543 COMhIUNICATIONS Editor’s Note The statements in the following letters represent the consensus of the Committee on Serology Standardization of the American Rheumatism Association. The letter from Dr. Epstein presents a minority opinion on antiglobulin (rheumatoid) factors. Serological Reactions of the Rheumatoid Factors Sir: There is a growing feeling that the antibodies commonly called rheumatoid factors found in high titer in the serum of patients with rheumatoid arthritis and the antiglobulin antibodies found in the serum of normal individuals may represent similar immune responses of different intensity. The myriad variability of these responses is expressed not only by differences in titer but also by the ability of these antibodies to react differently with rabbit or human gamma globulin, by differences in antibody specificity to inherited antigenic components of isologous gamma globulin (Gm groups), by single or multiple specificities of the antibodies (Snagg and Ragg), by differences in the various globulins participating in antibody production (IgM and IgC), and finally by the autoreactivity of some of the antibodies and the strictly isoreactivity of others. It is postulated that these antibodies may represent an immune potential of all capable individuals and that the intensity and duration of the response may reflect the different antigenic stimuli. This is not to disregard the opinions of those who feel that these antibodies are an intense secondary or auxiliary response to an unknown antibody of sharply defined specificity. There is little doubt that the measure of an antibody response of variable specificity, associated most closely with a disease of unknown etiology and ill-defined pathogenesis, has inherent difficulties in standardization. The suggestion has been made that a standardized test should include both the consideration of specificity and of sensitivity. The selected test must therefore be primarily one of great sensitivity capable of detecting all varieties of antiglobulin antibodies even when pres- ent in low titer. This test could be standardized so that “diagnostic titers” could be established. There are three test systems at the present time which could be standardized with these objectives in mind: (1) tube latex test, ( 2 ) F I1 hemagglutination test, and (3) the sensitized sheep cell test. It is of concern that this test be reproducible with due regard to ease of performance. Unless these criteria are met, the test will be slow to meet with acceptance. An alternate suggestion is to have two tests: ( 1 ) a quick simple test of great sensitivity to be used as a screening test (the slide latex test has met with wide acceptance for this purpose); ( 2 ) a test which will give a titer of reactivity (this test could either be a tube test or a slide test). The serology standardization committee of the American Rheumatism Association is attempting to solve this problem in two ways: (1) by evaluating all popular test systems, and ( 2 ) by providing a reference laboratory where individuals may send problem bloods for evaluation by a variety of test procedures, and where they may direct any questions they have about rheumatoid serology. In the meantime, members of the serology committee feel that clinicians and investigators should realize that these tests are measuring exaggerated responses of what may be observed in the normal state and that, as with the tests for antinuclear antibodies, positive results should not override or alter a diagnostic impression made on the basis of clinical signs and symptoms. MAFUONWALLER,PH.D. Medical College of Virginia Richmond, Va. The Present Status of Antinuclear Antibody Serology Sir: It has been 20 years since the original description of the lupus erythematosus (LE) cell phenomenon and more than 10 years since immunofluorescence technics have been applied to the detection of antinuclear antibodies. During this period clinicians have received considerable aid in the confirmation of the diagnosis of systemic lupus erythematosus by use of these technics. However, it has been fully appreciated that antinuclear antibodies, whether detected by the immunofluorescent technic or in LE cell preparations, may be associated with disease entities other than systemic lupus erythematosus. Indeed, if the 544 most highly sensitive tests are used, antinuclear antibodies of one or another immunoglobulin class and of specificity for a wide variety of nuclear antigens may be detected in sera from many asymptomatic individuals. The incidence of such serologic phenomena unassociated with clinical disease has been shown to increase with advancing age. Diagnostic criteria recently proposed for systemic lupus erythematosus do not include tests for antinuclear factor. This omission was brought about by the lack of data on antinuclear antibodies available at the time data were compiled for the purpose of defining the criteria. Not including antinuclear antibodies among the criteria for the diagnosis of systemic lupus erythematosus merely reflects the absence of data on tests for antinuclear factor at the time criteria were analyzed and in no way excludes such tests from being of diagnostic significance. The frequency of positive LE cell preparations in active cases of systemic lupus erythematosus rarely exceeds 80 per cent. The incidence of “false negative” tests is from 15 to 20 per cent. However, utilizing a variety of modifications for the indirect fluorescent technics, many investigators have reported from 95 to 100 per cent positive antinuclear antibody tests associated with active SLE. In centers in which tests are performed, the clinicians have learned that a negative test for antinuclear antibodies performed by the immunofluorescent technic practically excludes the diagnosis of active systemic lupus erythematosus. Although there are nearly as many different methods of performing the indirect immunofluorescent test for antinuclear antibodies as there are investigators performing the test, the following generalizations seem appropriate for 1969: 1. The indirect immunofluorescent technic for antinuclear antibodies when “properly performed” should be positive in nearly every case where the LE cell phenomenon is positive. 2. Tests for antinuclear antibodies should be performed on serum dilutions and a report submitted so that the clinician may infer that the antinuclear antibodies are either absent or are at a concentration below that usually associated with active systemic lupus erythematosus. 3. The pattern of nuclear fluorescence should be reported so that the clinician can interpret the significance of the pattern: e.g., sera yielding bright, high titer, antinuclear antibody with shaggy or peripheral patterns are frequently from patients with active SLE; bright, large, speckled fluorescence patterns are commonly seen in sera from patients with systemic sclerosis. 4. At the present time, a variety of nuclear substrates are in use in various laboratories. Since no data are available on the comparative merits of the various substrates, no recommendation can be COMMUNICATIONS made as to the substrate to be used. However, it should be noted that differences in the results from various laboratories may depend, in part, on a difference in the substrate employed. Efforts are under way under the auspices of the World Health Organization, Medical Research Council Division of Biologic Standards of Great Britain, the Serology Standardization Committee of the American Rheumatism Association, and others to attempt definite criteria for the performance of the indirect fluorescence technic for demonstration of antinuclear antibodies. The groups all agree that investigators, when submitting reports for publication, should provide data regarding: ( a ) nuclear substrate used; ( b ) the concentration( s ) of patient’s serum used; and ( c ) specifications of the conjugate, e.g., its specificity (monovalent or multivalent), the degree of conjugation of fluorescein isothiocyanate to the protein antibody, the units or milligrams per milliliter of antibody present, etc. All groups agree that commercial laboratories producing conjugates for use in the indirect fluorescent antibody technic should provide details described under ( c ) above with each batch of conjugate. There are a variety of other serologic tests available in some centers for the detection of antinuclear antibodies. Sera from patients with active SLE frequently have antibodies to DNA in high titer when these are checked by a variety of technics. It should be emphasized that the tests for antinuclear antibodies should in no way be used to alter a diagnostic impression made on the basis of clinical signs and symptoms. To date, there have been no laboratory tests without false positive and false negative clinical correlations. The clinician must also bear in mind that patients falling into no clear-cut clinical diagnostic category rarely have sera with clear-cut serologic abnormalities sufficient to alter the diagnostic impression from that suggested by the clinical signs and symptoms. Furthermore, since we are at present dealing with serologic abnormalities associated with diseases of unknown etiology and since our present modes of treatment are mainly symptomatic, the wise clinician should not allow himself to be tempted to use experimental or toxic therapy not indicated by clinical signs and symptoms even though these may be suggested b y abnormal laboratory test results. EUGENEI?. BARNETT, M.D. University of California at Los Angeles Los Angeles, Calif. NAOMIROTHFIELD,M.D. University of Connecticut Hartford, Conn.