Chapter 35 Keratocentesis and Vitreous Biopsy JOSEPH B. MICHELSON, MITCHELL H. FRIEDLANDER and G. RICHARD O'CONNOR Table Of Contents |
The dazzling panorama of new diagnostic laboratory techniques allows for
the identification and characterization of cells, proteins, and histopathologic
specimens and even for ultrastructural analysis of very small
samples obtained by paracentesis. Diagnostic paracentesis of the eye (keratocentesis
of the anterior chamber fluid) and vitreous
biopsy (paracentesis of the vitreous fluid in the posterior segment
of the eye) have definite value in the following scenarios:
Immune complexes and antibodies associated with Behçet's disease may be found. Polymerase chain reaction (PCR) analysis has suggested the presence of DNA from the infection. Tumor cells may be identified when a malignant infiltration of the eye (e.g., large cell lymphoma, leukemia, retinoblastoma, malignant melanoma) masquerades as a uveitis, or by the presence of tumor cell enzymes and antigens (Table 1). 1,2 TABLE 1. Diagnostic Paracentesis
CMV = cytomegalovirus; ELISA = enzyme-linked immunosorbent assay; HLA = human leukocyte antigen.
Although keratocentesis had been advocated historically as a treatment for active uveitis, it lost the attention of ophthalmologists until 1919, when Bruckner3 first examined the aqueous humor for diagnostic purposes. Laboratory techniques were revolutionized in the 20th century in areas such as: (a) evaluating very small aliquots of fluid (0.2 to 0.3 mL of aqueous or vitreous), and (b) identifying specific microbial organisms and the predominance of other cell types, antibodies, and proteins in these fluids (Figs. 1, 2, 3, 4, 5, and 6). These advancements have led to the development of diagnostic paracentesis for sight-threatening ocular inflammations that are difficult to diagnose. Witmer4 and O'Connor5 have provided strong evidence that samples of the aqueous humor reflect the antibody-producing capabilities of the iris and ciliary body, particularly when more specific antibody per unit of gamma globulin can be found on the aqueous humor than in the blood of the same patient.6–8 These determinations may be highly significant when one considers the fact that diseased tissue is being bathed in an antibody-containing fluid that is elaborated locally. For instance, in the case shown in Figure 1, the immunofluorescent antibody titer to toxoplasmosis is four times greater in the vitreous aspirate at the time of vitrectomy for repair of retinal detachment than in the plasma. These same considerations have long been recognized in syphilis of the central nervous system, wherein specific antibodies may be present in the cerebrospinal fluid but not in the blood. This is also the case with an unusual presentation of ocular coccidioidomycosis9 or toxocariasis.
Many forms of uveitis are characterized by specific types of inflammatory cells. Usually, however, one encounters mixtures of cell types in any given specimen, with the relative percentages of lymphocytes and polymorphonuclear leukocytes varying. There may be unusual numbers of eosinophils, or macrophages laden with lens material may be present. Thus, an enumeration of the cells and a careful analysis of their structure can be useful as a diagnostic aid (Figs. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20). Figure 15 demonstrates eosinophils that were aspirated from the anterior chamber of a patient with Toxocara canis endophthalmitis. Figure 12 demonstrates malignant cell infiltrate from the vitreous, showing the stained presence of monoclonal light chains being elaborated in the cytoplasm. Interleukin-10, detectable in the vitreous of intraocular lymphoma patients, is also directly indicative of both the clinical activity and the number of malignant cells as observed by cytopathology.
Precise identification and culture of bacterial and fungal pathogens from both the aqueous humor and the vitreous fluid can be obtained. Gram's stain and Giemsa's stain smears of centrifuged specimens from the aqueous humor and the vitreous humor frequently demonstrate the bacterial or fungal causative agent. Attempts to isolate bacteria and fungi and to identify them on Gram's stain or Giemsa's stain smears have been most rewarding in the following cases: (a) postoperative endophthalmitis, (b) infection after a penetrating injury of the eye, (c) drug abuse patients with endogenous endophthalmitis (Figs. 21, 22, 23, 24, and 25), (d) patients receiving hyperalimentation, and (4) patients who are immunocompromised as a result of exogenous immunosuppressive agents.
Studies have demonstrated the usefulness of ocular paracentesis for the identification of ocular infections in order to implement sight-saving treatment.10–16 Even acid-fast bacilli and viruses may be diagnosed in this fashion when emergency dictates (see Fig. 5).17 It is recommended that diagnostic paracentesis be performed in all cases of postoperative endophthalmitis, and it is safe to perform the postoperative procedure in the operating room with the safety of vitrectomy surgery. Further, any patient older than 65 who presents with a deteriorating uveitis (usually with vitreitis as the predominant infiltrate) of undetermined etiology should undergo paracentesis of the vitreous to rule out reticulum cell sarcoma (large cell lymphoma).18 Similarly, any patient suspected of being an intravenous drug abuser who presents with an endogenous endophthalmitis or uveitis should undergo diagnostic paracentesis to avoid allowing an intraocular infection to be borne by the bloodstream.19,20 |
TECHNIQUE OF KERATOCENTESIS |
Many techniques have been described for paracentesis of the eye. Most authorities
agree that aspiration of the aqueous fluid (keratocentesis) is best accomplished with the following technique:
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SPECIAL CONSIDERATIONS |
Suction of the vitreous in a patient with endophthalmitis is very tricky. The
vitreous tends to put traction on the retina, leading to unanticipated
tearing and splitting of the retina and ultimately retinal detachment. To
minimize traction on the retina caused by vitreous condensation
and fibrous tissue formations, it is best to aspirate vitreous in
the operating room under sterile conditions and under the controlled
suction and cutting of a vitreous instrument. This is highly preferable
to the unstable “sucking only” of a large-bore needle aspiration
at the pars plana. The cutting rate of the vitrectomy instrument
should be turned up higher than 300 cuts per minute, which further
serves to minimize tractional forces. Too much cutting of the vitreous, however, disturbs, disrupts, contorts, and destroys whatever cellular
elements and hyphae one may wish to obtain from the vitreous specimen. The
optimal degree of vitreous surgery should include aspiration
with a minimum of cutting of cellular elements, but with enough rapid
cutting so as not to prolong tractional forces. Such a vitreous sample
diluted by the irrigating solution is then passed through a disposable
membrane filter system. Adequate sterile technique must be maintained. The
procedure for obtaining the vitreous includes a stop-cock assembly
somewhere in the line before the machine receptacle is encountered
by the sterile line. This varies with each machine used, but some forethought
to the system allows for sterile maintenance of the fluid. Outpatient keratocentesis can be performed carefully at the slit-lamp with a minimum of complications. The same manipulative criteria must be observed as listed under Techniques (i.e., one should avoid the corneal endothelium and avoid rupturing the anterior lens capsule in phakic patients). Although the ideal conditions for vitreous aspiration are absolute sterility of the operating room and the more optimal aspiration/cutting ability of the vitreous instrument, in emergency situations it may be necessary to perform a pars plana paracentesis in the office setting. Even in such an emergency situation, however, it is almost always necessary to delay treatment until the patient can be taken to the operating room for a more controlled surgical evacuation of vitreous material. It is of both diagnostic and therapeutic benefit to the patient to achieve a more complete core vitrectomy, especially when cellular elements (e.g., large cell lymphoma, fungal endophthalmitis) are required to determine the diagnosis. |
CYTOLOGIC EXAMINATION OF THE AQUEOUS AND VITREOUS |
Cytologic examination of the aqueous and vitreous obtained with paracentesis
may be performed by a number of different methods. The simplest
method is the single-drop preparation, which consists of placing a small drop of fluid from the aspirating needle
directly onto the glass slide. To avoid dispersing the cells too
widely, the drop should not be spread. The drop should merely be allowed
to air dry. When dry, the preparation is fixed in absolute methanol
for 10 minutes and once again allowed to dry. The slide is finally placed
in dilute buffered Giemsa solution and allowed to stain for 1 hour. It
is then quickly rinsed with 95% ethanol and allowed to dry. A
cover slip is mounted with Canada balsam and the preparation examined
microscopically. The relative numbers of lymphocytes, polymorphonuclear leukocytes, and macrophages can be determined immediately. In viral infections and chronic hypersensitivity reactions, the cell type is predominantly mononuclear. In acute uveitis reactions, especially those involving the binding of complement to immune complexes (as in Behçet's syndrome), an abundance of neutrophils may be expected, and aqueous and vitreous can be examined by the Raji immune complex assay to determine the amount of immune complex in those fluids. In lens-induced uveitis, one may expect to see many macrophages as well as some neutrophils. In parasitic infections, numerous eosinophils may be seen (see Fig. 19). Bacteria or other organisms can be detected by the same examination. If bacteria are seen on the Giemsa preparation, a Gram's stain also should be performed on another single-drop preparation. Other methods of cytologic examination include techniques for concentrating the cellular elements. The simplest of these consists of passing the entire aspirate through a Millipore filter (mean pore size 0.45 γm). The filter disk may be stained and cleared with xylene before it is examined microscopically. Various centrifuges have been designed for the concentration of aqueous cells. Immediately upon aspiration, the cells should be fixed in a glutaraldehyde-paraformaldehyde mixture and postfixed in osmium tetroxide. They are centrifuged onto a thin sheet of araldite before sectioning. This technique results in the preservation of excellent cytologic detail. The secretory granules of the various types of polymorphonuclear leukocytes are easily identifiable, and organelles such as the Golgi apparatus can be seen readily. This technique also provides a method for examining viral inclusions in certain cells. Wet fixation of cells via a modification of the Papanicolaou technique may provide an ideal means of identifying intranuclear intracytoplasmic inclusions in affected cells. Studies have indicated that this method is far superior to the Giemsa technique for this purpose. Darkfield microscopic examinations of aqueous humor specimens have sparked much interest. Smith22 has identified mobile Treponema in the aqueous humor of patients suspected of having syphilitic uveitis. Correlative tests using fluorescent antibody methods seemed first to confirm the pathogenic T. pallidum in the anterior chamber of these patients, but subsequent examinations with adequately absorbed antisera seemed to indicate that some of the spirochetal forms previously observed were nonpathogenic treponemes (e.g., T. microdentium). Other difficulties with darkfield microscopy include the length of time necessary for observation and the frequent appearance of artifacts. |
SEROLOGIC EXAMINATION OF AQUEOUS AND VITREOUS HUMOR | ||||||||||||||||||||||||||||||||||||||||||||
The use of paracentesis for the procurement of diagnostic samples for serologic
testing is increasing. Microtiter methods for virtually all the
serologic tests used in the laboratory are being perfected for microtiter
specimens (Table 2). These are available for testing aqueous humor and liquid vitreous
samples and are particularly useful when tests of the aqueous or vitreous
are performed in conjunction with simultaneous tests on the patient's
serum. These include the new techniques of enzyme-linked immunosorbent
assay (ELISA) and PCR.
TABLE 2. Intraocular Inflammation Serology Measured In Microtiter Quantity
Ab = antibody; ACE = angiotensin-converting enzyme; Ag = antigen; CF = complement fraction; CIE = countercurrent immunoelectrophoresis; EIA = enzymal immunoassay; ELISA = enzyme-linked immunosorbent assay; FTA = fluorescent treponemal antibody; ID = immunodiffusion; IFA = indirect fluorescent antibody; IHA = indirect hemagglutination; PCR = polymerase chain reaction; RIA = radioimmunoassay; RPR = rapid plasma reagin: VDRL = Venereal Disease Research Laboratory (test for syphilis).
The ciliary body and iris may act as local factories for the production of antibodies. It is diagnostically significant whenever it can be established that the specific antibodies detected in the aqueous or vitreous are actually being made in the eye. One also must remember that the malignant B cell seen in the eye in large cell lymphoma (reticulum cell sarcoma) may be identified by the presence of antibodies on its surface (see Fig. 12) and cell-specific markers. One can demonstrate kappa or lambda light chains exclusively that are being produced on these cells as a manifestation of monoclonal infiltration. This is quite different from what is seen in a mixture of both kappa and lambda light chains, as manifested in a pure inflammation. Numerous qualitative reactions also are available for detection of specific antibodies in the aqueous and vitreous, and these have been adapted to microtiter quantitation by serial dilutions. These include paths of hemagglutination reactions for tuberculosis and, more frequently, ELISA, which have been developed for toxoplasmosis, toxocariasis, and herpesvirus and are available for most infectious pathogens. |
ENZYME-LINKED IMMUNOSORBENT ASSAY |
The development of solid-phase immunoassays during the past 10 years has
added greatly to our ability to detect small amounts of circulating
antibodies. One of the first assays to be developed was a radioimmunosorbent
technique in which fixation of a patient's antibodies to a
solid-phase antigen was detected by the use of radioactive iodine-labeled
antihuman globulin. This technique required maintenance of radioactive
materials in the laboratory as well as other potentially dangerous
procedures. The development of the ELISA test by Engvall and Perlmann23 provided a much safer method of fixation of antibodies to a solid-phase antigen. ELISA is used to detect antibodies to any antigen, and it screens for a wide number of bacterial, viral, and parasitic antigens, especially Toxocara, Toxoplasma, gonococcus, herpesvirus, and cytomegalovirus. The ELISA method is as follows:
Angiotensin-converting enzyme, which is diagnostic of granulomatous inflammation in general and suggestive of sarcoidosis in particular, can now be measured in such small amounts as aqueous specimens as well. |
SURGICAL SAMPLING |
No chapter on paracentesis of the eye would be complete without mentioning “sampling” of the uveal tissue to rule out certain malignancies. Although nodules of the iris may be benign in nature, and a consequence of inflammation as well as metaplasia or neoplasia, these lesions are more readily accessible for biopsy than choroidal or ciliary body lesions.25–30 Pioneering techniques of sophisticated biopsy surgery, initiated by Peyman and colleagues,30 have lent themselves to “eye wall biopsy,” leaving a candidate for possible enucleation for histopathologic evaluation intact. These specific surgical techniques have included iridocyclectomy (Fig. 26), iridochoroidectomy, eye wall resection, eye wall biopsy, endoretinal biopsy, and ab interno retinochoroidectomy.31 These techniques are especially valuable in the difficult diagnosis of large cell lymphoma infiltrate, atypical malignant melanoma of the choroid, acute retinal necrosis, viral retinitis, nematodes, therapeutic removal of malignant melanoma of the choroid or ciliary body, and metastatic cancer of the choroid. |
MOLECULAR GENETIC TECHNIQUES FOR CLINICAL ANALYSIS OF THE IMMUNE SYSTEM |
During the past two decades, advances in nucleic acid chemistry and recombinant
DNA technology have made it possible to analyze individual genes
rapidly and precisely. These techniques are now commonly used in research
and clinical laboratories. DNA is a remarkably sturdy molecule
that is easy to obtain and work with.32 It also provides access to information that allows us to investigate and
diagnose a variety of disease processes at a very fundamental level. These techniques include nucleic acid probes, short strands of bases with known sequences that can detect complementary base sequences. Hybridization assays can be used to evaluate cellular DNA by denaturing it and binding it to membranes, where it can be analyzed with probes to detect certain sequences. Examples of hybridization assays include the Southern blot assay, which determines the clonality of lymphoid cell populations. PCR is a powerful enzymatic technique that can exponentially replicate specific DNA sequences in the test tube. With this technique, it is now possible to assay vanishingly small samples that initially contain fewer than 10 copies of the sequence of interest. PCR is particularly useful for detecting viruses and other microorganisms in tissue specimens, because such organisms often can be recognized by their unique RNA or DNA sequences much more quickly and inexpensively than in culture. PCR has already assumed an important role in microbiologic diagnosis, and it seems likely that this role will expand in the future. Important organisms that can be assayed with this technique include Epstein-Barr virus, cytomegalovirus,33 human immunodeficiency viruses, and human T-cell leukemia viruses. DNA-based assays also are very useful for detecting large-scale chromosomal deletions or rearrangements characteristic of several hematologic malignancies. |