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| EYE PATHOLOGY UPDATE |
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| Year : 2011 | Volume
: 18
| Issue : 4 | Page : 261-267 |
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Biopsy pathology in uveitis
Jyotirmay Biswas, Radha Annamalai, Vaijayanthi Krishnaraj
Department of Ocular-Pathology and Uvea, Medical and Vision Research Foundation, Chennai, India
| Date of Web Publication | 23-Nov-2011 |
Correspondence Address:
Jyotirmay Biswas
Department of Ocular-Pathology and Uvea, Medical and Vision Research Foundation, 18, College Road, Chennai - 600 006
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/0974-9233.90126
 Abstract | | |
Uveitis is fraught with speculations and suppositions with regard to its etiology, progress and prognosis. In several clinical scenarios what may be perceived as due to a systemic infection may actually not be so and the underlying etiology may be an autoimmune process. Investigations in uveitis are sometimes the key in identification and management. Invasive techniques could be of immense value in narrowing down the etiology and help in identifying the cause. This article updates one on the invasive techniques used in biopsy such as anterior chamber paracentesis, vitreous tap and diagnostic vitrectomy, iris and ciliary body biopsy, choroidal and retinochoroidal biopsy and fine needle aspiration biopsy (FNAB). In populations where certain infections are endemic, the clinical scenario does not always respect a known presentation and the use of biopsy is resorted to as a sure way of confirming the etiology. Biopsies have a role in diagnosis of several inflammatory and infectious conditions in the eye and are pivotal in diagnosis in several dilemmas such as intraocular tumors and in inflammations. Appropriate and timely use of biopsy in uveitis could enhance the diagnosis and provide insight into the etiology, thus enabling precise management. Keywords: Anterior Chamber Paracentesis, Choroidal and Retinochoroidal Biopsy and Fine Needle Aspiration Biopsy, Iris and Ciliary Body Biopsy, Vitreous Tap and Diagnostic Vitrectomy
How to cite this article:
Biswas J, Annamalai R, Krishnaraj V. Biopsy pathology in uveitis. Middle East Afr J Ophthalmol 2011;18:261-7 |
| Introduction | |  |
Uveitis encompasses entities of varying duration, severity, location and above all a vast plethora of possible etiologies with quite similar and overlapping presentations.
Examination of a patient with uveitis needs to be meticulous as the incidence of association with systemic diseases is high. Uveitis is often diagnosed based on clinical features alone (e.g. Fuchs' heterochromic iridocyclitis, serpiginous choroiditis), with the help of ancillary tests (fundus fluorescein angiography, indocyanine green angiography, optical coherence tomography or with the help of laboratory tests (sarcoid, tuberculous and syphilitic uveitis). The importance and role of biopsy pathology is significant in masquerade syndromes, intraocular inflammation, infections, intraocular tumors and metastasis. With advancing surgical concepts and instrumentation, many of the biopsy procedures have become safe methods of reaching the correct diagnosis, thereby guiding appropriate treatment and also providing insights into the disease [Table 1].
| Anterior Chamber Paracentesis | | |
We describe herein a simple and safe technique, which can be performed in an outpatient department (OPD) setting, taking adequate aseptic precautions. The indication for an anterior chamber paracentesis may be many, but some of the common situations are in patients with masquerade syndromes [Figure 1], a hypopyon suspicious of infection [Figure 2], endophthalmitis and lens induced uveitis [Figure 3] and when performed can help in the diagnosis based on cytopathologic evidence [Figure 4]. | Figure 2: Anterior chamber aspirate showing multiple basophilic cells with pleomorphic nuclei suggestive of retinoblastoma (H and E, ×400)
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 | Figure 3: Granulomatous uveitis with mutton fat keratic precipitates, posterior synechiae, traumatic cataract and ruptured anterior capsule of the lens
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 | Figure 4: Anterior chamber aspirate of the same patient showing macrophages with engulfed matter (H and E, ×400)
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Steps
After using local antibiotic drops and a local anesthetic, a tuberculin or 2cc syringe with a 27 to 30 gauge needle is used. However, if fibrin is present or in case of granulomatous uveitis, it is preferable to use a large bore of 25-26 gauge needle. The needle entry into the anterior chamber is oblique through the stroma via the lower limbus. This acts as a valvular self-sealing paracentesis wound on withdrawal of the needle. One should avoid touching the corneal endothelium and particularly the lens in phakic patients and should stay over the peripheral iris at all times. Obtain a 0.1 to 0.3-ml of aqueous, and on withdrawal external pressure is applied to the entrance with sterile cotton tipped applicator. A drop of antibiotic is instilled in the conjunctival sac and the eye is patched for half an hour after which the patient is re-examined to ensure anterior chamber reformation.
The procedure is quite safe. In case of infectious uveitis and endophthalmitis, a portion of the material should be sent to microbiology for direct smear for bacteria, fungus, acid fast bacilli, culture for bacteria, fungus and mycobacteria and polymerase chain reaction. Remaining fluid should undergo cytospin study to obtain better cell recovery for microscopic examination.
The authenticity of aqueous humor analysis has been reported following analysis of samples using smears, cultures, polymerase chain reaction (PCR) and real time PCR. Rothova et al., [1] have reported the usefulness of AC tap in isolation of infection in posterior uveitis. Further studies supporting this have also been reported by Rao et al,[2] on the utility of PCR analysis of aqueous humour samples in necrotizing retinitis. Cytopathologic features of aqueous aspirate [3] help in the diagnosis of lens induced uveitis, masquerade syndrome, delayed endophthalmitis, parasitic uveits to name a few [Table 2].
Complications that can occur following AC tap are rare, nevertheless, one needs to be aware of the possibility of wound leak, hyphema, iritis and endophthalmitis.
PCR finds lot of application in ocular inflammation and uveitis and is now an important investigation. [4]
| Vitreous Tap and Diagnostic Vitrectomy | | |
Material obtained from such procedures can undergo cytologic evaluation, PCR, detection of intraocular antibodies (by ELISA), flow cytometry and culture. [5],[6],[7],[8],[9] Vitreous biopsy can be vitreous tap or diagnostic vitrectomy. Such vitreous biopsy is required in infective posterior uveitis, recalcitrant posterior uveitis, suspected large cell lymphoma (reticulum cell sarcoma) particularly in the elderly population [Figure 5]. Obtaining vitreous samples by tap is easier and can be done in the outpatient department [10] however, material obtained is small. A diagnostic vitrectomy when performed provides large amount of material (though diluted), but needs to be done always in an operation theater. A cytologic analysis of vitreous aspirate can be of conclusive evidence in phacoanaphylactic uveitis,endophalmitis and in specific large cell lymphoma (reticulum cell sarcoma) [Table 3].
Technique of vitreous tap
When performed in the out patient department, the technique is similar to that of anterior chamber paracentesis. As the perforation of the sclera is more painful than performing a keratocentesis, sub-conjunctival injection of 0.1 ml of 2% lidocaine can be given at the site of scleral perforation and entry into the vitreous space, 3.5-mm posterior to the limbus in phakic and 3.0 mm posterior to the limbus in aphakic/pseudophakic patients. It should be better done in the operation theater under special aseptic precautions.
The vitreous sampling is done using 25 to 23 gauge needles. Most eyes with long-standing intraocular inflammation have liquefied vitreous or fluid pockets within the vitreous. In such a situation, fine bored 25-guage needle can be used. When organization of vitreous is seen, 23-gauge needle is preferred. The needle is inserted in the vitreous cavity under direct visualization with slit lamp. The empty syringe withdraws the vitreous, and manipulating the stopcock, a similar quantity of antibiotic is injected into the vitreous cavity. After the injection the needle is slowly withdrawn from the eye. Complications with vitreous tap are vitreous hemorrhage, endophthalmitis, lens injury, retinal tears and secondary glaucoma.
Technique of vitreous sampling and biopsy
For analysis of the vitreous, it is essential to obtain undiluted vitreous specimen under sterile conditions intraoperatively. Newer techniques using vitrectomy with attachments and pneumovitrector have been described.
Doft et al., [11] obtained vitreous samples in eyes with endophthalmitis by directly connecting a syringe to the aspiration tube of the vitreous cutter. Others have used a collecting bottle with openings at both sides integrated into the aspiration system.
Smiddy et al., [12] obtained vitreous samples through a three-way stopcock through a manual aspiration.
Scholda and co-workers [13] have developed a new technique, in which a metal device is integrated into the aspiration system of the vitrectomy unit, which fits on standard laboratory plastic containers with integrated caps.
Peyman has described a full functional vitrectomy instrument (pneumovitrector) composed of an aspiration and cutting system combined with an infusion line for injecting air or gas into the vitreous cavity. [14]
Vitrectomy
A more thorough standard three-port vitrectomy can be performed, especially when therapeutically indicated in cases of endophthalmitis. The undiluted vitreous can be sampled and aspirated via a side-port.
All vitreous aspirated should be sent for examination as even the vitreous in the infusion line and vitrector can carry evidence of the pathology.
Tests and handling of aqueous and vitreous specimen
The specimen should be handled in such a way as to allow the maximum number of tests to be performed based on the clinical diagnosis. To maximize the chance of detecting the offending agents, the aqueous humor and vitreous specimen obtained should be divided in two equal volumes. One half of the specimen is used for the following tests
Microbiology
The samples should be immediately inoculated into blood agar, chocolate agar, brain-heart-infusion-broth (BHIB), thioglycolate fluid (maintained at body temperature), Sabouraud's agar, Brucella More Details agar and BHIB with gentamicin (maintained at room temperature for fungal isolation).
Polymerase chain reaction
It is a powerful method to amplify specific sequences of DNA from a large complex mixture of DNA. For example, one can design PCR primers to amplify a single locus from an entire genome. PCR done on these samples particularly the vitreous sample can be a significant indicator of the presence of the organism.
A minimum volume of 0.05 ml should be reserved for this test especially when P. acnes, fungal endophthalmitis and uveitis of viral etiology are suspected. The other half of the original sample can be processed for the following tests.
Nested PCR means that two pairs of PCR primers were used for a single locus. The logic behind this strategy is that if the wrong locus were amplified by mistake, the probability is very low that it would also be amplified a second time by a second pair of primers.
Real time polymerase chain reaction
In molecular biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (Q-PCR/qPCR/qrt-PCR) or kinetic polymerase chain reaction (KPCR), is a laboratory technique which is an evolution of PCR. In this technique, both evidence and quantification are done simultaneously and hence this investigation has the advantage of quantifying the load of the pathogen.
Cytology
The entire sample can be spinned down, the supernatant transpipetted and the pellet resuspended in formalin or glutaraldehyde. These pellets are passed through two or more millipore filters and a number of specific staining methods including immunohistochemistry are carried out to identify the infiltrating cell types. [15] The other technique includes cytocentrifuge by cytospin method (about 1000 revolutions for 5 min). Cytopathologic findings of vitreous aspirate are given in [Table 3].
Antibodies
The supernatant obtained after spinning down the cellular components within the aqueous humor should be subjected to ELISA. The local production of specific antibodies within the ocular fluids is an important indication for the possible etiology especially when toxocara or toxoplasma is suspected. [16]
In Europe, the estimation of the Goldmann Witmer coefficient is done as given below:
Titer of antibody in aqueous concentration of serum globulin ×Titer of antibody in serum concentration of aqueous globulin.
= Goldmann Witmer coefficient
| Results | | |
0.5 to 2 = No intraocular antibody production
2 to 4 = Suggestive of intraocular antibody production
> to 4 = Diagnostic of intraocular antibody production
Flow cytometric analysis
Flow cytometry (FCM) measures the physical and chemical properties of individual particles or cells moving in a single file in a fluid stream.
The most clearly defined application of FCM is in diagnostic surgical pathology. [17] It is an adjunct to histologic examination in the diagnosis of lymphoproliferative and leukemic processes. FCM is applied to the study of uveal melanomas, retinoblastomas and ocular lymphoid proliferations, especially masquerade syndrome. It is now also being used to provide valuable information regarding the ratio between the cytotoxic and helper T-cells, an indication for the immunologic events taking place during the course of the ocular disease.
In a recent study, Davis et al., [18] showed that in case of intraocular lymphoma positive predictive value (PPV) of cytology was 100%, but negative predictive value (NPV) was 60.9%. For infection, PPV for bacterial and fungal culture was 100% and NPV was 94.9%. CD 22 + ve B lymphocyte ≥ 20% of total cells on cytofluorographic analysis had a PPV of 88% for lymphoma.
| Iris and Ciliary Body Biopsy | | |
Biopsy of iris and ciliary body is usually performed in suspected tumors in these regions. These lesions include glioneuromas, medulloepitheliomas, iridociliary cysts, leiomyomas, malignant melanomas and nematode granulomas. [19] Indications of iris and ciliary body biopsy in uveitic conditions are few and include the following:
- Metastatic lesions to the iris and ciliary body masquerading as uveitis.
- Ruptured iris cysts mimicking anterior uveitis.
- Iris and ciliary body nodules secondary to granulomatous conditions like tuberculosis and sarcoidosis. Moorthy and co-workers reported three patients with coccidioidomycosis iridocyclitis diagnosed by biopsy of iris nodules. [20]
| Choroidal and Retinochoroidal Biopsy | | |
Lesions within the choroid can be difficult to differentiate clinically, although technological advances in non-invasive imaging have helped to monitor the size and the growth. The ophthalmologist has to consider the option of performing a choroidal or retinochoroidal biopsy. With advances in instrumentation and microsurgical techniques, endoretinal biopsy and chorioretinal biopsy can be performed easily and safely. Although the diagnosis of intraocular lymphoma can usually be made on the basis of diagnostic vitrectomy alone, sometimes it requires a more aggressive approach with choroidal biopsy, when pars plana vitrectomy and extensive medical examination fails to confirm the diagnosis.
Technique of retinal and choroidal biopsy
Trans-scleral choroidal biopsy
After conjunctival recession and hemostasis, the area of sclera overlying the lesion is marked. A half-thickness scleral trapdoor hinged posteriorly is created. 7-0 preplaced vicryl sutures are taken to allow rapid closure. Diathermy is applied to the circumference of the biopsy site. An opening of smaller dimension is made into the suprachoroidal space within the floor of the lamellar scleral dissection. This opening is enlarged to expose the choroid which is excised using Vannas scissors taking care to avoid perforation of the retina. The scleral trap door is then sutured. [21]
Internal approach for retinal, subretinal or chorioretinal biopsy
A vitreous biopsy is obtained at the beginning of the procedure before starting infusion. [22] The biopsy site in case of detached retina is selected at the junction of involved and normal retina. Unimanual bipolar endocautery is used at the margin of the proposed biopsy site. A biopsy specimen of at least 2 ×2 mm is then cut with 20 gauge vertical cutting scissors. The specimen is left attached at one corner, an intraocular forceps is used to remove it from the eye. After reattaching the retina, endolaser is applied to the biopsy site and long-acting gas or silicone oil tamponade is used. In case of attached retina, combined retinal and choroidal biopsy is obtained. Intense endo-diode laser is first applied around the margin of the biopsy site. Two minutes later a 2×2 mm biopsy specimen is cut within the margins of the laser burns with vertically cutting intraocular scissors until sclera is visible.
| Fine Needle Aspiration Biopsy | | |
Biopsy specimens are performed when the cause of a disease is uncertain or its extent or exact character is in doubt. Aspiration biopsy had been first described as early as 1847, though the first report was published in 1930. [23] Jacobiec et al., proposed fine needle aspiration biopsy (FNAB) for intraocular tumors in 1979. FNAB offers a histopathologic correlation to the clinical diagnosis in the case of atypical presentation of intraocular tumors. It aids in effective planning and management; it enables histopathological diagnosis without sacrificing the eye or having to resort to open biopsy methods
FNAB has been recommended in,
- Cases of suspected infectious subretinal lesions (abscesses or tuberculomas) mimicking as choroidal tumors. Gregor and coworkers diagnosed a Nocardia asteroides subretinal abscess following a trans-vitreal FNAB. [24]
- Cases where diagnosis is difficult, distinction between benign and malignant lesion is not clear, all ancillary tests are inconclusive and where therapeutic decisions will be made on the basis of cytological findings.
- Patients with metastatic disease of the choroid but with no primary.
- Cases where patient refuses recommended therapy until histopathological confirmation is obtained.
Techniques of fine needle aspiration biopsy
Approach
- Limbal route is used to approach anterior uveal lesions [25] eg. iris lesions, or in aphakic patients for posterior ciliary body lesions.
- In the posterior segment lesions, [26] the possible approaches are
Pars plana trans-vitreal approach
In this approach, the needle is passed from the pars plana region (3.5 mm from the limbus) in the quadrant opposite to the lesion, through the vitreous gel. For some of the eyes with tumors located posteriorly, a vitrectomy needs to be performed before aspiration.
Corneo-limbal approach
This approach through the zonules prevents dissemination of the tumor mass through the needle track. This approach is used in patients with retinoblastoma, a highly friable tumor, as the chance of needle track dissemination is extremely high. Through a corneolimbal approach the needle passes through multiple planes, thus wiping out the tumor cells as the needle is removed from the eye.
Sub retinal fine needle aspiration biopsy
This can be done in cases of sub retinal abscesses and tuberculomas, considering the site is approachable.
Complications
- The most common complication of FNAB is bleeding from the site of the needle track.
- Orbital dissemination of tumor cells and distant metastatic spread caused by tumor implantation along the needle track have been reported which are rare now.
- Iatrogenic retinal perforations are unavoidable by the indirect needle approach to the choroidal lesions and can theoretically cause a retinal detachment after FNAB.
Biopsy of intraocular lymphoma
Steroids should be stopped before diagnostic procedures to increase the diagnostic yield in suspected lymphoma cases. Samples should be obtained from the densest part of the infiltrate. [27] Also, it is worthy to note that lymphoma cells undergo apoptosis 90-100μ from their blood supply. If a distinct sub retinal mass is present, a direct sub retinal biopsy is better than a vitreous tap. The cells of lymphoma are fragile [Figure 6]. Due to the associated presence of inflammatory cytokines, they may degenerate and also the DNA degrades resulting in false negative results. [28] Samples may be placed in cell culture media to improve viability of cells. Interleukin 10 (IL- 10) levels have been found to be elevated in serum and vitreous of patients with non Hodgkins lymphoma while interleukin 6 (IL- 6) levels are elevated in the vitreous of patients with intraocular inflammation. Whitcup et al., found in a study that IL 10 levels exceeded the IL 6 levels in all 5 of study patients with primary central nervous system lymphoma involving the eye but in none of 13 patients with uveitis. [29] | Figure 6: Fine needle aspiration biopsy showing pleomorphic malignant cells in necrotic background (H and E, ×100)
Click here to view |
Biopsy in vasculitis
Biopsy of the temporal arteries is often performed for suspected inflammatory bowel disease (Crohn's disease and ulcerative colitis) which can present with uveitis both anterior non granulomatous or posterior type. Frequent biopsies are taken to assess the activity of disease and to assess changes that precede malignancy. The FNAB has a sensitivity and specificity rate of 84 and 98% respectively. [30]
| Conclusion | | |
In the future, newer association with infection, systemic disease and genetic patterns in uveitis will arise and needs to be explored further, focusing on the pathogenesis of the underlying ocular inflammation. However, while translating these research findings into clinical application, several points like cost effectiveness will have to be considered. The different immunological hypotheses of etiology of uveitis needs to be verified, and adopted or rejected and management done accordingly. Biopsy pathology in ocular inflammation has no doubt a well defined place in the investigation protocol especially considering the rarity of certain types and the overlapping features that may co exist in the same patient. The most important consideration is that biopsy proven pathogenesis and etiology is reliable and treatment can be instituted based on the histopathological outcome.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3]
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