S rum by serologic tests is now an accepted

Transcription

1 CTJRRENT COMMENT Antinuclear Antibodies : Diagnostic Significance and Methods By GEORGE J. FRIOU EARCH FOR antinuclear antibodies in se- S rum by serologic tests is now an accepted part of the routine diagnostic evaluation of patients with the rheumatic diseases. There is considerable confusion, however, in the selection of appropriate technics and in the interpretation of results. This confusion is not surprising since a variety of different immunologic technics have been used to detect antinuclear antibodies, and even within each general method, there have been many variations. Very little attempt has been made to standardize methods or to compare the results from different laboratories. In additon to the irnmunofluorescent technic, other methods applied have included complement fixation, antiglobulin consumption, and precipitin tests, as well as agglutination of sensitized inert particles. With the immunofluorescent technic alone, results vary greatly, depending chiefly on the nature of the material used as the source of antigen, as well as its method of preparation. The purpose of this review will be to attempt to summarize the available information regarding clinical significance of antinuclear antibodies, the selection of a technic for their detection, and interpretation of results. The chief usefulness of these tests relates to the diagnosis of systemic lupus erythematosus ( SLE ), and this ques- tion will be our principal concern here. For details of technic, reference is made to other sources of inf0rmation.l DIAGNOSTIC SIGNIFICANCE OF ANTINUCLEAR ANTIBODIES It is possible to distinguish antinuclear antibodies which react with different components of the cell nucleus, and because distinction of these different antibodies has some clinical significance, we will build our discussion around these differences. The most characteristic antinuclear antibody of SLE is antinucleoprotein (anti-dnp), the antibody which is responsible for the LE cell phenomenon. This antibody reacts with the DNA-protein complex which exists in normal chromosomal material2 A second antibody of major importance is the antibody which reacts with DNA which is not complexed with protein (anti-dna).3 The third antibody of importance is one which reacts with a nuclear antigen which is easily extractable in isotonic buffer solutions, and which does not contain DNA.4,5 The biochemical nature of this antigen has not been determined. Antibody reactive with this antigen occurs rather commonly under a variety of circumstances not associated with SLE. The principal facts are summarized in Table 1. It is readily apparent that the best results will be obtained by a combination of tests GEORGE J. FRIOU, M.D.: Director, Clinical Immunology and Rheumatic Disease Section, De- Supported by Grants AM05483 and AM09703 from the National Institutes of Health, Bethesda, partment of Medicine, University of Southern Md. California School of Medicine, Los Angeles, Calif. 151 ARTHRITIS AND RHEUMATISM, VOL. 10, No. 2 (APRIL 1967

2 152 FHlOU Table 1.-Diagnostic Significance of Antinuclear Antibodies (ANA) in SLE Antibody Significance If Present (specificity) Significance If Absent Anti-DNA (anti-desoxyribonucleic Very high. Rare in other conditions. Absent in to %, acid) Probably diagnostic. especially in less acute phase. Absence does not exclude SLE diagnosis. Anti-DNP (anti-desoxyribonucleo- Never completely diagnostic alone. Rarely if ever absent, protein) Strong support for diagnosis in compatible clinical syndrome of systemic disease. High titer more significant than low titer. Very significant in isolated pleurisy, therefore absence strongly against SLE diagnosis.* pericarditis or glomerulonephritis. Total ANA (anti-dna, anti-dnp, and antibody to phosphate extractable antigen not distinguished). Specificity poor. Other specific ANA None-not related to SLE None Ohlay be absent after long continued (month or more) large dose steroid therapy. Never absent in SLE therefore absence strongly against SLE diagnosis. enabling one to detect anti-dnp and anti- DNA individually, and that detection of the third antibody does not contribute to the clinical usefulness of these tests. Certain of the methods which will be discussed enable us to distinguish between the major antinuclear antibodies outlined above, whereas other methods do not accomplish this. The term total ANA is used here to indicate antinuclear antibodies when detected by a method which does not separate these different types of reactivity. An antinuclear antibody has been identified which reacts only with material within the nucleoli of cells, and there is some evidence that this antibody reacts with nucleolar RNA.4 Another antibody has also been detected which reacts with nuclear histone.c These two latter types of antibody are of relatively uncommon occurrence, and their significance is largely unknown. We shall be concerned chiefly with the first three antinuclear antibodies mentioned, because OE their frequency and clinical importance. It should be recognized that these designations represent general categories of antibody reactivity, and that there may be further differences in specificity within each group. This is known to be so for anti-dna, for example, since it has been demonstrated that different lupus serums may vary in their reactivity to various oligonucleotide fragments of the DNA molecule.7 From the practical point of view, however, it has been possible to make meaningful clinical distinctions based on the larger categories, and these finer individual variations in reactivity have not yet been shown to have importance. Antinucleoprotein (Anti-DNP) Antinucleoprotein, the factor responsible for the LE cell phenomenon, is always present in the serum of patients with untreated SLE, and always demonstrable by serologic tests. During quiescent or less active periods of disease, titers of this antibody tend to be lower than during exacerbations. Treatment with large doses of corticosteroids may cause titers to drop to very low

3 ANTINUCLEAR ANTIBODIES 153 levels, and at times the antibody may disappear completely. The relationship between titer and disease activity, as well as the effect of treatment, tends to vary considerably from patient to patient. This lack of exact correlation between clinical activity and antibody titer is one of the reasons to doubt the causal role of this antibody in the acute manifestations of the disea~e.~~~~~ LE cell tests are negative in 25 per cent of SLE patients, even though anti-dnp antibody can be detected by serologic methods. Occurrence of negative LE cell tests in SLE prevents the use of this test in exclusion of the diagnosis, and forces reliance on serologic technics for ANA for this purpose. The reason for the occurrence of negative LE cell tests when anti-dnp is present has not been fully explained. Negative LE cell tests may be due to the absence of the serum factor when this occurs after a number of weeks of treatment with large doses of corticosteroids. The LE cell test may also be negative in the presence of relatively high titers of anti-dnp, but in serum with low titers, negative tests are much more frequent, and extracellular material ( ECM ) having staining characteristics of the LE cell inclusion is more frequently seen. Low titer of antibody, or failure of phagocytic activity for some other reason, appear to be the two most frequent explanations for the occurrence of negative LE cell tests in the presence of anti-dnp.1 Since there is little reason to think that there is a direct cause and effect relationchip between this antibody and SLE, it might be expected that patients will be seen in whom antinucleoprotein cannot be demonstrated serologically. Such patients seem to be rare, and absence of this antibody in serum is therefore strong evidence against the diagnosis of systemic lupus, unless large doses of corticosteroids (40 to 60 mg. prednisone) have been used for at least several weeks. Anti-DNP can be demonstrated in serum from an appreciable percentage of cases of rheumatoid arthritis. Although the exact incidence varies in different published series, our own experience of about 15 per cent seems to be in line with the reports of others." Among patients with anti-dnp are those patients with rheumatoid arthritis having positive LE cell tests. No clear relationship between this finding and other features of the disease in most of these patients, such as severity of the arthritis or features suggestive of SLE, has been shown, although it is well known that occasional cases are seen in which a clear distinction between rheumatoid arthritis and SLE is difficult. Other clinical conditions in which anti- DNP is seen follow very much the pattern reported for the LE cell test, although frequency of anti-dnp is somewhat higher, These include acute drug hypersensitivity, active chronic hepatitis, dermatomyositis and scleroderma, some patients with necrotizing vasculitis of the small vessel type, and occasional patients with acute infectious hepatitis. In a large series of general medical patients we have seen positive anti-dnp tests in low titer in several patients with atypical pneumonia, two patients out of a large group with active pulmonary tuberculosis, and a total of several cases with anaplastic carcinoma and lymphoma Because titers of anti-dnp tend to be higher in SLE than in rheumatoid arthritis and other conditions, a high titer of this antibody has greater diagnostic significance than a low titer. Tests positive at low serum dilutions (i. e., undiluted or 1 : 2 ) are seen less frequently in SLE than in other situations where positive tests are encountered. Some exceptions to this general correiation are seen. Outside of those groups of patients in which positive LE cell tests are recognized to occur, the frequency of anti-dnp is extremely low.'? In patients with pleurisy

4 154 VHIOU of unknown cause, idiopathic pericarditis, and acute and chronic glomerulonephritis, other than SLE we have never seen positive tests for anti-dnp. In these latter groups of patients, therefore, the finding of anti-dnp is highly significant support for the diagnosis of SLE, while its absence is strongly against this diagnosis. Anti-Desoxyribonucleic Acid ( Anti-DNA) Anti-DNA, the antibody which reacts with protein free desoxyribonucleic acid, occurs in serum of about % of patients with SLE, usually in association with the more acute phases of the disease.*j4 Since it is absent from serum of about?4 of obvious cases of lupus, failure to demonstrate this antibody does not help in ruling out this possibility. On the other hand, it rarely occurs in any other situation, and therefore demonstration of anti-dna is very strong confirmation for the diagnosis. This antibody tends to appear and disappear more rapidly than anti-dnp, and usually is gone from the serum after a short period of corticosteroid therapy. Appearance of anti-dna, along with reduced levels of serum complement, are related to the acute phase of disease, and therefore may be useful in evaluating these patients. In our experience, the most useful information yielded by ANA tests is obtained by carrying out separate tests for both anti- DNP and anti-dna. Other Antinuclear Antibodies The third antinuclear antibody which occurs with considerable frequency is an antibody which reacts with a phosphateextractable antigen of cell nuclei not containing DNA. This antibody is important because it is found frequently, but it does not have diagnostic usefulness because its clinical significance has not been demonstrated. Use of a method which detects this antibody tends to complicate the interpretation of results. The exact identity of the antigen has not yet been determined, but it is distinct from those which have already been di~cussed.~~~ Antibody to the phosphate-extractable antigen occurs with higher frequency than anti-dnp. It has been found in all situations where anti- DNP antibody has been demonstrated and with relative frequency in still other diseases as well.15 Demonstration of this antibody is of no known diagnostic value. With certain immunologic methods, antibody to this antigen is not distinguished from anti-dnp and anti-dna. The results then reflect presence or absence of these three antibodies together, referred to here as "total ANA." Measurement of total ANA rather than the two specific antibodies, anti-dna and anti-dnp, yields poor specificity because of the frequency of occurrence in other conditions besides SLE.*"16 With methods detecting total ANA, absence of antibody is, however, strongly against the diagnosis of SLE, because antibody of one kind or another is always present in these patients. In addition to the three major factors already discussed, antibody to nucleoli4 and antibody to nuclear histone6 are also present in occasional serums. These factors occur infrequently and their clinical significance has not been determined. They will not be considered here in further detail. METHODS FOR DETECTING ANTINUCLEAR ANTIBODIES A summary of methods available for detection of antinuclear antibodies is presented in Table 2. A complete review of the most useful methods, with detailed instructions, is now avai1able.l In general, detection of a particular antibody depends more upon selection of the appropriate antigen than on the immunologic method used. Detection of anti-dna with purified DNA as antigen yields similar results, whether complement fixation, bentonite agglutination or immunofluorescent spot test is used.

5 ANTINUCLEAR ANTIBODIES 155 Table Z.-Methods Antibody Anti-DNA (Anti desoxyribonucleic acid) Anti-DNP ( Anti desoxyribonucleoprotein ) Total ANA ( Anti-DNA, anti DNP, and antibody to phosphate extractable antigen not distinguished) for Antinuclear Antibodies (ANA) Methods D NA-Bentonite Inert particle coated with DNA, agglutinated by antibody. Immunofluorescent spot Serum incubated with DNA spot, then tested for bound antibody with fluorescent anti human globulin. hmunofluorescence with tissue Serum incubated with liver section or leukocyte smear, then fluorescent anti globulin. Shaggy pattern. Requires fluorescence microscope. Complement fixation lmmunofluorescent spot Similar to DNA spot test above 1mmun.ofluorescence with tissue Similar to tissue technique for anti DNA Homogeneous pattern. Anti globulin consumption Anti DNP, if present, will coat suspended DNP particles. Coated particles remove antibody from an anti human globulin serum of known titer. Complement fixation LE cell test lmmunofluorescence with tissue Similar to tissue technique for anti-dna and anti-dnp, but no attempt made to distinguish different antibodies. Complement fixation with suspension of nuclei Anti globulin consumption with suspension of nuclei. Other ANA (Anti phosphate extractable antigen- speckled antigen ) Various methods. Clinical value not ( Anti-nucleolar ) established. ( Anti-histone) There are some differences between results with these different methods, but available information does not indicate that these are significant relative to clinical use. Agglutination of sensitized bentonite particles, for example, may be slightly more sensitive, usually yielding higher titers. It has not been determined, however, whether this increased sensitivity is really useful, or is simply a reflection of the sensitivity d agglutination tests to certain immunoglobulins, such as IgM. A nucleoprotein-latex test has also been described, in which latex particles coated with an extract thought to contain nucleoprotein are agglutinated by some serums. In our experience, results do not correlate well with either the clinical diagnosis of SLE nor the presence or absence of any recognizable antibody in serum. Results are considerably less reliable than the LE cell test itself, and the method is therefore not recommended. Total ANA is detected with antigen con-

6 156 FRIOU sisting of suspensions of whole nuclei, or tissue containing intact nuclear material. More specific tests, such as those for anti- DNP and anti-dna, utilize antigens which have been extracted from nuclei and partially or largely refined. Highly purified DNA can be obtained from commercial sources. The species of origin of antigen is in general not critical, since antinuclear antibodies are not species specific, but, for reproducibility of results with a particular method, the exact source and method of preparation of antigen is of utmost importance. Careful standardization and comparison of results obtained by different workers, through exchange of serum or antigenic material, is essential. Both complement fixation and immunofluorescence can be utilized for detection of anti-dna, anti-dnp, or total ANA. On the other hand, other technics are applicable primarily only to certain antigen-antibody systems. The use of bentonite particles coated with antigen (DNA) in an agglutination test has been limited to detection of anti-dna.17j8 This method may appeal to some workers because a very similar method has been widely used for detection of rheumatoid factor. These two applications differ only in a few details and may be carried out under very similar conditions using identical reagents, other than antigen. The antiglobulin consumption test is most often done in laboratories facile with immuno-hematologic technics, and under such conditions apparently gives results quite comparable to the other method^.^^.^^ The technic is suitable only for particulate or insoluble antigens, whereas complement fixation may be used with soluble antigens as well. Use of complement fixation is complicated by the occurrence of anticomplementary activity in serum from a significant proportion of SLE A microtechnic now availablezz yields very satis- factory results with greatly reduced quantities of reagents, by reducing all quantities ten times from a standard pr0cedure.l This method may be carried out with DNP or DNA as antigen. In laboratories already doing serum complement levels by the microtechnic, standardized reagents will be available. Precipitin tests have been used for anti- DNA,23 but sensitivity of the precipitin test is low and it is therefore not widely used for diagnostic purposes. The deciding factor in the quality of results obtained with any of these systems is the care in selection and preparation of antigenic material, with due importance given to efforts to standardize results. During the past several years there have been several reports on the significance of immunoglobulin class in relation to the different types of antibody reactivity already discussed, or in relation to various diagnostic categories. Results of existing reports are conflicting, and do not establish a basis for application of such technics in clinical diagnosis at the present time. A study now being completed in our laboratory confirms this, and indicates in addition that use of whole serum in such tests may falsely indicate presence of antinuclear activity in the IgM fraction, due to the presence of IgM anti-gamma activity reacting with IgG antinuclear antibody bound to antigen. These results suggest that it may be necessary to work with serum fractions, rather than whole serum, if meaningful data are to be obtained in such studies. Immunofluorescent Technic for ANA At present the indirect immunofluorescent method is most frequently applied for detection of antinuclear antibodies; details of this technic are pub1ished.l~~~ With this method it is possible to test a patient s serum against nuclear antigens of the individual s own leukocytes or other cells. so that the presence of true autoantibody

7 ANTINUCLEAR ANTIBODIES 157 activity may be demonstrated.s Practically, however, this is usually not an important question, and the patient s serum may be tested against nuclear material from other humans, or, since the antinuclear antibodies are not species-specific, against nuclear material from other species. High quality equipment for fluorescence microscopy is now readily available, and several commerical laboratories produce reagents which are of a quality satisfactory for use in routine diagnostic work. For those interested in other anti-tissue antibodies, the indirect immunofluorescent technic has an advantage in that the same fluorescent conjugates can be used to detect a variety of anti-tissue antibodies, such as antibodies to smooth muscle2: and liver cells2g in chronic liver disease, antibody to thyroid2 and to skeletal muscle,28 and the anti-perinuclear factor.29 DNA and DNP may be used in immunofluorescent spot tests for anti-dna and anti-dnp.s*30 These technics require only simple apparatus and are read by gross inspection, rather than by microscopy. They therefore serve as very satisfactory clinical tests. By placing spots of DNA and DNP adjacent to each other on the same slide, the de- termination of both antibodies can be carried out simu1taneously.l Our widest personal experience has been with immunofluorescence, especially with the spot tests for anti-dnp and anti-dna, but various other methods have also been used. We have found these two to be relatively simple to carry out, reproducible, and altogether the most satisfactory as clinical diagnostic tests. 1 mmunofluorescent Technic aith Tissuc Sections When using the immunofluorescent technic with tissue sections or cell smears, total ANA is determined when no attempt is made to distinguish between the different types of antibody on a mor- phologic basis.15,1g Considerable experience has now accumulated in the distinction between different antinuclear antibodies on the basis of the fluorescent pattern of distribution of antigen with which antibody reacts. In this approach tissue sections or smears are examined carefully under the fluorescent microscope after incubation with se- rum and immunofluorescent conjugate under carefully standardized conditions. Under optimal conditions, patterns of fluorescence may be seen which identify the antibody content of the particular serum. Thus, serum containing anti-dnp yields a characteristically even distribution of fluorescence in the nuclei. The term homogeneous has been applied to this pattern4 Another characteristic pattern of staining which is frequently seen is the one which we have described as shaggy. 31 This descriptive title was based on the appearance of nuclei of lymphocytes in peripheral blood smears following indirect immunofluorescent staining, using serum containing anti- DNA. In this pattern, the margin of the fluorescent nucleus is irregular and ragged, and it is apparent that antigen has extended beyond the nuclear membrane into the adjacent cytoplasm. In addition, the central part of the nucleus often has a dark or unstained appearance. The term peripheral has been applied by others to a pattern produced by anti- DNA.32 In the author s experience, atypical fluorescence involving the nuclear margin alone, in the absence of anti-dna in serum, may occasionally give a pattern which might be described as peripheral, although fluorescence is not seen extending into the adjacent cytoplasm in the manner seen in the shaggy pattern. The appearance of the shaggy pattern is also less characteristic in tissue sections than in preparations using peripheral blcod leukocytes. Nevertheless, in our experience and in that

8 1513 of others,8*s2 there is a good correlation between the appearance of a shaggy pattern of fluorescence as described above and the presence of anti-dna as detected by complement fixation. A third important pattern of fluorescence has been given the descriptive term speckled, and is produced by antibody to the phosphate-extractable antigen.4j In this pattern the nucleus shows an irregular mottled appearance distributed throughout the entire area of the nucleus. Failure to distinguish antibody to this antigen from anti-dna or anti-dnp yields a determination of total ANA, with resulting decreased specificity. There are technical problems which limit the usefulness of patterns of fluorescence in distinguishing different antibodies. Individual serums may contain all three of the major antibodies, and in such a situation, the appearance of the fluorescent pattern may be confusing. When anti-dna and anti-dnp are both present, anti-dna is usually in relatively low titer, so that when tested undiluted, the serum may yield a shaggy pattern; at higher dilution, this may change to homogeneous. A serum containing both anti-dnp and antibody to the speckled antigen may produce a homogeneous pattern at low dilution but speckled in the high dilution range. The latter antibody tends to be present at the highest titer. For this reason, tests for anti- DNA and anti-dnp are usually done with serum undihted or at 1:2, while tests for antibody to the phosphate-extractable antigen are done at an initial dilution of 1:lO. Occasional serums may contain still unidentified antibodies, causing further variation in patterns. In addition, there are artifacts which may be produced by changing salt concentrations in drying of tissue, or in other steps of preparation, which may simulate the shaggy pattern even though only anti-dnp is present. In summary, it is our feeling that patterns of fluorescence do provide an adequate basis for distinguishing the different antibodies in most cases, but that the technic is relatively tedious and cumbersome. Because of the technical problems referred to above, one must acknowledge that distinction cannot be made in occasional instances, and one should report the results as atypical in such instances. 1. Friou, C. J.: The LE cell factor and antinuclear antibodies. In Cohen, A. S. (Ed.): Laboratory Methods in Rheumatic Diseases. ( Chap. 5). Boston. Little, Brown and Company, : The significance of the lupus globulinnucleoprotein reaction. Ann. Intern. Med. 49:866, Seligmann, M., and Milgrom, F.: Mise en evidence par la fixation du complement de la reaction entre acide dksoxyribonucleique et serum de malade atteints de lupus erythhmateux dissemine. C. R. Acad. Sci. (Paris) 245:1472, Beck, I. S.: Variations in the morphological patterns of autoimmune nuclear fluorescence. Lancet 1:1203, Lachmann, P. J., and Kunkel, H. G.: Correlation of antinuclear antibodies and nuclear \taining patterns. Lancet 2:436, REFERENCES 6. Robbins, W., Holman, H., Deicher, H., and Kunkel, H.: Complement fixation with cell nuclei and DNA in lupus erythematosus. Proc. SOC. Exp. Biol. Med. 96:575, Levine. L.: Determinants of specificity of proteins, nucleic acids, and polypetides, Fed. Proc. 21:711, Casals, S. P., Friou, G. J., and Myers, L. L.: Significance of antibody to DNA in systemic lupus erythematosus. Arthritis Rheum. 7:379, Townes, A. S., Stewart, C. R., Jr., and Osler, A. G.: Immunologic studies of systemic lupus erythematosus: I. Quantitative estimations of nucleoprotein-reactive gamma globulin in sytsemic lupus erythematosus and other diseases. 11. Variations of nucleoprotein-reactive gamma globulin and hemolytic serum complement levels with disease activity. Bull. Hopkins Hosp. 112:183, 1963.

9 .INTINUCLEAR ANTIBODIES Golden, H. E., and McDuffie, F. C.: Role of lupus erythematosus (L.E.) factor and accessory serum factors in the production of extracellular nuclear material (ECM). Arthritis Rheum. 9:507, 1966 (Abst.) 11. Friou, G. J.: The L.E. cell phenomenon and antinuclear antibodies. Arthritis and Allied Conditions. In Hollander, J. L. (Ed.) ed. 7 (Chap. 7). Philadelphia, Lea & Febiger, 1966, p Unpublished observations. 13. Hasker. J., Mackay, I. R., and Miller, J. J., 111: The incidence of antinuclear factor in human disease. Australasian Ann. Med. 14:96, Seligmann, M.: DNA antibodies. Arthritis Rheum. 6:542, Pollak, V. E.: Antinuclear antibodies in families of patients with systemic lupus erythematosus. New Eng. 1. Med. 271:165, Barnett, E. V., Condemi, J. J., Leddy, J. R., and Vaughan, J. H.: Gamma2, gammal,, and gamma,, antinuclear factors in human sera. J. Clin. Invest. 43:1104, Bozicevich, J., Nasou, J. P., and Kayhoe, D. E.: Desoxyribonucleic acid (DNA)-bentonite flocculation test for lupus erythematosus. Proc. SOC. Exp. Biol. Med. 103:636, Kayhoe, D. E., Nasou, J. P., and Bozicevich, J.: Clinical evaluation of the DNA bentonite flocculation test for systemic lupus erythematosus. New Eng. J. Med. 263:5, Miescher, P. A., and Strassle, R.: New serological methods for the detection of the L.E. factor. Vox sang , hliescher, P. A.: Immune globulins in systemic lupus erythematosus. Proc. 7th Congr. Europ. Sac. Haemat. London, 1959, 2:534, Ceppelini, R., Polli. E., and Celada, F.: A DNA reacting factor in serum of a patient with lnpns erythematosus diffusus. Proc. SOC. Exp. Biol. Med. 96:572, Sever, J. L.: Application of a microtechnique to viral serological investigations. J. Immun. 88:320, Deicher, H. R. G., Holman, H. R., and Kunkel, H. G.: The precipitin reaction between DNA and a serum factor in systemic lupus erythematosus. J. Exp. Med. 109:97, Friou, G. J.: Immunofluorescence and antinuclear antibodies. Arthritis Rheum. 7:161, Johnson, G. D., Holborow, E. J., and Glynn. L. E.: Antibody to smooth muscle in patients with liver disease. Lancet 2:878, , -, and -: Antibody to liver in lupoid hepatitis. Lancet 2:416, Ralfour, B. M., Doniach, D., Roitt, 1. M., and Couchman, K. G.: Fluorescent antibody studies in human thyroiditis: Auto-antibodies to an antigen of the thyroid colloid distinct from thyroglobulin. Brit. J. Exp. Path. 42:307, Beutner, E. II., Witebsky, E., Ricken, D., and Adler, R. H.: Studies on auto-antibodies in myasthenia gravis. J.A.M.A. 182:46, Nienhuis, R. L. F., and Mandema, E.: A new serum factor in patients with rheumatoid arthritis: the antiperinuclear factor. Annals Rheum. Dis. 23:302, Friou, G. J.: Fluorescent spot test for antinuclear antibodies. Arthritis Rheum. 5:407, Casals, S. P., Friou, C. J., and Teague, P. 0.: Specific nuclear reaction pattern of antibody to DNA in lupus erythematosus sera. J. Lab. Clin. Med. 96:625, Gonzales, E. N. and Rothfield, N. F.: Immunoglobulin class and pattern of nuclear fluorescence in systemic lupus erythematosus. New Eng. J. Med. 274:1333, 1966.