the eye. Antiproliferative factors in vitreous may also contribute to the problems associated with in vitro growth; such factors have been shown to be present in aqueous humor [20]. Complete RPMI medium with 10% fetal calf serum and appropriate antigen or mitogen supports the proliferation of human peripheral-blood lymphocytes in vitro. There is little experience with cell culture of vitreal cells, so it is unknown if their requirements are more fastidious. Because the number of cells retrieved is likely to be small in any case, clonal expansion using the appropriate growth factors may be necessary to achieve sufficient numbers of cells for in vitro studies.
Cytokines Bioassays for soluble factors such as cytokines are best performed on cell-free supernatants since dead or dying cells can release these factors and confound results. Cellular proteases can also alter the peptide constituents of vitreous and may render some cytokines biologically or immunologically unreactive. Consideration should be given to the addition of protease inhibitors such as phenylmethane-sulfonyl fluoride to the cell-free supernatants. Freezing the supernatants is recommended if they are to be stored. Pure vitreous specimens will be necessary if quantitative results are desired. Mem-
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 2:06:32 AM
Cytology Cytological techniques require the vitreous specimen to be delivered promptly to the cytopathology laboratory. Delivery of the specimen by the surgeon is not an unreasonable precaution. Poor cellular morphology from autolysis due to excessive storage time in the vitreous-infusate solution can destroy the usefulness of an otherwise adequate specimen. Cytocentrifugation in a special apparatus can be used to make multiple slides for Papanicolau or Wright staining and also for immunoperoxidase staining of cell surface markers. Alternatively, the cell suspension can be centrifuged at 1,100 rpm for 8 min to create a `pellet' of cells, which may be invisible. The pellet is resuspended in about 75 µl or physiological solution for every slide desired. Fifty microliters of solution are then pipetted onto slides coated with a 1 % solution of gelatin and allowed to air-dry. Ten to 15 slides can usually be obtained from the typical vitreous specimen and compare favorably with those prepared by cytocentrifugation. Table 1 lists the antibodies and stains recommended for the examination of vitreous cells. Cell surface markers may also be determined by fluorescence-activated cell sorting (FRCS) [21].
Diagnostic Vitrectomy in Intermediate Uveitis
125
Table 1. Monoclonal antibodies for immunoperoxidase staining of vitreous cells Basic panel — uveitis and intraocular infections Lymphocyte markers CD3 Pan-T cell CD8 Suppressor/cytotxic T subset CD4 Helper/inducer T subset CD22 B lymphocytes CD 16 Natural killer cells Macrophage markers Leu-15, OKMI Macrophage /monocyte Class II antigens HLA-DR Activated lymphocytes HLA-DQ Activated lymphocytes Lymphoma panel Wright stain CD3 Kappa Lambda
Hematologic stain Pan-T cell Monoclonal and polyclonal light-chain Fc Monoclonal and polyclonal light-chain Fc
Microbial Culture Routine microbiological cultures are generally not performed in diagnostic vitrectomy for uveitis. However, the fastidious anerobic Propionibac-
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 2:06:32 AM
brane ultrafi ltration allows the partitioning of a very dilute specimen roughly according to molecular weight. Inhibitors which may interfere with bioassays of the cytokines can be removed in this fashion [7]. Lyophilization and reconstitution with tissue culture fluid can produce a concentrated solution of the cytokine of interest but at the risk of hyperton city or other aberrations which may affect the growth of the indicator cell lines or denature the peptide. ELISA techniques using pure vitreous avoid some of the pitfalls of bioassays but have the disadvantage of not providing any information concerning the biological activity of the immunologically detected molecule. Commercially available kits are able to detect picogram quantities of interleukins 1, 2, 3, 4 and 6 and tumor necrosis factor-a (R & D Systems, Minneapolis, Minn., USA). Other cytokines such as angiogenic factors and transforming growth factor-β require bioassays for detection. Neutralizing antibodies to many human cytokines are available (Genzyme, Boston, Mass., USA). Prostaglandins and leukotrienes have been measured in animal models of uveitis [22].
Davis/Chan/Nussenblatt
126
terium acnes bacterium was ultimately discovered to be the cause of many cases of delayed-onset intraocular inflammation following extracapsular cataract surgery by obtaining anaerobic cultures and by observing for growth over a 2-week period [23, 24]. Endogenous fungal infection or indolent bacterial infection is occasionally the cause of chronic inflammatory processes in unoperated eyes. Pure vitreous can be submitted for culture, but its viscosity probably renders it an inferior specimen for most culture techniques. Better results have been reported by passing the vitrectomy fluid through a sterile, self-contained 0.45-Μm filter unit [25]. Pie-shaped wedges of the filtering membrane are placed face up on culture plates or submerged in liquid growth medium. Good results have also been obtained by injecting 10 ml of the initial vitrectomy specimen into blood culture bottles in the operating room and subculturing the subsequent bacterial growth [26]. Mycobacteria and virus require inoculation onto special medium or live cells. Recovery of these organisms from the vitreous has only rarely been accomplished [27] and demonstration of these infections generally relies on histological techniques [28, 29].
Polymerise Chain Reaction Demonstration of specific microbial products in the intraocular fluid can be used as evidence for infection in cases where fastidious organisms cannot be grown in culture. The polymerise chain reaction (PCR) has extraordinary capability for detecting the DNA of microorganisms within the
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 2:06:32 AM
Immuniglobulins Quantitative immunoglobulin determinations using an ELISA technique require 10 [1 of pure aqueous or vitreous per antibody tested. A modification of standard nephelometry can be used to quantitate total IgG on microquantities of fluid. The total IgG is then used to control for passive diffusion of antibody into the eye by calculating a ratio of specific antibody to total IgG in the aqueous or vitreous humor divided by the ratio of specific antibody to total IgG in a simultaneous serum specimen. Polyclonal activation is excluded by dividing the quotient for the antibody of interest by a similar quotient generated for an irrelevant antibody. Values greater than 1 are taken as evidence of local intraocular production of antibody. Small quantities (approximately 20 Μ1) of aqueous humor have also been used in an immunofluorescence technique in patients with toxoplasmosis [30]. Specimens for this sort of testing can be collected and stored frozen, enabling them to be analyzed in reference laboratories if necessary.
Diagnostic Vitrectomy in Intermediate Uveitís
127
eye [31-33]. One hundred microliters or less of aqueous or vitreous fluid, stored frozen or analyzed fresh, may contain the single copy of DNA required for amplification by this technique. The sensitivity of the assay is also one of its drawbacks as false positives can occur. Exceptional care must be taken to avoid contamination of the specimen. Simultaneous control specimens are also helpful in protecting against the overinterpretation of results. PCR for mRNA has the potential to allow the detection of intraocular production of inflammatory mediators. RNA is considerably more labile than DNA and collection containers need to be specially treated to remove RNAse. Since PCR is usually considered a nonquantitative test, a positive result for a syngeneic mRNA will be more difficult to interpret than the presence of foreign DNA within the eye. The major current drawback to PCR is that it can take months to set up an assay for a single DNA sequence. In addition, it is currently considered a research technique; it use for clinical diagnostic purposes may not be appropriate.
The intraocular fluids of intermediate uveitis patients which have been analyzed come from several sources. Some fluids have been obtained in the course of surgery for other indications. Other fluids have been removed specifically for diagnostic purposes, usually to rule out intraocular lymphoma. The number of patients potentially requiring vitrectomy surgery who have intermediate uveitis is not trivial; retinal complications have been estimated to occur in 20-50% of patients [34]. Some experts have recommended vitrectomy surgery therapeutically for marked vitreous inflammation and macular edema unresponsive to periocular steroids or cryotherapy [16]. The number of specimens available for study is therefore potentially large although published results to date are small. Results of diagnostic vitrectomy in intermediate uveitis appeared by the late 1970s as part of small series of uveitis patients with various diagnoses. Both acute and chronic inflammatory cells have been reported in 6 uveitis patients of unspecified type [35]. Lymphocytes and macrophages were reported as the predominant cellular type in another group of 5 uveitis patients [36]. Granulomatous inflammation, characterized by epithelioid cells and multinucleated giant cells, was reported in 2 of 4 vitreous specimens from pars planitis patients [2]. An additional specimen contained lymphocytes, plasma cells and macrophages. A small number of specimens have been
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 2:06:32 AM
Results of Diagnostic Vitrectomy in Intermediate Uveitis
Davis/Chan/Nussenblatt
128
Table 2. Results of immunoperoxidase staining of vitreous cells in uveitis patients Diagnosis
Sarcoid Intermediate uveitis Intermediate uveitis Intermediate uveitis
vitreous blood vitreous blood vitreous blood vitreous blood vitreous blood vitreous blood
pan T
helper
suPeres- B cell sor
95 35 70 30 95 30 50 25 75 15 95 10
90 20 40 15 85 15 35 15 50 10 90 5
2 10 40 10 5 5 5 10 50
Diagnostic Vitrectomy in Intermediate Uveitis Janet L. Davis°, Chi-Chao Chanb, Robert B. Nussenblatt' of Ophthalmology, University of Miami, Bascom Palmer Eye Institute, Miami, Fla.; hNational Eye Institute, Laboratory of Immunology, Bethesda, Md., USA
The intraocular cells and snowbanks of intermediate uveitis are logical targets for biopsy and study. They are the visible evidence of the disease, correlate with its activity and may in fact be the effectors of damage to the eye [ 1 ]. The vitreous potentially contains a variety of clues as to the etiology of intermediate uveitis and of other vitreoretinal diseases in which peripheral neovascularization, cystoid macular edema and retinal vascular leakage play a part. Diagnostic vitrectomy has become a more common surgical procedure. It is ordinarily performed to confirm the fairly restricted list of diagnoses that can be made by the cytological or histological examination of small numbers of cells or tissue fragments. The usefulness of vitrectomy specimens in the diagnosis of intraocular lymphoma is proven [2-4]. The clinical indications for diagnostic vitrectomy are typically limited to cases where either neoplasia or bacterial or fungal infection is suspected. Cytological and histological diagnoses of a wider variety of diseases have been established using the technique, however, including lens and blood-induced glaucoma, metastatic tumors and amyloidosis [2]. In uveitis, a precise diagnosis is usually not confirmed by examination of vitrectomy specimens as chronic inflammatory cells are seen without other specific diagnostic features. Conventional pathological examinations have not been useful in discriminating between the various types of uveitis. Research techniques practiced on intraocular specimens removed incidentally in the course of other indicated surgery, such as lensectomy and vitrectomy for uveitis-associated cataract or retinal detachment repair [5-7], have expanded the number of laboratory investigations to which vitreous fluid can be subjected. As laboratory techniques are improved and useful diagnostic information is pro-
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 2:06:32 AM
a Department
Diagnostic Vitrectomy in Intermediate Uveitís
121
duced, the indications for diagnostic vitrectomy in intermediate uveitis may increase.
Diagnostic vitrectomy is generally performed by surgeons with specialized training in vitreoretinal surgery. In order to obtain adequate specimens with good visualization and control over the intraocular pressure, most surgeons prefer the standard three-port pars plana approach developed for more complicated vitreous procedures [8]. The exact sequence of surgery depends on other procedures planned at the time of vitrectomy and the objectives of the biopsy. Paracentesis of the anterior chamber may be performed as a first step in phakic eyes or pseudophakic eyes with posterior chamber lenses and intact posterior capsules. This should be performed before other fluids are infused into the eye if a pure aqueous humor specimen is desired. The anterior chamber should be reformed with balanced saline solution since there is a risk that even transient hypotony will produce miosis. About 200 µl of fluid can be obtained by careful aspiration into a 30gauge needle. Pure specimens of vitreous need to be removed before lensectomy by using the vitreous cutter through one of the superior sclerotomies while either (1) infusing air via an air pump into the preplaced pars plana cannula or (2) removing vitreous until the eye softens to the maximal safe extent. Simultaneous scleral depression can help control hypotony [9]. Air infusion is probably the safest procedure in eyes which are intended to remain phakic, but requires a high-minus contact lens and endoillumination to maintain a good view. In addition, the cannula tip must be visible in the vitreous cavity. Usually no more than 0.5-1.0 ml of pure vitreous can be removed from the eye if air is not simultaneously infused. If lensectomy is planned, this is usually performed before vitrectomy, using a fragmentation needle through the pars plans. Secondary infusion through a 20- to 23-gauge bent needle should be used until the medium is sufficiently clear to visualize the cannula tip. Lensectomy can compromise the quality of the vitreous specimen in two ways. First, infusion fluid must enter the eye, either through the posterior infusion cannula or through the secondary infusion line in the lens capsule. This will necessarily dilute the vitreous specimen and is undesirable if quantitative determinations are planned.
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 2:06:32 AM
Techniques of Diagnostic Vitrectomy
122
The second problem created by lens material is that it markedly interferes with the quality of cytological and histological preparations. This problem can be lessened by emptying the collection chamber of the vitrectomy instrument before proceeding with vitrectomy. There will usually still be enough residual material clinging to the sides of the container to affect the quality of the vitreous specimen. An alternative is to have an assistant manually aspirate the lens material into a separate syringe attached to the aspiration line. The syringe is connected by a three-way stopcock or by flexible tubing to the rigid aspiration port on the vitrectomy handpiece. The initial lensectomy specimen is discarded and the aspiration line flushed before performing the vitrectomy. If an absolutely pure vitreous specimen is not needed for quantitative determinations, it may still be useful to partition the vitreous specimen in two parts. The first portion of the vitrectomy always contains a less dilute specimen because it is easier to keep the tip of the vitrectomy instrument embedded in vitreous early in the case. It is a useful practice to aspirate the first 10 ml of vitreous mixed with infusate into a separate syringe. This will roughly correspond to the central core vitreous diluted with infused saline and provides a cell suspension of the appropriate density for preparation of slides by cytocentrifugation. After the central formed vitreous is removed, peripheral vitreous skirt can be removed. This results in a more dilute specimen which can be conveniently collected using automatic suction into the machine cassette. A low cutting rate (200 cuts/ min) has been recommended to ensure optimal specimen quality [9]. As complete a vitrectomy as possible without endangering the retina or lens is generally performed. Removing the central formed vitreous from a phakic eye of a uveitis patient does not much increase the complication rate beyond that associated with removal of only a small amount of fluid with the vitreous cutter. Furthermore, standard pars plana vitrectomy is a more controlled method than simple needle aspiration of the vitreous cavity with its inherent risks of creating vitreoretinal traction and retinal detachment. Uveitis patients may benefit from diagnostic vitrectomy in that the clearing of the central visual axis of opaque vitreous may allow better visual acuity. Reroyal of formed vitreous may also decrease the risk of tractional or rhegmatogenous retinal detachment. Subtotal vitrectomy is acceptable when removal of the residual vitreous skirt is not possible because of pupillary miosis or a compromised view of the periphery. In general, however, attempts should be made to remove as much of the vitreous as possible, including attempts to disinsert the posterior cortical vitreous from the retinal surface with a membrane pick or a soft-tipped extrusion needle [10].
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 2:06:32 AM
Davis/Chan/Nussenblatt
Diagnostic Vitrectomy in Intermediate Uveitis
1 23
In young intermediate uveitis patients, collapse of the globe due to massive loss of liquid vitreous from the eye may occur when the initial sclerotomy is made. The vitreous may be difficult to evaluate accurately preoperatively as to its degree of liquefaction. If pure vitreous specimens are necessary, an initial attempt can be made to aspirate through the pars plana with a 25-gauge needle on a tuberculin syringe before opening the 20-gauge sclerotomy for the infusion cannula.
Risks of Diagnostic Vitrectomy Pars plana vitrectomy ranks as a major ocular surgical procedure and should not be undertaken unless the information gained is likely to lead to a change in therapy or diagnosis for the patient or result in improved vision. Phakic patients run the immediate risk of damage to the crystalline lens during the surgical procedure or accelerated cataract formation after the surgery. Reported rates of cataract formation 1 year after vitrectomy range from 15% of patients [1] to 47% [12]. Conventional cataract extraction procedures are generally successful if cataracts do form [13]. Retinal complications of vitrectomy, including retinal detachment, occur in about 2% of patients in whom the retina is attached prior to surgery and in whom membrane peeling is not performed [14]. Uveitis patients may be at increased risk for this complication because inflammatory changes in the vitreous may produce abnormal vitreoretinal adhesions [15]. Both improved and persistent cystoid macular edema following vitrectomy for uveitis have been reported [16, 17]. Vitreous hemorrhage has been reported as a complication of vitrectomy surgery in intermediate uveitis patients with active neovascularization [17]. Endophthalmitis following vitrectomy has been reported but is rare [ 18, 19].
Cell Culture For techniques requiring live cells such as lymphocyte proliferative studies, the vitreal cells must be separated by centrifugation and transferred to a supportive growth medium as rapidly as possible. The length of survival of live cells in vitreous alone is unknown. There has been an impression that cells retrieved from vitreous specimens were of marginal viability. This may be due to the presence of many dead and degenerating cells in vivo rather
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 2:06:32 AM
Specimen Handling and Brief Methodology
Davis/Chan/Nussenblatt
124
than death after removal from the eye. Antiproliferative factors in vitreous may also contribute to the problems associated with in vitro growth; such factors have been shown to be present in aqueous humor [20]. Complete RPMI medium with 10% fetal calf serum and appropriate antigen or mitogen supports the proliferation of human peripheral-blood lymphocytes in vitro. There is little experience with cell culture of vitreal cells, so it is unknown if their requirements are more fastidious. Because the number of cells retrieved is likely to be small in any case, clonal expansion using the appropriate growth factors may be necessary to achieve sufficient numbers of cells for in vitro studies.
Cytokines Bioassays for soluble factors such as cytokines are best performed on cell-free supernatants since dead or dying cells can release these factors and confound results. Cellular proteases can also alter the peptide constituents of vitreous and may render some cytokines biologically or immunologically unreactive. Consideration should be given to the addition of protease inhibitors such as phenylmethane-sulfonyl fluoride to the cell-free supernatants. Freezing the supernatants is recommended if they are to be stored. Pure vitreous specimens will be necessary if quantitative results are desired. Mem-
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 2:06:32 AM
Cytology Cytological techniques require the vitreous specimen to be delivered promptly to the cytopathology laboratory. Delivery of the specimen by the surgeon is not an unreasonable precaution. Poor cellular morphology from autolysis due to excessive storage time in the vitreous-infusate solution can destroy the usefulness of an otherwise adequate specimen. Cytocentrifugation in a special apparatus can be used to make multiple slides for Papanicolau or Wright staining and also for immunoperoxidase staining of cell surface markers. Alternatively, the cell suspension can be centrifuged at 1,100 rpm for 8 min to create a `pellet' of cells, which may be invisible. The pellet is resuspended in about 75 µl or physiological solution for every slide desired. Fifty microliters of solution are then pipetted onto slides coated with a 1 % solution of gelatin and allowed to air-dry. Ten to 15 slides can usually be obtained from the typical vitreous specimen and compare favorably with those prepared by cytocentrifugation. Table 1 lists the antibodies and stains recommended for the examination of vitreous cells. Cell surface markers may also be determined by fluorescence-activated cell sorting (FRCS) [21].
Diagnostic Vitrectomy in Intermediate Uveitis
125
Table 1. Monoclonal antibodies for immunoperoxidase staining of vitreous cells Basic panel — uveitis and intraocular infections Lymphocyte markers CD3 Pan-T cell CD8 Suppressor/cytotxic T subset CD4 Helper/inducer T subset CD22 B lymphocytes CD 16 Natural killer cells Macrophage markers Leu-15, OKMI Macrophage /monocyte Class II antigens HLA-DR Activated lymphocytes HLA-DQ Activated lymphocytes Lymphoma panel Wright stain CD3 Kappa Lambda
Hematologic stain Pan-T cell Monoclonal and polyclonal light-chain Fc Monoclonal and polyclonal light-chain Fc
Microbial Culture Routine microbiological cultures are generally not performed in diagnostic vitrectomy for uveitis. However, the fastidious anerobic Propionibac-
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 2:06:32 AM
brane ultrafi ltration allows the partitioning of a very dilute specimen roughly according to molecular weight. Inhibitors which may interfere with bioassays of the cytokines can be removed in this fashion [7]. Lyophilization and reconstitution with tissue culture fluid can produce a concentrated solution of the cytokine of interest but at the risk of hyperton city or other aberrations which may affect the growth of the indicator cell lines or denature the peptide. ELISA techniques using pure vitreous avoid some of the pitfalls of bioassays but have the disadvantage of not providing any information concerning the biological activity of the immunologically detected molecule. Commercially available kits are able to detect picogram quantities of interleukins 1, 2, 3, 4 and 6 and tumor necrosis factor-a (R & D Systems, Minneapolis, Minn., USA). Other cytokines such as angiogenic factors and transforming growth factor-β require bioassays for detection. Neutralizing antibodies to many human cytokines are available (Genzyme, Boston, Mass., USA). Prostaglandins and leukotrienes have been measured in animal models of uveitis [22].
Davis/Chan/Nussenblatt
126
terium acnes bacterium was ultimately discovered to be the cause of many cases of delayed-onset intraocular inflammation following extracapsular cataract surgery by obtaining anaerobic cultures and by observing for growth over a 2-week period [23, 24]. Endogenous fungal infection or indolent bacterial infection is occasionally the cause of chronic inflammatory processes in unoperated eyes. Pure vitreous can be submitted for culture, but its viscosity probably renders it an inferior specimen for most culture techniques. Better results have been reported by passing the vitrectomy fluid through a sterile, self-contained 0.45-Μm filter unit [25]. Pie-shaped wedges of the filtering membrane are placed face up on culture plates or submerged in liquid growth medium. Good results have also been obtained by injecting 10 ml of the initial vitrectomy specimen into blood culture bottles in the operating room and subculturing the subsequent bacterial growth [26]. Mycobacteria and virus require inoculation onto special medium or live cells. Recovery of these organisms from the vitreous has only rarely been accomplished [27] and demonstration of these infections generally relies on histological techniques [28, 29].
Polymerise Chain Reaction Demonstration of specific microbial products in the intraocular fluid can be used as evidence for infection in cases where fastidious organisms cannot be grown in culture. The polymerise chain reaction (PCR) has extraordinary capability for detecting the DNA of microorganisms within the
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 2:06:32 AM
Immuniglobulins Quantitative immunoglobulin determinations using an ELISA technique require 10 [1 of pure aqueous or vitreous per antibody tested. A modification of standard nephelometry can be used to quantitate total IgG on microquantities of fluid. The total IgG is then used to control for passive diffusion of antibody into the eye by calculating a ratio of specific antibody to total IgG in the aqueous or vitreous humor divided by the ratio of specific antibody to total IgG in a simultaneous serum specimen. Polyclonal activation is excluded by dividing the quotient for the antibody of interest by a similar quotient generated for an irrelevant antibody. Values greater than 1 are taken as evidence of local intraocular production of antibody. Small quantities (approximately 20 Μ1) of aqueous humor have also been used in an immunofluorescence technique in patients with toxoplasmosis [30]. Specimens for this sort of testing can be collected and stored frozen, enabling them to be analyzed in reference laboratories if necessary.
Diagnostic Vitrectomy in Intermediate Uveitís
127
eye [31-33]. One hundred microliters or less of aqueous or vitreous fluid, stored frozen or analyzed fresh, may contain the single copy of DNA required for amplification by this technique. The sensitivity of the assay is also one of its drawbacks as false positives can occur. Exceptional care must be taken to avoid contamination of the specimen. Simultaneous control specimens are also helpful in protecting against the overinterpretation of results. PCR for mRNA has the potential to allow the detection of intraocular production of inflammatory mediators. RNA is considerably more labile than DNA and collection containers need to be specially treated to remove RNAse. Since PCR is usually considered a nonquantitative test, a positive result for a syngeneic mRNA will be more difficult to interpret than the presence of foreign DNA within the eye. The major current drawback to PCR is that it can take months to set up an assay for a single DNA sequence. In addition, it is currently considered a research technique; it use for clinical diagnostic purposes may not be appropriate.
The intraocular fluids of intermediate uveitis patients which have been analyzed come from several sources. Some fluids have been obtained in the course of surgery for other indications. Other fluids have been removed specifically for diagnostic purposes, usually to rule out intraocular lymphoma. The number of patients potentially requiring vitrectomy surgery who have intermediate uveitis is not trivial; retinal complications have been estimated to occur in 20-50% of patients [34]. Some experts have recommended vitrectomy surgery therapeutically for marked vitreous inflammation and macular edema unresponsive to periocular steroids or cryotherapy [16]. The number of specimens available for study is therefore potentially large although published results to date are small. Results of diagnostic vitrectomy in intermediate uveitis appeared by the late 1970s as part of small series of uveitis patients with various diagnoses. Both acute and chronic inflammatory cells have been reported in 6 uveitis patients of unspecified type [35]. Lymphocytes and macrophages were reported as the predominant cellular type in another group of 5 uveitis patients [36]. Granulomatous inflammation, characterized by epithelioid cells and multinucleated giant cells, was reported in 2 of 4 vitreous specimens from pars planitis patients [2]. An additional specimen contained lymphocytes, plasma cells and macrophages. A small number of specimens have been
Downloaded by: Université de Paris 193.51.85.197 - 1/11/2020 2:06:32 AM
Results of Diagnostic Vitrectomy in Intermediate Uveitis
Davis/Chan/Nussenblatt
128
Table 2. Results of immunoperoxidase staining of vitreous cells in uveitis patients Diagnosis
Sarcoid Intermediate uveitis Intermediate uveitis Intermediate uveitis
vitreous blood vitreous blood vitreous blood vitreous blood vitreous blood vitreous blood
pan T
helper
suPeres- B cell sor
95 35 70 30 95 30 50 25 75 15 95 10
90 20 40 15 85 15 35 15 50 10 90 5
2 10 40 10 5 5 5 10 50
![[Pars plana vitrectomy in uveitis].](/assets/img/hold.png)
