Antinuclear
Antibody Profile in Italian
Patients with
Connective Tissue Diseases R.
NERI,
A.
TAVONI, R. CRISTOFANI*, C. LEVANTI, G. SODINI,
A.
D’ASCANIO, C. VITALI,
C. FERRI and S. BOMBARDIERI Clinical Immunology Unit, University of Pisa; *Department of Public Health and Biostatistics, University
of Pisa; and IFC-CNR,
Pisa, Italy
In the present work we report data on the specificity of antinuclear antibodies (ANA) in a large series of Italian patients suffering from a broad spectrum of connective tissue diseases (CTD), by using a series of homogeneous and validated techniques. The present study confirms, on the one hand, generally accepted concepts, i.e. that certain autoantibodies are strictly associated to certain disease states (such as anti-PCNA and anti-Sm in systemic lupus erythematosus, Jo1 in polymyositis, and ACA and Scl-70 in scleroderma); the presence of ’marker’ antibodies is, however, restricted to a relative minority of CTD patients. The application of a new methodological approach that considers the entire profile of ANA can greatly augment their diagnostic relevance and may provide useful indications for their interpretation, allowing us to establish for the first time the diagnostic usefulness not only of marker autoantibodies but also of certain associations between non-marker autoantibodies. Finally, the application of a more appropriate and powerful statistical tool (multiple correspondence analysis) has further emphasized the clear relationship existing between antibody specificities and certain disease states.
Key Words: Antinuclear antibodies Connective tissue disease Counterimmuno-
electrophoresis
Introduction
Italian patients with CTD. ANA were detected by means of a series of techniques that were recently validated during the course of multicenter Consensus Finding
Many autoantibodies that react with different nuclear components (ANA) such as nucleic acids, histones, and non-histone proteins have been described in the sera from patients with connective tissue diseases (CTD), and their detection and characterization appear to be very useful in the diagnosis and management of these diseases’-’. Some of these autoantibodies are considered to be markers of a particular disease state (such as antidsDNA2, anti-Sni&dquo; and anti-PCNA7.8 for systemic lupus erythematosus (SLE), or Sci-70 and anticentromere antibodies (ACA) for systemic sclerosis (SSc) and the CREST variant of scleroderma)&dquo;’. However, in most cases only an association among different specificities or a particular antibody profile may be indicative of a particular CTD or clinical subset4, 12-14. While there is general on the above concepts, discordant have been reported concerning the prevalence of the autoantibodies,. These differences often are not ascribable to genetic or environmental factors&dquo;, but rather to the different patient selection, or to the differing sensibility or specificity of various laboratory tests&dquo;&dquo;. In the present work we report data on the specificity of ANA in a large series of
Studies 19. Patients and methods Patients A total of 1457 sera from 615 consecutive patients with various CTD who were attending the Clinical Immunology Unit of Pisa were tested between 1987 and 1989. The patients included 155 with SLE, 78 with primary Sj6gren’s syndrome ~i °5~~~ 18 with mixed connective tissue disease 23 with idiopathic Raynaud’s phenomenon (Ray), 87 with rheumatoid arthritis (RA), 33 with unclassified connective tissue disease (UCTD), nine with polymyositis 54 with mixed cryoglobulinemia (MC), and 157 with SSc. The diagnoses of SLE, RA and MCTD were made according to well-established criteria2°-22. The diagnosis of 1 °SS was based on clinical symptoms of dry eyes and dry mouth, and confirmed by the objective demonstration of xerophthalmia (positive Schirmer’s test) or
keratoconjunctivitis sicca (positive rose Bengal staining) and
Correspondence: Rossella Neri, M.D., Clinical Immunology Unit, University of Pisa, via Roma 67, 56100 Pisa, Italy.
on
the presence of salivary-gland involvement and/or hydrostatic sialography) in
(positive lip biopsy 221
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the absence of other associated diseases. For SSc, three subsets were considered in which the distinction hinged primarily on the distribution of cutaneous involvemene3: the first (type I) with sclerodactyly only, the second (type II) with skin stiffness proximal to the metacarpophalangeal joints with sparing of the trunk, the third (type III) with diffuse stiffness of the skin including the trunk. The diagnosis of Ray was made when a positive cold photoplethysmography test was not associated with the presence of signs and/or symptoms typical of CTD, and after careful exclusion of traumatic or compressive local factors. The diagnosis of PM was based on the presence of at least two of the following: elevated serum muscle enzymes, abnormal electromyogram, and/or typical muscle biopsy in the absence of other CTD. MC was defined as previously reported2B and in particular in the presence of typical symptoms (arthralgias, purpura with or without systemic involvement) and signs (cryoglobulins with rheumatoid factor activity) and after the exclusion of other known autoimmune, infectious or neoplastic diseases. Patients with UCTD included those with clear features of a diffuse connective tissue disorder, but who did not meet the diagnostic criteria for any other defined disease. Ninety-two sera from 92 healthy subjects were used as controls.
Methods Anti-extractable nuclear antigen (ENA) antibodies were detected by counterimmunoelectrophoresis (CIE) as. described by Bunn et al.2s A commercial preparation of lyophilized rabbit thymus extract (Pel Freeze Biologicals, Roger, Ark.) was used as the source of antigens for the detection of anti-Sm, anti-RNP, anti-La/SSB, anti-PCNA, ~.nti-Sl and anti-Jol antibodies. Extraction was carried out according to the method described by Kurata and T~.n~~; the material was adjusted to a final concentration of 5 mg/ml and stored in aliquots at - 70°C until use. Human spleen extract was used for anti-Ro/SSA antibody detection; it was prepared according to Venables et al.17. Briefly, human spleen homogenate dialysed phosphate buffered saline was centrifuged for 20 min at 9500 x g. The ultracentrifuged supernatant was then added to a DEAE-52 cellulose column. The fraction eluted from ion-exchange chromatography with 0.35 M NaCI, pH 7.2, was used as the source of Ro/SSA (at a concentration of 0.5 mg/ml). ~,nti-S~l-’7~ antibodies were detected by CIE, essentially as described above but with care taken.to use fresh and more highly concentrated (lOmg/ml) rabbit thymus extract. Reference sera, kindly provided by Dr M. l~,ei~hlin (USA) and Dr ~.1~.~. Hughes (UK), were used to define antibody specificity. Indirect immunofluorescence was employed for the anti-dsDNA and ACA
detection using Crithidia luciliae8 and as
HEp-2 cell lineS21
substrate, respectively.
Statistics Pearson’s correlation coefficient and the chi-square test were used to determine both the correlation between antibodies and the correlation between diseases and the presence and degree of antibody specificities. Multiple correspondence analysis (MCA) was then used to analyse the data; this is a descriptive method, a particular type of factor analysis that is specific for categorical data30. It summarizes in a single graph all the information contained in the two sets of variables (in this case, disease and antibody specificity) that are to be correlated. Briefly, MCA involves translating the data by means of a complete disjunctive code that replaces each level of response with a new variable, which is assigned a value of 0 or 1. If ~ variables are recorded on n subjects we obtain a table with n rows and p columns. From a geometrical point of view each disease corresponds to a point in a multidimensional space, each dimension of which represents a different antibody specificity. Similarly, each antibody specificity is represented by a point in this space, each dimension of which corresponds to a different disease. In this multidimensional space the diseases (and antibody specificities) are arranged in ‘clouds’ of points that can be visualized only by their pro-
jection on appropriate planes. We initially found the direction of maximal variance of the cloud; this direction is called the first factorial axis (fl); we then searched for a second direction uncorrelated to the first one, which is the second factorial axis (f2), and so on. The final aim is to replace the original variables by a smaller number of quantitative variables, the factorial axes, that can optimally summarize the information. As each axis explains a decreasing percentage of the ’multidimensional’ variance that underlies the information, we can consider that part of the total variance explained by the first three factorial axes. In conclusion, the results of MCA can be represented by a series of plots that relate to pairs of factorial axes and show the projection for the modalities of antibody specificities and diseases on the factorial planes. The distance of the points frorn. the origin of the axes is an expression of the contribution of the variables to the factorial axis (and is proportional to the significance of the variable), while the proximity of the variables to each other is an indication of the strength of their
relationship. Results The most
patients
important epidemiologic findings on the reported in Table L Table II shows the
are
222
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Table I
SLE
Main
epidemiological data
for 615 Italian
patients
with various CTD.
systemic lupus erythematosus; MCTD mixed connective tissue disease, 1 °SS primary Sj6gren’s syndrome; PM polymyositis; MC mixed cryoglobulinemia; RA rheumatoid arthritis; Ray idiopathic Raynaud’s phenomenon; SSc systemic sclerosis; UCTD unclassified connective =
=
=
=
=
=
=
=
=
tissue disease.
SLE
systemic lupus erythematosus; MCTD mixed connective tissue disease; 1°SS = primary Sj6gren’s syndrome; PM polymyositis; MC mixed cryoglobulinemia; RA = rheumatoid arthritis; Ray idiopathic Raynaud’s phenomenon; SSc systemic sclerosis; UCTD unclssified connective =
=
=
=
=
=
=
tissue disease. I
When multiple specimens were a~rai3abl~ for the same patient, the prevalence was calculated for the first specimen drawn by date.
The most common and significant serological correlations found are shown in Table III. Certain ANA specificities were closely related, while other specificities were mutually exclusive. Furthermore, although anti-Sm antibodies never occurred in the absence of anti-RNP, and anti-La/SSB rarely occurred without Ro/SSA, both anti-RNP and anti-Ro/SSA could be found alone.
prevalence of specificities in the different CTD. When multiple sera from the same patient were available, this prevalence was always calculated for the first drawn by date. Anti-dsDNA antibodies were more frequent in with SLE (63%) than in females (30%) and the simultaneous presence of anti-Sm and anti-RNP antibodies was found in 9% of the female and in 18% of the male patients. Anti-La/SSB antibodies were not found in the male patients. Patients with SSc had an extremely distinct ANA profile, characterized by the presence of particular specificities rarely found in other CTD, such as Scl-70 and ACA. In type III scleroderma, the presence of antiScl-70 antibodies was higher in males (83%) than in
Table III
Serological relationships.
females (56Vo). UCTD patients represented
a serologically heteroand geneous group practically all of the abovementioned specificities were present. All sera from normal subjects were negative. .
223
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Diagnostic relevance of single antibody specificities antibody associations.
Table IV or
SLE = systemic lupus erythematosus; PM = pc~lymyositis; SSc = systemic sclerosis; MCTD = mixed connective tissue disease; lOSS primary Sj6gren’s syndrome; UCTD = unclassified connective tissue disease; others other connective tissue diseases. =
=
’Chi-square: 13.44; ~~hi-~quar~: 62.5; ’chi-square:
Table IV shows the
7.79.
antibody specificities and anti-
body associations that were found only in certain disease states and therefore could have great diagnostic r~levance (marker antibodies), and antibody specificities and antibody associations that were strongly indicative of only certain clinical conditions. Serial measurements of the ANA profile were available for a large proportion of the patients with SLE and I °SS; qualitative changes (positive to negative and vice
versa) occurred much more often in SLE than in 1 ass (Figure 1). In SLE, the prevalence of the ANA specificities increased consistently when multiple specimens were examined during the course of the illness; these changes were much more pronounced for anti-dsDNA, anti-RNP and anti-Sm than for anti-Ro/SSA and antiLa/SSB. Finally, we attempted on the basis of the above data to define more precisely the relationship between the antibodies and diseases means of a more comprehensive statistical approach, i.e. MCA. Figure 2 is a graphic representation of the results of the MCA analysis, showing the projection of the variables (diseases and specificities) on the factorial planes. In Figure .2a the first axis, characterized by the SLE patients, shows a clear-cut distinction between the SSc variants (right) and SLE, MCTD and 1°SS (left); the controls, Ray, MC and RA are in between. The second axis, characterized by the MCTD patients, shows the distinction between 1 °~ a on
Figure 1 Variation in ANA positivity on follow-up. The time interval between multiple samples is not always the same but in most cases it is approximately 3 months.
the
hand and SLE and MCTD on the other. This demonstrates the clc~se association of 1 °SS with graph anti-La/SSB antibodies and, to a lesser extent, with anti-Ro/SSA antibodies. Also evident is the association of SLE with anti-dsDNA, anti-Sm and anti-RNP antibodies, and MCTD with anti-RNP antibodies. Examination of the contribution of the diseases and autoantibodies to the first and third axes (Figure 2b) helps to clarify further the distinction between the SLE and sciernderrna variants; in addition, it allows a better definition of the association between the type I form of scleroderma and ACA, and between the type II and type III forms and anti-Scl-70 antibodies. In the figure the one
variables
are not
represented because they
tended to concentrate in a cloud close to the origin of the axes.
Discussion The analysis of this large series of patients suffering from a broad spectrum of CTD confirms the high diagnostic value of the ANA profile. Although in’soane studies up to lt3~l~ of the controls tested were found to
224
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majority, if not all, of the CTD that have been studied. The present work, which analyses a large number of CTD by means of well-established and generally validated techniques, makes it possible to confirm the high diagnostic value of certain antibody associations. Antibodies directed against certain antigens such as RNP or Ro/SSA, while not specific per se, can assume diagnostic significance when considered together. For instance, the presence of anti-Sl alone, or anti-dsDNA alone or in association with anti-La/SSB-Ro/SSA, or the contemporaneous presence of over four different specificities can all be considered highly specific for SLE. In addition, the association of anti-La/SSB-Ro/ SSA is closely linked to i ~’S~, while the presence of anti-RNP alone was almost invariably found only in MCTD. A consideration of the entire profile could greatly augment the diagnostic relevance of ANA and may provide useful indications for their interpretation. In view of the results obtained here, it appears rather surprising that this global approach has up to now been somewhat overlooked in spite of the extensive literature on the ,
subject. Although the prevalence of the various ANA in our series is, on the similar to other large series that have been reported 12, 34, certain discrepancies deserve special attention. For instance, anti-Sm antibodies were found in only 1 2 Vo of our SLE patients, but this percentage increased when multiple specimens from the same patient were taken into account. This incidence is certainly lower than that reported for the North American population’, &dquo; but is in keeping with most recent European studies’6. Both environmental, ethnic37 and methodological factors&dquo; may help to explain these differences. Finally, the prevalence and specificity of the autoantibodies found in patieqts with scleroderma and scleroderma variants are worth noting. In our series, anti-Scl-70 antibodies were present in a much higher frequency than has been reported previously&dquo;, &dquo; although comparable results were recently reported by others-&dquo;.
Figure2 Projection of the variables on the factorial planes (a) fl-f2 and (b) fl-f3 showing the relationship between antibody specificities and diseases. The distance of the points from the origin of the axes is proportional to the significance of the variable, while the proximity of the variables to each other is an indication of the strength of their relationship. SLE systemic lupus erythematosus; MCTD mixed connective tissue disease; t°SS primary Sj6gren’s syndrome; MC mixed cryoglobulinen-da; RA rheumatoid arthritis; Ray idiopathic Raynaud’s phenomenon; I systemic sclerosis type I; 2 systemic sclerosis type II; 3 systemic sclerosis type III; NHS normal human subjects; ACA anticentromere antibodies. =
=
=
=
=
=
=
=
=
=
=
have ANA16,31, most authors report the total absence or negligible incidence of specific ANA 12, which is consistent with our findings. The present study confirms, on the one hand, gener-
In contrast, the level of ACA in the three subsets was generally lower than that reported in the literature. It is that these results the frequency of ACA and Scl-70 antibodies in SSc may be due
ally accepted i.e. certain autoantibodies are restricted to certain disease states, such as
to
the lack of uniform classificative and to
methodological problems. Their co-existence has been reported in a previous stud~~3, but as an exceptional
anti-PCNA and m SLE~, JOI32 in PM, and ACA and-70 in scleroderma 33 . The observation that certain are often with one
study we subdivided the patients according recently proposed criteria. Although our data confirm that these two antibodies are in general restricted to scleroderma patients, neither of them can be considered to be specific for any of the scleroderma
occurrence.
In
our
to
another (Sm-RNP, Ro/SSA-La/SSB) or are mutually (RNP-La/SSB, ACA-Scl-70) is’ also not new
exclusive
underlines the existence of serological subsets within the same syndrome. The presence of ’marker’ antibodies is, however, restricted to a relative minority of CTD patients. The most specificities are found in the
variants and thus able to discriminate between the three subsets. In spite of this fact, we confirmed that ACA were more frequent in the limited form of the disease,
225
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while anti-Scl-70 antibodies were more prevalent in the type III form. It is interesting to note from a serological standpoint that the type III form is not s~rolc~gi~ally distinct from the type II form. The absence of serological markers in Ray, as found by us and also described in some other studies’, is in some contrast with other series where ANA have been found in variable percentages in this condition&dquo;. These negative results are probably due to the more rigorous patient selection methods used by us, which made it possible to exclude patients with initial CTD. Finally, special attention should be directed to the group of patients with UCTD. This is a heterogeneous group showing a serological profile that is sometimes similar to SLE, scl~rc~derm.a or 1 asps. It is therefore likely that it represents, at least in certain cases, an early stage of welldefined CTD. Finally, the application of MCA to a large series of unselected patients with well-established forms of CTD made it possible to define more appropriately the precise relationship between ANA and other CTD. Although the application of this statistical approach did not disclose any unexpected data, it further emphasizes the clear relationship that exists between antibody specificities and certain disease states.
immunotaxonomy of connective tissue diseases. Scand J Rheumatol 56S: 49-57. 1985; 13. Passaleva A, Massai G, Ricci M. Advances in the laboratory
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(Received 16 September 1991) (Accepted 12 December 1991)
Antibody Profile in Italian
Patients with
Connective Tissue Diseases R.
NERI,
A.
TAVONI, R. CRISTOFANI*, C. LEVANTI, G. SODINI,
A.
D’ASCANIO, C. VITALI,
C. FERRI and S. BOMBARDIERI Clinical Immunology Unit, University of Pisa; *Department of Public Health and Biostatistics, University
of Pisa; and IFC-CNR,
Pisa, Italy
In the present work we report data on the specificity of antinuclear antibodies (ANA) in a large series of Italian patients suffering from a broad spectrum of connective tissue diseases (CTD), by using a series of homogeneous and validated techniques. The present study confirms, on the one hand, generally accepted concepts, i.e. that certain autoantibodies are strictly associated to certain disease states (such as anti-PCNA and anti-Sm in systemic lupus erythematosus, Jo1 in polymyositis, and ACA and Scl-70 in scleroderma); the presence of ’marker’ antibodies is, however, restricted to a relative minority of CTD patients. The application of a new methodological approach that considers the entire profile of ANA can greatly augment their diagnostic relevance and may provide useful indications for their interpretation, allowing us to establish for the first time the diagnostic usefulness not only of marker autoantibodies but also of certain associations between non-marker autoantibodies. Finally, the application of a more appropriate and powerful statistical tool (multiple correspondence analysis) has further emphasized the clear relationship existing between antibody specificities and certain disease states.
Key Words: Antinuclear antibodies Connective tissue disease Counterimmuno-
electrophoresis
Introduction
Italian patients with CTD. ANA were detected by means of a series of techniques that were recently validated during the course of multicenter Consensus Finding
Many autoantibodies that react with different nuclear components (ANA) such as nucleic acids, histones, and non-histone proteins have been described in the sera from patients with connective tissue diseases (CTD), and their detection and characterization appear to be very useful in the diagnosis and management of these diseases’-’. Some of these autoantibodies are considered to be markers of a particular disease state (such as antidsDNA2, anti-Sni&dquo; and anti-PCNA7.8 for systemic lupus erythematosus (SLE), or Sci-70 and anticentromere antibodies (ACA) for systemic sclerosis (SSc) and the CREST variant of scleroderma)&dquo;’. However, in most cases only an association among different specificities or a particular antibody profile may be indicative of a particular CTD or clinical subset4, 12-14. While there is general on the above concepts, discordant have been reported concerning the prevalence of the autoantibodies,. These differences often are not ascribable to genetic or environmental factors&dquo;, but rather to the different patient selection, or to the differing sensibility or specificity of various laboratory tests&dquo;&dquo;. In the present work we report data on the specificity of ANA in a large series of
Studies 19. Patients and methods Patients A total of 1457 sera from 615 consecutive patients with various CTD who were attending the Clinical Immunology Unit of Pisa were tested between 1987 and 1989. The patients included 155 with SLE, 78 with primary Sj6gren’s syndrome ~i °5~~~ 18 with mixed connective tissue disease 23 with idiopathic Raynaud’s phenomenon (Ray), 87 with rheumatoid arthritis (RA), 33 with unclassified connective tissue disease (UCTD), nine with polymyositis 54 with mixed cryoglobulinemia (MC), and 157 with SSc. The diagnoses of SLE, RA and MCTD were made according to well-established criteria2°-22. The diagnosis of 1 °SS was based on clinical symptoms of dry eyes and dry mouth, and confirmed by the objective demonstration of xerophthalmia (positive Schirmer’s test) or
keratoconjunctivitis sicca (positive rose Bengal staining) and
Correspondence: Rossella Neri, M.D., Clinical Immunology Unit, University of Pisa, via Roma 67, 56100 Pisa, Italy.
on
the presence of salivary-gland involvement and/or hydrostatic sialography) in
(positive lip biopsy 221
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the absence of other associated diseases. For SSc, three subsets were considered in which the distinction hinged primarily on the distribution of cutaneous involvemene3: the first (type I) with sclerodactyly only, the second (type II) with skin stiffness proximal to the metacarpophalangeal joints with sparing of the trunk, the third (type III) with diffuse stiffness of the skin including the trunk. The diagnosis of Ray was made when a positive cold photoplethysmography test was not associated with the presence of signs and/or symptoms typical of CTD, and after careful exclusion of traumatic or compressive local factors. The diagnosis of PM was based on the presence of at least two of the following: elevated serum muscle enzymes, abnormal electromyogram, and/or typical muscle biopsy in the absence of other CTD. MC was defined as previously reported2B and in particular in the presence of typical symptoms (arthralgias, purpura with or without systemic involvement) and signs (cryoglobulins with rheumatoid factor activity) and after the exclusion of other known autoimmune, infectious or neoplastic diseases. Patients with UCTD included those with clear features of a diffuse connective tissue disorder, but who did not meet the diagnostic criteria for any other defined disease. Ninety-two sera from 92 healthy subjects were used as controls.
Methods Anti-extractable nuclear antigen (ENA) antibodies were detected by counterimmunoelectrophoresis (CIE) as. described by Bunn et al.2s A commercial preparation of lyophilized rabbit thymus extract (Pel Freeze Biologicals, Roger, Ark.) was used as the source of antigens for the detection of anti-Sm, anti-RNP, anti-La/SSB, anti-PCNA, ~.nti-Sl and anti-Jol antibodies. Extraction was carried out according to the method described by Kurata and T~.n~~; the material was adjusted to a final concentration of 5 mg/ml and stored in aliquots at - 70°C until use. Human spleen extract was used for anti-Ro/SSA antibody detection; it was prepared according to Venables et al.17. Briefly, human spleen homogenate dialysed phosphate buffered saline was centrifuged for 20 min at 9500 x g. The ultracentrifuged supernatant was then added to a DEAE-52 cellulose column. The fraction eluted from ion-exchange chromatography with 0.35 M NaCI, pH 7.2, was used as the source of Ro/SSA (at a concentration of 0.5 mg/ml). ~,nti-S~l-’7~ antibodies were detected by CIE, essentially as described above but with care taken.to use fresh and more highly concentrated (lOmg/ml) rabbit thymus extract. Reference sera, kindly provided by Dr M. l~,ei~hlin (USA) and Dr ~.1~.~. Hughes (UK), were used to define antibody specificity. Indirect immunofluorescence was employed for the anti-dsDNA and ACA
detection using Crithidia luciliae8 and as
HEp-2 cell lineS21
substrate, respectively.
Statistics Pearson’s correlation coefficient and the chi-square test were used to determine both the correlation between antibodies and the correlation between diseases and the presence and degree of antibody specificities. Multiple correspondence analysis (MCA) was then used to analyse the data; this is a descriptive method, a particular type of factor analysis that is specific for categorical data30. It summarizes in a single graph all the information contained in the two sets of variables (in this case, disease and antibody specificity) that are to be correlated. Briefly, MCA involves translating the data by means of a complete disjunctive code that replaces each level of response with a new variable, which is assigned a value of 0 or 1. If ~ variables are recorded on n subjects we obtain a table with n rows and p columns. From a geometrical point of view each disease corresponds to a point in a multidimensional space, each dimension of which represents a different antibody specificity. Similarly, each antibody specificity is represented by a point in this space, each dimension of which corresponds to a different disease. In this multidimensional space the diseases (and antibody specificities) are arranged in ‘clouds’ of points that can be visualized only by their pro-
jection on appropriate planes. We initially found the direction of maximal variance of the cloud; this direction is called the first factorial axis (fl); we then searched for a second direction uncorrelated to the first one, which is the second factorial axis (f2), and so on. The final aim is to replace the original variables by a smaller number of quantitative variables, the factorial axes, that can optimally summarize the information. As each axis explains a decreasing percentage of the ’multidimensional’ variance that underlies the information, we can consider that part of the total variance explained by the first three factorial axes. In conclusion, the results of MCA can be represented by a series of plots that relate to pairs of factorial axes and show the projection for the modalities of antibody specificities and diseases on the factorial planes. The distance of the points frorn. the origin of the axes is an expression of the contribution of the variables to the factorial axis (and is proportional to the significance of the variable), while the proximity of the variables to each other is an indication of the strength of their
relationship. Results The most
patients
important epidemiologic findings on the reported in Table L Table II shows the
are
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Table I
SLE
Main
epidemiological data
for 615 Italian
patients
with various CTD.
systemic lupus erythematosus; MCTD mixed connective tissue disease, 1 °SS primary Sj6gren’s syndrome; PM polymyositis; MC mixed cryoglobulinemia; RA rheumatoid arthritis; Ray idiopathic Raynaud’s phenomenon; SSc systemic sclerosis; UCTD unclassified connective =
=
=
=
=
=
=
=
=
tissue disease.
SLE
systemic lupus erythematosus; MCTD mixed connective tissue disease; 1°SS = primary Sj6gren’s syndrome; PM polymyositis; MC mixed cryoglobulinemia; RA = rheumatoid arthritis; Ray idiopathic Raynaud’s phenomenon; SSc systemic sclerosis; UCTD unclssified connective =
=
=
=
=
=
=
tissue disease. I
When multiple specimens were a~rai3abl~ for the same patient, the prevalence was calculated for the first specimen drawn by date.
The most common and significant serological correlations found are shown in Table III. Certain ANA specificities were closely related, while other specificities were mutually exclusive. Furthermore, although anti-Sm antibodies never occurred in the absence of anti-RNP, and anti-La/SSB rarely occurred without Ro/SSA, both anti-RNP and anti-Ro/SSA could be found alone.
prevalence of specificities in the different CTD. When multiple sera from the same patient were available, this prevalence was always calculated for the first drawn by date. Anti-dsDNA antibodies were more frequent in with SLE (63%) than in females (30%) and the simultaneous presence of anti-Sm and anti-RNP antibodies was found in 9% of the female and in 18% of the male patients. Anti-La/SSB antibodies were not found in the male patients. Patients with SSc had an extremely distinct ANA profile, characterized by the presence of particular specificities rarely found in other CTD, such as Scl-70 and ACA. In type III scleroderma, the presence of antiScl-70 antibodies was higher in males (83%) than in
Table III
Serological relationships.
females (56Vo). UCTD patients represented
a serologically heteroand geneous group practically all of the abovementioned specificities were present. All sera from normal subjects were negative. .
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Diagnostic relevance of single antibody specificities antibody associations.
Table IV or
SLE = systemic lupus erythematosus; PM = pc~lymyositis; SSc = systemic sclerosis; MCTD = mixed connective tissue disease; lOSS primary Sj6gren’s syndrome; UCTD = unclassified connective tissue disease; others other connective tissue diseases. =
=
’Chi-square: 13.44; ~~hi-~quar~: 62.5; ’chi-square:
Table IV shows the
7.79.
antibody specificities and anti-
body associations that were found only in certain disease states and therefore could have great diagnostic r~levance (marker antibodies), and antibody specificities and antibody associations that were strongly indicative of only certain clinical conditions. Serial measurements of the ANA profile were available for a large proportion of the patients with SLE and I °SS; qualitative changes (positive to negative and vice
versa) occurred much more often in SLE than in 1 ass (Figure 1). In SLE, the prevalence of the ANA specificities increased consistently when multiple specimens were examined during the course of the illness; these changes were much more pronounced for anti-dsDNA, anti-RNP and anti-Sm than for anti-Ro/SSA and antiLa/SSB. Finally, we attempted on the basis of the above data to define more precisely the relationship between the antibodies and diseases means of a more comprehensive statistical approach, i.e. MCA. Figure 2 is a graphic representation of the results of the MCA analysis, showing the projection of the variables (diseases and specificities) on the factorial planes. In Figure .2a the first axis, characterized by the SLE patients, shows a clear-cut distinction between the SSc variants (right) and SLE, MCTD and 1°SS (left); the controls, Ray, MC and RA are in between. The second axis, characterized by the MCTD patients, shows the distinction between 1 °~ a on
Figure 1 Variation in ANA positivity on follow-up. The time interval between multiple samples is not always the same but in most cases it is approximately 3 months.
the
hand and SLE and MCTD on the other. This demonstrates the clc~se association of 1 °SS with graph anti-La/SSB antibodies and, to a lesser extent, with anti-Ro/SSA antibodies. Also evident is the association of SLE with anti-dsDNA, anti-Sm and anti-RNP antibodies, and MCTD with anti-RNP antibodies. Examination of the contribution of the diseases and autoantibodies to the first and third axes (Figure 2b) helps to clarify further the distinction between the SLE and sciernderrna variants; in addition, it allows a better definition of the association between the type I form of scleroderma and ACA, and between the type II and type III forms and anti-Scl-70 antibodies. In the figure the one
variables
are not
represented because they
tended to concentrate in a cloud close to the origin of the axes.
Discussion The analysis of this large series of patients suffering from a broad spectrum of CTD confirms the high diagnostic value of the ANA profile. Although in’soane studies up to lt3~l~ of the controls tested were found to
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majority, if not all, of the CTD that have been studied. The present work, which analyses a large number of CTD by means of well-established and generally validated techniques, makes it possible to confirm the high diagnostic value of certain antibody associations. Antibodies directed against certain antigens such as RNP or Ro/SSA, while not specific per se, can assume diagnostic significance when considered together. For instance, the presence of anti-Sl alone, or anti-dsDNA alone or in association with anti-La/SSB-Ro/SSA, or the contemporaneous presence of over four different specificities can all be considered highly specific for SLE. In addition, the association of anti-La/SSB-Ro/ SSA is closely linked to i ~’S~, while the presence of anti-RNP alone was almost invariably found only in MCTD. A consideration of the entire profile could greatly augment the diagnostic relevance of ANA and may provide useful indications for their interpretation. In view of the results obtained here, it appears rather surprising that this global approach has up to now been somewhat overlooked in spite of the extensive literature on the ,
subject. Although the prevalence of the various ANA in our series is, on the similar to other large series that have been reported 12, 34, certain discrepancies deserve special attention. For instance, anti-Sm antibodies were found in only 1 2 Vo of our SLE patients, but this percentage increased when multiple specimens from the same patient were taken into account. This incidence is certainly lower than that reported for the North American population’, &dquo; but is in keeping with most recent European studies’6. Both environmental, ethnic37 and methodological factors&dquo; may help to explain these differences. Finally, the prevalence and specificity of the autoantibodies found in patieqts with scleroderma and scleroderma variants are worth noting. In our series, anti-Scl-70 antibodies were present in a much higher frequency than has been reported previously&dquo;, &dquo; although comparable results were recently reported by others-&dquo;.
Figure2 Projection of the variables on the factorial planes (a) fl-f2 and (b) fl-f3 showing the relationship between antibody specificities and diseases. The distance of the points from the origin of the axes is proportional to the significance of the variable, while the proximity of the variables to each other is an indication of the strength of their relationship. SLE systemic lupus erythematosus; MCTD mixed connective tissue disease; t°SS primary Sj6gren’s syndrome; MC mixed cryoglobulinen-da; RA rheumatoid arthritis; Ray idiopathic Raynaud’s phenomenon; I systemic sclerosis type I; 2 systemic sclerosis type II; 3 systemic sclerosis type III; NHS normal human subjects; ACA anticentromere antibodies. =
=
=
=
=
=
=
=
=
=
=
have ANA16,31, most authors report the total absence or negligible incidence of specific ANA 12, which is consistent with our findings. The present study confirms, on the one hand, gener-
In contrast, the level of ACA in the three subsets was generally lower than that reported in the literature. It is that these results the frequency of ACA and Scl-70 antibodies in SSc may be due
ally accepted i.e. certain autoantibodies are restricted to certain disease states, such as
to
the lack of uniform classificative and to
methodological problems. Their co-existence has been reported in a previous stud~~3, but as an exceptional
anti-PCNA and m SLE~, JOI32 in PM, and ACA and-70 in scleroderma 33 . The observation that certain are often with one
study we subdivided the patients according recently proposed criteria. Although our data confirm that these two antibodies are in general restricted to scleroderma patients, neither of them can be considered to be specific for any of the scleroderma
occurrence.
In
our
to
another (Sm-RNP, Ro/SSA-La/SSB) or are mutually (RNP-La/SSB, ACA-Scl-70) is’ also not new
exclusive
underlines the existence of serological subsets within the same syndrome. The presence of ’marker’ antibodies is, however, restricted to a relative minority of CTD patients. The most specificities are found in the
variants and thus able to discriminate between the three subsets. In spite of this fact, we confirmed that ACA were more frequent in the limited form of the disease,
225
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while anti-Scl-70 antibodies were more prevalent in the type III form. It is interesting to note from a serological standpoint that the type III form is not s~rolc~gi~ally distinct from the type II form. The absence of serological markers in Ray, as found by us and also described in some other studies’, is in some contrast with other series where ANA have been found in variable percentages in this condition&dquo;. These negative results are probably due to the more rigorous patient selection methods used by us, which made it possible to exclude patients with initial CTD. Finally, special attention should be directed to the group of patients with UCTD. This is a heterogeneous group showing a serological profile that is sometimes similar to SLE, scl~rc~derm.a or 1 asps. It is therefore likely that it represents, at least in certain cases, an early stage of welldefined CTD. Finally, the application of MCA to a large series of unselected patients with well-established forms of CTD made it possible to define more appropriately the precise relationship between ANA and other CTD. Although the application of this statistical approach did not disclose any unexpected data, it further emphasizes the clear relationship that exists between antibody specificities and certain disease states.
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21.
22.
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(Received 16 September 1991) (Accepted 12 December 1991)
