Screening and Laboratory Diagnosis of Autoimmune Diseases ...
嚜燙creening and Laboratory Diagnosis
of Autoimmune Diseases
Using Antinuclear Antibody
Immunofluorescence Assay
and Specific Autoantibody Testing
Paul P. Doghramji, MD
Educational support provided by Quest Diagnostics, Inc.
Introduction
Autoimmune diseases occur when the immune system
Laboratory screening and diagnostic
testing for disease classification
attacks the normal tissue within joints, vasculature,
The recommended ANA screen approach uses HEp-2
and other organ systems, causing inflammation, pain,
human tissue culture cells in an IFA. In this assay, the
diminished mobility, fatigue, and other non-specific
patient*s blood sample is mixed with HEp-2 cells fixed
symptoms.1 The strong overlap of signs and symptoms
to a slide. ANAs present in the sample react with the
among the autoimmune diseases can lead to delays in
cells and treatment with a fluorescent anti-human IgG
diagnosis and appropriate treatment. According to a
antibody allows visualization of antibody binding under
survey by the Autoimmune Diseases Association, it takes
fluorescence microscopy. This test screens for a large
up to 4.6 years and nearly 5 doctor visits to receive a
number of known autoantibodies, approximately 150,
proper autoimmune disease diagnosis.2
directed against nuclear antigens and cell cytoplasm.
Laboratory testing, in addition to clinical assessment, is
necessary for differential diagnosis and disease classification
A positive screen result is followed by evaluation of
antibody titer and pattern (consult side bar below).
of autoimmune diseases. However, research shows that
With a positive ANA IFA screen and titer, the clinician
primary care physicians tend to overuse common
needs to determine the root cause of the positivity. This
autoantibody tests for autoimmune conditions, which can
can be accomplished through a reflex to an algorithm
limit the positive predictive value and clinical utility of such
of specific antibody tests to help identify autoantibodies
testing.3 To facilitate appropriate referral to specialists,
associated with specific autoimmune diseases.
if necessary, laboratory testing should be reserved for
patients who have signs and symptoms consistent with
one or more autoimmune disease (Table 1).
An ANA reflex algorithm tests for specific antibodies in a
clinically logical sequence. With a combination of ANA IFA
plus a reflex algorithm of specific antibody testing, positive
The antinuclear antibody (ANA) immunofluorescence
results automatically reflex to a tier of disease-specific
assay (IFA) is a first-line screening test for patients with
autoantibodies. Testing begins with the most prevalent
a suspected autoimmune disease. This test is the gold
autoimmune diseases and continues until a positive result
standard because of its high sensitivity compared to
is found, or all tests in the algorithm have returned a
other assays.4,5 Positive results should prompt clinicians
negative result. This algorithm/reflex approach provides the
to continue investigating the cause of a positive ANA IFA
clinician with a rational approach to confirming a diagnosis
and narrow the field of potential culprits. The following
in a patient with a suspected autoimmune disease, with a
describes how ANA IFA in combination with specific
single blood draw.
autoantibody testing can be used in the differential
diagnosis of a suspected autoimmune disease.
Titer and Pattern
If the ANA IFA screen is positive, testing for antibody titer and pattern can help evaluate the presence and type of
autoantibody disease. ANA titers are determined by diluting the liquid portion of the blood sample in saline at a
ratio of 1:40 to 1:1280. The titer is thus the highest dilution that yields a positive ANA result. Any titer above 1:40
is considered positive, and titers above 1:80 are consistent with an autoimmune disease. To assess the nuclear and
cytoplasmic staining patterns of samples with positive results, patient antibodies react with indicator cells and the
localization of patient antibodies is visualized by a second fluorescein antihuman IgG antibody evaluated under
a fluorescence microscope. These patterns may provide additional information to rule out or further implicate a
suspected condition and can guide the selection of additional testing for specific autoantibodies.
2
Figure 1.
Use of ANA (IFA) and Specific Antibody Testing Cascade (Test Code 16814)
Figure 1. Use of ANA (IFA) and Specific Antibody Testing Cascade (Test Code
16814) for the Diagnosis
for the Diagnosis of Rheumatic Disease6-10
of Rheumatic Disease
ANA Screen (IFA)
Negative
Positive
Autoimmune
disease less likely.
May consider
rheumatoid
arthritis disease
testing.
Tier 1
Chromatin, dsDNA, RNP, Sm,
and Sm/RNP antibodies
Titer and Pattern
Negative
Positive
Tier 2
Jo-1, Scl-70, SS-A,
and SS-B antibodies
Negative
Positive
Tier 3
Centromere B and
ribosomal P antibodies
Negative
No evidence of
rheumatic disease shown
by analytes tested
Positive
Antibody
Test
Centromere B
Ribosomal P
Antibody
Test
SS-A
SS-B
Scl-70
Jo-1
CREST
Syndrome
+
每
Antibody
Test
dsDNA
Chromatin
Sm
Sm/RNP
RNP
Sj?gren*s
Syndrome
+
+
每
每
Systemic Lupus
Erythematosus
+ (high specificity)
+ (high sensitivity)
+ (high specificity)
Systemic
Sclerosis
每
每
+
每
+
+
Mixed Connective
Tissue Disease
每
每
每
+ (high titer)
+ (high titer)
Polymyositis
每
每
每
+
Neuropsychiatric
SLE
每
+
The acronym
refers
a syndrome
defined
bybypresence
ofcalcinosis,
calcinosis,
Raynaud*s
phenomenon,
esophageal
dysmotility,
sclerodactyly,
TheCREST
acronym
CRESTto
refers
to a syndrome
defined
presence of
Raynaud's
phenomenon,
esophageal
dysmotility, sclerodactyly,
and
and telangiectasia.
dsDNA
indicates
DNA;
Sm/RNP
antibody,
Smith/ribonucleoprotein
SS-A and
-B antibodies,
telangiectasia.
dsDNA
indicates double-stranded
double-stranded DNA;
Sm/RNP
antibody,
Smith/ribonucleoprotein
antibody; SS-A antibody;
and -B antibodies,
Sj?gren's
Sj?gren*s Syndrome
and
B antibodies;
antibody,
scleroderma
(topoisomerase
I) antibody;
Jo-1 antibody,
histidyl-tRNA
Syndrome AAand
B antibodies;
Scl-70 Scl-70
antibody,
scleroderma
(topoisomerase
I) antibody; Jo-1 antibody,
histidyl-tRNA
synthetase antibody;
and SLE,synthetase
antibody; and
SLE,lupus
systemic
lupus erythematosus.
systemic
erythematosus.
was developed
by Quest
Diagnosticsbased
based on
6-10.1-5.
It is It
provided
for informational
purposes only
and is notonly
intended
This figure This
wasfigure
developed
by Quest
Diagnostics
onreferences
references
is provided
for informational
purposes
andasis not
advice.
A physician*s
test selection
interpretation,
and patient
management
decisions
should
be b
ased on his/her
education,
intended asmedical
medical
advice.
A physician*s
testand
selection
and diagnosis,
interpretation,
diagnosis,
and
patient
management
decisions
should be
clinical expertise,
and clinical
assessment
of the patient.
based on his/her
education,
expertise,
and assessment of the patient.
References
1. Kavanaugh A, Tomar R, Reveille J, et al. Guidelines for clinical use of the antinuclear antibody test and tests for specific autoantibodies
to nuclear antigens. Arch Pathol Lab Med. 2000;124:71-81.
2. Satoh M, Chan EKL, Sobel ES, et al. Clinical implication of autoantibodies in patients with systemic rheumatic diseases.
Expert 1
Rev
Clin Immunol.
2007;3:721-738.
Figure
above
illustrates
an example of an algorithm
(In the cell, Sm and RMP proteins form
3. Stinton LM,
Fritzler
clinical
approachtiers
to autoantibody
testing in systemic
autoimmune
approach
with
a MJ.
set Aof
reflexing
for suspected
If the
first tierrheumatic
yields adisorders.
positive
Autoimmun Rev. 2007;7:77-84.
a complex.)
result, the results are
rheumatic
disease,
more common
group ofand
autoimmune
reported
and the testing
stops.clinics
If the
first tier criteria
of antibody
AM,aAlarc車n
GS, et al. Derivation
validation of systemic
lupus international
collaborating
classification
4. Petri M, Orbai
for systemic
lupus erythematosus. Arthritis Rheum. 2012;64:2677-2686.
6-10
diseases.
With
this algorithm, samples with a positive
testing is negative, the testing reflexes to the 2nd tier,
5. Cappelli S, Randone SB. ※To be or not to be,§ ten years after: evidence for mixed connective tissue disease as a distinct entity.
IFA Semin
resultArthritis
are tested
for 2012;41:589-598.
five autoantibodies associated with
Rheum.
which includes four autoantibodies associated with
systemic lupus erythematosus (SLE) and mixed connective
Sj?gren*s Syndrome, systemic sclerosis, and polymyositis:
tissue disease: dsDNA, chromatin (nucleosomal), Smith
SS-A, SS-B, Scl-70, and Jo-I antibodies. If a positive result
(Sm), ribonuclear protein (RNP), and Sm/RNP antiodies.
is determined for any of these autoantibodies, the results
3
Table 1. Common Signs and Symptoms of Autoimmune Rheumatic and Related Diseasesa,b
Sign or Symptom
Gout
JIA
MCTD
PM/DM
Pseudogout
RA
Sarcoidosis
Joint-/muscle-related
Joint pain, stiffness,
or inflammation
Muscle weakness
Myalgia
Skin-/hair-related
Alopecia
Rash
Raynaud*s phenomenon
Skin lesions
c
General
Anorexia
Cough
Ear involvement
d
Eye involvement
Fatigue
Fever
GI involvement
Malaise
Nasal symptoms
Nervous system involvement
Respiratory involvement
Weight loss
Other
Adenopathy
Anemia
Dysphagia
Swelling of hands
(Continued)
4
Table 1. Common Signs and Symptoms of Autoimmune Rheumatic and Related Diseasesa (Continued)
Sign or Symptom
SjS
SLE
SpA
AS
ReA
PsA
EA
SSc
Systemic Vasculitis
GPA
EGPA
MPA
Joint-/muscle-related
Joint pain, stiffness,
or inflammation
Muscle weakness
Myalgia
Skin-/hair-related
Alopecia
Rash
Raynaud*s phenomenon
Skin lesions
General
Anorexia
Cough
Ear involvement
d
Eye involvement
Fatigue
Fever
GI involvement
Malaise
Nasal symptoms
Nervous system involvement
Respiratory involvement
Weight loss
Other
Adenopathy
Anemia
Dysphagia
Swelling of hands
indicates common; indicates less common but not rare.
AS=ankylosing spondylitis, EA=enteropathic (inflammatory bowel disease-associated) arthritis, EGPA=eosinophilic granulomatosis with
polyangiitis, GPA=granulomatosis with polyangiitis, JIA=juvenile idiopathic arthritis, MCTD=mixed connective tissue disease,
MPA=microscopic polyangiitis, PM/DM=polymyositis/dermatomyositis; RA=rheumatoid arthritis, PsA=psoriatic arthritis,
ReA=reactive arthritis, SjS=Sj?gren*s Syndrome, SLE=systemic lupus erythematosus, SpA=spondyloarthropathies, SSc=systemic sclerosis.
b
This is not a complete list of signs and symptoms; some conditions have more signs and symptoms than could be presented here.
c
In dermatomyositis.
d
External ear in gout; middle ear in GPA.
a
5
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