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Laboratory diagnosis of autoimmune diseases

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COVER STORY

Laboratory diagnosis
of autoimmune diseases
By Marc Golightly, Ph.D. and Candace Golightly, MS

Introduction Systemic diseases

The rheumatic and autoimmune diseases can generally be The clinical manifestations of the basic systemic autoimmune
classified into two groups: those that are systemic in nature diseases, while being very complex, have been classified ac-
(i.e. systemic lupus erythematosus, Sjögren’s syndrome, scle- cording to generally accepted guidelines.1,2 While this is be-
roderma, rheumatoid arthritis, autoimmune vasculitis, mixed yond the scope and purpose of this review, the diseases’ com-
connective tissue disease, and various other overlap syn- plexity must be appreciated, since this clinical complexity is
dromes); and those that are more organ or tissue directed often proportional to the complexity of test results seen in
(autoimmune thyroid disease, myasthenia gravis, and certain the laboratory. The most common systemic autoimmune dis-
skin diseases such as bullous pemphigoid). Each of these eases are systemic lupus erythematosus, Sjögren’s syndrome,
groups presents unique problems to the diagnostic labora- scleroderma, rheumatoid arthritis, autoimmune vasculitis, and
tory, complicated by the fact that they may occur in combi- mixed connective tissue disease. The disease manifestations
nation with each other. Testing for these highly variable dis- are highly variable, may progress, and in some cases evolve
eases can be complex and is often misunderstood. In today’s into or coalesce with other autoimmune diseases in many dif-
healthcare environment, it is critical that the proper tests be ferent combinations (generally called “overlap syndromes”).3
performed not only from the point of view of reducing pa- As might be expected, the symptoms range in severity and
tient care costs, but also for laboratory issues such as compli- may progress or evolve from being extremely mild to severe
ance, staff workloads, and cost containment. In this review, and protracted, even fatal. While no single pathogenic
we will consider generalized autoimmune testing and algo- mechanism has been elucidated for autoimmune diseases, they
rithms for their use as well as more specific testing. In addi- share the commonality of autoantibodies being produced to
tion, a discussion of the value and utility of the newer tech- nonorgan specific antigens on modified or unmodified struc-
nology applied to the field will be discussed. tures on nucleated cells and serum proteins. Whether the
autoantibody is the inciting event, or is produced as a result
of tissue insult and subsequent release of autoantigens yield-
CONTINUING EDUCATION
EDUCATION ing a break in B cell tolerance, or both, is not clear.4,5
To earn CEUs, see test on page 22. Antinuclear antibodies and specific
autoantibody reactivities
LEARNING OBJECTIVES
One of the major laboratory screening tests for detection of
Upon completion of this article systemic rheumatic diseases for the last four decades has been
the reader will be able to: the detection of antinuclear antibodies (ANA) (see Table 1).
1. Distinguish between systemic and organic-specific Since the ANA uses a whole cell substrate, which includes
autoimmune diseases and name two specific most nuclear and cytoplasmic antigens, this test can provide
diseases of each type. much information regarding the possible autoantibody
2. Name two methodologies used to detect anti- system(s) present, given the pattern of reactivity seen in the
nuclear antibodies (ANA); to detect rheumatoid positive ANA. This can then be utilized to direct which ad-
factor. ditional tests the clinician should order. Correct interpreta-
3. Describe three positive ANA patterns and state the tion of the ANA by the laboratory personnel performing the
disease process commonly associated with each. test and by the clinician is key to eliminating unneeded test-
4. Identify four methodologies used to determine ing (discussed below). 6 While the ANA is often positive in
specific autoantibody reactivity and cite two connective tissue diseases, it is also often positive in other
performance-related issues. nonautoimmune diseases such as infectious disease (e.g. lep-
rosy, EBV, and viral hepatitis), neoplastic diseases (e.g. leu-
CE test published through an educational grant from kemia, lymphoma, melanoma, and other solid tumors), and
other diseases such as primary biliary cirrhosis. In fact, a sig-
nificant number of healthy individuals may have a positive
ANA. However, positive ANAs in these nonconnective tis-
sue diseases usually occur at a lower titer. Therefore, a posi-

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 AUTOIMMUNE DISEASES

tive ANA is not diagnostic by itself and must be correlated the specific autoantibodies present. It should be noted that
with clinical findings.7 This can create problems for inexpe- more than one autoantibody may be present, which may trans-
rienced physicians, resulting in inappropriate referral to the late to more than one ANA pattern in a particular sample.
rheumatology service. For these reasons, a positive signifi- This can occur in the classic autoimmune diseases, but is es-
cance range for results should be established by running a pecially true in the overlap syndromes where different com-
number of normal specimens from the catchment area of test- binations of autoimmune processes may combine into one
ing. An accompanying cautionary statement in the reporting disease. When this occurs, results should be reported and
of low positive results (below the significant level) may also titred, as they may be significant to the disease process or
alleviate some of these unnecessary referrals. prognosis.3 It is in the pattern meaning and recognition where,
The testing methodology for ANAs is available in both besides the positive/significant cutoff levels, most of the un-
the indirect fluorescent (IFA) and ELISA assay formats. The certainty and confusion exist in ANA testing, both at the tech-
decision on which assay to perform should be based on the nical and clinician level. Due to the subtleties and subjectiv-
testing institution’s patient referral pattern. In those institu- ity in scoring the patterns and for continuity, it is often
tions which perform large volumes and which historically advantageous to have the patterns and titers scored blindly
have a low percentage of positive specimens, the ELISA may by two technologists.
be more cost effective. For those institutions with a high posi- The most common autoantibodies (and antigens recog-
tive rate (e.g., a large rheumatology division), an IFA is often nized) and their associated ANA pattern are listed in Table
more cost effective, since all ELISA positives should be run 2. Some patterns are more specific to the disease process in-
on the IFA to determine the pattern. A high positive rate volved than others. For example, the homogeneous, nucle-
would result in a large number of specimens being tested by olar, and centromere patterns are fairly specific for SLE, scle-
both assays. This then becomes a simple case of “do the math.” roderma, and CREST variant of scleroderma respectively.
The ANA, when performed by IFA, yields distinctive pat- On the other hand, the speckled pattern is the most nonspe-
terns of staining in the nucleus or cytoplasm of the cell. These cific.6 Thus the specific antigenic reactivity of an autoanti-
patterns suggest which autoantibody may be present, which body causing a positive ANA should be determined in a re-
in turn will direct further testing (if required) to elucidate flexive manner, based on the ANA pattern observed, if
Continues on page 14

Images from WebPath, courtesy of Edward C. Klatt, M.D., Florida State University College of Medicine
In the antinuclear antibody (ANA) test, serum is incubated with a tissue The “speckled” pattern of staining is more characteristic of the presence
substrate to which autoantibodies to nuclear antigens bind. A fluoro- of autoantibodies to extractable nuclear antigens, particularly ribonucleo-
sceinated antibody is added and the tissue is observed under fluorescence protein. This pattern is not very specific, but may be seen with “mixed
microscopy. Seen here is the “homogenous” pattern of a positive ANA. connective tissue disease” without serious renal or pulmonary disease.

The “nucleolar pattern” of staining in which the bright fluorescence is Sometimes when performing the ANA test, the substrate cells demonstrate
seen within the nucleoli of the Hep2 cells. This pattern is more suggestive particular patterns of staining. This is the “rim” pattern that is more
of progressive systemic sclerosis. characteristic of SLE.

www.mlo-online.com MLO ■ July 2002 13

COVER STORY

required and
pertinent to di- ANA Positivity with Connective Tissue Disease
agnosis/prog- Connective Tissue Disease Approximate Percent
nosis). For ex- Positive ANA Test
ample, testing
for anti-DNA Systemic Lupus Ery
thmatosis 90 - 100
antibodies, Sjögre n’s Syndrome 50 - 85
Scleroderm a 88
which give a
Rheumatoid Arthritis 25 - 55
homogeneous
Juvenile Arthritis 22
pattern, in a
Mixed Connective Tissue Disease >95
specimen that Overlap Syndromes Variable*
had a positive
speckled ANA * Positivity with overlap syndromes depends on actual syndromes
making up the overlap; multiple systems/patterns may be
would not be present
justified, nor
would testing Table 1
for anti-Sm,
RNP, SS-A, or SS-B antibodies in a tis Foundation and the Centers for Dis-
positive ANA that had a homogeneous ease Control; however, only nine par-
pattern. Testing for all would create an ticipated. The researchers found that
unwarranted healthcare expense.10 Fig- while some manufacturers produced
ure I is an algorithm for further testing kits demonstrating exceptional accu-
when a significant ANA titer is found.6 racy, performance was not uniform, in
At the present time, antibodies to na- that the same manufacturer also pro-
tive DNA and the extractable nuclear duced a kit demonstrating poor accu-
antigens (SM, RNP, SS-A, and SS-B) racy. 9 Another study found that the
are the most common secondary tests ELISAs tended to produce more false
performed. Occasionally, anti-Scl-70 positives and weak positives than the
and Jo-1 tests are performed but usu- traditional immunodiffusion assays,
ally not before the others. While Scl- thus giving ELISAS a lower positive
70 antibodies are typically found in pa- predictive value.10 The establishment of
tients with scleroderma, they may be significance levels often deals with this
also found in a subset of SLE patients problem, however. Based on these stud-
and additionally correlate with antibody ies, laboratorians should be aware that
levels to doublestranded DNA. The assay performance parameters are de-
presence of these antibodies may cor- pendent upon the method and manu-
relate with increased risk for pulmonary facturer; good performance with one
hypertension and nephritis.8 analyte does not guarantee the same
The methodologies used to deter- performance with the others. There-
mine specific autoantibody reactivity fore, while inconvenient, laboratories
are ELISAs, immunodiffusion assays, may need to use different manufactur-
countercurrent electrophoresis, and re- ers for different analytes. Likewise, cli-
cently, immunoblotting. The first two nicians should be aware that inter-
methods are used by the majority of manufacturer variation exists and
laboratories for antibodies to DNA, across-the-board comparisons between
Sm, RNP, SS-A, SS-B, and Scl-70. results from different laboratories will
While ELISA is the best method for be difficult.
quantitation of the amount of these an- While the antibody-antigen systems
tibodies, a recent study has demon- listed in Table 2 are those most com-
strated that there are some perfor- monly assayed for in the clinical labo-
mance issues, which warrant careful ratory, some of these have been further
selection of the kit to be used.9 In this defined into their subunit components,
study, 20 manufacturers were ap- and many entirely new autoantibodies
proached to participate in blind testing have been added to the list. Currently,
of reference samples from the Arthri- there are approximately 50 auto-
Continues on page 16

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COVER STORY

Antigens Associated With Autoimmune Disease

Autoantibody/Antigens ANA Pattern Disease Association
Recognized
Double & Single St
randed DNA Homogenous/Rim SLE & low level in other rheumatic disease
Histones Homogenous/Rim Drug-induced lupus, SLE
Deoxynucleoprotein Homogenous/Rim Drug-induced lupus, SLE
Smith (Sm) Speckled Diagnostic of SLE
Nuclear RNP Speckled High titer — Mixed connective tissue disorder/SLE
SS-A (Ro) Speckled or Negative (low titer) Sjögren’s syndrome SLE
SS-B (La) Speckled Sjögren’s syndrome
Centromere Centromere CREST variant of Scleroderm a
RNA Polymerase I Nucleolar Scleroderma — high prevalence
Fibrillarin Nucleolar Scleroderm a
DNA Topoisomerase I (Scl-70) Nucleolar Scleroderm a
PM-ScL Nucleolar Polymyositis
Mitotic Spindle MSA Carpal tunnel syndrome, SLE
Apparatus, NuMA Sjögren’s
Jo-1 Cytoplasmic Polymyositis
Nucleolus
Table 2

antigens, predominately nuclear pro- various tissues, such as the synovium, referred to as having overlap syn-
teins, which are involved in the autoim- with an ensuing complement activation dromes. The recognition of overlap
mune rheumatic diseases.3 The ma- and inflammatory response resulting in syndromes or an evolving overlap syn-
jority of these are not routinely utilized tissue damage, and in some cases, vas- drome is important since treatment
diagnostically. For example, antibodies cular damage. It should be noted that RF may need to be directed at some clini-
to the various subunits of Sm and the can occur in the other autoimmune dis- cal symptoms which may respond to
RNP complexes can be detected by eases (i.e., SLE, Sjögren’s syndrome, and treatment. Unfortunately, the many
Western or immunoblotting tech- occasionally in scleroderma and poly- various combinations that occur often
niques. However, the utilization of myositis).12 This is especially true in the complicate the laboratory diagnosis.
these assays in the general laboratory Sjögren’s syndrome overlap syndromes Further complicating this is the fact
is presently limited due to the complex- and the rheumatoid arthritis overlap that overlap syndromes have been de-
ity of testing and interpretation and the syndromes (RA/SLE overlap and Scle- scribed in organ-specific autoimmune
lack of a clearly defined advantage to roderma/RA overlap).3,13 Like the ANA, diseases such as autoimmune liver dis-
diagnosis and/or prognosis in most pa- the presence of RF is not totally specific eases.14 In many of these cases, the less
tients.11 There are, of course excep- and can occur in some nonautoimmune frequently assayed autoantibodies and
tions, and these seem to be primarily diseases as well (e.g., chronic active techniques are often utilized to help
in helping to define the overlap syn- hepatitis, sarcoidosis, chronic infectious with the diagnosis. While an in-depth
dromes, which in turn has relevance to diseases, neoplasia, and syphilis).12 In description of the laboratory diagno-
prognosis.3 As further clinical and labo- addition, RF has been reported to occur sis of overlap syndromes is beyond the
ratory studies are performed, their use with increasing prevalence with age in scope of this review, it is the subject of
in the routine clinical laboratory may the absence of disease. Latex agglutina- a review by Jury, D’Cruz, and Mor-
become justified and common. tion and nephelometry assays are the row.3
While the ANA is considered a ba- methods most often used.
sic screen for SLE and related disor-
Organ-specific autoimmune diseases
ders (see Figure 1), rheumatoid factor
Overlap syndromes Organ-specific autoimmune diseases
(RF) is considered the screen and hall- Many patients with autoimmune rheu- occur in most major organ systems, in-
mark test for rheumatoid arthritis matic disease either cannot be catego- cluding but not limited to, endocrine,
(RA)2. Rheumatoid factor is an autoan- rized into one of the classic autoim- hematologic, cardiac and vascular, gas-
tibody that has reactivity to IgG. Typi- mune diseases, or start with one of the trointestinal, liver, kidney, skin, lung,
cally, RF is of the IgM class; however, classically recognized diseases and and brain. When a specific organ sys-
IgA and IgG rheumatoid factors are evolve into something else. Often these tem is the target of an organ-specific
known to exist. The pathogenesis of patients share symptomatology of or autoantibody, tissue damage similar to
rheumatoid arthritis involves the depo- more of the classic autoimmune dis- that which occurs in systemic autoim-
sition of RF complexed with IgG in the eases and, as previously discussed, are mune diseases can occur, resulting in
Continues on page 20
16 July 2002 ■ MLO www.mlo-online.com
COVER STORY

Figure 1. Autoimmune Testing Algorithm

organ failure or malfunction. If the organ’s receptors are

Dr. Marc Golightly is professor of pathology in the Department of Pathology at
bound by the autoantibody, that receptor may be either stimu- the School of Medicine, State University of New York at Stony Brook. Candace
lated or blocked. The resulting clinical manifestations are Golightly is clinical assistant professor in the Department of Clinical Labora-
then characteristic of over- or understimulation of the or- tory Sciences, School of Health Technology and Management, State Univer-
gan, respectively. sity of New York at Stony Brook.

The detection of these organ-specific autoantibodies, of- References

ten directed against cellular receptors, can aid in establish-
1. Tan EM, Cohen AS, Fries JF, et al. The 1982 revised criteria for the classification
ing the diagnosis and monitoring these patients. While the of systemic lupus erythematosus. Arthritis Rheum;25:271-5, 1982.
detection of many are straightforward (e.g. thyroglobulin, 2. Arnett FC, Edworthy SM, Bloch DA, et al. The American Rheumatism Association
1987 revised criteria for the classification of rheumatoid arthritis. Arthritis
microsomal, mitochondrial, and smooth muscle antibodies.), Rheum;31:315-324. II -II r-I) II, 1987.
many are not and involve complex bioassays (such as those 3. Jury EC, D’Cruz D, and Morrow WJW. Autoantibodies and overlap syndromes in
autoimmune rheumatic disease. J Clin Path;54:340-347, 2001.
used to examine hormone receptor binding). The majority
4. Steinman L. Escape from “horror autotoxicus”: Pathogenesis and treatment of
of these bioassays are not standardized and may demonstrate autoimmune disease. Cell. 80(1):7-10, 1995.
considerable variation in sensitivity. Detection and 5. Radic MZ, Weigert M. Origins of anti-DNA antibodies and their implications for
B-cell tolerance. Annals of the New York Academy of Sciences. 764:384-96, 1995.
quantitation of autoantibodies to poorly defined antigens are 6. Breen C. and Golightly M. “ Autoimmune and Rheumatic Diseases “ in Saunders
also problematic as the antigens used in the assays often vary Manual of Clinical Laboratory Science. pp 295-304. Ed. by C. Lehmann. W.B. Saunders
Co., Philadelphia, Pa. 1998.
(a problem common to some of the systemic autoantibodies
7. Callegari PE. Williams WV. Laboratory tests for rheumatic diseases. When are
as well). Finally, the interpretation of positive organ-specific they useful?. Postgraduate Medicine. 97(4):65-8, 71-4, 1995.
autoantibodies (even the well defined tests) must be viewed 8. Elicha Gussin HA, Ignat GP, Varga J, and Teodorescu M. Anti-topoisomerase I
(anti-Scl-70) antibodies in patients with systemic lupus erythematosus. Arthritis &
with caution, since the presence of these specific autoanti- Rheumatism. 44(2):376-383, 2001.
bodies does not always correlate with a specific disease or 9. Tan EM, Smolen JS, McDougal JS, Fritzler MJ, Gordon T, Hardin JA, Kalden JR,
Lahita RG, Maini RN, Reeves WH, Rothfield NF, Takasaki Y, Wiik A, Wilson M, and Koziol
disease activity.6,12,15 JA. A critical evaluation of enzyme immunoassay kits for detection of antinuclear au-
toantibodies of defined specificities. II. Potential for quantitation of antibody content.
Conclusions J of Rheumatology. 29(1):68-74, 2002.
10. Griesmacher A, and Peichl P.. Autoantibodies associated with rheumatic dis-
As the pathology and clinical understanding of systemic and eases. Clinical Chemistry & Laboratory Medicine. 39(3):189-208, 2001.
organ-specific autoimmune diseases become better under- 11 Margaux J, Hayem G, Palazzo E, et al. Clinical usefulness of autoantibodies to
U I snRNP proteins in mixed connective tissue disease and systemic lupus erythema-
stood, it has become apparent that the testing has become tosus. Rev Rhum Engl Ed. 65:378-86, 1998.
more complex and will most likely continue to evolve. The 12. Fye K, and Sack K. Rheumatic Diseases in Basic and Clinical Immunology. Eds
Stites, D, Terr, AI, and TG Parslow. Eighth edition. Appleton and Lange, Norwalk, Con-
ultimate goal of this testing in today’s healthcare environ- necticut. 1994.
ment is to establish the diagnosis, predict the prognosis and 13 Hamasaki K, Mimura T, Kanda H, et al. Development of systemic lupus erythema-
disease course, and monitor treatment, all in a cost effective tosus in a rheumatoid arthritis patient with anti-ribosomal P protein antibody. Lupus;
6:734-736, 1997.
manner. A good understanding of the testing, positive result 14. Cervera R, Khamashta MA, and Hughes GVR. Overlap syndromes. Ann Rheum
patterns, and disease associations is required to accomplish Dis;49:947-948, 1990
15. Bach JF. Organ-specific autoimmunity. Immunology Today. 16(7):353-5, 1995.
this goal. l

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