The clinical laboratory should not be viewed as the target for a battery of tests routinely ordered for all patients with uveitis. Instead, the laboratory evaluation should be seen as but one step in an organized workup of each patient, consisting of the following steps:

1. Naming
   
2. Meshing
   
3. Office testing
   
4. Specific and nonspecific clinical laboratory tests
   
5. Specialty consultations
   
6. Therapeutic tests
   
7. Diagnosis and treatment
   

TABLE 1. Classification of Uveitis

Naming Example A. Mr. Smith is a 30-year-old white man with severe acute recurrent nongranulomatous iridocyclitis, active in the right eye and quiescent in the left, with recent arthritis of the left knee and ankle as well as episodes of urethritis that have been culture negative in the past.

Naming Example B. Mr. Yonida is a 37-year-old Japanese man with a history of chronic bilateral diffuse granulomatous uveitis, headaches, hearing loss, and vitiligo. He has no known history of ocular trauma.

Naming Example C. Laurie Jones is a 9-year-old black girl with chronic nongranulomatous bilateral iridocyclitis, posterior synechiae, band keratopathy, and arthritis of the right wrist.

Naming Example D. Mr. Williams is a 28-year-old white homosexual man with acute unilateral granulomatous iridocyclitis and retinochoroiditis, weight loss, cough, and a history of intravenous drug abuse.

DEMOGRAPHICS: AGE, SEX, AND RACE

Obvious clues may be obtained from straightforward demographic data. Juvenile rheumatoid arthritis, retinoblastoma, Kawasaki's syndrome, and toxocariasis, for example, are seen primarily in children. Pars planitis, multiple sclerosis, and Fuchs' heterochromic iridocyclitis usually occur in young adults. Reiter's syndrome, ankylosing spondylitis, acute multifocal posterior placoid epitheliopathy, birdshot choroiditis, Vogt-Koyanagi-Harada syndrome, and Behçet's syndrome are most likely to occur in middle age, while large cell lymphoma or choroidal melanoma is found in older patients. Toxoplasmosis and sarcoidosis may present in childhood, youth, or middle age. Tuberculous or luetic uveitis can be found at any age. Primary uveitis of any origin rarely if ever makes its first appearance in old age.

Uveitis associated with juvenile rheumatoid arthritis is found much more frequently in females, while uveitis associated with ankylosing spondylitis and Reiter's syndrome is usually seen in males. Most patients with Behçet's syndrome and uveitis are males. Sympathetic ophthalmia and fungal endophthalmitis are mostly reported in males since they are more likely to have penetrating injuries or abuse intravenous drugs.

Ankylosing spondylitis, Reiter's syndrome, and other HLA-B27-associated arthritides are most common in whites. Sarcoidosis occurs most commonly in blacks. Behçet's and Vogt-Koyanagi-Harada syndromes are most prevalent in Asians; most of these patients who are not Asian or American Indian will have this lineage buried somewhere in their family history. In the United States, most patients with Behçet's syndrome are of Mediterranean ancestry. In the Mediterranean countries, Behçet's syndrome is more common and more virulent than it is in the United States. Coccidioidomycosis is more common and more severe in Filipinos and blacks than in other races comparably exposed to this New World fungus.

GEOGRAPHY, DIET, AND DOMESTIC AND PERSONAL FACTORS

Histoplasmosis is highly localized to the Mississippi-Ohio-Missouri River Valleys, as well as river valleys between 45^° North and 45^° South latitudes worldwide. It is not found in Australia, New Zealand, Europe, England, or Japan.³ Although a syndrome identical to presumed ocular histoplasmosis has been identified in England, a specific pathogen has not been identified. Coccidioidomycosis is found primarily in the southwest United States and Central and South America. Although tuberculosis is far less prevalent in Western countries than a generation ago, extremely high new active case rates are found in parts of East Asia, Hong Kong, Japan, Africa, and South America.

A history of the ingestion of raw or undercooked meat is important when considering a diagnosis of ocular toxoplasmosis. In some parts of Western Europe and Brazil where this practice is common, positive Toxoplasma antibodies are found in most of the population.

Contact with feline feces containing oocysts of Toxoplasma gondii may occur in children who play in sandboxes frequented by stray cats, or in adults who clean kitten litter boxes. These practices should be avoided, particularly by pregnant women. A history of contact with an unwormed puppy or kitten is significant in children with suspected ocular toxocariasis.

Patients exposed to sewers or rodent urine may be at risk for leptospirosis, while farmers and veterinarians are far more likely than the general population to develop brucellosis. Persons who ingest unpasteurized milk are at risk of tuberculosis and brucellosis.

Exposure to infectious agents may tip-off the diagnosis of tuberculous, luetic, cytomegalic, or Reiter's uveitis. Thus, a thorough history of sexual activity and preferences, previous venereal diseases, genitourinary symptoms, and family members with contagious diseases may obviate an expensive diagnostic workup. Similarly, a history of drug abuse should be tactfully obtained when necessary. Associated illnesses, including viral syndromes, rashes, or arthritis, may also provide invaluable hints in best naming a patient's uveitis and eventually formulating a differential diagnosis.

TABLE 2. Recognized Uveitic Entities

A comparison of patient characteristics to the clinical characteristics of each uveitic entity will reveal profiles similar enough to be included in a preliminary differential diagnosis. This comparison should be seen as a superimposing of clinical profiles. This is what is meant by meshing. The degree of fit or mesh is assessed, and the proposed diagnoses ranked in order of likelihood. Thus, the preliminary differential diagnosis following meshing in each of the four previous examples would be:

Example A:

1. Reiter's syndrome
   
2. Ankylosing spondylitis
   
3. Immunogenic uveitis
   

Example B:

1. Vogt-Koyanagi-Harada syndrome
   
2. Behçet's syndrome
   
3. Sympathetic ophthalmia
   

Example C:

1. Juvenile rheumatoid arthritis
   
2. Sarcoidosis
   

Example D:

1. Cytomegalovirus inclusion disease
   
2. Toxoplasmosis
   
3. Tuberculous or luetic uveitis
   
4. Fungal endophthalmitis
   
5. Acute retinal necrosis syndrome
   

SKIN TESTING FOR DELAYED HYPERSENSITIVITY

Intradermal injection of 0.1 ml of soluble antigens prepared from a number of infectious agents can be performed in the office during the initial visit. The most useful test antigen is the tuberculin purified protein derivative (PPD). The standard dose is 5 TU (US tuberculin units) or intermediate strength, equivalent to 0.0001 mg (Aplisol, Parke-Davis, Morris Plains, NJ). Current lots of PPD are standardized for biologic activity against PPD-Seibert (PPD-S), a reference bulk lot prepared in 1940. Second strength (250 TU) can be given to patients who do not react to an initial 5-TU injection, whereas first strength (1 TU) should be given to patients who the clinician suspects may have a strong reaction and are therefore at risk for sloughing and ulceration at the injection site (Tubersol, in first and second strength, available through Squibb/Connaught, Princeton, N J). There is no evidence of booster effects or repeated skin testing leading to conversion from negative to positive.⁴ Intracutaneous injection, the Mantoux test, is the most reliable test available. Injection of 0.1 ml of PPD antigen into the volar or dorsal surface of the forearm is made just below the skin surface with a short (1/2-inch) No. 27 gauge needle, bevel upward, to produce a discrete 6- to 10-mm wheal or bubble. The use of insulin syringes with intrinsic needles will avoid repeated waste of antigen extract in the needle hub of standard tuberculin syringes. If no bubble appears due to a deep injection, the test should be immediately reapplied at a site at least 5 cm away. Skin tests are read 48 to 72 hours following injection. Induration measuring 10 mm or more is considered positive: this indicates a previous infection with Mycobacterium tuberculosis, although previous infection with M. bovis or photochromogenic mycobacteria may also produce a positive result. Patients may also develop coexistent erythema, a wheal and flare that usually fades after 12 to 18 hours, which is evidence of an immediate hypersensitivity to the same test antigen. Uveitis patients with induration less than 10 mm, or even erythema alone, have had favorable clinical responses to oral antituberculous therapy.⁵ On the other hand, patients with proven ocular tuberculous infection have had insignificant⁶ or negative⁷ PPD skin tests.

Other commercially available skin test antigens include histoplasmin (Parke-Davis, Morris Plains, N J) and coccidioidin (Spherulin 1:100, Berkeley Biologicals, Berkeley, CA). Although the use of histoplasmin skin testing in uveitis is no longer universally recommended, greater than 80% of patients with presumed ocular histoplasmosis syndrome have positive skin tests.⁸ Some authors believe that histoplasmin skin testing may increase the danger of activating a macular lesion in susceptible patients.

Skin test reagents for other infectious diseases are not commercially available and must be obtained through individual laboratories actively engaged in production and purification of the specific antigen. These include brucellosis, tularemia, blastomycosis, toxoplasmosis, nontuberculous mycobacterial disease, and cryptococcosis antigens, all of which are subject to strict safety controls limiting their use.⁹ Control antigens to which normal, presumably exposed, adult patients should react, include Candida extract (Dermatophyton O, 1: 100, Hollister-Stier, Spokane, WA), Trichophyton, mumps, and streptokinase-streptodornase (Lederle Diagnostics, Pearl River, NY).

Patients with sarcoidosis and a number of other conditions (Table 3) are often anergic to all skin test materials.¹⁰ In patients taking corticosteroids, a negative test should be repeated or delayed until the medication is discontinued because systemic corticosteroids may reduce or eliminate all skin test reactions. A positive test, however attenuated, is still significant in patients on oral corticosteroid therapy. A self-addressed postcard clearly diagramming the position and dosage of all skin tests applied can be expeditiously returned after 48 hours by patients who do not have convenient access to the uveitis clinic (Fig. 1). Skin tests should be read only by health care personnel who understand diameter measurements of erythema and induration.

TABLE 3. Clinical Conditions Associated With Anergy

  Technical Errors in Skin Testing

  Improper dilutions of test antigens
  Bacterial contamination
  Exposure to heat or light
  Adsorption of antigen to container walls
  Faulty injection: too deep or leaking
  Improper reading of reaction

  Congenital cellular immunodeficiency:

  Thymic/parathyroid aplasia (DiGeorge's syndrome)
  Mucocutaneous candidiasis

  Ataxia-telangiectasia
  Nezelof's syndrome
  Severe combined immunodeficiency
  Wiskott-Aldrich syndrome

  Acquired immunodeficiency syndrome
  Sarcoidosis
  Chronic lymphocytic leukemia
  Hodgkin's disease and lymphomas
  Carcinoma
  Immunosuppressive medications
  Rheumatoid diseases
  Uremia
  Alcoholic and biliary cirrhosis
  Surgery

  Influenza, mumps, measles and viral infections
  Viral vaccinations
  Typhus
  Miliary and active tuberculosis
  Disseminated mycotic infection
  Lepromatous leprosy
  Scarlet fever

Since a positive reaction to skin tests requires a succession of immunologic processes, a positive control skin test rules out a major defect in cell-mediated immunity. Bacille Calmette-Guérin (BCG) has been used in the past to test a patient's ability to react to a new antigen, but it should not be used in immunocompromised patients. Although BCG vaccination is routinely recommended in some European and Third World countries, it is given in the United States only to PPD-negative contacts of ineffectively treated or persistently untreated cases and other unusually high-risk groups. Another approach to rule out anergy is skin sensitization to dinitrochlorobenzene (DNCB) or dinitrofluorobenzene (DNFB) by one cutaneous application of a high concentration of the substance, usually on the volar aspect of the forearm. Three weeks thereafter a much smaller dose can be used to confirm sensitization.¹¹ Virtually all normals can be sensitized in this manner. More advanced techniques to assess lymphocyte function include in vitro lymphocyte stimulation testing utilizing various antigens compared with known mitogen controls,¹² assays for interleukins and other cytokines, as well as flow cytometry and cell sorting with fluorochrome conjugated antibody labels for a variety of leukocyte subset surface markers.¹³

Approximately 80% of patients with sarcoidosis react positively to skin testing with the Kveim antigen. This antigen is prepared from human sarcoid granulomas, is difficult to obtain, requires several months of incubation followed by a biopsy at the injection site for histologic verification, does not have absolute specificity, and entails a risk of hepatitis or retrovirus transmission. False-positive and false-negative results may occur, limiting its usefulness.

BEHÇET'S SKIN PUNCTURE TEST

Patients with active systemic Behçet's disease show increased dermal sensitivity to needle trauma, owing to their unusual leukotactic tendency. The appearance of a pustule 24 to 36 hours after an intradermal needle puncture, with or without the injection of 0.1 ml of normal saline, is almost diagnostic for Behçet's disease.¹⁴ Erythema and infiltration may appear within a few hours, and occasionally a microabscess can be produced. This test may not be positive in patients with ocular manifestations when the systemic disease is in remission. No universal agreement on the usefulness of this test exists because only 10% of Behçet's patients demonstrate a positive response.¹⁵

MACULAR FUNCTION AND THE PHOTOSTRESS TEST

Macular edema is a common complication of intraocular inflammation and can lead to the formation of irreversible cysts and macular holes. Early macular disease can be detected by progressive loss of visual acuity, Amsler grid testing, tangent screen examination, careful contact lens or 90-diopter lens biomicroscopy, and fluorescein angiography. Maximal visual acuity after shining a bright light into the eye of a normal person recovers in less than 180 seconds. The photostress test is considered abnormal when there is marked asymmetry, or if the recovery time is greater than 180 seconds in either eye. The photostress test is extremely sensitive and may detect a maculopathy not demonstrable by any other measure. The recovery time is not prolonged in patients with optic nerve disease.¹⁶

TEAR FUNCTION TESTING

Aqueous tear deficiency states associated with uveitis include sarcoidosis, Sjögren's syndrome, and other systemic connective tissue diseases. Schirmer's tear strip testing or tear lysozyme levels can be used to quantify aqueous tear production. Tear lysozyme concentration can be measured with Micrococcus lysodeikticus bioassay plates, spectrophotometric, viscometric, turbidimetric, and immunologic methods. In contrast to classic keratitis sicca, patients with sarcoidosis and aqueous tear deficiency may have normal or elevated tear lysozyme levels.¹⁷

Other tear enzymes, such as lactoferrin, can be used to estimate tear production (Lactoplate, Eagle Vision, Memphis, TN). A tear-soaked filter paper disc is used in office-based tests in order to measure a standardized tear volume.¹⁸ Patients with active ocular sarcoidosis, even in the absence of systemic disease, are likely to have elevated tear angiotensin-converting enzyme (ACE) activity.¹⁹ Tear ACE levels provide a noninvasive test for diagnosing and monitoring this disease. Cytomegalovirus has been recovered on occasion from the tears of patients with active systemic infections.

CONJUNCTIVAL BIOPSY

A conjunctival biopsy containing discrete, noncaseating, subepithelial granulomas can be diagnostic for sarcoidosis. Disagreement exists as to whether there is value in obtaining a random, or “blind,” conjunctival biopsy to rule out sarcoidosis. Some reports demonstrated that 28% of random specimens were positive in selected patients who were suspicious for sarcoidosis without obvious conjunctival lesions.^20,21 Conjunctival biopsy is more likely to be diagnostic when granulomas are present in the inferior fornix, a large volume of tissue is removed, and serial sections are carefully examined. Random conjunctival biopsies yielded noncaseating granulomas in 55% of patients who already had or were later documented to have systemic sarcoidosis by positive biopsy from another site.²² In the usual clinical setting, however, it is superfluous to obtain a conjunctival biopsy from a patient who is already known to have sarcoidosis.

Conjunctival biopsy is much safer than bronchoscopy or even lacrimal gland biopsy, and it is reasonably specific when interpreted along with other clinical and laboratory data.²³ Biopsy of cutaneous lesions or minor salivary glands²⁴ is also safe and may be useful in suspected sarcoidosis.²⁵

AQUEOUS PARACENTESIS

Occasionally, anterior chamber fluid can provide information essential in establishing the etiology of a patient's uveitis. Paracentesis may prove useful if specific antibodies to infectious agents, such as Herpes simplex, Toxoplasma, Toxocara, Leptospira, Treponema pallidum, or Mycobacterium tuberculosis are found in greater concentration in the aqueous humor than in the serum. Angiotensin-converting enzyme levels in the aqueous may be elevated in sarcoidosis.²⁶ The value of aqueous ACE determinations may be limited by poor specificity due to the presence of high intrinsic ACE levels in normal ocular tissues, particularly retinal vessels.²⁷ Aqueous eosinophilia is seen in ocular toxocariasis²⁸ or in cysticercosis²⁹ associated with hypopyon. In retinoblastoma, the aqueous to plasma lactate dehydrogenase (LDH) ratio is often elevated to 50:1 or more, whereas the ratio is less than 1:1 in pseudoretinoblastoma patients.³⁰ Aqueous cytology may also be diagnostic for retinoblastoma, particularly when associated with pseudohypopyon, but the danger of extraocular seeding must always be considered when tapping eyes with suspected tumors.

In phacoanaphylactic endophthalmitis, anterior chamber exudates consist of polymorphonuclear granulocytes and monocytes.³¹ This exudate can provoke secondary open-angle glaucoma.³² Phacoanaphylactic endophthalmitis may occur after accidental trauma, surgical lens discission, extracapsular cataract extraction, or spontaneous lens rupture in hypermature cataract. Phacolytic open-angle glaucoma occurs when macrophages and amorphous lens material obstruct the trabecular meshwork.³³ Phacolytic glaucoma may follow traumatic or inapparent lens injury. A milder form of lens-induced uveitis probably occurs in all cases of lens trauma and may be granulomatous or nongranulomatous. This macrophage response, also called phacotoxic or phacogenic uveitis, corresponds to a foreign body reaction. Aqueous aspirates may contain macrophages and epithelioid cells laden with cytoplasmic periodic acid-Schiff (PAS) positive granules, as well as plasma cells and lymphocytes. The spectrum of aqueous cytology may assist the ophthalmic pathologist in providing a diagnosis of suspected lens-induced uveitis.

Cytologic specimens taken from body fluids are best fixed by rapid air drying, or with absolute methanol or 80% ethyl alcohol. Staining with Wright's, Giemsa (Eastman Kodak, Rochester, NY), or Diff-Quik (American Scientific Products, McGaw Park, IL) will allow maximal resolution of cellular characteristics. In viral infections and chronic hypersensitivity reactions, mononuclear cells predominate. In acute reactions, such as HLA-B27 related and Behçet's syndrome uveitis, polymorphonuclear leukocytes abound. A Gram stain should be performed when infection is suspected. A Papanicolaou stain may best identify herpetic intranuclear and intracytoplasmic inclusions.³⁴ When a paracentesis is performed to rule out intraocular spread of a known neoplasm Such as lymphoma or leukemia, the specimen should be compared with a diagnostic bone marrow or lymph node biopsy. This allows more certain matching of the neoplastic cell types.

In Fuchs' heterochromic iridocyclitis, progressive iris atrophy is often accompanied by abnormal vessels in the angle. An anterior chamber tap may therefore produce a filiform hemorrhage, known as Amsler's sign. This test is not clinically productive and is not recommended, especially since the diagnosis is usually readily apparent from the characteristic constellation of physical findings. In addition, simple gonioscopy will adequately demonstrate these fine chamber angle vessels.

Aqueous paracentesis is a safe office procedure when Close attention is paid to patient control and education, meticulous aseptic technique, and respect for the iris and lens structures. Topical 4% lidocaine (Xylocaine), adequate assistance, a wire speculum, povidone-iodine (Betadine) irrigation, ocular control with a fine-toothed forceps at the temporal limbus, and a No. 30 gauge needle fitted to a 1-ml plastic tuberculin syringe will minimize potential hazards. The tap can be done at the slit lamp Or with surgical loupes with the patient in the supine position. Afterward, the eye is patched for several hours, and topical antibiotics prescribed four times a day over the following 4 days. Patients are always rechecked 24 to 48 hours postoperatively.

Vitreous aspiration in the outpatient setting can also be safely performed in selected circumstances. Aspiration through the pars plana with a No. 18 gauge blunt-tipped needle following retrobulbar anesthesia can be done with the patient in a semireclining position or at the slit lamp. When therapeutic antibiotic or corticosteroid injections are anticipated following the vitreous tap, a three-way Luer-Lok stopcock avoids the problem of needle replacement. Care should be taken to constantly monitor the depth of needle penetration in order to avoid retinal or optic nerve injury. Indications are the same as for diagnostic surgical vitrectomy.

FLUORESCEIN ANGIOGRAPHY

Angiography is useful in delineating macular edema, vasculitis, disc edema, inflammatory foci, or old scars found in uveitis. One must differentiate leaking fluorescence, indicative of inflammatory activity, from that which radiates through thinned pigment epithelium (transmitted fluorescence), which fades fairly rapidly over 10 to 30 minutes and does not indicate inflammatory activity. Transmitted fluorescence may be seen at the earliest stages of uveitis³⁵ or represent old, inactive, destruction lesions. Fluorescence that persists may arise from stained sclera or from a pool of fluorescein in the choroid or under the sensory retina.³⁶ Areas of chorioretinitis due to toxoplasmosis and toxocariasis are often severe and demonstrate prominent fluorescence, which may increase with time (staining). In the early stages, however, the fluorescence from the choroid may be obstructed by the opaqueness of the retinitis (blocked fluorescence). The nonfenestrated retinal vessels resemble blood vessels in the central nervous system in that their vascular endothelium is impermeable to fluorescein dye.³⁷ In contrast, the choroidal vessels, like blood vessels outside the central nervous system, are permeable to the dye.

Visualization of the ciliary processes is possible with a special three-mirror contact lens with a ridge to depress the ciliary body region. Following intravenous injection, fluorescein can be seen to enter the posterior chamber through the prebasal cortex of the vitreous body. Thus, inflammation of the cornea ciliaris is relatively easily recognized.³⁸ Even without this lens, leakage from the corona ciliaris can be detected if the anterior part of the vitreous becomes greenish after 10 to 60 minutes. Direct observation obviates the problem of differentiating true from pseudofluorescence on black-and-white photographic film.³⁹

Intravenous fluorescein may also aid in anterior segment examination. In iritis, the anterior chamber becomes dark green. In cyclitis, the retrolental space may fluoresce. The fundus camera may also demonstrate accentuated fluorescence of iris vessels, especially in blue irides⁴⁰ during inflammatory episodes. Iris angiography may also detect early vascular damage in Fuch's heterochromic iridocyclitis,⁴¹ recent maturity-onset diabetes, or hyperlipoproteinemia.⁴² Fluorescein injections may also be used to delineate foci of episcleritis and scleritis and to define active areas of clinically suspicious necrotizing scleritis.⁴³

The standard intravenous dose is 5 ml of 10% fluorescein. Within 3 to 5 minutes, the fluorescein is equally distributed throughout the blood. It is eliminated, largely by way of the kidney, in about I hour. Toxicity is rare, but nausea, the most common side-effect, occurs within 15 to 30 seconds after injection and subsides within 1 minute. Nausea and vomiting can usually be prevented by an intramuscular injection of 25 mg promethazine hydrochloride (Phenergan). Pyrexia has been observed after administration of at least one batch of fluorescein as a result of the incorporation of pyrogens. The estimated incidence of reactions to fluorescein is 0.6%, of which most are allergic, and only one fourth are severe. Reactors may develop itching and hives, wheezing with laryngeal edema, or anaphylactic shock. One reported patient died of a myocardial infarction several hours following a severe reaction.⁴⁴ Oxygen and an emergency cart should always be available whenever fluorescein is administered.⁴⁵ Patients who are known to develop urticaria following fluorescein can be pretreated with antihistamines if angiography is judged essential and anesthesia personnel are readily available. If allergic hypersensitivity is suspected, an intradermal skin test with 0.1 ml of fluorescein is suggested. Extravasated fluorescein causes immediate severe pain that lasts only a few minutes and is somewhat relieved by application of an ice pack. Rarely, skin sloughing⁴⁶ or eczematous dermatitis⁴⁷ may result.

VISUAL FIELDS

Visual fields are especially useful in differentiating heredodegenerative disease from uveitis. For example, primary retinitis pigmentosa has a different field than the bone spicule fundus pigmentation seen in luetic uveitis. Because many uveitic syndromes are associated with neurologic disease, a visual field may also be helpful in distinguishing neurologic papillitis from juxtapapillary retinitis. Since many uveitic entities are associated with secondary glaucoma, attention should be given to the possibility of progressive visual field loss.

ULTRASONOGRAPHY

Ultrasonography is noninvasive and most useful when the media are opaque. This test can characterize inflammatory foci, such as choroidal thickening, uveal tumors, orbital masses or infiltrates, intumescent or dislocated lenses, vitreous opacities, cyclitic membranes, choroidal or retinal detachments, some foreign bodies, and posterior scleritis.

Sound waves with frequencies greater than 18 KHz produce echoes as they strike interfaces between acoustically different structures. Reflected sound waves are converted into an electric potential by a piezoelectric crystal and displayed on a cathode ray tube. The A-scan mode provides a one-dimensional image of acoustic density and is useful as an aid to interpretation of the two-dimensional B-scan. The immersion method of ultrasonography allows greater resolution of anterior segment structures, more flexibility in the selection of transducer frequencies, and consecutive serial sections of the globe. Contact ultrasonography is more convenient and portable, requires less expensive equipment, and allows the examination of infants without anesthesia or direct compression of the globe.

ELECTRORETINOGRAPHY AND ELECTRO-OCULOGRAPHY

Electroretinography (ERG) measures the action potential evoked by light stimulation of the retina. Because this is a mass response the reading may be normal when retinal lesions are focal. The test is useful in detecting disease with extensive retinal pathology, such as central retinal artery or vein occlusion, retinal detachment, hypovitaminosis A, siderosis, diabetic retinopathy, anemia, chloroquine toxicity, hyperthyroidism, retinitis pigmentosa, acute diffuse chorioretinitis, and retinal destruction seen with Behçet's syndrome, cytomegalovirus retinitis, or acute retinal necrosis. Electroretinography is especially useful with opaque media and may signal retinal involvement before the clinician can make a diagnosis.⁴⁸ Most studies indicate that there is no specific electroretinographic depression of diagnostic value in uveitis, although there is evidence that electroretinographic changes do correlate well with clinical and morphologic alterations. Corticosteroid administration may actually enhance electroretinographic responses.⁴⁹

Electro-oculography (EOG) measures the standing potential between the positive cornea and the negative retina. A decrease in the ratio between the response in the light- and dark-adapted retina indicates a disorder of the retinal pigment epithelium. These electrical changes are seen in chloroquine retinopathy, diabetic retinopathy, siderosis retinae, flecked retina, vitiliginous maculopathy, and shallow retinal pigment epithelial detachments.

ANTIBODY TESTING METHODS

Host production of specific antibodies to infectious agents allows the laboratory to incriminate an ever-expanding repertoire of organisms in clinical and uveitic disease. Normal human infants are born with very low levels of serum immunoglobulins that they have themselves synthesized; virtually all of an infant's IgG has been transferred transplacentally from maternal pools.⁵⁰ After birth, the overall level of IgG decreases until significant autologous IgG synthesis begins, usually at about 2 months of age. Total IgG gradually increases, approaching normal adult levels during late adolescence. On the other hand, a newborn must produce its own IgM, which is too large a molecule to cross the placenta. Thus, detectable IgM titers in any age group are indicative of in utero or postnatal exposure. The time interval between an early, or acute, and a late, or convalescent, serum specimen must be sufficient to allow for the development of a significant titer change: a fourfold serial dilution increase is generally adequate to establish re-exposure in an adult. The time interval may also be dependent on the etiologic agent being tested, the type of antibody test employed, the antibody isotype being assayed, or modulation by antimicrobial therapy.⁵¹ Previous skin testing may also stimulate antibody production, for example, with Histoplasma capsulatum or Brucella species. Accordingly, serum collected later than 3 to 4 days after skin testing may be invalid for that particular organism. A wide variety of ligand-binding assays are available to the modern clinical laboratory. A brief description of a few of the more common techniques should be instructive.

Radioimmunoassay (RIA) was the first ligand assay developed. RIA uses a specific binding molecule, usually a protein antigen or an antibody, to react with the molecule of interest, called the analyte. Calibrated amounts of binder and radiolabeled analyte are mixed with a series of sequentially diluted specimens, such as patient serum. The central event in RIA is the competition between label and the unlabeled analyte for binding sites on an antibody. On equilibration, the bound and free fractions are partitioned and radioactivity counted. Final analyte concentration is derived from a standardized calibration curve. Numerous variations of this test are now in use, including solid phase or sandwich assays in which binder molecules are coated to polystyrene beads or 96-well microtiter plates.

The enzyme-linked immunosorbent assay (ELISA) employs a solid phase antigen component to detect antibodies. The antibody being measured binds to this component, and a second enzyme-labeled antibody directed against the antibody to be measured is added during a second incubation. Thereafter, substrate specific for the enzyme tagged to the secondary antibody is added for a third incubation, which results in a colorimetric alteration of the enzyme molecule. This last incubation is halted with an enzyme-specific inhibitor, and optical density readings are taken with a spectrophotometer at the optimal wavelength for the particular enzyme employed.

Immunofluorescent assay (IFA) systems employ fluorochrome-labeled antigens and antibodies incubated with specimens in much the same way enzyme-labeled antibodies are used in the ELISA. The amount of light emitted at a given wavelength by a fluorescent specimen can be precisely measured by a microfluorometer. Antinuclear and anti-DNA antibodies as well as serum antibodies and complement can be quantified by IFA. Both ELISA and IFA techniques can be enhanced for greater sensitivity by employing a biotinylated secondary antibody and an additional incubation with enzyme- or fluorochrome-labeled avidin. Monoclonal antibodies have greatly improved the specificity of immunoassay techniques.

Complement fixation (CF) is a widely used two-stage test. In the first stage, antigen and antibody are reacted in the presence of precisely measured complement. Antigen-bound antibody, if present, will fix the free complement to Fc fragment receptors. In the second stage, residual unbound complement is measured by lysis of cells susceptible to complement lysis called erythrocyte amboceptors.

Agglutination techniques use stable cell lines or antigen-coated particle suspensions that can be directly or indirectly clumped into a lattice, or agglutinated, by serum antibody. These tests are in widespread use and can be simply assessed with the unaided eye. In general, complement fixation and agglutination techniques are sensitive to 1 μg/dl, ELISA to less than 1 μg/dl, and quantitative IFA and RIA to less than 1 pg/dl.⁵² The older CF and agglutination tests for many antibodies are being replaced gradually in many laboratories by the more rapid and sensitive ELISA, RIA, and IFA methods.

Immunohistochemical techniques for detection and localization of antigens are useful following clinical specimen collection from epithelial surfaces, secreted fluids, or biopsy material. Direct labeling with immunofluorescent or enzyme-tagged antibodies, or indirect methods using biotin, avidin/streptavidin amplification systems, or the three-step peroxidase-antiperoxidase (PAP) method are widely used in surgical pathology.⁵³

Flocculation refers to the precipitation of fleecy or fluffy masses from a solution, usually serum. Heat-inactivated serum (56°C for 30 minutes) is mixed with antigen diluted serially in buffer on a rotating glass slide and read microscopically at 100 x. Rigid volumetric standardization is required to preserve interlaboratory comparisons and may be a source of interpretation difficulties when patients have sequential tests performed at different centers. A tube flocculation test that can be read macroscopically is also available for the VDRL test.⁵⁴

LUETIC SEROLOGY

Laboratory testing for syphilis is indicated in a broad variety of ocular diseases. Scrapings of mucocutaneous lesions to confirm a diagnosis of primary or secondary syphilis can be examined directly by phase-contrast or darkfield microscopy and confirmed by direct immunofluorescence, or DFATP, testing. In the absence of obvious superficial lesions, serology is required to make a diagnosis. These tests fall into two categories: nontreponemal and treponemal. Nontreponemal, or nonspecific, antibody tests measure IgM and IgG directed against a phospholipid antigen called cardiolipin, or diphosphatidyl-glycerol, produced co-incidentally during luetic infections. These are also known as Wasserman, or reaginic, antibodies. The simplest and most practical of these tests is the VDRL test, employing a slide microflocculation technique, and the RPR (rapid plasma reagin) circle card test. Positive tests are reported as the highest serum dilution producing a reaction. These nontreponemal tests are widely used for screening, as well as for quantitative assessment of treatment response. The quantitation VDRL titer is both helpful in diagnosis and in following the course of treatment. If positive, titers are low in primary lues, usually below 1:32, while in secondary lues it is virtually always greater than 1:32. A high or rising titer is essentially diagnostic of syphilis, despite the long list of diseases producing false-positive results (Table 4).⁵⁵False-positive test results occur in 10% to 30% of the population, depending on a given laboratory's location.

TABLE 4. Disorders Producing Ocular Disease as Well as Biologic False-Positive Serologic Reactions for Syphilis

  Nontreponemal Tests

  Leprosy
  Tuberculosis
  Leptospirosis
  Relapsing fever
  Yaws
  Lymphogranuloma venereum
  Vaccinia
  Measles
  Varicella
  Infectious mononucleosis
  Narcotic addiction
  Systemic lupus erythematosus
  Rheumatoid arthritis
  Sjögren's syndrome
  Polyarteritis nodosa
  Systemic sclerosis
  Hyperglobulinemia
  Pregnancy
  Bacterial endocarditis
  Atypical mycoplasmal pneumonia
  Scarlet fever
  10% of persons over age 80

  Leprosy
  Relapsing fever
  Yaws
  Narcotic addiction
  Systemic lupus erythernatosus
  Rheumatoid arthritis
  Sjögren's syndrome
  Crohn's disease

The VDRL and RPR tests are therefore very sensitive but somewhat nonspecific. Following successful treatment of early syphilis, the titer declines approximately fourfold at 3 months, and eightfold at 6 months. Titers should be negative within 1 year of treatment of seropositive primary syphilis, within 2 years for secondary syphilis, or within 5 years for late latent syphilis. If titers do not return t₀ negative, persistent active infection or reinfection should be suspected, especially if the titer is greater than 1:4. If secondary syphilis is clinically suspected despite a negative nontreponemal test, a prozone phenomenon due to excess antibody might be responsible and additional dilutions should be ordered. The VDRL rises more slowly than the fluorescent treponemal antibody (FTA) test and thus may miss an early primary infection. Therefore, an FTA test should be obtained whenever lues is clinically suspected, despite a negative VDRL test. The VDRL is the only test that can be reliably employed in evaluating cerebrospinal fluid.

In lues, as in all infections, it is best to use an antigen specific for the organism believed to be infecting the patient. These treponemal, or specific, tests measure antitreponemal IgG and remain positive throughout life; therefore they cannot be used to distinguish between active or prior disease. The Treponema pallidum immobilization test (TPI), introduced in 1949, was the first treponemal test developed. Because of technical difficulty and poor standardization, it is rarely used now, although it has served as a valuable benchmark for the development of newer tests. The fluorescent treponemal antibody absorption test (FTA-ABS) is an indirect immunofluorescent test found to be reactive in about 80% of patients with primary syphilis. The VDRL test is positive in only 50% of patients with primary lues, while both the FTA and VDRL tests are positive in all patients with secondary syphilis. The specificity of the FTA-ABS test is greatly enhanced by first absorbing the specimen with nonpathogenic treponemal strains. The FTA-ABS can be used to rule out a biologically false-positive nontreponemal test, although even this test is susceptible to false-positive reactivity, particularly in the presence of lupus erythematosus (see Table 4). Specific measurement of treponemal antibody can also be performed by microhemagglutination with the MHA-TP test, or hemagglutination with the HATTS. All of the specific treponemal tests are interpreted as reactive, borderline, or nonreactive. The automated MHA-TP is more rapid and less expensive than the FTA-ABS but may be less sensitive during primary and early secondary disease.

TOXOCARA SEROLOGY

Serologic tests in suspected ocular Toxocara infections are crucial in eliminating a diagnosis of retinoblastoma. A sensitive ELISA test is available throughout the United States through state laboratories and the Centers for Disease Control (CDC) in Atlanta. The presence of any antibody, even in undiluted serum, may be significant⁵⁶ Titers to Toxocara canis, the common dog worm, as well as the common ascarids of cats, Toxocara mystax, Toxocara leonina, and Toxocara cati, can be ordered.

TOXOPLASMOSIS SEROLOGY

About 40% of the adult population worldwide has been exposed to toxoplasmosis, but this can be much higher in areas where hygiene is poor or raw meat is routinely ingested. Infection leads to IgA, IgG, and IgM antibody production, which despite high titers is not necessarily protective. Complement fixation, latex agglutination, indirect hemagglutination, direct and indirect IFA, immunodiffusion, ELISA tests, and the Sabin-Feldman dye test have all been employed in the quantification of anti-Toxoplasma titers.⁵⁷The dye test was well documented and reliable, but it lacked standardization, required a great deal of technician time, required live Toxoplasma organisms, and was only available in a few centers. The ELISA⁵⁸ and indirect IFA test are now the most clinically useful.⁵⁹ Titers may vary considerably among these tests, so laboratory methodology must be kept consistent when a suspected flare-up is evaluated by sequential serology. Nevertheless, any titer of specific anti-Toxoplasma antibody may be significant. State laboratories will often only report positive tests if a titer of greater than 1: 16 is found. There have been, however, cases of pathologically proven toxoplasmic retinitis in which serum antibody levels were positive only in undiluted specimens. The initial diagnosis of ocular toxoplasmosis is usually based on the characteristic focal, necrotizing retinal lesion; positive serum antibodies are used to confirm the clinical impression.

HERPETIC SEROLOGY

Human herpetic infections result from primary inoculation or reactivation of latent neural virions and include herpes simplex (HSV) types 1 and 2, varicella-zoster virus (VZV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV).

Acute retinal necrosis syndrome, or Kirisawa's uveitis,⁶⁰ entails anterior uveitis, vitreous inflammation, and progressive necrotizing vaso-occlusive retinitis with a poor prognosis for visual recovery.⁶¹ The disease may be unilateral or bilateral and generally occurs in otherwise healthy patients. Retinal necrosis syndromes have been attributed to HSV-1,⁶² HSV-2,⁶³ CMV,⁶⁴ and VZV⁶⁵ infections. Significant acute anti-herpes titer elevations, if present, can be useful in the diagnosis of active retinitis, as appropriate intravenous antiviral therapy should be started immediately.⁶⁶ If CMV infection is implicated by acute serology in a patient started on empiric acyclovir without clinical improvement, a switch to ganciclovir may be indicated; CMV is far less sensitive to acyclovir than the other herpesviruses.⁶⁷ Convalescent serum titers are often inconclusive and arrive from the laboratory long after the retinitis has taken its toll. Aqueous antibodies to HSV-1 in acute retinal necrosis syndrome may indicate local antibody production, antibody sequestration within the eye, or damage to the blood-ocular barrier.⁶⁸

Anti-HSV antibody clearly does not prevent reactivation of herpes labialis, and titers will often show no increase whatsoever with recurrent oral episodes. Seroconversion in paired sera may provide evidence of a primary HSV infection. In general, significant HSV titer rises occur only with primary infections.

In the serodiagnosis of VZV infection, acute and convalescent phase sera should be tested in parallel in the same run. The value of VZV serology is limited somewhat by the fact that heterotypic antibody liter rises to VZV may occur in certain patients with HSV infection who have experienced a prior infection with VZV. This is most likely a heterotypic antibody response to common antigens in the two viruses.⁶⁹ Thus a fourfold VZV titer rise is significant only in the absence of a concomitant HSV titer rise in the same specimens. Many clinicians will order paired sample testing for HSV-I, HSV-2, VZV, and CMV herpesvirus types in appropriate patients.

CMV recovery from urine, throat, or other body fluids is the preferred diagnostic method for congenital infection⁷⁰ and may be of use in ocular disease. Serodiagnosis in infants is complicated by the presence of transplacental maternal IgG. Although there are some technical difficulties with detection of CMV-specific IgM, this test is excellent for the rapid confirmation of an acute or congenital infection. Up to 20% of patients remain persistently IgM positive, indicating a latent CMV carrier state that regularly becomes re-established in host tissue. In suspected adult disease, a fourfold titer rise indicates a recent infection, due to either reinfection or reactivation. Since CMV titers can remain elevated for extended periods, elevated but unchanged acute and convalescent titers may indicate a recent infection in which the acute specimen was obtained relatively late in the patient's illness.

CMV antibodies are not helpful in the diagnosis of retinitis in patients with acquired immunodeficiency syndrome (AIDS), especially since the clinical picture is so typical.⁷¹ Furthermore, CMV antibodies are very common in patients at risk for AIDS and CMV serum titers may be negative in AIDS patients despite the presence of CMV retinopathy.⁷²

CMV-positive titers should be confirmed by culture whenever possible. In acute CMV infections, characterized by fever, thrombocytopenia, hepatosplenomegaly, pneumonitis, and lymphadenopathy, virus is recoverable from most patients' urine and from 50% of throat swabs. Isolation from buffy coat cells, spun urine, saliva, subretinal fluid, or biopsy specimens can be diagnostic. Isolation does not, however, necessarily establish CMV as the responsible organism; asymptomatic viremia has been described, and biopsy specimens often contain other pathogens, rendering the clinical interpretation tenuous. Direct specimen examination for CMV by exfoliative cytology, histopathology, immunofluorescence, electron microscopy, or DNA hybridization techniques can also assist in the diagnosis of CMV infection. Fresh spun urine should be passed through a membrane filter, and trapped cells should be stained with hematoxylin-eosin or Papanicolaou reagents. Characteristic large cells with prominent eosinophilic inclusions are seen in positive preparations. Similar cells are also rarely seen in HSV infections.

EBV isolation is usually not clinically useful due to the ubiquity of EBV found in healthy persons, technical difficulties in culturing the organism, and a long incubation period. Serologic studies are the method of choice. In primary infections with symptoms compatible with infectious mononucleosis, a positive heterophil antibody is diagnostic. This IgM antibody agglutinates sheep red blood cells but not guinea pig kidney. A slide spot test is now used for screening and confirmed by the Paul-Bunnell test. The heterophil antibody rises rapidly, remains high, and then falls rapidly, generally after 4 weeks of illness.

Humoral immunity to primary EBV infection appears so rapidly that 80% of patients usually have reached peak titers by the time they consult a physician.⁷³ Thus, paired specimens are of limited value. Nevertheless, effective laboratory diagnosis can be made on a single acute-phase serum by simultaneous testing for antibodies to several EBV-associated antigens. The profile is sufficiently distinct in most cases to determine whether the patient is still susceptible, has a current infection, has a recent primary infection within the past 2 to 3 months, had a past infection, or may be having reactivated or persistent EBV infection (Table 5). There are four principal antigens: viral capsid antigen (VCA), Epstein-Barr nuclear antigen (EBNA), and the two early antigen components, either diffuse (EA/D) or restricted (EA/R). In infectious mononucleosis, the VCA-IgG and VCA-IgM rise in tandem during the vital incubation period, usually 4 to 5 weeks from the time of exposure.⁷⁴ Early antigen titers rise with the onset of clinical illness, usually 5 to 10 weeks after exposure. In a typical case of mononucleosis, the early antigen liters rise to a peak, then fall to undetectable levels 6 to 12 months after clinical resolution. The anti-EBNA antibody begins to rise slowly after 2 months and remains positive throughout life, as does the VCA-IgG. VCA-IgM, on the other hand, falls to negligible levels following clinical resolution, usually 6 to 12 months after the onset of symptoms. Thus, the combination of anti-early antigen IgG and anti-VCA IgM antibodies indicates a recent or persistent infection.⁷⁵

TABLE 5. Serologic Profile in Epstein-Barr Virus Infection

EBV infection is associated with Burkitt's lymphoma and nasopharyngeal carcinoma, in which antibody titers are particularly high, as well as B-cell lymphoma, oral hairy leukoplakia, chronic malaise and lethargy syndrome,⁷⁶ Sjögrens syndrome,⁷⁷ lymphoproliferative syndromes in immunocompromised hosts, and various aplastic bone marrow conditions.⁷⁸ Ocular manifestations of EBV infection include nummular keratitis,⁷⁹ stromal keratitis,⁸⁰ follicular conjunctivitis,⁸¹ severe vitritis,⁸² punctate outer retinitis,⁸³ and multifocal choroiditis with panuveitis,⁸⁴ a syndrome mimicking ocular histoplasmosis⁸⁵ first described by Nozik and Dorsch.⁸⁶ EBV has been cultured from conjunctival and tear samples,⁸⁷ although etiology has not been proven in any ocular syndrome.⁸⁸ EBV serology may be helpful in characterizing patients with these ophthalmic problems and in eliminating other diagnostic possibilities.

HUMAN IMMUNODEFICIENCY VIRUS

Although the diagnosis of AIDS cannot be made in a person who is antibody-negative for human immunodeficiency virus (HIV), positive serology does not establish a diagnosis, as there are many healthy persons who are positive for the antibody. An ELISA test is available for routine documentation of HIV-specific antibodies, with informed consent required in nonmilitary settings.⁸⁹ The screening tests for HIV antibodies are calibrated to be extremely sensitive, so a significant number of false-positive results occur. All positive tests should be confirmed with a Western blot analysis, in which purified, electrophoretically separated HIV antigens incubated with patient serum produce a characteristic pattern.⁹⁰ Opportunistic infections such as Mycobacterium tuberculosis, Treponema pallidum, fungi, Toxoplasma gondii, and the herpesviruses define the syndrome and may cause uveitis. CMV retinitis is the most common form of ocular inflammation seen in AIDS and is a poor prognostic sign.⁹¹A number of immunologic abnormalities have been documented in AIDS, including lymphopenia (<600/mm³), a substantial reduction in the percentage of T cells (<30%), and a reversed helper-to-suppressor T-cell ratio (<0.5).⁹² T-cell subsets (Table 6) can be accurately quantified with immunofluorescent labeling, flow cytometry, and cell-sorting techniques.⁹³

TABLE 6. Reference Range for the Lymphocyte Panel

CHLAMYDIAL SEROLOGY

Chlamydial organisms have been isolated from the joints and the anterior chamber from one patient with Reiter's syndrome.⁹⁴ Although chlamydial urethritis⁹⁵ and conjunctivitis⁹⁶ have been implicated in the pathogenesis of Reiter's syndrome, the presence of antibodies is so common that an elevated titer is of minimal use. A significant convalescent IgG titer rise or high IgM titers might provide a clue to the cause of new-onset Reiter's syndrome in selected patients. The microimmunofluorescence test, an indirect antibody test using whole purified chlamydial elementary bodies, provides antibody titers with serotype specificities.⁹⁷ Serotype-specific monoclonal reagents are now available for serologic testing in a research setting.⁹⁸

FUNGAL DIAGNOSIS

Antibody titers are of little use in the diagnosis of ocular fungal diseases. In candidal endophthalmitis, vitreous biopsy, blood cultures, and clinical evaluation are the diagnostic methods of choice. Similarly, in cryptococcosis, aspergillosis, and mucormycosis, smears and cultures from the vitreous or the cerebrospinal fluid provide the diagnosis. Presumed ocular histoplasmosis syndrome is a clinical diagnosis made by examination, fluorescein angiography, and geographic history; the histoplasmin skin test is meaningless in hyperendemic areas and may even exacerbate the maculopathy. A skin test may also falsely elevate the antibody titer. Complement fixation antibody titers are not helpful⁹⁹ and may be negative in ocular histoplasmosis. Patients with ocular coccidioidomycosis are usually desperately ill because of generalized infection by the time they complain of ocular symptoms. Complement fixation serology and a skin test using Coccidiodes immitis antigen are available, but, once again, they are not very useful clinically. Biopsy of cutaneous lesions in a patient with choroiditis and apical pulmonary lesions has provided a diagnosis of latent disseminated blastomycosis.¹⁰⁰

BORRELIA TITERS IN LYME DISEASE

Lyme disease is characterized by fever, malaise, inflammatory arthropathy, hepatosplenomegaly, respiratory involvement, rash, meningeal signs, and an antecedent tick bite. It is also associated with hemorrhagic conjunctivitis, dendritiform keratitis, iritis, iridocyclitis, retinal vasculitis and hemorrhages, optic nerve inflammation, bilateral diffuse choroiditis, and exudative retinal detachments.¹⁰¹ Various combinations of systemic as well as ocular manifestations occur. Either ELISA or indirect IFA tests for IgG and IgM directed against the tick-borne causative agent, Borrelia burgdorferi, are available through state laboratories and the CDC in Atlanta. Many patients with the classic cutaneous manifestation of early Lyme disease, erythema chronicum migrans, have elevated IgM responses. Sometimes, however, several weeks of illness are required before the levels of either IgM or IgG antibody become elevated.¹⁰² In these cases, testing of both acute and convalescent sera may increase the chances of obtaining a positive result. Because the antibody response may be aborted altogether by early antibiotic therapy, early Lyme disease cannot be documented in all patients. After the first 5 or 6 weeks of illness, almost all patients have an elevated IgG response, and virtually all patients with arthritis have elevated titers. Titers can be performed on the serum as well as the cerebrospinal fluid. Small amounts of IgM rheumatoid factor are produced at certain times in many patients with Lyme disease.¹⁰³

Relapsing fever is due to Borellia recurrentis or Borellia novyi infection and may be accompanied by uveitis. These Borellia species are transmitted by other members of the Ornithodoros tick family or by the human body louse. All of the Borellia spirochetes can be identified in Giemsa- or Wright-stained blood smears drawn during a febrile episode.

LEPTOSPIRA ANTIBODIES

Leptospirosis is not thought to be a common cause of uveitis in the United States, although Witmer¹⁰⁴ believes that 2% of his patients with uveitis may be suffering from this disease. The leptospiral spirochete is a well-known cause of uveitis in horses. An agglutination test utilizing commercially available killed organisms is available through most state public health laboratories. A tentative diagnosis of ocular leptospirosis should not be made in the absence of serum antibodies to Leptospira icterohemorrhagiae, the major human pathogen. A titer of 1:400 or more is diagnostic. The organisms may be isolated from blood or urine in the early, febrile stages. Many mammals serve as reservoirs, where chronic renal infection leads to passage in the urine. Occasionally, small epidemics of Weil's disease are seen in sewer workers and others exposed to rat urine or contaminated water supplies. Signs and symptoms develop after an incubation period of 10 to 12 days and vary from a mild fever to severe illness, including jaundice, renal failure, and meningitis. Uveitis has not been a frequent component of the disease in the United States, but mild anterior iritis, membranous vitreous opacities, retinal and papillary hemorrhage, and chorioretinal exudate have been described.

BACTERIAL SEROLOGY

Serologic testing for antibody responses to gram-negative and gram-positive organisms is not generally believed to be useful in the diagnosis of uveitis syndromes. The rare diagnosis of neisserial endophthalmitis is made by identification of organisms by culture and Gram's stain. Although serum antituberculous antibodies may be useful in identifying pulmonary tuberculous infection,¹⁰⁵ tuberculous serology alone is not useful in isolated ocular tuberculosis. As focal extrapulmonary manifestations assume a greater proportion of tuberculous disease in the United States,¹⁰⁶ ophthalmologists will have to rely more on clinical judgment, diagnostic therapeutic trials with anti-tuberculosis chemotherapy, a precise history, and skin testing rather than serology and the traditional chest roentgenogram in making a diagnosis of tuberculous uveitis.

BRUCELLOSIS ANTIBODY

Brucellosis has been incriminated as a cause of recurrent iritis, nodular choroiditis, and, rarely, a severe endophthalmitic panuveitis.¹⁰⁷ A standard tube agglutination test with acute and convalescent titers should show a fourfold rise in order to establish a presumptive diagnosis of Brucella uveitis. Like leptospirosis, blood cultures may be positive in the early stages of the disease. Brucellosis is usually found in hoofed farm animals: goats, sheep, cattle, and swine. Pregnant animals are particularly susceptible and frequently abort. Human transmission occurs by direct contact with infected animals or consumption of unpasteurized milk. Acute symptoms include fever, chills, and weakness. Chronic infections are characterized by fever, malaise, depression, and abscesses in the bones, spleen, kidneys, or brain.

ANTINUCLEAR ANTIBODIES

Plasma cells, the activated B cells of patients with autoimmune diseases, produce antibodies directed against their own tissues. In some diseases, including lupus, Sjögrens syndrome, mixed connective tissue disease, scleroderma, polymyositis, and dermatomyositis, the antinuclear antibodies (ANAs) have a primary pathogenic role.

A variety of serologic tests are now available to characterize the specificities of these innumerable antibodies. The LE (lupus erythematosus) cell test was the first such laboratory examination for autoimmune disease and is still used as a screening tool in some centers. Membrane-free leukocyte nuclei are used as antigen and mixed with patient serum. The subsequent antigen-antibody reaction results in the appearance of free, round, purplish nuclei. Viable polymorphonuclear (PMN) leukocytes complete the reaction by phagocytosing the nuclear material and forming intracytoplasmic PMN inclusions, which are read on a microscope slide. This test was first described in 1945.¹⁰⁸

By 1961, the indirect immunofluorescent ANA test had become popular, especially since different patterns of staining corresponded to different diseases (Table 7). This test is an indirect slide-mounted IFA employing human leukocyte nuclei as antigen. The report includes the highest dilution at which fluorescence was detected, the upper limits of normal for the test in that particular laboratory, and the pattern of nuclear staining detected. The ANA test is positive in a number of disease states (Table 8). Unfortunately, the test is limited by poor standardization, changing patterns with changing titers, a high degree of variability even in clinically stable patients, considerable observer bias, a lack of specificity, and the fact that titers often bear little relationship to disease activity.

TABLE 7. Patterns of Nuclear Immunofluorescence and Their Disease Associations

TABLE 8. Prevalence of Positive Immunofluorescent Antinuclear Antibody Tests in Various Clinical Disease States

It has been reported that with repeated testing, up to 80% of patients with juvenile rheumatoid arthritis who have chronic nongranulomatous iridocyclitis will show significant ANA titers.¹⁰⁹ Children with rheumatoid arthritis and a negative ANA rarely develop uveitis. Early onset, pauciarticular juvenile rheumatoid arthritis is very likely to be ANA positive and HLA-DR5 positive.¹¹⁰ In these patients, there is a 53% chance of developing uveitis.¹¹¹ A subset of children with juvenile rheumatoid arthritis will be ANA negative. These are usually boys with a positive HLA-B27 who go on to develop classic HLA-B27 style recurrent, alternating, acute, unilateral iridocyclitis.¹¹²

Both immunodiffusion and counterimmunoelectrophoresis have been used to isolate a variety of saline extractable nuclear antigen (ENAs), many of which are listed in Table 9. These techniques employ an agarose gel with two wells; one contains antigen and the other serum. Precipitin line formation between the wells defines a positive reaction. Many laboratories will perform anti-RNP and Sm titers when a screening test for extractable nuclear antigens is positive. Specific anti-ENA titers may be specially ordered through the Scripps Foundation in La Jolla, California. Autoantibodies to extractable nuclear antigens are most likely to be identified when the immunofluorescent ANA test shows a speckled pattern; these include the Sin, RNP, SS-A, SS-B, PM-1, and RANA specificities. Sjögrens syndrome A antigen has been associated with retinal vasculitis.¹¹³ Anti-ENA titers will become increasingly useful as specific disease profiles are better characterized and automated panels become universally available.

TABLE 9. Disease Associations of Specific Antinuclear Antibodies

LUPUS ANTICOAGULANT

The lupus anticoagulant is an acquired serum immunoglobulin that prolongs several coagulation measurements. Lupus anticoagulants are antio phospholipid antibodies found in at least 10% of patients with systemic lupus erythematosus¹¹⁴ as well as in patients with infectious, drug-induced, malignant, and other autoimmune disorders, and otherwise healthy persons.¹¹⁵ The traditional indirect assay for lupus anticoagulant relies on the ability of some antiphospholipid antibodies to prolong in vitro clotting tests, such as the activated partial thromboplastin time and prothombin time.¹¹⁶ There are two other types of tests that detect antiphospholipid antibodies. The first includes the standard tests for reagin found in syphilis, such as the VDRL or Wasserman complement fixation tests. This reactivity explains the biologic false-positive tests for syphilis found in patients with lupus.¹¹⁷ The other test is the anticardiolipin antibody test, performed by RIA¹¹⁸ or ELISA using partial thromboplastin as the antigen.¹¹⁹ The anticardiolipin IgG antibody appears to be the most predictive and specific test for recurrent arterial or venous thrombosis, recurrent fetal loss,¹²⁰ and thrombocytopenia,¹²¹ the three clinical disorders with which the lupus anticoagulant is associated. Other features associated with lupus anticoagulant include livedo reticularis, migraine, chorea, and cardiac valve lesions.¹²² The IgM anticardiolipin antibodies are relatively nonspecific.

Despite the association with impaired coagulation, thrombotic systemic and ocular disease occurs in lupus anticoagulant positive patients. Symptoms of disturbed vision may bring these patients to medical attention.¹²³ Branch retinal artery occlusion, ischemic optic neuropathy, transient visual loss, transient diplopia, vertebrobasilar insufficiency, retinal vein occlusion, and homonymous visual field loss have been reported. Histologically, the classic retinopathy seen in lupus is not an inflammatory vasculitis but a focal intravascular thrombosis,¹²⁴ corroborating the phenomenon seen in the skin¹²⁵ and central nervous system. ¹²⁶ Patients with unexplained visual symptoms and occlusive findings should be evaluated for hypercoagulation with testing for anticardiolipin antibodies, partial thromboplastin time, and VDRL. Management of these patients, although far from settled, consists of oral anticoagulants supplemented with immunosuppressive drugs and/or corticosteroids to lower anticardiolipin antibody levels in patients who continue to have thrombosis despite adequate anticoagulant therapy.

RHEUMATOID FACTOR

Rheumatoid factors are 7S IgG, 7S IgM and 19S IgM autoantibodies directed against the patient's own IgG. The union of autoantibodies with normal IgG is followed by complement fixation at the tissue site and subsequent chemotaxis of leukocytes and platelets. The resultant occlusive vasculitis is believed to contribute to rheumatoid nodule formation, synovitis, sclerouveitis, and other manifestations of rheumatoid arthritis. Recent studies suggest a possible relationship between EBV and rheumatoid arthritis: the rheumatoid-associated nuclear antigen (RANA) is expressed only by a human lymphoblastoid cell line containing EBV.¹²⁷ Rheumatoid factor is found in significant titer in a number of disease states, most commonly rheumatoid arthritis (Table 10). Rheumatoid factor is negative in classic pauciarticular juvenile rheumatoid arthritis and the seronegative spondyloarthropathies, including Reiter's syndrome, psoriatic arthritis, arthritis associated with ulcerative colitis and regional enteritis, and ankylosing spondylitis.

TABLE 10. Rheumatoid Factor Positive Latex Fixation Tests in Various Clinical Disease States

FECES FOR PARASITES

Intestinal parasites are a rare cause of uveitis in the United States: for example, only one case of Ascaris ocular invasion has been proven. Entamoeba histolytica, Escherichia coli, E. nana, and Giardia lamblia have been described as producing a cystic macular lesion. Although Toxocara canis and T. cati are recognized causes of uveitis, humans are not natural hosts. Ingested ova from contaminated soil or uncooked vegetables such as lettuce advance to the larval stage in the human intestinal epithelium, penetrate the portal vasculature and lymphatics, and spread to the liver, where eosinophilic granulomas may be produced. Some larvae survive, infect the lungs, enter the heart, and thereafter disseminate throughout the body. The larvae never mature in the human host; thus neither ova nor mature Toxocara parasites are found in human feces.

BEHÇET'S IMMUNOFLUORESCENCE TEST

In patients with Behçet's syndrome, antibodies to cytoplasmic antigen have been identified in the stratified squamous epithelium of human and guinea pig lip mucosa.¹²⁸ An IFA test using lip epithelium antigen and secondary fluorescein conjugated goat antihuman immunoglobulin antibody is positive in over 75% of patients with Behçet's syndrome, with a false-positive rate of 6.5%.¹²⁹ This test may prove useful in selecting out those patients with Behçet's syndrome from those with other ocular vasculitides.

ERYTHROCYTE SEDIMENTATION RATE

The erythrocyte sedimentation rate (ESR) is a measurement of the degree to which erythrocytes settle in 1 hour in a calibrated standard vertical tube. It is a nonspecific but useful test, indicating to the ophthalmologist that there is some systemic inflammatory activity in a patient. Erythrocytes normally settle slowly, but erythrocyte aggregates settle rapidly because the proportional increase in their total mass exceeds the proportional increase in their volume. Erythrocyte aggregation is caused by electrostatic forces, which are increased during inflammatory episodes by acute phase reactants in serum. Erythrocyte disorders may confuse interpretation of the test. In anemia, for example, the ESR increases, probably because frictional forces between sedimenting aggregates are reduced. In sickle cell disease, abnormal erythrocyte shape retards the ESR. Numerous other conditions can alter the ESR (Table 11).¹³⁰

TABLE 11. Factors Influencing the Erythrocyte Sedimentation Rate

The Westergren method is most commonly employed in clinical laboratories. It requires a pipette, 0.015 M sodium citrate solution as an anticoagulant/diluent, and 5 ml of whole blood. Anticoagulated venous blood is diluted 4:1 with sodium citrate and placed in a 200-mm glass tube with a 2.5-ram internal diameter. After exactly 1 hour, the distance from the meniscus to the top of the column of erythrocytes is recorded as the ESR. The modified Westergren method uses edetic acid (EDTA) as an anticoagulant and is especially convenient because the same tube of blood can be used for the ESR and other hematologic studies.

The ESR is higher in women than in men and gradually increases with age (Table 12);¹³¹ it may be as high as 40 mm/hr in some persons over age 65 without obvious cause. There is a useful empirical formula for normal ESR values:¹³² for men, age in years is divided by 2; and for women, age in years plus 10 is divided by 2. The upper limit of normal is arbitrary, and some laboratory directors choose a value well above 2 standard deviations above the mean in order to minimize false-positive results. Wintrobe's technique is also employed, but it is less accurate than the Westergren method. The zeta sedimentation rate (ZSR) uses a centrifugal device (Zetafuge, Coulter Diagnostics, Hi-aleah, FL) that spins capillary tubes in a vertical position for four 45-second cycles, resulting in a controlled compaction and dispersion of erythrocytes. The ZSR is rapid, requires only a 100-μl sample, and, unlike the ESR, is not affected by anemia. ¹³³

TABLE 12. Normal Westergren Erythrocyte Sedimentation Rates

C-REACTIVE PROTEIN

Acute phase reactants are proteins that become elevated in response to stressful or inflammatory states such as infection, injury, surgery, trauma, or tissue necrosis. They include globulins, α₁-antitrypsin, α₁-acid glycoprotein, haptoglobin, ceruloplasmin, fibrinogen, and C-reactive protein (CRP). The CRP is a highly sensitive acute phase indicator and can be measured by ELISA or other immunologic methods. CRP has a molecular weight of 144 kd and reacts with numerous other substances, including DNA, nucleotides, various lipids, and polysaccharides, including the pneumococcal C-polysaccharide. There is still no consensus among physicians for the widespread use of CRP,¹³⁴ because other clinical parameters may be just as sensitive, including fever, leukocytosis and the ESR. CRP has been used as a marker for disease activity in the seronegative spondyloarthropathies.¹³⁵

ANGIOTENSIN-CONVERTING ENZYME

Angiotensin-converting enzyme (ACE) is produced by a variety of cells, including capillary endothelial cells, lung tissue, and activated monocytes, macrophages, and epithelioid cells found in noncaseating granulomas. ACE is a dipeptidyl carboxypeptidase, catalyzing the conversion of the decapeptide angiotensin 1 to the pressor angiotensin 2, the cleavage of bradykinin to an inactive metabolite, and a number of other reactions. The serum ACE concentration is probably a reflection of the total amount of granulomatous tissue in the body. Serum ACE levels are elevated in about 75% of patients with active, untreated systemic sarcoidosis and about 40% of chronic, untreated patients.¹³⁶ False-positive results have occurred in many other syndromes (Table 13).¹³⁷

TABLE 13. Condition With Elevated Serum Angiotensin-Converting Enzyme

  Acute untreated systemic sarcoidosis (75%

  Chronic untreated systemic sarcoidosis (40%)
  Gaucher's disease
  Diabetic retinopathy
  Amyloidosis
  Leprosy

  Tuberculosis
  Mycotic infections
  Histoplasmosis
  Berylliosis
  Silicosis
  Asbestosis
  Neonatal respiratory distress syndrome

  Primary biliary cirrhosis
  Chronic persistent hepatitis
  Chronic aggressive hepatitis
  Fatty liver
  Obstructive jaundice

  Lymphomatoses
  Immunoblastic sarcoma
  Carcinomatosis
  Histiocytic medullary reticulosis

Because ACE levels may be reduced in patients taking systemic corticosteroids, their use in assessing systemic disease activity in sarcoidosis is limited. The test is commonly negative in patients with proven sarcoidosis that is in remission,¹³⁸ but results will again rise during disease relapse. Thus, serum ACE is a valuable laboratory test only when interpreted within the complete clinical context. When combined with the gallium scan or a chest roentgenogram, serum ACE can be a very sensitive test for noninvasive diagnosis of presumed sarcoidosis¹³⁹ and is useful in the assessment of disease activity level in patients with a previously established diagnosis of sarcoidosis.¹⁴⁰ An elevated serum ACE in some patients with granulomatous uveitis even in the absence of diagnostic clinicoradiographic or histologic findings of sarcoidosis is strongly presumptive of sarcoid uveitis.¹⁴¹ Normal serum values by the standard spectrophotometric method are 12 to 35 nmol/min/ml in men and 11 to 29 nmol/min/ml in women. Normal levels are significantly higher in children and intermediately elevated in adolescents.¹⁴² Patients with active isolated ocular sarcoidosis and no systemic activity often have normal serum ACE levels.

LYSOZYME

Lysozyme, or muramidase, is a basic, cationic low-molecular weight enzyme present in tears, saliva, and nasal secretions. It reduces the local concentration of susceptible bacteria by attacking the mucopeptides in bacterial cell walls. Lysozyme originates from phagocytic cells and is normally present in serum at a concentration of 1 to 2 μg/ml. It is actively secreted by monocytes and macrophages. Lysozyme is also secreted by the sarcoid granuloma.¹⁴³ In adults, serum lysozyme levels generally parallel elevated serum ACE levels,¹⁴⁴ although lysozyme may be elevated with a normal ACE in occasional patients with sarcoidosis. The lysozyme test is nonspecific and may be elevated in other diseases, including tuberculosis, leprosy, osteoarthritis, and pernicious anemia. In tuberculosis, lysozyme activity is elevated in over 50% of patients, while the ACE is elevated in only 10%.¹⁴⁵ Serum lysozyme is depressed by systemic corticosteroid administration but, unlike serum ACE, is not normally higher in children when compared with adults. The serum lysozyme value may be elevated, however, in patients older than 60,¹⁴⁶ SO age must be taken into considerationwhen interpreting both ACE and lysozyme levels. Both ACE and lysozyme can be sensitive noninvasive tools in approaching a patient with granulomatous uveitis when the entire clinical presentation is carefully considered.¹⁴⁷

Lysozyme levels are elevated in tuberculous pleural effusion specimens¹⁴⁸ and may prove to be a useful marker in aqueous paracentesis specimens taken from patients with presumed tuberculous uveitis. Most laboratories quantify lysozyme levels spectrophotometrically, but turbidometric, viscometric, immunologic, and bioassay methods are also available.¹⁴⁹

MAJOR HISTOCOMPATIBILITY ANTIGENS

The human major histocompatibility complex, containing DNA encoding human leukocyte antigen (HLA) genes, is located on the short arm of chromosome 6. The HLA sites localize to heterodimeric (two different chains) cell surface molecules belonging to the immunoglobulin superfamily.¹⁵⁰ Class I HLA molecules include the A, B, and C loci and are found on the surface of virtually every cell. Class II HLA molecules include the D, DR, DQ, and DP loci and are restricted to lymphocytes, macrophages, and other immunocompetent cells. Humans possess two haplotypes, usually inherited as a unit from each parent. Each haplotype has three class I and four class II alleles. Because HLA expression is codominant, it is theoretically possible to type two antigens at each of these seven loci unless the subject is homozygous for one or more loci or antisera are not yet available for a rare HLA type. There are currently 124 recognized HLA specificities.¹⁵¹

The class I molecules provide a recognition target for lymphocytes responsible for cell-mediated immunity. Thus, class I antigens are recognized during graft rejection and they restrict the cytotoxic response against virus-infected cells to lymphocytes expressing the same class I antigens as the infected cells. Class II molecules are essential to antigen presentation and normal interactions between immunocompetent cells.

Genetically predetermined susceptibility to 530 distinct diseases has been linked to certain specific HLA markers in over 4,000 separate clinical studies.¹⁵² A summary of these disease associations pertinent to uveitis is found in Table 14. None of the HLA markers is, of course, specific for a given disease. The presence of an HLA antigen m a given patient, however, is suggestive of its associated disease and may provide the clinician with an additional clue in establishing a definitive diagnosis. The relative risk of 69.1 for HLA-B27 and ankylosing spondylitis, which was the first such association ever described,^153,154 indicates that an HLA-B27-positive white person is about 69 times more likely to develop ankylosing spondylitis than someone not carrying this antigen. The HLA-B27 antigen itself characterizes a unique clinical picture in uveitis,¹⁵⁵ making it an important diagnostic tool, unlike the situation in rheumatologic practice in which HLA-B27 typing is less valuable.¹⁵⁶ HLA-B27-positive patients with acute anterior uveitis are more likely to be younger at the age of onset, to be male, to show frequent unilateral alternating eye involvement, to have severe symptoms with each episode (including fibrinoid anterior chamber reactions), to have a higher incidence of ocular complications, to lack mutton-fat keratic precipitates, and to have an associated seronegative spondyloarthropathy. There is also an exceptionally strong association

TABLE 14. HLA Disease Associations

The HLA type may also help define prognosis. In presumed ocular histoplasmosis syndrome, HLA-B7 is strongly associated with disciform macular lesions,¹⁵⁸ but not with peripheral atrophic scars.¹⁵⁹ HLA-DR2, however, is associated with both macular and peripheral scarring,¹⁶⁰ suggesting a distinct genetic predisposition to macular neovascularization in this disease.

Patients with pauciarticular juvenile rheumatoid arthritis are particularly prone to uveitis. HLA-DR5, which is associated with the early-onset form of this disease most frequently seen in girls, characterizes patients more likely to develop a chronic, bilateral disease associated with band keratopathy and the presence of antinuclear antibodies. HLA-B27, on the other hand, is associated with later-onset pauciarticular juvenile rheumatoid arthritis and concomitant uveitis similar to the classic HLA-B27 pattern.¹⁶¹ Uveitis occurs in 53% of patients with HLA-DR5 related early-onset juvenile rheumatoid arthritis found mostly in females¹⁶² and in 25% of patients with the HLA-B27-related, later-onset form¹⁶³ usually found in males.

In general, HLA tests are not ordered on a routine basis and complete histocompatibility typing is never ordered as part of an evaluation for uveitis. Specific HLA types can, however, provide more information to the astute clinician who requires additional data to confirm a suspected diagnosis.

HEMATOLOGIC TESTING

The standard complete blood cell count (CBC) may vary between laboratories but generally consists of a white blood cell count, a differential white blood cell count, a platelet count, and a red blood cell count with hematocrit. Included with the red blood cell count are the following indices: mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), and mean corpuscular hemoglobin content (MCHC). Complete blood cell counts can now be performed by automated multichannel flow cytometers, employing the principles of voltage pulse counting and size analysis together with photosensitive detectors to measure hemoglobin and light scatter. Newer devices use laser light sources to improve accuracy and versatility (Coulter Diagnostics, Hialeah, FL). Normal values are given in Table 15.

TABLE 15. Normal Complete Blood Cell Count

The complete blood cell count is usually obtained as part of the general evaluation of the status of a patient's health rather than as part of a specific diagnostic evaluation. The total eosinophil count is often of value when an allergic or parasitic disease is suspected. A significant eosinophilia is defined by a total count of greater than 500 cells/mm³, or a differential count greater than 5%. Eosinophilia may also be present in a number of other conditions, listed in Table 16.

TABLE 16. Causes of Eosinophilia

  Allergic disorders:

  Hay fever
  Asthma
  Angioneurotic edema
  Serum sickness

  Trichinella
  Toxocara
  Visceral larva migrans
  Ascaris
  Shistosoma
  Toxoplasma
  Malaria
  Amebiasis

  Hodgkin's disease
  Lymphomas
  Brain tumors
  Mycosis fungoides
  Myelogenous leukemia
  Pulmonary and bone carcinoma
  Polycythemia vera

  Psoriasis
  Pemphigus
  Scabies

  Addison's disease and hypoadrenalism

  Colitis
  Protein-losing enteropathy

URINALYSIS

The urinalysis is also used to assess the general health status of the patient. A urine specimen is a cost-effective, painless liquid tissue biopsy of the urinary tract that rapidly provides a great deal of information. The urinalysis is a useful screening test for renal, genitourinary, metabolic, and hepatic diseases. Among the most significant conditions detected by chemical means in a macrourinalysis are proteinuria, glucosuria, ketonuria, and the presence of the pigments hemoglobin and bilirubin. A microurinalysis of centrifuged urine sediment should be performed on fresh or refrigerated specimens. White cells, red cells, urinary casts, and crystalluria can all be quantified under the microscope. The presence of a suspected infection can be rapidly confirmed by urine dip-stick nitrite and leukocyte esterase tests (Chemstrip, Boehringer-Mannheim Diagnostics, Indianapolis). A urine culture should probably be performed in all cases of presumed urinary tract infection including cases of suspected Reiter's syndrome with or without genitourinary symptoms. Chlamydia trachomatis may be isolated from the urethra, urine, conjunctiva, or rectum of patients with Reiter's syndrome, although iridocyclitis usually develops after the acute infectious phase. Only a few patients with Reiter's uveitis, however, have responded to tetracycline therapy.

Urine CMV cultures as well as CMV cytology are central to the diagnosis of CMV infection. Hypercalciuria is twice as common as hypercalcemia in sarcoidosis.¹⁶⁴ Thus, a 24-hour urinary calcium determination may be helpful, however inconvenient and nonspecific. Twenty-four hour urine collections for uric acid can be a more sensitive screening test than serum uric acid in establishing a diagnoses of gout. Finally, the ova of Endolimax nana have been found in the urine of a patient with exudative chorioretinopathy.

SERUM CHEMISTRY

Increased sensitivity to vitamin D or increased activity of 1,25-dihydroxy vitamin D in sarcoidosis increased intestinal calcium reabsorption and subsequent hypercalcemia and hypercalciuria.¹⁶⁵ Hypercalcemia occurs in about 25% of patients with systemic sarcoidosis, but the test is only rarely positive in patients with ocular sarcoidosis who are undergoing systemic remission. The serum calcium concentration is also elevated in a number of other conditions,¹⁶⁶ by far the most common of which are primary hyperparathyroidism and malignancy (Table 17).

TABLE 17. Causes of Hypercalcemia

  Primary hyperparathyroidism
  Malignancies

  Thiazide therapy
  Sarcoidosis

  Milk-alkali syndrome
  Immobilization
  Hyperthyroidism

  Adrenal insufficiency
  Renal disease
  Familial benign hypercalcemia

Results of liver function studies may be elevated in sarcoidosis. Baseline liver function studies should be ordered prior to beginning a course of isoniazid for tuberculosis. Otherwise, multiparameter serum chemistry should be ordered only when screening for other specific diseases is appropriate.

SERUM PROTEIN ELECTROPHORESIS

Serum placed in an agarose gel and subjected to an electric current results in the migration of the various protein components at different rates. The migration is based on each protein's molecular weight, shape, and electrical charge. The four major fractions, in order of declining net negative charge, are albumin, α-globulin, ß-globulin, and γ-globulin, all of which can be further separated by additional techniques. Nearly all of the serum antibodies are found in the γ -globulin fraction.

A reduced albumin:globulin ratio is found with increased serum globulin, which occurs in lupus erythematosus, rheumatoid arthritis and other collagen vascular diseases, chronic infections, acute hepatic disease, multiple myeloma, sarcoidosis, and some malignant tumors. The normal values for the serum albumin:globulin ratio are from 1.5: 1 to 2.5: 1. Normal values for albumin are 4.0 to 4.5 g/dl, and for globulin they are 1.5 to 3.0 g/dl.

HEMOGLOBIN ELECTROPHORESIS

Patients with obliterative vasculitis or peripheral proliferative retinopathy should be evaluated for hemoglobinopathy when clinically indicated.¹⁶⁷ The hemoglobin electrophoresis is diagnostic for abnormalities in one of the four polypeptide chains found in the hemoglobin molecule, usually due to the substitution of a single amino acid. The sickling disorders, in which mutant hemoglobin proteins S and C are inherited as alleles of normal hemoglobin A, must be considered in the differential diagnosis of retinal vasculitis and peripheral ischemia before a specific diagnosis of infection, collagen vascular disease, or Eales' disease can be made. Sickle trait (AS) affects 8% of black Americans; 0.4% have sickle cell disease (SS), and 0.2% have hemoglobin SC disease. Thalassemia, persistent fetal hemoglobin F, rarely causes retinopathy. Rapid screening for sickle cell disease and trait is now possible with an inexpensive, highly specific and sensitive test using a monoclonal antibody specific for hemoglobin S (Joshua, Pacific Hemostasis, Curtis-Matheson Scientific, Ventura, CA).

SERUM COMPLEMENT

Complement molecules comprise an integrated system of normally inactive enzymes that remain in the circulation to kill foreign cells directly by opsonization and cytolysis and indirectly by neutrophil activation, leukocyte chemotaxis, smooth muscle contraction, mast cell degranulation, and the release of vasoactive amines. Antibody molecules have complement binding sites that are exposed on formation of antigen-antibody complexes. When this site becomes available, complement protein C1 is activated and quickly catalyzes activation of the next complement protein, and so on down the cascade to C9. Complement molecules so catalyzed aggregate to punch chemical holes in bacterial cell membranes, eventuating swelling and cytolysis of the intruder.¹⁶⁸

The complement system consists of some 20 protein molecules, functionally grouped into the classic pathway, the alternate or properdin pathway, and the final common pathway. The classic, or lytic, pathway is activated by IgG or IgM, DNA, C-reactive protein, and staphylococcal protein-A. Classic pathway immune complexes are frequently associated with autoimmune phenomena. The alternate pathway is activated by bacteria, certain fungal infections, and virus-induced IgA and IgG antibodies. The final common pathway consists of components C5 through C9, also known as the membrane attack complex, and is activated by either the classic or alternate pathways. Deficiencies in various complement components result in a wide variety of diseases (Table 18).¹⁶⁹

TABLE 18. Human Disorders Associated With Inherited Complement Deficiencies

Laboratory tests of complement function include CH₅₀, C3, and C4 levels. The complement hemolytic 50%, or CH₅₀, indicates the amount of serum needed to lyse 50% of a suspension of sheep red blood cells coated with anti-red blood cell antibody. Although normal values rule out a complement deficiency, a low value is not specific, as it may reflect selective deficiency of a single factor or an overall activation of the complement system followed by consumption of all components. Normal range for the CH50 is 100 to 200 units/ml. C4 levels reflect activity in the classic pathway, and C3 levels reflect activity in the alternate pathway. Normal range for C4 is 10 to 20 mg/dl, and that for C3 is 100 to 200 mg/dl.

CIRCULATING IMMUNE COMPLEXES

Immune complex disease, or type III hypersensitivity, results from the deposition of toxic antigen-antibody aggregates precipitated into otherwise normal tissue, particularly blood vessels. Physiologic immune complex formation is an essential process serving to eliminate foreign substances and debris. Immune complex disease often follows persistent infections, leading to post-streptococcal glomerulonephritis, arthritis, chronic hepatitis, luetic nephrotic syndrome, or skin lesions such as erythema nodosum leprosum. Serum immune complexes are frequently found in systemic lupus erythematosus and other vasculitic syndromes. In the eye, rheumatoid sclerouveitis, Behçet's disease,¹⁷⁰ sarcoidosis,¹⁷¹ acute retinal necrosis,¹⁷² and toxoplasmosis¹⁷³ are associated with type III hypersensitivity, although direct correlations with circulating immune complexes are not established.¹⁷⁴

Until recent years, the presence of circulating immune complexes could only be indirectly inferred from depressed serum complement levels. Numerous methods are now available to quantify circulating immune complexes.¹⁷⁵ These include physical methods, such as gel filtration; methods employing interactions with rheumatoid factor, conglutin, or complement C1q; and methods using interaction with cell receptors, such as the platelet aggregation test, the Raji cell bioassay, or the phagocytosis inhibition test. Most experts concede that serum immune complex determinations remain in the realm of emerging rather than established clinical use.

CRYOGLOBULINS

Precipitation of serum immunoglobulins in the cold has been noted in a wide variety of conditions, including myeloma and other myeloproliferative neoplasms, rheumatoid arthritis, Sjögren's syndrome, lupus erythematosus, Waldenströms macroglobulinemia, hepatitis, cytomegalovirus infections, infective endocarditis, mononucleosis, and leprosy.¹⁷⁶ Cryoglobulins are quantified by centrifugation of whole serum in a hematocrit tube at 4°C. The precipitate formed may be isolated, dissolved in an acidic buffer, and the cryoglobulin level is then estimated by spectrophotometric absorbance at 280 nm. Further characterization into three types is then done by immunoelectrophoresis. Although this is a nonspecific test, it should be remembered that cryoglobulins may cause serious laboratory errors by precipitating at ambient temperatures and thus removing other serum constituents. Entrapped complement and immunoglobulins are not restored by redissolving the cryoprecipitate.

RADIOLOGY

When an ophthalmologist orders a roentgenogram the radiologist should know exactly which diagnoses are being considered. Chest roentgenograms are useful when conditions such as tuberculosis, sarcoidosis, or histoplasmosis are suspected. Active pulmonary sarcoidosis characteristically shows bilateral hilar adenopathy with or without parenchymal disease; hilar adenopathy characteristically precedes the onset of peripheral infiltrates. Chest roentgenographic abnormalities are found in about 80% of patients with ocular sarcoidosis. Active pulmonary tuberculosis may reveal characteristic wedge-shaped segmental infiltrates, cavitary lesions, calcified granulomas, or diffuse parenchymal nodules accompanied by ipsilateral hilar enlargement. Lordotic views of the relatively oxygen-rich apices may demonstrate old scarring or concentrated active lesions due to the aerobic preferences of mycobacteria.

Ocular and orbital plane films may show calcification of various parts of the uvea; for example, late degenerative choroidal changes, fine stippling in retinoblastoma, or intraocular calcification seen in retinopathy of prematurity and hyperparathyroidism. Intraocular foreign bodies can be identified and localized by plain films as well as ultrasonography and computed tomography.

Sacroiliac joint films should be ordered when ankylosing spondylitis or other seronegative spondyloarthropathies, such as Reiter's syndrome or psoriatic arthritis, are suspected. It is important to specifically order a sacroiliac series, not a lumbar spine series, since both a straight antero-posterior projection and the more sensitive oblique tunnel view of the joints will be taken.

Sacroiliac disease occurs in 20% of patients with Reiter's syndrome. When a patient has psoriasis with sacroiliitis, iridocyclitis is also likely to occur, whether or not the HLA-B27 antigen is positive.¹⁷⁷

Painful, swollen joints can be x-rayed if juvenile or adult rheumatoid arthritis, Reiter's syndrome, or sarcoidosis is suspected. In juvenile rheumatoid arthritis, the knee is most commonly affected. Since children with pauciarticular manifestations develop the worst iridocyclitis, a routine knee roentgenogram is recommended, even though there is no clinically suspected arthritis. In Reiters syndrome, the hands, feet, heels, and sacroiliac joints are most commonly affected. Weight-bearing joints are also afflicted in ankylosing spondylitis and sarcoidosis. Permeating, lytic, and destructive bony changes are also seen occasionally in sarcoidosis.¹⁷⁸

Radiographic studies for cerebral calcification, although helpful, are nonspecific and do not differentiate between toxoplasmosis and CMV inclusion disease, as the roentgenographic findings can be the same for both infections. This is not surprising when one realizes that Toxoplasma gondii and CMV are both neurotrophic intracellular pathogens and may both be seen together within the same host cell.

Upper gastrointestinal small bowel series and barium enema roentgenograms may identify anomalies associated with uveitis, including Whipple's disease, Crohn's disease, ulcerative colitis, and abdominal abscess. Occasionally, paranasal sinus and dental films may be of value in locating clinically suspected loci of infection in otherwise idiopathic uveitis.

NUCLEAR MEDICINE

Gallium citrate can identify inflammatory disease and tumors throughout the body.¹⁷⁹ Gallium is taken up by mitotically active liposomes of granulocytes. The actual scan is performed 48 hours following the intravenous injection of radiolabeled gallium citrate. For the purpose of sarcoid diagnosis, a limited scan of the head, neck, and chest is sufficient and less expensive than a total body scan. Although radiation doses are extremely minute, careful dosimetric consultation with a clinical radiation physicist should be undertaken before performing this test in pregnant women and young children.

The gallium scan is more sensitive than routine chest roentgenograms for showing pulmonary involvement in sarcoidosis.¹⁸⁰ It lacks specificity, however, as numerous other pulmonary diseases show abnormal uptake, including tuberculosis, carcinoma, lymphoma, and silicosis. Increased lacrimal gland uptake of gallium occurs in over 80% of patients with active sarcoidosis,¹⁸¹ although this is also nonspecific; as many as 25% of patients with increased lacrimal gland uptake have no other evidence of the disease. Thus, gallium scans may lead to a false-positive diagnosis of sarcoidosis by physicians unfamiliar with these limitations. When combined with determination of serum ACE and lysozyme levels, a skin test panel, chest roentgenogram, and other tests tailored to a careful history and examination, the gallium scan can be instrumental in establishing a diagnosis of ocular sarcoidosis.

Technetium scans have been used in the diagnosis of Sjögren's syndrome and correspond well to minor salivary gland biopsy findings.¹⁸²

INTERNAL MEDICINE

The referring ophthalmologist should employ common sense when seeking the advice of an internist. A specific description of the patient's illness and suspected diagnoses is essential if the internist is to provide meaningful assistance, avoiding shotgun laboratory tests and an unhelpful, unfocused general systemic examination. A routine referral for a general medical evaluation is not recommended. The ophthalmologist who takes charge of a coordinated effort to use the laboratory and consultative specialists will provide the best service to patients with uveitis. It may be best to suggest the tests believed to be most important to a uveitis workup and allow the internist to order them all from one place. This will avoid duplication and permit the internist to add constructively to the diagnostic evaluation. In more urgent situations this may not be practical, but a telephone communication prior to formal consultation can be immensely helpful, as well as educational to both parties. The training of the medical consultant selected for referral will depend on the nature of the patient's illness.

A medical consultation should be sought when prolonged systemic corticosteroid therapy is anticipated, especially when a patient has concomitant diabetes or hypertension. Having assistance in the management of a patient who may have potentially lethal side-effects from corticosteroids, potent nonsteroidal anti-inflammatory drugs, or immunosuppressive cytotoxic agents is extremely comforting even to the most experienced uveitis specialist. Medical consultation is also essential when systemic disease accompanies uveitis; pulmonary sarcoidosis, tuberculosis, Behçet's syndrome with central nervous system involvement, or progressive ankylosing spondylitis should not be treated by an ophthalmologist. Infectious disease, pulmonary, or oncology subspecialists should be consulted when appropriate.

A neurologic consultation may be helpful when treating a patient with vasculitis and the possibility of multiple sclerosis. A lumbar puncture test, best performed by an experienced neurologist, may be useful if central nervous system involvement in syphilis, large cell lymphoma, Vogt-Koyanagi-Harada disease, or cryptococcosis is suspected.

Pediatricians should be consulted when a child with uveitis may have an associated systemic medical problem, such as juvenile rheumatoid arthritis, mucocutaneous lymph node syndrome (Kawasaki's disease), or sarcoidosis. The use of systemic corticosteroids, antibiotics, and immunosuppressive agents in children presents a unique set of potential complications best followed by a physician intimately familiar with pediatric diseases and drug dosages.

Rheumatologists can be extremely helpful when systemic inflammatory disease, vasculitis, or arthritis accompanies uveitis. Arthritis is found in patients with sarcoidosis, Behçet's syndrome, Reiter's syndrome, ankylosing spondylitis, psoriatic arthritis, inflammatory bowel disease, juvenile rheumatoid arthritis, and rheumatoid arthritis. Systemic vasculitic syndromes with ocular involvement include systemic lupus erythematosus, Reiter's syndrome, Wegener's granulomatosis, Behçet's syndrome, giant cell arteritis, Kawasakis disease, classic polyarteritis nodosa, and infections such as tuberculosis.¹⁸³ The consulting rheumatologist may be extremely helpful in suggesting an appropriate oral nonsteroidal anti-in-flammatory agent or an immunosuppressive drug with which to augment topical corticosteroid therapy.

Dermatologic involvement may occur with several uveitis syndromes. An accurate description of these lesions may help finalize a diagnosis in different cases. Reiter's syndrome includes keratoderma blennorrhagica and circinate balanitis. Genital ulcerations occur in Behçet's syndrome. Sarcoidosis is often accompanied by erythema nodosum. Acute, recurrent, unilateral, alternating nongranulomatous iridocyclitis typical of the HLA-B27 pattern is seen with psoriasis. Cutaneous biopsy may be diagnostic in sarcoidosis or blastomycosis.

DENTISTRY

Behçet's syndrome is characterized by aphthous ulcers found on the tongue, lips, soft and hard palate, and oral mucosal membranes. These lesions are often multiple and tender and may leave small mucosal scars. More subtle lesions challenge the dentist's diagnostic acumen. Oral mucosal lesions also afflict patients with Reiter's syndrome, but these are generally asymptomatic. Patients with Sjögren's syndrome can develop severe gingivostomatitis and candidal infections and may rapidly lose all dentition without aggressive intervention. Traditional teaching stated that dental infections were a common cause of uveitis. This is rare today but should not be overlooked in cases in which other diagnostic possibilities have been exhausted.

ANTITUBERCULOUS THERAPY

Isoniazid has been given to PPD-positive patients with active but clinically stable presumed ocular tuberculosis while all ocular medications remain unchanged. If clinical improvement occurs over a period of 2 to 3 weeks the uveitis can be attributed to mycobacterial infection¹⁸⁴ and the treatment is continued for 1 year. This approach is invaluable since there is no specific diagnostic test for ocular tuberculosis other than isolation of the slow-growing bacillus or identification of acid-fast organisms from biopsy specimens. Serious adverse reactions to isoniazid given for chemoprophylaxis are uncommon,¹⁸⁵ even in patients over the age of 35.¹⁸⁶ Thus isoniazid therapy should be used without hesitation when tuberculous uveitis is the leading diagnostic possibility. Transmission of resistant strains of Mycobacterium tuberculosis have been documented throughout the United States.¹⁸⁷ The three variables that are most helpful in estimating the likelihood of drug resistance are a history of prior antituberculous therapy, country of origin, and duration of residence in the United States.¹⁸⁸ Recent immigrants from Latin America and Asia are more likely to harbor isoniazid-, rifampin-, or streptomycin-resistant organisms than American-born patients or immigrants residing in the United States for more than 10 years. Because of drug resistance, we recommend that antituberculous clinical therapeutic trials routinely consist of treatment with two or more agents simultaneously. Selection of the agents can be made with the assistance of a medical consultant; rifampin is the second drug of choice. Adult doses are isoniazid, 300 mg/day, and rifampin, 600 mg/day, both given in a single oral dosage. Pediatric doses are isoniazid, 10 mg/kg/day up to 300 mg, and rifampin, 10 mg/kg/day up to 600 mg.¹⁸⁹ Treatment may be covered with supplemental vitamin B₆ (pyridoxine hydrochloride), 25 mg/day, in patients who are malnourished, predisposed to neuropathy (diabetes or alcoholism), and adolescents. Additional agents include streptomycin, pyrazinamide, ethambutol, p-aminosalicylic acid, cycloserine, and kanamycin.¹⁹⁰ With tuberculosis developing in an increasing number of AIDS patients,¹⁹¹ increased awareness and clinical flexibility will be necessary to control this old nemesis.

CORTICOSTEROID THERAPY

In the attempt to differentiate neoplastic or degenerative syndromes from those that are inflammatory, oral corticosteroid therapy is not reliable.¹⁹² Prednisone may have a temporarily beneficial effect in all three processes, including fungal infections, because corticosteroid treatment tends to make all types of inflammation better. This approach, however, leads only to delay in diagnosis and prolongation of the disease process. Any patient about to receive oral corticosteroids should be evaluated for tuberculosis with a careful history and PPD skin test¹⁹⁰ in order to avoid the small but undeniable risk of endogenous reactivation.

DIAGNOSTIC VITRECTOMY

There are certain forms of ocular inflammation that lend themselves to diagnostic vitrectomy.¹⁹³ The patients are usually elderly with bilateral chronic vitreous inflammation suggestive of large cell lymphoma, also known as histiocytic lymphoma or reticulum cell sarcoma.¹⁹⁴ Since this disease carries a poor prognosis for survival, a diagnostic vitrectomy performed with standard pars plana techniques is recommended. The vitreous aspirate should be taken immediately to the cytology laboratory.¹⁹⁵ Specimen handling should be planned in advance and the pathology staff notified. If possible, the vitreous should be obtained by aspiration alone, because vitreous cutters may distort the cells. These older patients usually have a fluid vitreous, facilitating the aspiration. The lens should be avoided in all cases where diagnostic vitrectomy is performed. The lenses are usually clear in such cases, and lens material mixed with the vitreous contents can be confusing to the cytologist. Intraocular large cell lymphoma can also be diagnosed by choroidal biopsy¹⁹⁶ using controlled systemic hypotensive anesthesia.¹⁹⁷

Diagnostic vitrectomy is therapeutic in many circumstances. Patients with toxocaral endophthalmitis may benefit from pars plana vitrectomy when chronic inflammation does not respond to medical measures or when such inflammation causes permanent structural changes that threaten or interfere with central vision.¹⁹⁸ Diagnostic core vitrectomy with intravitreal administration of antimicrobial agents may be indicated in fungal or bacterial endophthalmitis,¹⁹⁹ anaerobic bacterial endophthalmitis,²⁰⁰ or acute retinal necrosis syndrome.²⁰¹ Selected cases of culture-proven postoperative Staphylococcus epidermidis endophthalmitis, on the other hand, may not require aggressive therapy with intravitreal antibiotics and vitrectomy to obtain a satisfactory visual outcome.²⁰² Indolent postoperative Propionibacterium acnes endophthalmitis may require removal of residual lens material, lens capsule, intraocular lens, as well as vitrectomy with intravitreal and systemic antibiotics.²⁰³ Vitrectomy may be both diagnostic and therapeutic in Whipple's disease²⁰⁴ and amyloidosis.²⁰⁵

LENSECTOMY

The treatment of all forms of lens-induced uveitis calls for suppression of the inflammation with high doses of topical, periocular, and systemic corticosteroids, followed by removal of the lens or any remnants of lens material. In both phacoanaphylactic and phacolytic uveitis, some inflammation remains as long as lens material remains in the eye. Concomitant topical and systemic glaucoma therapy to control intraocular pressure prior to surgery is also important. Once the eye has become quiet, the type of surgery performed will depend on the clinical picture. If the lens capsule has already been ruptured, or the case follows cataract extraction, an extracapsular technique with careful removal of all lens material is indicated.²⁰⁶ If there is nuclear material in the posterior segment following posterior capsular rupture, then a pars plana vitrectomy should be performed. An intact capsule in the face of glaucoma and hypermature cataract with zonular disarticulation may be best treated with an intracapsular extraction. Cytology and cultures taken at the beginning of these cases before further disruption of lens material has occurred can be helpful in determining the mechanism of inflammation. Finally, patients with cataract and uveitis may benefit from combined lensectomy and vitrectomy procedures.²⁰⁷

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