Multiparameter ELISA testing for autoantibodies in rheumatology

CLINICAL

Perspective on A u t o i m m u n i t y David E. Normanseli University of Virginia Health Sciences Center, Charlottesville, Virginia

lmost 10% of the population suffers from some form of autoimmune disease, in which an immune response is mounted against normal body constituents. Why these diseases occur is not known, but autoantibodies are often present in the circulation, particularly in older females. Although the presence of such autoantibodies doesn't necessarily mean that there is ongoing autoimmune disease, if autoimmune disease is present, then the presence and level of specific autoantibodies can be used as a marker for the disease. In certain cases the level of specific autoantibodies can be used to follow the progress of the disease. Most autoimmune processes are very poorly understood. Even when there is a clear involvement of antibodies, such as in

A

autoimmune hemolytic anemia, and even when the antibodies are responsible for the features of the disease, as in the destruction of erythrocytes by antibody and complement, the stimulus for the initial production of the antibodies is frequently not known; it is presumed to be either a cross-reaction or the result of a cellular immune response to an unknown antigen with production of antibodies to the released cellular components. Among the many questions about autoimmunity are (a) what is the nature of the autoantigens involved, Co) is there a relationship to an infectious agent, (c) how is genetic susceptibility involved, and (d) why do the normal mechanisms guarding against autoimmunity sometimes fail? Although there are no complete answers to these

questions, some progress has been made, especially regarding some of the antigens involved and the autoantibodies that react with them. Many of the antigens involved are intracellular proteins that appear to be important in cellular processes, for example, the spliceosome. This issue of Clinical Immunology Newsletter will focus on the nature of some of these autoantigens and their antibodies, with special reference to systemic lupus erythematosus. In the first article, Dr. Marius Teodorescu will discuss laboratory testing for autoantibodies, particularly a multiparameter ELISA for ANAs and strategies for defining specific testing pathways. In the second article, Dr. John Davis will discuss how data generated by the dicontinued on page 50

M u l t i p a r a m e t e r ELISA T e s t i n g for Autoantibodies in Rheumatology Marius Teodorescu University of lllinois College of Medicine, Chicago, Illinois

The ANA and Related Tests: Communication Between Clinical and Laboratory Immunologists here are probably over 100 different combinations of tests, policies, or "habits" involving tests for antinuclear antibodies. Since a similar end-result of all these combinations for patient diagnosis and management is very unlikely, the potential value of these tests is

T CIMNDC 12(4)49--64

Elsevier

largely unrealized. In addition, there is very little standardization and generally poor communication between laboratory and clinical immunologists. The strategy proposed here for the use of ELISA autoantibody profile tests in the clinical context is based in part on published systematic studies and in part on the expericontinued on page 50 0197-1859,/92/$0.00 + 3.00

50 C L I N I C A L I M M U N O L O G Y

Newsletter

agnostic laboratories are used in patient management and how we in the laboratory can help the clinician.

Multiparamctgr ELISA Testing for A u t o a n t l b o d i e s in Rheumatology continued from page 49

ence of our laboratory. The tests discussed are the ANA, the ANA specificity prof'des-- ANA/6 (ssDNA, dsDNA, Sin, RNP/Sm, SSA, and SSB) or ANA/8 (ANA/6 plus histone and Sol 70)--- IgM, IgG, IgA rheumatoid factor, and IgM, IgG, IgA antieardiolipin antibody. When an ANA test is requested by the clinician, usually without providing a diagnosis or at least a suspicion, it is most likely performed by immunofluorescence (F-ANA) on Hep 2 cells. With very few exceptions, the laboratory reports the results without information as to the type of ceils used or the Ig class of the antibody detected. When a test is positive (15%-70%, depending on laboratory "habits," standardization, and/or patient population), some clinicians do not request any additional immunologic tests. They base their diagnosis and therapeutic decisions only on clinical data and/or investigations thought to reflect organ damage (e.g., biopsy, urine tests, spinal fluid analysis). Others request a second round of tests for specific antinuclear antibodies, sometimes based on titer and pattern of F-ANA. Although much is sometimes made of patterns, they have little real meaning since the well known patterns (speckled, homogeneous, nucleolar, etc.) may be obtained by staining the cells with different monoclonal antibodies specific only for phospholipids. 13 The ANA test, included among the diagnostic criteria for SLE, is extremely sensi-

I

Vol. 12' No. 4, 1992

In the next issue, Dr. Robert Lahita will discuss three antigen-antibody systems which occur in lupus: the Ku-anti Ku sys-

tern, the Smith D-anti Smith D system and an apolipoprotein-anti apolipoprotein system. CIN

tive (96%--98%), but has low diagnostic specificity (40%); thus, it is used primarily to exclude SLE.23 Positive ANA test results are reported in patients with a wide variety of conditions, in healthy elderly (10%-37%), and in many nonelderly normals. The rate of positive tests m normals depends also on the class of antibody identiffed (IgG specific tests are less frequently positive), the use of the international standard. 11 the technician reading the slides, etc. Nevertheless. antinuclear antibodies are

positive") and abnormal ("pathologic") ANA. a second round of tests may be requested and frequently involves anti-dsDNA and sometimes other tests: anti-Sm, RNP, SSA and SSB, Scl 70. anti-histone, etc. The laboratory performs the tests by different methods or combinations of methods: double diffusion, counterimmunoelectrophoresis, ELISA. dot-blot or western blot, hemagglutination. radioimmunoassay, immunoftuorescence. etc. 24 In most instances the laboratory provides no information as to the method used for each one of the tests or the Ig classes detected. In contrast, some laboratories found it cost effective to screen by F-ANA and specimens with over 6.2 I.U. of ANA are reflex-tested by ANA/6 or ANA/8: a final result is issued. By this procedure the labor intensive titration of ANA is eliminated. In fact. the value of the end-point titration is only relative, since patients with very high titers may not have any condition associated with antinuclear antibodies and patients with titers as low as 1/80 may have active SLE.

T h e precision and accuracy of a quantitative ELISA is influenced primarily by the intrinsic differences in antibody heterogeneity between the standard (heterologous standard) and the analyte (patient serum antibody). present at various levels in normal donors and they may play a significant role in immune homeostasis. Indeed. western blotting of nuclear antigens showed substantial differences in the specificity of IgG antibodies for different protein bands in normal and SLE sera. SLE lgG antibodies reacted at various intensities with upwards of 55 proteins (HeLa cell extract), whereas normal sera recognized only 17. Furthermore, it was clear that IgG from SLE patients recognized proteins that were distinct from those identified by IgG from n o r m a l s . 19

To differentiate between normal ("false-

Testing for Spgcific Antinuclear Autoantibodies Tests for specific antinuclear antibodies are requested when 1) clinical data strongly suggests SLE or a related condition and the F=ANA test is negative; 2) there are insufficient criteria for formal diagnosis of SLE; 3) there is a low clinical probability f o r S L E b u t a positive ANA test and, therefore, a need for "reassurance": 4) there is the need for a quantifiable immunological parameter to follow with clinieal evolution; or 5) as a reflex follow-up Of a positive F-ANA screening test. Since diagnosis relies both on the pres-

CLINICALIMMUNOLOGTNEWSLETTER(I$SN 0197-1859) is issued monthly in one indexed volume per year by Elsevier Science Publishing Co., Inc.. 655 Avenue of the Americas, New York, NY 10010. Subscription price per year. $145.00. For surface airlift add $43.00. Second-class postage paid at New York, NY, and at additional mailing off'rues. P~stmast~r: Send address changes to

Clinical lnmumology Newsletter,Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010. NOTE: No responsibility is assumed by the Publisher for any injury and/or damage to persons or p r o p e ~ as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, produclS, instructions or ideas contained in the material he~in. No suggested test or procedure should be carried out unless, in the reader's jud~'nem, its risk is justified. Because of rapid advances in the medical sciences, w e recommend that the independent verification of diagnoses and drag doses should be made. Discussions, views and recommendations as to medical procedures. choice of drags and drag dosages are the responsibility of the authors.

0 1 9 7 - 1 8 5 9 / 9 2 / $ 0 . 0 0 + 3.00

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C L I N I C A L I M M U N O L O G Y Newsletter 51

Vol. 12, No. 4, 1992

U of I R h e u m / I m m u n e

Lab

Report:

Patient: MS. PATIENT, Physician: DR. XYZ Inst: C L I N I C Drawn: 0 4 / 3 0 / 9 2 Reported: 0 4 / 3 0 / 9 2

EL-ANA8 A n a l y t i c a l Sample ID: 98765 R e p o r t e d By:

R e s u l t s e x p r e s s e d as U n i t s / m l Test

P a t i e n t Normal High Result Max Value

(ELISA m e t h o d - I g G )

ssDNA Quantitative dsDNA Quantitative Sm Q u a n t i t a t i v e RNP/Sm - Quantitative SSA (Re) Q u a n t i t a t i v e SSB (La) Q u a n t i t a t i v e Histone - Quantitative Sci-70 Quantitative

872 553 386 785 234 125 436 26

99 81 140 105 65 44 139 49

i000 i000 1200 1200 1200 I000 i000 500

1,000

600 -

400 -

200

I

I i

i

ssDNA

dsDNA

i Normal Range I

Sm

!

RNP/Sm

SSA

~Test

Result

SSB

T h e r a T e s t Labs, Inc. E L - A N A p r o f i l e (c) 1991 Interpretation (valid o n l y in c l i n i c a l context) [] N o r m a l [] S j o g r e n ' s S y n d r o m e [] C h r o n i c I n f l a m m a t i o n [] M C T D ~] SLE [] S c l e r o d e r m a Active Disease [] D r u g I n d u c e d SLE

If

Histone

Sci-70

[] 2% of "normals" ~ Suggest Retest in 4 to 6 w e e k s [] I n t e r p r e t a t i o n d e f e r r e d to c l i n i c i a n

Figure 1. Profile of eight autoantibodies (ANA/8) in patients with positive ANA suspected of SLE (I'ERIS© progrean, TheraTest Labs Inc., Chicago, IL). The values in the small open columns represent nomml ranges. The dots represent levels of autoantibodies. The absorbance values obtained in the absence of antigen (specimen blanks) were subtracted and the absorbance values convened to units/ml. The antigens on solid phase were ssDNA: denatured, single stranded call thymus DNA; dsDNA: double stranded bacteriophage lanabda DNA; Sin: Sm antigen from calf thymus; RNP:RNP/Sm complexes from bovine thymus; SSA: SSA antigen from bovine spleen; SSB: antigen from bovine thymus; histone: total bovine thymus histone; Scl 70 was obtained from bovine thymus.

ence and on the absence of certain autoantibodies, detection of a particular antibody is most useful when it is incorporated into a uniformly sensitive and specific assay system that measures all autoantibodies. ELISA appears to be most suitable for this

purpose 24 and is becoming the method of choice in many clinical laboratories. Unfortunately, faulty design or use of ELISA may generate invalid results. For example, since a binding agent may be needed to attach dsDNA to plastic, the same agent has © 1992 Elsevier Science Publishing Co., Inc.

to be present in the solution as a competitor. By disregarding this basic principle, some investigators 2 have concluded that ELISA is unreliable for anti-DNA antibody tests. In general, the higher the test sensitivity of an immunoassay the lower its diagnostic specificity. Although ELISA has high sensitivity, it can maintain specificity through appropriate adjustment of various parameters: antigen purity, type of enzyme and substrate, amount and specificity of second antibody, period of incubation, use of external standards to perform quantitative analysis, etc. 9 ELISA grade native nonhistone antigens are usually purified by immunoaffinity chromatography from mammalian tissues. They may be also obtained from recombinant DNA expression systems, provided they contain all components, maintain antigenic determinants, and are not contaminated with bacterial antigens and/or [3-galactosidase from the fusion protein. For dsDNA, plasmid or bacteriophage DNA are the most appropriate sources. 5 High affinity IgG antibodies are most often associated with active disease, 23 an apparent drawback for ELISA, which detects both high and low affinity antibodies. However, when the epitope density of the antigen on the solid phase is sufficiently low, high affinity antibody is preferentially detected. 21 This finding may explain the good correlation between the IgG autoantibody level determined by ELISA and disease activity. 5 The precision and accuracy of a quantitative ELISA are influenced primarily by the intrinsic differences in antibody heterogeneity between the standard (heterologous standard) and the analyte (patient serum antibody).9 Differences in the diversity of epitopes identified either on the same molecule or on different polypeptides in a complex antigen (e.g., Sm, RNP) and the affinity heterogeneity of an antibody population make standardization only a relative process. Thus, ELISA for ANAs is most valuable when multiple antibodies are detected and when it is used to compare serum samples from the same individual undergoing longitudinal observation. The ELISA method used to generate ANA/6 or ANA/8 profiles5 (Figures 1 and 0197-1859/92/$0.00 + 3.00

52 C L I N I C A L I M M U N O L O G Y Newsletter

I,ooo

1 .ooo

400

400

20O

200

ssDNA 1

Vol. 12, No. 4, 1992

dsDNA

Sm

RNP/Sm

SSA

S~B

His~one

Sc 1-70

multiparameter specificity test for antiphospholipid antibodies, which is an issue that needs further evaluation. There are practical advantages to performing multiple tests by ELISA: only one work-station for all immunoassays, the same pipetting for all tests, and predetermined order of antigens or antibody classes on the plate to minimize error.

ssDNA

dsDNA

Sm

RNP/Sm

SSA

SSB

Anti-ssDNA, Anti-dsDNA, and A n t i - H i s t o n e Antibodies

For over 25 years anti-DNA antibodies have been known to correlate with disease activity in SLE, particularly renal disc ease. 23 Furthermore. an elevation in anti8o dsDNA antibodies has been reported to predict flare s. 2~ - Anti-dsDNA is detected routinely by any of the following methods: Crithidia luciliae immunofluorescence (the most widely used and probably the q~ least useful); Farr radioimmunoassay with plasmid, E. coil bacteriophage, or calf thymus DNA: ELISA or dot-blot with calf thymus or phage DNA, etc. Any of these ssDNA dsDNA Sm RNP/Sm SSA SSB Histone Sci-70 SSDNA dSDNA Sm RNP/Sm SSA SSB tests may be qualitative, semiquantitative, or quantitative. Some detect only anti-dsDNA while others may detect both anti-ssDNA and anti-dsDNA. Most often the clinician is not informed as to the type of test used or as to its specificity and sensitivity: some of the manufacturers of test kits gwe no information as to the type of DNA used. When interpreting the results of anti200 dsDNA antibody, two main issues deserve consideration: a) contamination of dsDNA 7 - ] with ssDNA, and b) class and/or affinity SsDNA dsDNA Sm RNP/Sm SSA SSB SSB Historic Sci-70 0 SS~NA ds~NA S~m RNP/Sm (or avidity) of the antibody measured unFigure 2. Muitipammeterprofiles of autoantibodies in patients with various conditions. See legendfor Figure 1 der the conditions of the assay, lgG antifor setup and the description of the antigens. Clinical diagnosesfor these profileswere: A: SLE; B: proliferative dsDNa has high specificity constituting a glomemlonephritis and vascditis in an F-ANA negative patient; C: Sj6gren's syndrome; D: subacute. F-ANA- "gold standard" for SLE, whereas anti-ssnegative.SLE; E: seleroderma: F: MCTD. See legend to Figure 1. DNA, like anti-SSA, SSB, RNP may be found in a variety of other autoimmune conditions) "8'24 However, high values of anti-ssDNA are "almost entirely associated graphically (Figures 1 and 2). International 2) may be summarized as follows. Wells with active SLE. Simultaneous measureunits are used for anti-dsDNA; for other of microtiter plates are coated with antiment of anti-ssDNA and anti-dsDNA autoantibodies the units are on the same gens and are prearranged in a fixed sespecificities is most useful as part of an scale as for anti-dsDNA, which allows for quence (Figure 1). At least one well does ANA/6 or ANA/8 (Figure 2). plotting on the same graph. not contain antigen and is used as a blank To measure anti-dsDNA antibodies reliThe same method is used to detect rheuto correct for the variable concentration of ably, the preparation of dsDNA must be matoid factor and anticardiolipin antibody, IgG in the serum. After subtracting the free of ssDNA. Certain DNA preparations. although the parameters measured simultablank absorbance value, the computer calnotably calf thymus, are unstable even neously are different antibody classes culates s i m u l t a n e o u s l y the unit levels for when attached to solid phase so that the rather than specificities, However, some inall antibodies against calibrators and the resingle strands must be redigested with S 1 vestigators have advocated the use of a suits are displayed both numerically and

1,000

0197-t 859/92/$0.00 + 3.00

1,oo(

©'i992 Elsevier ~cieneePtCt~shingCo.. Inc.

C L I N I C A L I M M U N O L O G Y Newsletter 53

Vol. 12, No. 4, 1992

looo

,.12oo,

,soNI

800

[]

DN

I

_

l l osm

1000 800

[]

RNP/Sm

800

,,sA

6oo

[~

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400

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i

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Figure 3. Autoantibody levels followed during treatment in a patient with lupus nephritis. The antibody levels were determined by ELISA. 5 Anti-dsDNA is presented in international units.

nuclease immediately before using the DNA in the test. For unclear reasons, lambda bacteriophage DNA remains stably double stranded. 5 Whatever the source of DNA for ELISA or Farr assay, the possibility of a reaction with ssDNA remains. Thus, it is preferable to have a continuous quality control by detecting in parallel anti-ssDNA (denatured) and anti-dsDNA antibodies. Since almost all anti-dsDNA antibodies react with ssDNA but anti-ssDNA does not react with dsDNA, 23 the magnitude of the antibody reaction in terms of absorbance with ssDNA is either greater than or equal to that produced with dsDNA. If the "double stranded" character of the DNA on the solid phase has been maintained, control sera containing only anti-ssDNA should react with ssDNA (denatured) but not dsDNA (Figure 2D). The narrow focus on anti-dsDNA as the sole indicator of disease activity may have significant drawbacks. For example, antidsDNA has the tendency of decreasing rapidly under oral steroid treatment along with improvement of overall clinical presentation; other antibodies are much less influenced. However, oral steroid treatment does not prevent kidney failure, which is the end-result in about 80% of patients with nephritis. Thus, it appears that the utility of anti-dsDNA antibody as best indicator of disease activity needs reevaluation. In my opinion as long as one of the six autoantibodies persists the disease is only in apparent rather than real remission and

the progression of organ damage continues silently. Anti-ssDNA is found in over 90% of patients with active SLE, whereas antidsDNA (bacteriophage lambda DNA) is found in 75%. 5 Whether it is single or double, the pathogenic consequences are the same, since both antibody specificities are found in kidney deposits of immune complexes. In other words, it is far more preferable to detect anti-ssDNA antibody than no antibody at all, which is often the case of the low sensitivity (or rater limited spectrum) C. luciliae immunofluorescence test. The presence of anti-ssDNA antibody may complete the information on an ANA/6 profile (Figure 2D) and change its "pattern." When it is the only antibody detected, it suggests that "something is wrong" that needs further evaluation for a precise diagnosis. For example, antissDNA is found frequently in patients with anti-cardiolipin antibody, most likely the result of a cross-reaction.2° Anti-histone antibody measured by immunofluorescence is frequently obscured by the presence of anti-DNA antibody. By ELISA almost all patients with drug induced SLE (procainamide) and over 80% of patients with active SLE have elevated levels of anti-histone antibodies. ELISA can be used to detect antibodies against total histones, histone fractions, or antiDNA-histone complexes. Antibody levels in idiopathic SLE are generally lower than in drug-induced lupus in which the anti© 1992 Elsevier Science Publishing Co., Inc.

body is directed mainly against the H2a/H2b complex. 27 Similar to anti-ssDNA, anti-histone antibody is a sensitive chronicity marker in SLE, i.e., it indicates that the patient is only in "relative" remission.

Antibodies to Non-Histone Nuclear Antigens: Sm, RNP, SSA, SSB, and Scl 70 Anti-Sm and anti-RNP antibodies are distinct sets of antibody specificities that react with subcellular particles composed of polypeptides containing small nuclear RNA (U RNAs). Native immunoaffinity purified Sm antigen is readily available, whereas native RNP can only be obtained as an RNP/Sm complex. Therefore, two broad antibody specificities are measured: anti-Sm and anti-Sm/RNP complex5 (Figure 2A). Bovine thymus and spleen offer an abundant supply of these antigens. Detection of antibodies to the RNP/Sm antigen complex and not to Sm in the same specimen indicates the presence of only anti-RNP antibody (Figure 2F). If the serum reacts with both antigen preparations (Figure 2A), the level of anti-Sm is clearly identifiable, whereas the level of anti-RNP is only roughly estimated. However, when the serum reacts with both antigens it may be assumed that both antibody specificities are present, since anti-RNP is found together with anti-Sm antibodies in all or almost all patients. 4 Indeed, addition of relatively high concentration of purified 0197-1859/92/$0.00 + 3.00

54 C L I N I C A L I M M U N O L O G Y Newsletter U.of I. Rheum/Immuno

Lab

Report:

Vot. 12, No. 4, 1992 A C A Quantitative

Patient: BB ID0401, Physician: AC Inst: U.of I. Drawn: 05/14/92 R e p o r t e d : 05/15/92

(curve)

Sample ID: 3389 Reported By:

Results expressed as Units/ml Test

Patient Normal High Result Max Value

(ELISA method)

Quant ACA Quant ACA Quant ACA

(Curve) (Curve) (Curve)

- IgS - IgG - IgA

14

~

13o

95 40

15 6

200 200

i00-

8O

60

40-

20-

0

IgM Normal

IgG Range

IgA ~Test

Result

Interpretation (valid only in clinical context) Anti-phospholipid syndrome [] Suggest Retesting in [] IgM(+) X L M H Neg 4 to 6 weeks [] IgG(+) L x M H Neg [] IgA(+) L X M H Neg

Figure 4. The reportof resultsfor anticardiolipinantibody(I'ERIS©programprintout).

Sm antigen to sera that contained both anti- , anti-ssDNA and anti-dsDNA. Anti-Sm is a diagnostic marker for Sm and anti-RNP/Sm antibodies reduced SLE 23 more frequently found in Afrothe reaction with RNP/Sm antigen coated Caribbean I and Afro-American patients wells by only 20%. 5 Testing of several inthan in Caucasians; overall it is positive in dividual patient sera also showed a maxiabout 30% of patients with active SLE. mum of 20% competitive inhibition Anti-RNP, although very frequently found (unpublished data). Moreover, when pain SLE (>80%), is also found in most contents with both antibodies are followed nective tissue diseases. The highest levels longitudinally, anti-Sm becomes undetectare seen in mixed connective tissue disease able first, whereas anti-RNP/Sm may per(MCTD) where it is the only antibody desist. In parallel testing within the same tected on ANA/6 or ANA/8 (Figure 2F). ELISA system, a positive result for both Anti-Sm antibody level fluctuates with anti-Sin and anti-RNP/Sm offers an interchanges in disease activity,5'23 but also apnal control analogous to that obtained with omT:Issgtg~o.oo + 3.oo

© 1992EiS~vier'S~g;i~eVligi¢~fn'i' Co.. r.~.

pears to follow rather than precede clinical flares; under treatment it decreases more slowly than anti-dsDNA (Figure 3). Since underlying organ damage is likely to subside much more slowly than overt clinical symptoms, anti-Sm may be a valuable indicator of therapeutic efficacy, superior to anti-dsDNA (Figure 3): anti-dsDNA responds readily even after minimal therapeutic action. Elevation of anti-RNP antibodies has been reported to precede flares in both SLE 26 and in MCTD. 12 It also decreases with treatment and with decreased disease activity (Figure 3). The SSA and SSB antigens are a pair that behave immunologically very much like the Sm-RNP pair. SSA consists of 52 and 60 kDa polypeptides that associate with a species of hYl-hY5 RNA and SSB is a 46-48 kDa phosphorylated ribonucleoprotein involved in processing products of RNA polymerase III. 23 These two antibodies are often associated with primary Sjtgren's syndrome (Figure 2C), SLE, and RA. The overlap between SLE and Sjtgren's is also reflected in organ involvement. since about 30% of SLE patients have the lymphocytic infiltration of the salivary glands similar to that observed in Sjtgren's syndrome. 16 Anti-SSA is frequently seen in SLE without anti-SSB (Figure 2D). However. the presence of anti-SSB antib~gly is almost invariably associated with anti-SSA 23 (Figure 2C). Therefore. if a test specimen has anti-SSB and not anti-SSA antibodies, it may simply be due to technical reasons: a) the test sensitivity for anti-SSB is higher than for antiSSA: b) the anti-SSB levels are borderline; c) some SSA determinants are not available for appropriate interaction with antibody in the test system. Thus, for clinical purposes, if only anti-SSB is identified, it may be assumed that anti-SSA is also present in the serum. Anti-SSA has been shown to play a significant role in lupus nephritis for patients who may have this antibody and lack antidsDNA14: SSA antigen has been identified in glomerular deposits of immune complexes. 28 For diagnosis it appears that the presence of anti-SSA or anti-SSA and antiSSB may give some mdication regarding the likelihood of kidney disease, 28 since anti-SSA was detected by ELISA in 53% of nephritis patients while only 9% had co-

C L I N I C A L I M M U N O L O G Y Newsletter 55

Vol. 12, No. 4, 1992

oYer

2000

1500

over

ovw

200

400

150

-

Anti-Phospholipid Antibodies

300

0 ~ 1000-

•

200

50 •

I00

100

5O0

I

IgM

IgG

IgA

Yeues within n orrne limits

Figure 5. Report of results for lgM, lgG. and IgA rheumatoid factor. For lgM the results are in international units.

existing anti-SSB. Moreover, in SjOgren's syndrome, where nephritis is rare, the coexistence of anti-SSA and anti-SSB in high. 23 Anti-SSA has been associated frequently with cutaneous manifestations of SLE, either as subacute SLE (i.e., ANA negative lupus [Figure 2D]) or as a neonatal rash. This association may be due to the fact that exposure of cells to UV light causes expression of SSA on the cell surface of keratinocytes.6 Anti-SSA antibodies have also been associated with congenital heart block~ However, since a large proportion of babies born to anti-SSA positive mothers do not have heart block, the heart block may or may not be due to anti-SSA antibody. The levels of anti-SSA may vary with disease activity, and, analogous to antidsDNA, anti-SSA antibodies have been reported to increase significantly prior to a flare of lupus nephritis, x7 However, the anti-SSA decreases much slower than other antibodies under treatment (Figure 3); it appears that as long as anti-SSA persists, real remission has not been obtained and a careful follow-up of creatinine levels is indicated. When measured by ELISA, anti-SSB levels also appear to change with disease activity5 (Figure 3). Scl 70 is the 100 kDa DNA topoisomerase I and is found in about 30% of patients with scleroderma23 with relatively high degree of specificity. So far there is no documentation as to changes in anti-ScI 70 levels in relationship to disease activity.

These antibodies may sometimes be responsible for a positive ANA. 13 The anticardiolipin antibody (ACA) test is performed on patients who have thromboembolic manifestations (mainly CNS), recurrent fetal wastage, pulmonary hypertension, etc., or on patients with SLE. The frequent association between lupus anticoagulant and anticardiolipin antibodies appears to be only temporal, since different antibody molecules are responsible for the two phenomena, z5 Clinical manifestations associated with anti-phospholipid antibodies appear to correlate better with antibodies to a combination of phospholipids 1° than to an individual phospholipid, including cardiolipin. In active SLE, 40% to 60% of patients usually have low to moderate elevations of IgG anti-cardiolipin antibody, whereas in primary antiphospholipid syndrome the levels are moderate to high (Figure 4).

Rheumatoid Factor In addition to ANA, rheumatoid factor (RF) is one of the most frequently used tests in clinical immunology. RF reacts with human or rabbit Fc of IgG (E epitopes). Classical rheumatoid factor is an IgM antibody and agglutinates latex particles coated with human IgG or sheep red cells treated with rabbit anti-sheep red cell antibody. 3 Nephelometry with human IgG as the antigen is the most widely used method. The ELISA test for rheumatoid factor (Figure 5),4 which we have used routinely, detects IgM, IgG, and IgA rheumatoid factors directed against rabbit IgG. The use of rabbit IgG and the detection of all three classes offer superior specificity for rheumatoid arthritis. 3 IgG RF has been associated with vasculitis in RA TMand IgA RF has been described as being associated with erosive disease. Along with radioimmunoassay, ELISA is considered the method of choice for detecting rheumatoid factor. 7 The method used to detect all three classes follows the principle of technical simplicity of ANA/6 and ACA and may become a routine assay in the clinical laboratory. Briefly, serum is added to minitubes containing diluent for IgM, IgG, and IgA, all organized in a prearranged design for

© 1992 Elsevier Science Publishing Co., Inc.

easy transfer to the antigen-coated plate. The rack with IgM and IgA tubes is placed in the refrigerator and the tubes for IgG (which contain pepsin) are placed at 37°C. The following day the pepsin digestion is stopped, all tubes are reunited on the same rack, and, with the help of a multichannel pipette, the contents are transferred to the plate and developed with specific conjugates.

Multiparameter ELISA Testing and Diagnosis The diagnostic value of the multiparameter autoantibody profile exceeds that of any individual autoantibody test performed separately. It also far exceeds the value of F-ANA titration, although it is only slightly more expensive. The best use of resources is to screen by F-ANA or similar procedure and test as reflex by ANA/6 or ANAJ8 test. Since all antigens and the blank are on the same strip of wells in a fixed order, the number of strips used is equal to the number of specimens. This approach also eliminates the need for handling of a new specimen from the same patient. Profiles may be interpreted by the laboratory based on the presence or absence of various combinations of antibodies. The patterns created by multiparameter ANA testing (Figures 1 and 2) may be correctly interpreted by the laboratory in most cases, although a results is valid only in clinical context. Here are some rules of interpretation for new, untreated patients. (The name of the antibody is replaced by the antigen.) SLE sera may generate as a multitude of profiles; so far we have not seen a patient with active SLE with normal profile on the panel (>6,000 assays) (Figures 1 and 2): 1) high ssDNA; 2) dsDNA + ssDNA (Figure 2B); 3) Sm and RNP/Sm (Figure 2A); 4) any four to seven antibodies on ANA/6 or ANA/8, with at least one at high level (Figure 1); 5) any combination of two or three antibodies with moderate to high ssDNA; 6) histone in addition to two other antibodies on ANA/8, without Scl 70; 7) SSA only, or SSA and ssDNA (Figure 2D) (mainly in subacute, sometimes ANA negative SLE). IgG ACA is present in about 60% at low to moderate levels in SLE. When the ACA levels are high the possibility of anti-phospholipid antibody 0197-1859/92/$0.00 + 3.00

56 C L I N I C A L I M M U N O L O G Y Newsletter

Vol. 12. No. 4, 1992

CLINICAL

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probability-- exclusion--of SLE. Explanations in the text.

syndrome as the cause of clinical manifestations rather than SLE should be considered (Figure 4). Drug-induced SLE appears as 1) moderate to high histone, 2) low to moderate ssDNA, and 3) low to moderate ssDNA plus histone. At the same time all other values are within normal limits. MCTD may appear as high RNP/Sm and normal levels for the remaining tests I

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on ANA/6 or ANA/8 (Figure 2F). If Sc170 is also positive it is likely that the patient has or may develop scleroderma. Scleroderma may appear as 1) Sc170 at any level with no other antibody (Figure 2E), 2) Sc170 plus low to moderate RNP and/or SSA, and 3) all normal on ANA/6 or ANA/8. In some patients the serology overlaps with SLE along with clinical presentation and organ involvement. . . . . . I!. I1_.! I i] I L~. I IIII © 1992 Elsevier Sd6nce Publishing Co.,Inc.

Sjdgren's may have 1) SSA and SSB at any level (Figure 2C), 2) low ssDNA plus SSA and SSB, and 3) SSA only without SSB. In addition, high levels of IgM and IgG RF are frequently seen. Chronic inflammation (unspecified): low to moderate ssDNA. Rheumatoid arthritis sera have. with some exceptions, a negative ANAJ6 or ANA/8 profile and a positive combination of different RF classes: 1) mild, with low incidence of erosion. IgM only; 2) longstanding, usually associated with vasculitis, IgM RF, IgG RF, and in some low level ssDNA: 3) long-standing, usually erosive RA. all three classes of RF: 4) RF and SSA and SSB in patients with secondary SjOgren's: and 5) RF and low levels of RNP. In some patients the profile of RA and SLE overlap and they have clinical features of both diseases. When a patient is diagnosed as RA and the serology is that of SLE, the diagnosis needs reevaluation. Primary anti-phospholipid antibody syndrome is a complex and poorly defined clinical condition, generally associated with thromboembolic manifestations: therefore, the routine requests yield few positive results, which may appear as 1) high lgM ACA. 2) moderate to high lgM and IgG, 3) moderate to high lgA only (rare), or 4) all three classes with at least one at moderate level. The test has to be positive on at least three separate occasions to consider this diagnosis. Some patients have low to moderate anti-ssDNA. The advantages of specificity profile testing may be summarized as follows. . The polymorphism of autoantibodies in SLE and related conditions does not allow for any predictions of specificity to be made in a given ANA positive serum. The presence of more than three antibodies on an ANA/6 has an 80% chance of indicating active SLE, which is a parameter missed by separate autoantibody testing, Both multiple autoantibodies and no autoantibody are significant findings for diagnosis and management: a) when five or s~x antibodies are found it is an indication of severe disease (Figure 1) that requires careful management: b) patients with active SLE and with all six autoantibodies within normal limits have not yet been

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Vol. 12, No. 4, 1992

seen (in over 6,000 profiles examined), i.e., the diagnosis of SLE is almost excluded in atypical clinical presentations. F-ANA (vide infra) may be negative in 2%-4% of SLE patients. 2. Complement consumption tests may be performed as a reflex, mainly on specimens with an SLE profile. 3. The same sensitivity is used for all tests. For example the profile in Figure 2F with anti-RNP as the only antibody at high level is strongly indicative of MCTD. What is significant is not only the presence of this antibody but also the conspicuous absence of the others, a characteristic finding in this condition. The value of this profile is illustrated by a case: L.W., a 32-year-old female with high ANA titer (1:1280), arthritis, fatigue, hair loss, night sweating, and severe CNS manifestations (interpreted as psychosis). She was started on high dose prednisone and cytoxan by a clinician who examined the patient and had available only the information mentioned above. The patient was sent to our clinic and a routine ANA/6 showed a profile as in Figure 2F, i.e., compatible with MCTD, not with SLE. This prompted a careful evaluation that revealed, in addition to MCTD, severe hyperthyroidism, which was responsible for her CNS manifestations. Steroids and cytoxan were stopped and she had a benign course. 4. The antibodies to be followed longitudinally during treatment of SLE are identified (Figure 3). Since the levels of all autoantibodies found in SLE have been correlated with target organ or tissue damage, repeated measurements during treatment may offer objective parameters.

Algorithm for Multiparameter Testing in Diagnosis and Management Arthralgias, arthritis, fatigue, vasculitis, etc., are common manifestations in several autoimmune rheumatic conditions: e.g., SLE, rheumatoid arthritis (RA), MCTD, Sjtgren's; some of these conditions may be in "pure form" or may overlap clinically and seroiogically. In addition, SLE has a highly "polymorphic" clinical presentation and sometimes a vague suspicion triggers a request for ANA and rheumatoid

factor and, in patients with thromboembolic or CNS manifestations, anticardiolipin antibodies (Figure 6). Based on initial examination a hypothesis of high or low probability for SLE, RA, etc., is made and various laboratory tests are requested. A simple algorithm can be designed for the effective use of multiparametric ELISA tests, which realizes its full potential through good communication between clinicians and the laboratory immunologists. The algorithm is described for SLE; the use of tests for rheumatoid factor and antiphospholipid antibodies follows a similar format (Figure 6). When the diagnosis of SLE is of low probability, a routine ANA and RF is ordered. If the ANA test result is negative (<6.2 I.U.), the diagnosis of SLE is very unlikely.23 If the result is positive, an ANA specificity profile is performed as a reflex (ANA/6 or ANA]8, depending on clinical suspicion). If the autoantibody profile suggests SLE or related condition (vide supra), additional investigations may be necessary. However, since for each antibody the cut-off for normal blood bank donors is at the 98th percentile, some ANA/6 or ANA]8 profiles may be false-positive, which is established only after a complete clinical evaluation. The ANA screen tests is frequently positive in patients without demonstrable SLE or related connective tissue disorders and in normal individuals. As many as 87% of patients sent to an arthritis center for positive ANA had negative ANA/6 and in final analysis did not have any condition in which antinuclear antibodies are likely to be involved62; thus, the "real" are differentiated from the "false" positive ANA tests. When the clinical probability of SLE is high, the ANA profile test is best performed first or in parallel with the ANA screening test. This concept is based on the following rationale. If the clinical probability is high and the F-ANA is negative, a situation represented by the ANA-negative SLE, the clinician disregards the laboratory results and requests an ANAJ6 or ANA/8 profile. In ANA negative SLE, an ANM6 may reveal elevations in anti-ssDNA and/or anti-SSA antibodies 14 or, rarely, anti-dsDNA. In fact, we have seen a case of a 53-year-old man with negative F-ANA (on HEP2 cells in two separate © 1992ElsevierSciencePublishingCo., Inc.

laboratories), vasculitis, and proliferative glomemlonephritis on the biopsy who had significant levels of anti-dsDNA (Figure 2B). If the ANA screen is positive and the ANA/6 profile is completely normal, the SLE patient may have inactive or sometimes "burned out" disease or kidney failure and is unlikely to require or benefit from any immunosuppressive treatment. Also, the original data are reevaluated to generate alternate diagnoses. When there is a high probability of SLE and the ANA/6 profile is that characteristic to active SLE, complement consumption tests (C3, C4, CH 50, complement fragments) may be performed as a reflex. Once the diagnosis of SLE is accepted, there are two questions relevant to the clinical laboratory: 1) what is the magnitude of damage of the involved organs, and 2) what test can be used to monitor this damage longitudinally. For an individual patient, when a correlate between serological and biochemical parameters and tissue damage is identified, repeated measurements (at 4- to 12-week intervals) during treatment may minimize the need for repeated invasive diagnostic procedures. Since practically all autoantibodies on an ANA/6 may increase before flares, repeat profile test (6 to 12 weeks) for an SLE patient "in remission" may be useful to indicate tleatment before another cycle of organ damage. Since survival in SLE is directly proportional to the number of flares, with patients with only one flare having almost 100% survival after 10 years, 22 preventing clinical flares may alter the final outcome. Further studies are needed to establish statistical validity of this preemptive approach to therapy. CIN

References 1. Amett F, Hamilton RG, Roebber MG, Harley JB, Reichlin M: increased frequencies of Sm and nRNP autoantibodies in American blacks compared to whites with systemic lupus erythematosus. J Rheumatol 15:1773-1776, 1988. 2. Brinkman K, Termaat R, Van den Brink H, Berden J, Smeenk R: The specificity of the anti-dsDNA ELISA. A closer look. J Immunol Methods 139:91, 1991. 3. CarsonDA: Rhetunatoid factor.In Kelley WN, Harris ED, Ruddy S, Sledge CB (eds): Textbook of Rheumatology,second edition, Philadelphia, Saunders, pp 665--679, 1985. 0197-185%/92/$0.00+ 3.00

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4. Faith A, Pontesilli O, Unger A, Panayi GS, Johns P: F_LISAassays for IgM and IgG rheumatoid factor. J Imrnnnol Methods 55:169-177, 1982. 5. Froelich CJ, Wallman J, Skosey JL, Teodorescu M: Clinical value of an integrated ELISA system for the detection of 6 autoantibodies (ssDNA, dsDNA, SM, RNP/Sm, SSA, and SSB). J Rhetnnatol 17:192-200, 1990. 6. Furaukawa F, Kashihara-Sawarni M, Lyons M, Norris D: Binding of autoantibodies to the extractable nuclear antigens SSA/Ro and SSB/La is induced on the surface of human keratinocytes by ultraviolet light: implications for the pathogenesis of photosensitive cutaneous lupus. J Invest Dermatol 94:77-85, 1990. 7. Goddard DH, Moore ME: Common tests for rheumatoid factors: poorly standardized but ubiquitous. Arthritis Rheum. 31:432-435,1988. 8. GripenbergM, Linder E, Kurki P, Engvall E: A solid phase enzyme-linkedimmunoabsorbent assay (ELISA)for the demonstration of antibodies againstssDNA in patient sera. Scand J Immunol 7:151-157, 1978. 9. Hamilton R, Adkinson N Jr: Quantitative aspects of solid phase Lmmunoassays. In Kemeny DM, ChaUacombe SJ (eds): ELISA and Other Solid Phase Inununoassays. New York: John Wiley & Sons, p 57, 1988. 10. Harris E, Pierangeli S, Simpson P: A more specific phospholipid antigen for the antiphospholipid syndrome. Arthritis Rheum 33(S):S163, 1990. 11. Hollingsworth PN, Bonifacio E, Dawkins RI: Use of standard curve improves precision and concordance of ANA measurement. J Clin Lab Immunol 22:197-200, 1987. 12. Houtman P, Kallenberg CGM, Limborg T, van Rijswijk M: Are anti-nRNP levels spe-

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cific guides for assessing disease activity in patients with mixed connective tissue disease. In Peeters H (ed): Protides of the Biological Fluids. Oxford: Pergamon Press, pp 357-358, 1985. 13. Kioke T, Ichikawa K: Proceedings, Second International Conference on SLE. Professional Postgraduate Services International: USA, UK, Mexico, Singapore, 146 pp. 1989. 14. Maddison P, Provost T, Reichlin M: Serologic findings in patients with "ANA" negative lupus. Medicine (Baltimore) 60:.87-94, 1981. 15. McNeil H, Chersterman C, Krilis S: Anticardiolipin antibodies and lupus anticoagulants comprise separate antibody subgroups with different phospholipid binding characteristics. Br J Hemato173:506-513, 1989. 16. Ramos-Neimbo F, Alarcon-Segovia D: Development of sicca symptoms in systemic lupus erythematosus patients with existing subclinical abnormalities of lacrianal and/or salivary glands. Arthritis Rheum 22:935936, 1979. 17. Scopelitis E, Biundo J, Alspaugh M. AntiSSA antibody and other antinuclear antibodies in SLE. Arthritis Rheum 23:287-293, 1980. 18. Scott DGI, Bacon PA, Allen C, Elson C J, Wallington T: IgG rheumatoid factor, complement and immune complexes in rheumatoid synovitis and vasculitis: comparative and serial studies during cytotoxic therapy. Clin Exp lanmuno143:54-63, 1981. 19. Shapiro P, McAllister G, Lemer E, Lemer M: Healthy individuals and patients with systemic lupus erythematosus have unique, person-specific spectra of antibodies detectable on immunoblots. Clin hmnunol hnmunopathol 59:129-138, 1991. 20. Shoenfeld Y, Rauch J, Massicotte H, Stollar

A C l i n i c i a n ' s V i e w o f Autoi

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B: Polyspecificity of monoclonal lupus autoantibodies. N Engl JMed 308:414--421. 1983. Steward MW, Lew AM: The hnportance of antibody affinity in the performance of immunoassay for antibody. J Inununol Methods 78:173--190, 1985. Swaak AJG. Nossent JC, Bronsveld W, van Rooyen A, Nieuwenhuys EJ. Theuns L. Smeenk RJT: Systemic lupus erythematosus. I. Outcome and survival: Dutch experience with 110 patients studies prospectively. Ann Rheum Dis 48:447454, 1989. Tan EM: Antinuclear antibodies: Diagnoslac markers for autoinarnune diseases and probes for cell biology. Adv Inununol 44:93-151. 1989. Teodorescu M, Froelich C: Laboratory evaluation of systemic lupus erythematosus. In Lahita R (ed): System Lupus Erythematosus, 2nd edition. 1992 (in press). ter Borg E, Horst G. Hummel E. Limburg P. Kallenberg CGM: Predictive value of rises in double-stranded DNA antibody levels for disease exacerbations in SLE: A long term prospective study. Arthritis Rheum. 33:634-643. 1990. ter Borg E, Horst G. Limburg P, van Vcnrooij W. Kallenberg CGM: Changes in levels of antibodies against the 70 kDa and A polypeptides of the U1RNP complex in relation to exacerbations of systemic lupus erythematosus. J Rheumatol 18:363-367~ 19:)1_ Totoriis M, Tan E. McNally E, Rubin R: Association of anti-histone (H2A-H2B) complex antibody with symptomatic procainamide-indueed lupus erythematosus, N Engl J Meal 318:1431-1436, 1988. Wasicek C. Reichtin M: Clinical and serological differences between SLE patients with antibodies to Ro and La. J Clin Invest 69:835-843. 1982.

nity

John S. Davis IV University of Virginia Health Sciences, Center, Charlottesville, Virginia T would like to briefly outline some I I views on the usefulness of selected roll laboratory tests in the evaluation and management of patients with suspected autoimmune diseases. Though there is no specialty (or subspecialty) of medicine that does not encompass patients with autoimmune conditions, I will stick to those con0i97-1959/92/$0.00 + 3.00

ditions that are most familiar t o m e as a rheumatologist, namely, those that fall within the framework of what we have come to call the connective tissue diseases, or rheumatic diseases. The range of factors and phenomena is wide: antibodies; sensitized lymphocytes; vascular and perivascular infiltrates; circu© 1992Elsevier SciencePublishing Co., Incl

lating immune complexes (CICs) and deposits o f immune complexes; evidence of complement consumption and activation; and a variety of circulating cytokines and cytokine receptors that may be found in a wide spectrum o f conditions. For the clinician who is evaluating and managing a probable autoimmune condition, it is these