This test is easily ordered in any clinical immunology laboratory. The terms “p- and c-ANCA” are standard terminology, and the laboratory will perform the test according to its routine method.

Antinuclear Antibodies

We order this test in the setting of scleritis or peripheral ulcerative keratitis, in young people with anterior uveitis in whom we suspect juvenile idiopathic (rheumatoid) arthritis, and in retinal or choroidal vasculitis. The Antinuclear antibodies (ANA) test looks for any antibodies against nuclear proteins such as DNA, RNA, histones, and others. We generally should not have ANA present, although with increasing age many people develop them at a low level. The test is performed by layering the patient’s serum over epithelial cells, such that any antinuclear antibodies in the serum will bind to the epithelial cell nuclei and can be detected with fluorescein-labeled antibodies directed against all human antibodies (the nuclei should fluoresce if ANA are present).

The result is reported as a titer, this being the greatest dilution at which fluorescence was still observed. Titers above 1:160 are significant and are commonly seen in systemic lupus erythematosus (SLE) and juvenile idiopathic arthritis.

Other immune diseases, notably RA, scleroderma, and mixed connective tissue diseases can also cause elevated ANA. It is worth noting that a pattern of nuclear fluorescence is sometimes reported (homogenous, nucleolar, speckled, etc.) and that only the “speckled” pattern is informative, being specific for SLE. A negative test more or less excludes SLE, while a high titer suggests a more severe disease. This test is easily obtained in most laboratories and is usually on most preprinted laboratory forms.

The ANA titer fluctuates with disease activity and is useful in following patients during treatment.

Complements C3 and C4 (C3, C4)

Diseases with activation of the complement pathways will generally show decreased levels of C3 and C4. C4 is part of the classic pathway, and C3 is at the convergence of the classic and alternate complement pathways. Complement activation thus consumes C3 and C4, causing decreased serum levels. We order C3 and C4 levels when we suspect SLE, usually in patients with scleritis, peripheral ulcerative keratitis (PUK), or retinal vasculitis.

Most laboratories can perform these tests, and they are ordered by writing “complement C3 and C4” on the laboratory slip. Results are reported in mg/dL with a RR.

C-Reactive Protein (CRP)

This blood test is standard in any hematology laboratory and is generally on preprinted laboratory forms. Note: related test called the “high sensitivity C-reactive protein” or hs-CRP is used in evaluating risk factors for heart disease. It is sometimes simply called “cardiac CRP.” Many laboratories will include both types of CRP on preprinted test order forms.

Double-Stranded DNA Antibody (ds-DNA Ab)

This serum test is useful in identifying and following patients with SLE. This antibody correlates fairly well with SLE disease activity and moderately well with glomerulonephritis. We do not routinely order this test but find it helpful in patients with scleritis, PUK, and retinal vasculitis in which the ANA is elevated and we suspect SLE. Results should be negative in drug-induced lupus, so this test is also helpful in evaluating patients in whom drug-related elevated ANA is suspected, as occurs with infliximab (Remicade) therapy.

This test is easily obtained in clinical laboratories by writing “ds-DNA” antibodies on the lab slip. Results are reported as a titer, and greater than 1:10 is 95% specific for SLE.

Erythrocyte Sedimentation Rate

ESR is helpful in determining the presence of a potentially severe systemic inflammatory process in patients with scleritis, PUK, and retinal vasculitis. The test measures the distance which the patient’s erythrocytes (RBCs) fall in a column in 1 hour, and the result is thus reported in units of mm/hour. RBCs normally fall slowly, but they fall much faster if the patient’s blood contains increased levels of blood proteins like fibrinogen and other acute phase reactants which cause RBC aggregation. Acute phase reactants are increased in inflammatory diseases. A markedly elevated ESR (indicating rapidly aggregating, fast-falling RBCs), generally over 80 mm/hour, suggests a potentially severe systemic inflammatory or infectious process or malignancy. Low or intermediate levels are not helpful. We do not order this test in the workup of uveitis, but we routinely order it for scleritis, PUK, and retinal vasculitis.

This test is routine in nearly all hematology laboratories and is usually listed on preprinted laboratory forms as “ESR” or occasionally “Sed rate.”

Fluorescent Treponemal Antibody—Absorption

This blood test looks for antibodies to Treponema pallidum. A patient’s serum is placed over a slide containing T. pallidum, and fluorescein-labeled antibodies are used to detect any patient antibodies that have bound there. The result is reported as a titer, and any titer is considered significant and usually indicative of syphilis. Lyme, leprosy, and ANA elevation can cause false positives. This test generally remains positive after treatment and so is not useful in assessing disease activity. It is rarely used on cerebrospinal fluid.

This test is easily obtained, although many laboratories treat this as a confirmatory test for Rapid Plasma Reagin (RPR) or Venereal Disease Research Laboratories (VDRL). It is thus important to indicate that either of these tests is not needed before performing the Fluorescent treponemal antibody (FTA) or that the FTA is necessary regardless of what the RPR or VDRL shows. Many laboratories may substitute another treponemal assay, frequently the microhemagglutination-Treponema pallidum, or MHA-TP, in place of FTA. This is fine, since all treponemal assays work quite nearly the same way as the FTA and are interpreted the same way. The MHA-TP may be falsely elevated in mononucleosis.

Human Leukocyte Antigen (HLA), or Major Histocompatibility Complex

Class II HLA designation works similarly. The major classes are DR, DQ, and DP, and these are expressed only on antigen-presenting cells. Humans have two copies of the DP and DR molecules and four copies of the DQ, for a total of eight major Class II HLA molecules. Serotypes and subtypes are indicated with numbers as with class I antigens.

Some HLA molecules are linked with certain diseases. Common HLA types involved in ocular inflammation are

1. HLA-B27, which is associated with recurrent anterior uveitis
   
2. HLA-A29, associated with Birdshot retinochoroidopathy
   
3. HLA-B^*0501, associated with Behcet disease
   
4. HLA-DR^*0405, associated with Vogt-Koyanagi-Harada (VKH) syndrome

In evaluating patients with ocular inflammation, one usually orders a test for the single HLA type in question, by simply writing, for example, “HLA-B27” on a laboratory slip. Testing is usually performed with polymerase chain reaction. The result is returned as “present” or “absent” or some similar terminology. Rarely, one will want to know all of a patient’s HLA serotypes, or all those in either class, in which case an entire panel can be ordered.

Lysozyme (Muramidase)

Lysozymes are proteins that assist in damaging bacterial cell walls. These compounds are found in the granules of neutrophils and in many bodily secretions (and, incidentally, in very high levels in egg whites). The serum lysozyme level can be used in establishing the diagnosis of sarcoidosis. It is reported as a concentration in μg/mL. We generally only use it to shore up the diagnosis in strongly suspected cases; it is not sufficiently specific to be useful routinely. Lysozyme levels are elevated in several forms of leukemia and can be elevated in TB as well.

Rheumatoid Factor

This test is useful in patients with scleritis or peripheral ulcerative keratitis, because it suggests the presence of a systemic immune process, most notably RA. Rheumatoid Factors (RF) are heterogeneous antibodies of the IgM class that are directed at the Fc region of human IgG class immunoglobulins. We generally have very low levels of RF antibodies, although this increases with age and even some normal individuals have them. Elevated RF nevertheless suggests immune activation in some form. The result is reported as a titer or as international units (IU) with a laboratory RR. Elevated RF is seen most commonly in RA, although this test is far from perfect, and many RA patients do not have RF, while Sjogren syndrome, SLE, and scleroderma patients often do. Methyldopa can increase RF titers, as can viral infections and chronic bacterial diseases.

RF is a standard blood test in most laboratories and is generally found on preprinted laboratory “menu” forms, occasionally listed as “RA,” usually for “rheumatoid antibodies.”

Venereal Disease Research Laboratories or Rapid Plasma Reagin Tests

We generally do not use nontreponemal tests, since uveitis tends to occur in the later stages of syphilis at which patients are often seroreverted and hence the test becomes nonreacative. Almost any laboratory can perform a VDRL or RPR, which are essentially interchangeable.

OTHER TESTS

Purified Protein Derivative

The purified protein derivative (PPD) is a skin test used to determine if an individual has been exposed to the tubercle bacillus, which causes TB. In this test, a small amount of biologically standardized reagent is injected subcutaneously, generally in the skin of the dorsal forearm. The reagent is commercially available, and most uveitis clinics stock it. The standard test involves injecting 0.l mL of reagent, containing 5 tuberculin units (TU—an internationally established measure of biologic activity), although other TU concentrations are available for special situations. The arm is examined 48 to 72 hours later, and induration at the injection site indicates prior exposure to the tubercle bacillus.

The size of induration at the injection site is determined. For most individuals, a PPD is considered positive if the induration is 15 mm or greater. In patients with impaired immunity or in whom the pretest likelihood of disease is high, 5 mm induration is considered significant. This group includes patients taking immunosuppressive medications including corticosteroids at a dose equivalent to prednisone 15 mg per day for a month, patients with active human immunodeficiency virus (HIV) infection, or patients with radiographic evidence of healed TB, as well as close contacts of patients with known TB.

There is an intermediate group in whom 10 mm of induration is considered positive. These are patients whose medical history puts them at risk for progression to active TB, such as those with diabetes, silicosis, or carcinoma of the head, neck, or lung. In addition, this group includes patients with a somewhat elevated pretest likelihood of TB, including immigrants from TB-endemic countries or intravenous drug users. Children under 4 years of age are also included in this group.

A positive PPD test obliges the physician to rule out active TB. By itself, a PPD does not indicate active disease.

Quantiferon Gold (QFT-g)

This is a whole blood test used to diagnose infection with Mycobacterium tuberculosis. It was approved by the U.S. Food and Drug Administration in 2005, being a modification of an earlier test called Quantiferon. The test is based on the fact that an infected individual’s white blood cells will produce interferon gamma (IFN-γ) if exposed to certain M. tuberculosis antigens. In the test, the patient’s whole blood is incubated with two such antigens for 16 to 24 hours, and the level of INF-γ released is quantified. This test may eventually take the place of PPD, but its use in the setting of diseases that affect immune system function (HIV infection, malignancy, diabetes mellitus) is not clearly delineated.

At the time of this writing, laboratory experience with this test is quite variable, and one should contact the receiving laboratory for its collection protocol.

Urinalysis

We evaluate patients for proteinuria when we suspect a systemic vasculitis, since renal involvement of the disease generally informs the prognosis and treatment. A simple Urinalysis (UA) achieves this.