Diagnosing PM/DM is challenging due to rarity of the disease, their similar clinical presentation and the possibility of overlap syndromes. A comprehensive strategy for serological testing comprises of parallel determination of both MSA and MAA thereby reducing the time to diagnosis. Current immunoassays techniques (Immunoblot) targeting MAA & MSAs have – The ability to include many parameters simultaneously which ensures higher detection rate; Become readily accessible & hence an opportunity for laboratories to contribute significantly in the disease diagnosis. MSA are highly specific for PM/DM, and many of them are also associated with a unique clinical subset of PM/DM, making them useful clinical diagnostic/prognostic biomarkers & continue to evolve.

1. Myositis – Review Of Autoantibodies
DR RAJESH V BENDRE
CHIEF OF LABORATORY
MD(PATH), DNB(PATH), DPB, MUMBAI

2.  Idiopathic inflammatory myopathies (IIM) represent a heterogeneous
group of autoimmune diseases with systemic involvement, affecting both
adults & children, often leading to severe impairment of the quality of life.
 Hallmark symptoms of IIM are muscle inflammation, proximal muscle
weakness, arthritis, cutaneous rashes, calcinosis, ulceration & in some
cases associated with malignancy and interstitial lung disease (ILD).
 They occur with a prevalence of 1 to 6 per 100,000 and with men to
women ratio as 1 to 2.
 They can be divided into –
 Polymyositis in adults (around 30%),
 Dermato-myositis in adults (around 30%),
 Paraneoplastic polymyositis with cancer of lungs, ovaries, breast,
gastrointestinal tract and in myeloproliferative diseases (around 8%),
 Infantile/juvenile myositis/dermatomyositis with accompanying
vasculitis (around 7-10%),
 Myositides in association with autoimmune diseases (Overlap or CTD)
such as rheumatoid arthritis, lupus erythematosus, mixed connective
tissue disease (MCTD) (15-20%).
 Rare forms as Inclusion body myositis(IBM)
INTRODUCTION-

3.  Except for classical dermatomyositis (DM), the diagnosis is mostly NOT straightforward and
usually requires testing of auto-antibodies, histological evaluation of a skeletal muscle biopsy,
muscle MRI and EMG.
 Disease Management is equally challenging since several sub-specialities are required for
optimal care, including rheumatologist and/or neurologist, dermatologist, pulmonologist,
cardiologist, physiotherapist etc.
 Hence, Early specific diagnosis is crucial
INTRODUCTION-

4. 2017 European League Against Rheumatism/American College of Rheumatology
Classification Criteria for Adult and Juvenile Idiopathic Inflammatory Myopathies
Probable IIM: Total
score ≥ 5.5 without
muscle biopsy,
≥ 6.7 with muscle biopsy.
Definite IIM: Total score
≥ 7.5 or more without
muscle biopsy,
≥ 8.7 with muscle biopsy.
Possible IIM: Total score
≥ 5.3 without muscle
biopsy,
≥ 6.5 with muscle biopsy.

5. Sub-classification: Meet probable or definite IIM criteria + the following
based on classification tree -

6. Sub-classification: Meet probable or definite IIM criteria + the following
based on classification tree -

7. Autoimmunity, May 2006; 39(3): 161–170
Anti-NT5c1A Autoantibodies as Biomarkers in Inclusion Body Myositis. Front. Immunol.,
April 2019
Myositis Autoantibodies
 In IIMs, apart from anti-Jo-1 autoantibody which was discovered
more than thirty years ago, the percentage of patients in whom an
autoantibody could not be recognized (the so called “serologic gap”)
was still high until recently.
 Due to heterogeneity and unavailability of reliable assays in all the
laboratories, the clinical use of serology lagged behind and
autoantibodies are not part of the most recent IIM Classification
Criteria
 Autoantibodies that are found in PM/DM are often classified into
myositis-specific autoantibodies (MSA) and myositis-associated
autoantibodies (MAA)
 MSA is defined as autoantibody specificities that are
considered relatively specific for PM/DM
 MAA is more vaguely defined than MSA as “autoantibody
specificity found in PM/DM but not specific for this diagnosis
and may be found in other SARD”
 Majority of MSA target Aminoacyl-tRNA-synthetase (ARS) is a
cytoplasmic enzyme involved in aminoacylation. While newly
identified target Cytosolic 5′-nucleotidases (NT5Cs) (play a central
role in regulation of purine nucleotide pool) major NT5Cs acting in
skeletal muscle, is cytosolic 5′-nucleotidase I (NT5C1)- which has
been identified as target in Inclusion Body Myositis (2013)

8.  Specific autoantibodies in systemic autoimmune rheumatic diseases (SARD) are clinically useful biomarkers associated with a particular
disease and/or clinical manifestations.
 These autoantibodies can be detectable years before clinical manifestation or diagnosis and thus have predictive value for the development of
the disease
 An estimated 50% of patients with polymyositis or dermatomyositis have one of the known MSA-myositis-specific antibodies
Myositis Autoantibodies

9. Myositis Autoantibodies
 Classic PM/DM-specific autoantibodies known are anti-Jo-1 & Mi-2.
 In recent years, new autoantibodies have been described in PM/DM with strong
specificities & clinical impact, such as antibodies to –
 Transcription intermediary factor 1γ (TIF1γ) that are frequently found in
cancer-associated DM
 Melanoma differentiation-associated gene 5 (MDA5), small ubiquitin-like
modifier activating enzyme (SAE1) associated with clinically amyopathic DM
(CADM) with progressive interstitial lung disease (ILD)
 NXP2 (MJ-p140-MU 140 kD protein,MORC3- microrchidia family CW-type
zinc-finger 3) are detected in 18% to 25% of cases of juvenile PM/DM (JDM)
and in only around 1% of adult cases. Often characterized by accompanying
calcinosis
 EJ, OJ often associated with severe muscle weakness, higher recurrence
rate & progressive interstitial lung disease (ILD)
 cN-1A (Cytosolic 5'-Nucleotidase 1A, MUP44 AB) often associated with
sIBM (29 to 52%), testing method ELISA, shows asymmetric muscle (distal
and quadriceps) involvement; disease progresses slowly and is treatment
refractory
 Anti-3-hydroxy-3-methylglutaryl-coenzyme A (anti-HMG-CoA) antibodies
have been found in patients with immune-mediated necrotizing
myopathy (IMNM)
 AMA-M2- often associated with cardiac involvement in the form of
myocarditis, arrhythmias, cardiomyopathy

10.  Anti-nuclear autoantibodies (ANA) determination by IIF is considered an accessible screening method for many
MSAs and MAAs. Furthermore, the recognition of particular IIF patterns can hint to some specific autoantibodies.
 However, using ANA IIF as the sole screening method for MSAs/MAAs, is not recommended because of low sensitivity, very
low specificity and/or lack of antigen expression by HEp-2 cells
 Immunoblot (IB) assays can simultaneously test for many autoantibodies, however, point to be noted is that there
is denaturation of proteins during gel preparation, but it does not impact recognition of linear epitopes.
 Commercial multiplex IBs, like dot blots or line blot assays (LIA), based on recombinant or synthetic peptides have been
increasingly available, benefitting from pure antigens not requiring a gel passage
 ELISAs are standardization, large-scale reproducibility and quantitative results.
 The disadvantage of the conjugation of antigens to a substrate resides on the possible loss of conformational epitopes
and/or the formation of neo-epitopes, which may in turn impact the test performance
 Immunoprecipitation is the gold standard as it evaluates the binding of the autoantibodies to the RNA/DNA and
protein complexes in their native conformation, yielding the best sensitivity and specificity.
 The major limitations of IP are due to technical difficulties, costs and use of radioactive reagents
Myositis Autoantibodies- Testing Methodology-

11.  To serologically investigate samples of
patients with suspected PM or DM,
screening with indirect immunofluorescence
tests (IIFT) should be followed by a
confirmation of results by monospecific tests.
 Because antibodies against the cytoplasmic
antigens (Jo-1, SRP, PL-7, PL-12, EJ, OJ
and Ro-52) are sometimes not clearly
detectable with IIFT, performance of both the
screening and confirmatory test is
recommended.
 Immunoblot is an ideal confirmatory method-
as it enables qualitative, monospecific &
simultaneous detection of many different
antibodies. Line blots allow antigens with
widely differing properties to be combined on
one test strip, allowing profiles to be
assembled according to the disease
application.
ANA PATTERNS- RELATED TO MYOSITIS AUTOANTIBODIES-
(A) Autoantibodies against Jo-1 show a fine granular cytoplasmatic fluorescence.
(B) Autoantibodies against PM-Scl show a homogeneous fluorescence in the nucleoli.
(C) Anti-Ku autoantibodies show a granular pattern in the nucleoplasm.
(D) Anti-PL-7 autoantibodies show a homogeneous fluorescence in the cytoplasm.
Immunoblot (IgG)- Against 16 Antigens (MSA & MAA)
Myositis Autoantibodies- Testing Methodology- IIF & Immunoblot

12. Data - 2017 to 2019
@Tertiary Care Hospital
Laboratory
MYOSITIS ANTIBODY PROFILE (IgG 16Ag)
Child (N=45) Mi-2 α Mi-2 β TIF 1γ MDA5 NXP2 SAE1 Ku
PM-Scl-
100
PM-Scl-
75
JO-1 SRP PL-7 PL-12 EJ OJ
Ro-
52
Total Positive 2 5 5 8 8 0 0 4 3 1 1 0 3 1 0 6
%Positive 4% 11% 11% 18% 18% 0% 0% 9% 7% 2% 2% 0% 10% 2% 0% 10%
Data - 2017 to 2019
@Tertiary Care Hospital
Laboratory
MYOSITIS ANTIBODY PROFILE (IgG 16Ag)
Adult (N=100) Mi-2 α Mi-2 β TIF 1γ MDA5 NXP2 SAE1 Ku
PM-Scl-
100
PM-Scl-75 JO-1 SRP PL-7 PL-12 EJ OJ
Ro-
52
Total Positive 12 4 2 3 2 0 1 2 2 39 11 0 10 2 0 13
%Positive 12% 4% 2% 3% 2% 0% 1% 2% 2% 39% 11% 0% 10% 2% 0% 13%
Myositis Autoantibodies- Hospital based Lab Data –
Using Immunoblot (IgG)- Against 16 Antigens
Autoantibodies- Children- commonest NXP2, MDA5, TIF 1γ , Mi-2 β; lower positivity noticed for Jo-1, SRP, PL-12
Autoantibodies- Adult- commonest Jo-1, SRP, Mi-2 α , PL-12; lower positivity noticed for NXP2, MDA5, TIF 1γ , Mi-2 β
Co-existence of Anti Ro-52 with Anti-Jo1 noted

13. CONCLUSION-
 Diagnosing PM/DM is challenging due to rarity of the disease, their similar clinical
presentation and the possibility of overlap syndromes.
 A comprehensive strategy for serological testing comprises of parallel determination of both
MSA and MAA thereby reducing the time to diagnosis.
 Current immunoassays techniques (Immunoblot) targeting MAA & MSAs have –
 The ability to include many parameters simultaneously which ensures higher detection rate
 Become readily accessible & hence an opportunity for laboratories to contribute significantly in the
disease diagnosis
 MSA are highly specific for PM/DM, and many of them are also associated with a unique
clinical subset of PM/DM, making them useful clinical diagnostic/prognostic biomarkers &
continue to evolve – eg- cN-1A (Cytosolic 5'-Nucleotidase 1A, MUP44 AB) shown to be
associated with Sporadic Inclusion Body Myositis (33%)

14. REFERENCES-
 1. A Comprehensive Overview on Myositis-Specific Antibodies: New and Old Biomarkers in
Idiopathic Inflammatory Myopathy. Satoh et al. Clin Rev Allergy Immunol. 2017 February ;
52(1): 1–19.
 2. Autoantibodies in Myositis. How to Achieve a Comprehensive Strategy for Serological
Testing Mende M et al. Mediterr J Rheumatol 2019;30(3):155-61
 3. Current Classification and Management of Inflammatory Myopathies. Schmidt, Journal of
Neuromuscular Diseases 5 (2018) 109–129
 4. New Myositis Classification Criteria—What We Have Learned Since Bohan and Peter.
I.Lundberg et al Current Rheumatology Reports (2018) 20: 18
 5. EUROLINE Autoimmune Inflammatory Myopathies 16 Ag (IgG), Test instruction
 6. https://understandingmyositis.org/myositis-antibody-testing/

15. THANK YOU