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Antinuclear antibody determinations in Ro(SSA)-positive, antinuclear antibody—negative lupus and Sjögren's syndrome patients
C||n|ca| a n d | a b o r a | o r y s l u d | e s Antinuclear antibody determinations in Ro(SSA)-positive, antinuclear antibody-negative lupus and Sjrgren's syndrome patients Normand Dorr, M.D., Daniel Synkowski, M.D., and Thomas T. Provost, M.D.
Montreal, Quebec, Canada, and Baltimore, MD Antinuclear antibody determinations were performed on the sera of twenty-eight Ro(SSA)-positive patients, who failed to demonstrate significant antinuclear antibody titers on mouse liver, with the use of various heterologous and homologous substrates. These studies demonstrated the following: (1) The existence of substrate specificity among various tissues and cells employed in the detection of antinuclear antibodies in Ro(SSA)-positive patients. (2) The existence of a significant group of Ro(SSA)-positive lupus patients with prominent cutaneous findings and systemic disease who, despite the employment of a variety of antinuclear substrates, failed to demonstrate significant antinuclear antibody titer. (J Am ACAD DERmATOL8:611-615, 1983.)
It has recently been recognized that a predominantly dermatologic group of lupus patients, many satisfying four or more of the American Rheumatism Association (ARA) preliminary criteria for the diagnosis of systemic lupus erythematosus (SLE), fail to demonstrate significant antinuclear antibody (ANA) titer. 1-7 Indirect immunofluorescent tests for ANA were performed in most of these studies with both undiluted and diluted sera,
From the Department of Dermatology, Johns Hopkins Medical Institutions. Supported by National Institutes of Health Grants Nos. 5-ROIAM25650 and 5-KO4-AM00524, a gift by the Estre Lauder Corporation, and a grant from the Society of Investigative Dermatology. Presented in pm-t at the National Meeting of the Society for Investigative Dermatology, Washington, DC, May 7, 1982. Accepted for publication Sept. 17, 1982. Reprint requests to: Dr. Normand Dor6, University of Montreal Department of Dermatology, Hotel-Dieu Hospital, 3840 St. Urbain, Montreal, Quebec, Canada H2W 1T8.
using various substrates, i.e., mouse liver, 1"~-7 human granulocytes, 4 rat liver, and mouse kidney a Many of the tested ANA-negative lupus patients possessed the anticytoplasmic antibody Ro(SSA) ~''~'~ and demonstrated a characteristic photosensitive lupus dermatitis. ~-'~ Recently, Sontheimer et al s described a distinct clinical subset of lupus patients with a characteristic widespread, photosensitive lupus dermatitis. These patients, termed "subacute cutaneous lupus erythematosus patients," are similar, clinically and serologically, to the Ro-positive, ANA-negarive, lupus patients reported by Provost et al? Indeed, 66% of Sontheimer's patients were Ro(SSA)positive. 9 However, unlike Provost et al's experience, 63% were considered to have a significant ANA. The substrate employed for the A N A was a commercial human tumor cell line (KB cells).8 In addition, exchange of sera with Dr. Thomas Burnham indicated that three of five of Provost et al's a original ANA-negative, Ro-positive lupus 611
612
Journal of the American Academyof Dermatology
Dord et al
(3128)
16(
(16/281
(9/28)
1B]28)
""
.
iiii
.....
Rat liver
Human spleen
ANA
NB cells
Hep-2 cells
S UBS TRATES
Fig. 1. Significant ANA titer distribution on various substrates using Ro(SSA)-positive ANA-negative lupus and Sj6gren's syndrome sara. patients were ANA-positive on human spleen imprints with the use of undiluted sara. Therefore in order to examine the discordant findings in the prevalence of the positive ANA, in this Ro-positive, ANA-negative group of patients, we investigated the possible role of substrata specificity as an explanation for the A N A negativity. The results of this study indicate that substrata specificity plays a prominent role in detecting the presence o f a significant ANA titer in a large group of Ro-positive patients with cutaneous lupus. MATERIALS AND METHODS Selection of patients Twenty-eight patients possessing only Ro(SSA) antibodies and failing to demonstrate a significant ANA titer on mouse liver substrata (i.e., ~>20) were studied employing rat liver, human spleen, KB, and Hap-2 cells as substrates for the ANA determination. Seventeen of these patients had clinical features of SLE, three had SLE with Sj6gren's syndrome, and eight had primary Sj6gren's syndrome. I m m u n o l o g i c technics The ANAs were detected employing indirect immunofluorescence technics according to the method of Coons? UAll sara were tested for ANA at 1/20, 1/40, 1/80, and 1/160 serum dilutions, employing rat liver and human spleen imprints as the tissue substrata, KB cells and Hap-2 cells were studied at 1/40, 1/80, and
1/160 serum dilutions. The screening dilutions employed were based on our previous experience and/or the supptier's recommendations. The KB cells were purchased from Meloy Laborato. ries Inc., Springfield, VA, and Hep-2 cells from Antibodies Incorporated, Davis, CA. Polyvalent rabbit antiserum to human immunoglobulins (F/P ratio of 3.6; protein concentration of 17.2 mg/ml; Meloy Laboratories, Springfield, VA) was employed to test for the ANA. Precipitin antibodies against nuclear and cytoplasmic macromolecules were detected with the use of gel double immunodiffusion in Ouchterlony plates containing 0.6% agarose. Saline extracts of rabbit thymus and human spleen were the source of antigens, and the patients' sara were tested with reference sara as previously described. ~ Antibodies to native deoxyribonucleic acid (DNA) (nDNA) were assayed by indirect immunofiuorescence using Crithidia lucitiae as the substrata. ~2 RESULTS All patients who were selected for this study demonstrated the Ro(SSA) antibody but failed to demonstrate a significant antinuclear antibody titer upon testing on mouse liver. On repeated testing, no other lines of precipitation were seen in gel double immunodiffusion (i.e., nRNP, Sm, La, or undefined antibody systems). Native DNA determinations were negative. Despite failing to demonstrate a significant ANA (i> 20 titer on mouse liver substrata), 3 of 28 (11%) of Ro-positive patients' sara demonstrated a significant positive antinuclear antibody titer on rat liver; 16 of 28 (57%) demonstrated a significant titer on human spleen imprints; 9 of 28 (32%) demonstrated a significant titer on KB cells, and 8 of 28 (28%) demonstrated a significant titer on Hap-2 cells (Fig. 1). In the same figure, one can appreciate the titer distribution of the four different substrates. Note that the significant ANA determinations in the Ro-positive Sj6gren and SLE patients are relatively low-titered. (Only 3 of 38 patients possessed a 160 or greater ANA titer.) Also note that the end point titer of the sara tested on KB cells and Hap-2 ceils are almost all localized at a titer of 40 (the recommended manufacturers' screening dilution).
Volume 8 Number 5 May, 1983
Ro(SSA)-positive, ANA-negative lupus and Sjbgren's
613
Table I. Detectability of a significant ANA titer on various substrates using Ro(SSA)-positive ANA-negative (mouse liver) sera
Substrate Rat liver Human spleen KB-cells HEP-2 cells Patients studied (28)
I
Percentage positive
ANApositivity + 1
+ + + 1
+ + + + 1
+ + 3
We did not specifically study the pattern of ANA obtained on different substrates. However, one pattern recognized frequently on human spleen imprints and sometimes on other substrates was a particulate pattern composed of three to ten small, dotlike structures within the nucleus. Table I gives the various combinations of positive results on the four different substrates. Note that only 1 patient had a positive ANA on all substrates tested, implying that 27 of 28 Ro-positive patients' sera (96%) demonstrated some ANA substrate specificity. Moreover, 7 of 28 (25%) of these Ro(SSA)-positive patients (6 SLE; 1 Sj6gren's) failed to demonstrate a positive ANA on all four ANA substrates tested. Although human spleen imprints had a higher specificity, 12 of 28 Ro(SSA)-positive patients (42%) failed to demonstrate a significant antinuclear antibody titer (ANA-negative) on this substrate. DISCUSSION The existence of ANA-negative SLE recently reported by several investigators 1-6 appears to delineate a new subset of SLE. Fessel, 4 however, in a review of the literature, noted that a small group of ANA-negative SLE patients had been well described previously. The prevalence of this SLE patient population displaying a persistently insignificant ANA determination is estimated at 5% to 10%. l'4 However, a number of these ANAnegative patients subsequently develop typical serologic findings of SLE, i.e., positive ANA, anti-DNA antibodies, etc.~'a Most important, these ANA-negative lupus patients generally possess prominent cutaneous findings and are frequently seen by dermatologists.
. + + + 2
.
.
.
+ + 1
+ 1
. + + . 2
.
. +
.
. 2
+ -
-
7
7
.
11 57 32 28
Table II. Reasons for a negative (insignificant) ANA determination in SLE patients 1. 2. 3. 4. 5. 6. 7. 8.
Technical errors Solubility of nuclear antigen Prozone phenomenon Immune complex formation Antibody bound to tissue Anti-ssDNA antibody Anticytoplasmic antibody Ro(SSA) Substrate specificity
A number of reasons have been advanced to explain the negative ANA in this group of SLE patients (Table II). Technical error, including solubility of the nuclear antigens, prozone phenomenon, an ANA hidden within circulating immune complexes, and an A N A antibody bound in tissue are commonly cited. 4 Other factors responsible for a negative ANA have been demonstrated recently by Wasieek et aP and Maddison et al. t These investigators described the existence of ANA-negative SLE patients who had antibodies directed either against ssDNA or the cytoplasmic macromolecule Ro. The ssDNA antibodies cannot be detected by routine ANA determinations because of the lack of available single-stranded determinants in resting nuclei of tissue sections employed as ANA substrates. The presence of anti-ssDNA is detected using an antissDNA specific radioimmunoassay. The anticytoplasmic antibody Ro(SSA) has been found in 25% of ANA-positive SLE and in approximately 40% of Sj6gren's syndrome patients. This antibody system is highly specific for connective tissue diseases; in fact, of 72 patients whose sera contained anti-Ro antibody, 80% had
614
D o r d et al
SLE. 7 In addition, 62% (41/66 patients) of ANA-negative SLE patients have been found to possess anti-Ro antibodies. 1 The Ro antibody is identical to the SSA antibody system detected in Sj6gren's patients by Alspaugh and Tan. 7a Of importance, Maddison et al ~previously noted a difference in substrate specificity in ANA detection in these ANA-negative, Ro-positive lupus patients. In their study, 8 of 15 patients (53%) demonstrated a nuclear fluorescence on mouse liver substrate with tmdiluted sera, while 14 of 20 patients (70%) demonstrated nuclear fluorescence on KB cell substrate with 1 : 20 serum dilution, In addition, other investigators' data suggest the presence of ANA substrate specificity in Ropositive lupus patients. Sontheimer et al, 8-'~using a commercial human tumor cell (KB cells) substrate at 1/80 dilution, found, in 81% of their clinical subset of patients termed "subacute cutaneous lupus erythematosus" (SCLE), a positive ANA test; 67% o f these patients had the Ro antibody.~.'~ Moreover, two of the patients with SCLE, having four or more ARA criteria for the diagnoses of SLE, were ANA-negative. Two points in relation to the high prevalence of a positive ANA in Sontheimer's report deserve comment. First, 37% of Sontheimer's patients were nDNA-positive. No mention was made of nRNP, Sm determinations. Conceivably, the ANA positivity in this group could be secondary to the presence of Sin, RNP, and/or nDNA antibodies. Second, the selection of a more "sensitive" substrate like KB cells could also explain the higher incidence of ANA. Indeed, at least 10% of their control subjects had a positive ANA on KB cells at a 1/20 dilution. Substrate specificity in the ANA determination also has been reported previously in lupus, 1'1~'1"Lscleroderma/~ and with other ANA-positive sera. 1~':~7 Expecting a positive ANA when testing for anticytoplasmic antibody may seem paradoxical! The cytoplasmic localization of the antigen Ro, however, has been confirmed by immunologic ~ and biochemical studies. ~s In addition, anti-Ro antibodies are also found frequently in association with La(SSB;Ha) antibodies. This antibody has a nuclear and cytoplasmic localization. ~'~8 In fact, Maddison et aP noted in their study of ANA-
Journal of the American Academyof Dermatology
negative SLE patients that when nuclear fluorescence was most marked on KB cells, the test sera contained both anti-Ro and anti-La(SSB)antibodies. However, in this study none of the patients had anti-La antibodies as determined by gel double-diffusion studies. The positive ANA found in our Ro-positive patients, however, could be conceivably explained by either a subthreshold level of La(SSB) antibodies or the presence of other uncharacterized antibodies not detected by the gel double-diffusion technic. From a practical point of view, determination of ANAs on mouse liver substrate is done in most laboratories at a 1/20 screening dilution, whereas the manufacturer's recommended screening dilutions for testing on the more sensitive substrates, such as KB cells and Hep-2 cells, are 1/40. The reason for these screening dilutions is very simple. By testing patients at a lower dilution, we will increase the number of false-positive results. Indeed, Richardson and Epstein ~:~ stressed the point that the low prevalence of SLE in the general population assures a low predictive value if the ANA test is highly sensitive. For example, previous large surveys of sera of nonnal controls, especially elderly individuals, have indicated that a certain proportion contain a posifi~ce ANA and low titers of many autoantibodies. 2°-2a Studies by Castadeno et al za found that twenty-two of twenty-five (88%) healthy blood donors had a low-titered pos- • itive ANA on calf thymus. Of importance, none were positive at titers greater than 1/8. Finally, for the physician confronted with a single patient, it is appropriate to question the predictive value of a positive ANA. The clinician must be aware that the ANA determination is not a simple test. There are a number of inherent problems, including a multitude of substrates, now available on the market. These substrates have their inherent specificity and sensitivity. Errors can also be made in determining an ANA during tissue processing, and the new fluorescent scopes have undoubtedly enhanced the sensitivity of detection of a positive ANA. Therefore, the clinician must also be aware of the substrate and significant titer for the substrate employed by his laboratory. The dermatologist especially must be aware that a group of ANA-negative, Ro-positive or ssDNA-
Volume 8 Number 5 May, 1983
Ro(SSA)-positive, ANA-negative lupus and Sjfigren' s
p o s i t i v e l u p u s , w i t h p r o m i n e n t skin d i s e a s e and s y s t e m i c features, exist. T h e s e r o l o g i c a b n o r m a l i t i e s in t h e s e p a t i e n t s c a n b e i d e n t i f i e d with c e r t a i n t y o n l y b y g e l d o u b l e - d i f f u s i o n technics and/or radioimmunoassay, respectively.
11.
12.
REFERENCES l. Maddison PJ, Provost TT, Reichlin M: Serological findings in patients with "ANA-negative" systemic lupus erythematosus. Medicine 60:87-94, 1981. 2. Bohan A: Seronegative systemic lupus erythematosus. I Rheumatol 6:534-540, 1979. 3. Gladman DD, Chalmers A, Urowitz MB: Systemic LE with negative LE cells and antinuclear factor. J Rheumatol 5:142-147, 1978. 4. Fessel WJ: ANA-negative systemic lupus erythematosus. Am J Med 64:80-86, 1978. 5. Provost TT, Ahmed AR, Maddison PJ, Reichlin M: Antibodies to cytoplasmic antigens in lupus erythematosus. Serologic marker for systemic disease. Arthritis Rheum 20:1457-1463, 1977. 6. Wasicek CA, Maddison PJ, Reichlin M: Occurrence of antibodies to single-stranded DNA in ANA negative patients. Clin Exp Immunol 37:190-195, 1979. 7. Maddison P, Mogavero H, Provost TT, Reichlin M: The clinical significance of autoantibodies to a soluble cytoplasmic antigen in systemic lupus erythematosus and other connective tissue disease. J Rheumatol 6:189-195, 1979. 7a. Alspaugh MA, Maddison PJ: Reso[ution of the identity of certain antigen-antibody systems in systemic lupus erythematosus and Sj6gren's syndrome: An interlaboratory collaboration. Arthritis Rheum 22:796-798, 1979. 8. Sontheimer RD, Thomas JR, Gilliam JN: Subacute cutaneous lupus erythematosus. A cutaneous marker for a distinct lupus erythematosus subset. Arch Dermatol 115:1409-1415, 1979. 9. Sontheimer RD, Stastny P, Maddison P, et al: Immunological and HLA association in subacute cutaneous erythematosus (SCLE). J Invest Dermatol 74:260, 1980. (Abst.) 10. Coons AH, Kaplan MH: Localization of antigen in tissue cells; improvement in method for detection of antigen by
13.
14.
15. 16.
17. 18. 19. 20, 21. 22. 23.
615
means of fluorescent antibody. J Exp Med 91:1-13, 1950. Clark GM, Reichlin M, Tomasi TB" Characterization of a soluble cytoplasmic antigen reactive with sera from patients with systemic lupus erythematosus. J Immunol 102:117-122, 1968. Aarden LA, de Groot ER, Feltkamp TE: Immunology of DNA. III. Crithidia luciliae, a simple substrate for the determination of anti-dsDNA with the immunofluorescence technique. Ann NY Acad Sci 254:505-515, 1975. Cleymae~ JE, Nakamura RM: Indirect immunofluorescence antinuclear antibody tests: A comparison of sensitivity and specificity of different substrates. Am J Clin Pathol 58:388-393, 1972. Zubrmiecki AS, Dorsch CA: Comparison of mouse liver sections and cultured mouse fibroblasts as substrates for the detection of antinuclear antibodies. Am 3 Clin Pathol 66:892-898, 1976. Blaszczyk M, Beutner EH, Rogozinski T, et aI: Substrate specificity of antinuclear antibodies in scleroderma. J Invest Dermatol 68:191-193, 1977. Bergquist NR, Danietson D: Evaluation of two different antigen preparations in the fluorescent antibody test for antinuclear antibodies (ANA). A comparison between two laboratories. Acta Pathol Microbiol Seand 81:446452, 1973. Halion N, Eckert HL, Stewart J: Evaluation of cellular substrates for antinuclear antibody determinations. J Clin Microbiol 2:42-45, 1975. Lemer MR, Boyle JA: Two novel classes of small ribonueleoproteins detected by antibodies associated with lupus erythematosus. Science 211:400-402, 1981. Richardson B, Epstein WV: Utility of the fluorescent antinuelear antibody test in a single patient. Ann Intern Med 95:333-338, 1981. Weir DM, Holborow EJ, Johnson GD: A clinical study of serum antinuelear factor. Br Med J 1:933-937, 1961. Alexander WRM, Bremmer JM, Duthie JJR: Incidence of the anti-nuclear factor in human sera. Ann Rheum Dis 19:338-350, 1960. Zeligman M, Caunat A, Howard M: Studies on antinuclear antibodies. Ann NY Acad Sei 124:816-832, 1965. Castanedo JP, White JG, Williams RC Jr: Anti-nuclear antibodies in normal human subjects. Arthritis Rheum 10:431-443, 1967.
Montreal, Quebec, Canada, and Baltimore, MD Antinuclear antibody determinations were performed on the sera of twenty-eight Ro(SSA)-positive patients, who failed to demonstrate significant antinuclear antibody titers on mouse liver, with the use of various heterologous and homologous substrates. These studies demonstrated the following: (1) The existence of substrate specificity among various tissues and cells employed in the detection of antinuclear antibodies in Ro(SSA)-positive patients. (2) The existence of a significant group of Ro(SSA)-positive lupus patients with prominent cutaneous findings and systemic disease who, despite the employment of a variety of antinuclear substrates, failed to demonstrate significant antinuclear antibody titer. (J Am ACAD DERmATOL8:611-615, 1983.)
It has recently been recognized that a predominantly dermatologic group of lupus patients, many satisfying four or more of the American Rheumatism Association (ARA) preliminary criteria for the diagnosis of systemic lupus erythematosus (SLE), fail to demonstrate significant antinuclear antibody (ANA) titer. 1-7 Indirect immunofluorescent tests for ANA were performed in most of these studies with both undiluted and diluted sera,
From the Department of Dermatology, Johns Hopkins Medical Institutions. Supported by National Institutes of Health Grants Nos. 5-ROIAM25650 and 5-KO4-AM00524, a gift by the Estre Lauder Corporation, and a grant from the Society of Investigative Dermatology. Presented in pm-t at the National Meeting of the Society for Investigative Dermatology, Washington, DC, May 7, 1982. Accepted for publication Sept. 17, 1982. Reprint requests to: Dr. Normand Dor6, University of Montreal Department of Dermatology, Hotel-Dieu Hospital, 3840 St. Urbain, Montreal, Quebec, Canada H2W 1T8.
using various substrates, i.e., mouse liver, 1"~-7 human granulocytes, 4 rat liver, and mouse kidney a Many of the tested ANA-negative lupus patients possessed the anticytoplasmic antibody Ro(SSA) ~''~'~ and demonstrated a characteristic photosensitive lupus dermatitis. ~-'~ Recently, Sontheimer et al s described a distinct clinical subset of lupus patients with a characteristic widespread, photosensitive lupus dermatitis. These patients, termed "subacute cutaneous lupus erythematosus patients," are similar, clinically and serologically, to the Ro-positive, ANA-negarive, lupus patients reported by Provost et al? Indeed, 66% of Sontheimer's patients were Ro(SSA)positive. 9 However, unlike Provost et al's experience, 63% were considered to have a significant ANA. The substrate employed for the A N A was a commercial human tumor cell line (KB cells).8 In addition, exchange of sera with Dr. Thomas Burnham indicated that three of five of Provost et al's a original ANA-negative, Ro-positive lupus 611
612
Journal of the American Academyof Dermatology
Dord et al
(3128)
16(
(16/281
(9/28)
1B]28)
""
.
iiii
.....
Rat liver
Human spleen
ANA
NB cells
Hep-2 cells
S UBS TRATES
Fig. 1. Significant ANA titer distribution on various substrates using Ro(SSA)-positive ANA-negative lupus and Sj6gren's syndrome sara. patients were ANA-positive on human spleen imprints with the use of undiluted sara. Therefore in order to examine the discordant findings in the prevalence of the positive ANA, in this Ro-positive, ANA-negative group of patients, we investigated the possible role of substrata specificity as an explanation for the A N A negativity. The results of this study indicate that substrata specificity plays a prominent role in detecting the presence o f a significant ANA titer in a large group of Ro-positive patients with cutaneous lupus. MATERIALS AND METHODS Selection of patients Twenty-eight patients possessing only Ro(SSA) antibodies and failing to demonstrate a significant ANA titer on mouse liver substrata (i.e., ~>20) were studied employing rat liver, human spleen, KB, and Hap-2 cells as substrates for the ANA determination. Seventeen of these patients had clinical features of SLE, three had SLE with Sj6gren's syndrome, and eight had primary Sj6gren's syndrome. I m m u n o l o g i c technics The ANAs were detected employing indirect immunofluorescence technics according to the method of Coons? UAll sara were tested for ANA at 1/20, 1/40, 1/80, and 1/160 serum dilutions, employing rat liver and human spleen imprints as the tissue substrata, KB cells and Hap-2 cells were studied at 1/40, 1/80, and
1/160 serum dilutions. The screening dilutions employed were based on our previous experience and/or the supptier's recommendations. The KB cells were purchased from Meloy Laborato. ries Inc., Springfield, VA, and Hep-2 cells from Antibodies Incorporated, Davis, CA. Polyvalent rabbit antiserum to human immunoglobulins (F/P ratio of 3.6; protein concentration of 17.2 mg/ml; Meloy Laboratories, Springfield, VA) was employed to test for the ANA. Precipitin antibodies against nuclear and cytoplasmic macromolecules were detected with the use of gel double immunodiffusion in Ouchterlony plates containing 0.6% agarose. Saline extracts of rabbit thymus and human spleen were the source of antigens, and the patients' sara were tested with reference sara as previously described. ~ Antibodies to native deoxyribonucleic acid (DNA) (nDNA) were assayed by indirect immunofiuorescence using Crithidia lucitiae as the substrata. ~2 RESULTS All patients who were selected for this study demonstrated the Ro(SSA) antibody but failed to demonstrate a significant antinuclear antibody titer upon testing on mouse liver. On repeated testing, no other lines of precipitation were seen in gel double immunodiffusion (i.e., nRNP, Sm, La, or undefined antibody systems). Native DNA determinations were negative. Despite failing to demonstrate a significant ANA (i> 20 titer on mouse liver substrata), 3 of 28 (11%) of Ro-positive patients' sara demonstrated a significant positive antinuclear antibody titer on rat liver; 16 of 28 (57%) demonstrated a significant titer on human spleen imprints; 9 of 28 (32%) demonstrated a significant titer on KB cells, and 8 of 28 (28%) demonstrated a significant titer on Hap-2 cells (Fig. 1). In the same figure, one can appreciate the titer distribution of the four different substrates. Note that the significant ANA determinations in the Ro-positive Sj6gren and SLE patients are relatively low-titered. (Only 3 of 38 patients possessed a 160 or greater ANA titer.) Also note that the end point titer of the sara tested on KB cells and Hap-2 ceils are almost all localized at a titer of 40 (the recommended manufacturers' screening dilution).
Volume 8 Number 5 May, 1983
Ro(SSA)-positive, ANA-negative lupus and Sjbgren's
613
Table I. Detectability of a significant ANA titer on various substrates using Ro(SSA)-positive ANA-negative (mouse liver) sera
Substrate Rat liver Human spleen KB-cells HEP-2 cells Patients studied (28)
I
Percentage positive
ANApositivity + 1
+ + + 1
+ + + + 1
+ + 3
We did not specifically study the pattern of ANA obtained on different substrates. However, one pattern recognized frequently on human spleen imprints and sometimes on other substrates was a particulate pattern composed of three to ten small, dotlike structures within the nucleus. Table I gives the various combinations of positive results on the four different substrates. Note that only 1 patient had a positive ANA on all substrates tested, implying that 27 of 28 Ro-positive patients' sera (96%) demonstrated some ANA substrate specificity. Moreover, 7 of 28 (25%) of these Ro(SSA)-positive patients (6 SLE; 1 Sj6gren's) failed to demonstrate a positive ANA on all four ANA substrates tested. Although human spleen imprints had a higher specificity, 12 of 28 Ro(SSA)-positive patients (42%) failed to demonstrate a significant antinuclear antibody titer (ANA-negative) on this substrate. DISCUSSION The existence of ANA-negative SLE recently reported by several investigators 1-6 appears to delineate a new subset of SLE. Fessel, 4 however, in a review of the literature, noted that a small group of ANA-negative SLE patients had been well described previously. The prevalence of this SLE patient population displaying a persistently insignificant ANA determination is estimated at 5% to 10%. l'4 However, a number of these ANAnegative patients subsequently develop typical serologic findings of SLE, i.e., positive ANA, anti-DNA antibodies, etc.~'a Most important, these ANA-negative lupus patients generally possess prominent cutaneous findings and are frequently seen by dermatologists.
. + + + 2
.
.
.
+ + 1
+ 1
. + + . 2
.
. +
.
. 2
+ -
-
7
7
.
11 57 32 28
Table II. Reasons for a negative (insignificant) ANA determination in SLE patients 1. 2. 3. 4. 5. 6. 7. 8.
Technical errors Solubility of nuclear antigen Prozone phenomenon Immune complex formation Antibody bound to tissue Anti-ssDNA antibody Anticytoplasmic antibody Ro(SSA) Substrate specificity
A number of reasons have been advanced to explain the negative ANA in this group of SLE patients (Table II). Technical error, including solubility of the nuclear antigens, prozone phenomenon, an ANA hidden within circulating immune complexes, and an A N A antibody bound in tissue are commonly cited. 4 Other factors responsible for a negative ANA have been demonstrated recently by Wasieek et aP and Maddison et al. t These investigators described the existence of ANA-negative SLE patients who had antibodies directed either against ssDNA or the cytoplasmic macromolecule Ro. The ssDNA antibodies cannot be detected by routine ANA determinations because of the lack of available single-stranded determinants in resting nuclei of tissue sections employed as ANA substrates. The presence of anti-ssDNA is detected using an antissDNA specific radioimmunoassay. The anticytoplasmic antibody Ro(SSA) has been found in 25% of ANA-positive SLE and in approximately 40% of Sj6gren's syndrome patients. This antibody system is highly specific for connective tissue diseases; in fact, of 72 patients whose sera contained anti-Ro antibody, 80% had
614
D o r d et al
SLE. 7 In addition, 62% (41/66 patients) of ANA-negative SLE patients have been found to possess anti-Ro antibodies. 1 The Ro antibody is identical to the SSA antibody system detected in Sj6gren's patients by Alspaugh and Tan. 7a Of importance, Maddison et al ~previously noted a difference in substrate specificity in ANA detection in these ANA-negative, Ro-positive lupus patients. In their study, 8 of 15 patients (53%) demonstrated a nuclear fluorescence on mouse liver substrate with tmdiluted sera, while 14 of 20 patients (70%) demonstrated nuclear fluorescence on KB cell substrate with 1 : 20 serum dilution, In addition, other investigators' data suggest the presence of ANA substrate specificity in Ropositive lupus patients. Sontheimer et al, 8-'~using a commercial human tumor cell (KB cells) substrate at 1/80 dilution, found, in 81% of their clinical subset of patients termed "subacute cutaneous lupus erythematosus" (SCLE), a positive ANA test; 67% o f these patients had the Ro antibody.~.'~ Moreover, two of the patients with SCLE, having four or more ARA criteria for the diagnoses of SLE, were ANA-negative. Two points in relation to the high prevalence of a positive ANA in Sontheimer's report deserve comment. First, 37% of Sontheimer's patients were nDNA-positive. No mention was made of nRNP, Sm determinations. Conceivably, the ANA positivity in this group could be secondary to the presence of Sin, RNP, and/or nDNA antibodies. Second, the selection of a more "sensitive" substrate like KB cells could also explain the higher incidence of ANA. Indeed, at least 10% of their control subjects had a positive ANA on KB cells at a 1/20 dilution. Substrate specificity in the ANA determination also has been reported previously in lupus, 1'1~'1"Lscleroderma/~ and with other ANA-positive sera. 1~':~7 Expecting a positive ANA when testing for anticytoplasmic antibody may seem paradoxical! The cytoplasmic localization of the antigen Ro, however, has been confirmed by immunologic ~ and biochemical studies. ~s In addition, anti-Ro antibodies are also found frequently in association with La(SSB;Ha) antibodies. This antibody has a nuclear and cytoplasmic localization. ~'~8 In fact, Maddison et aP noted in their study of ANA-
Journal of the American Academyof Dermatology
negative SLE patients that when nuclear fluorescence was most marked on KB cells, the test sera contained both anti-Ro and anti-La(SSB)antibodies. However, in this study none of the patients had anti-La antibodies as determined by gel double-diffusion studies. The positive ANA found in our Ro-positive patients, however, could be conceivably explained by either a subthreshold level of La(SSB) antibodies or the presence of other uncharacterized antibodies not detected by the gel double-diffusion technic. From a practical point of view, determination of ANAs on mouse liver substrate is done in most laboratories at a 1/20 screening dilution, whereas the manufacturer's recommended screening dilutions for testing on the more sensitive substrates, such as KB cells and Hep-2 cells, are 1/40. The reason for these screening dilutions is very simple. By testing patients at a lower dilution, we will increase the number of false-positive results. Indeed, Richardson and Epstein ~:~ stressed the point that the low prevalence of SLE in the general population assures a low predictive value if the ANA test is highly sensitive. For example, previous large surveys of sera of nonnal controls, especially elderly individuals, have indicated that a certain proportion contain a posifi~ce ANA and low titers of many autoantibodies. 2°-2a Studies by Castadeno et al za found that twenty-two of twenty-five (88%) healthy blood donors had a low-titered pos- • itive ANA on calf thymus. Of importance, none were positive at titers greater than 1/8. Finally, for the physician confronted with a single patient, it is appropriate to question the predictive value of a positive ANA. The clinician must be aware that the ANA determination is not a simple test. There are a number of inherent problems, including a multitude of substrates, now available on the market. These substrates have their inherent specificity and sensitivity. Errors can also be made in determining an ANA during tissue processing, and the new fluorescent scopes have undoubtedly enhanced the sensitivity of detection of a positive ANA. Therefore, the clinician must also be aware of the substrate and significant titer for the substrate employed by his laboratory. The dermatologist especially must be aware that a group of ANA-negative, Ro-positive or ssDNA-
Volume 8 Number 5 May, 1983
Ro(SSA)-positive, ANA-negative lupus and Sjfigren' s
p o s i t i v e l u p u s , w i t h p r o m i n e n t skin d i s e a s e and s y s t e m i c features, exist. T h e s e r o l o g i c a b n o r m a l i t i e s in t h e s e p a t i e n t s c a n b e i d e n t i f i e d with c e r t a i n t y o n l y b y g e l d o u b l e - d i f f u s i o n technics and/or radioimmunoassay, respectively.
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REFERENCES l. Maddison PJ, Provost TT, Reichlin M: Serological findings in patients with "ANA-negative" systemic lupus erythematosus. Medicine 60:87-94, 1981. 2. Bohan A: Seronegative systemic lupus erythematosus. I Rheumatol 6:534-540, 1979. 3. Gladman DD, Chalmers A, Urowitz MB: Systemic LE with negative LE cells and antinuclear factor. J Rheumatol 5:142-147, 1978. 4. Fessel WJ: ANA-negative systemic lupus erythematosus. Am J Med 64:80-86, 1978. 5. Provost TT, Ahmed AR, Maddison PJ, Reichlin M: Antibodies to cytoplasmic antigens in lupus erythematosus. Serologic marker for systemic disease. Arthritis Rheum 20:1457-1463, 1977. 6. Wasicek CA, Maddison PJ, Reichlin M: Occurrence of antibodies to single-stranded DNA in ANA negative patients. Clin Exp Immunol 37:190-195, 1979. 7. Maddison P, Mogavero H, Provost TT, Reichlin M: The clinical significance of autoantibodies to a soluble cytoplasmic antigen in systemic lupus erythematosus and other connective tissue disease. J Rheumatol 6:189-195, 1979. 7a. Alspaugh MA, Maddison PJ: Reso[ution of the identity of certain antigen-antibody systems in systemic lupus erythematosus and Sj6gren's syndrome: An interlaboratory collaboration. Arthritis Rheum 22:796-798, 1979. 8. Sontheimer RD, Thomas JR, Gilliam JN: Subacute cutaneous lupus erythematosus. A cutaneous marker for a distinct lupus erythematosus subset. Arch Dermatol 115:1409-1415, 1979. 9. Sontheimer RD, Stastny P, Maddison P, et al: Immunological and HLA association in subacute cutaneous erythematosus (SCLE). J Invest Dermatol 74:260, 1980. (Abst.) 10. Coons AH, Kaplan MH: Localization of antigen in tissue cells; improvement in method for detection of antigen by
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