autoantibody assessment in rheumatic disease dana ... · 19 year old female presenting with 2 year...
TRANSCRIPT
Autoantibody Assessment in Rheumatic Disease
Dana Ascherman, M.D.Division of Rheumatology
Case #1 19 year old female presenting with 2 year history of
Raynaud’s-type vasospasm
slight fatigue, mild arthralgias
ROS otherwise negative
Physical examination:• no oral ulcerations• no skin rash, peri-ungual blush• no synovitis
Case #1
Any additional physical exam maneuvers?
Should we order autoantibodies?
Case #2 37 year old female presenting with intermittent fever,
polyarthralgia/polyarthritis x 6 months
slight fatigue, mild xerophthalmia, Raynaud’s, difficulty climbing stairs
ROS otherwise negative
Physical examination:• afebrile• slightly diminished tear pool• sclerodactyly; no skin rash• no synovitis, but multiple tender joints• 4+/5 deltoid, iliopsoas, quadriceps strength
Case #2
Labs: leukopenia, mild anemia, ESR=47
ANA positive 1:640 in speckled pattern
What tests to order next?
Case #3 43 year old male referred for evaluation of ILD and
possible autoimmune disorder
Developed “pneumonia” in 2007—no response to antibiotics
Diagnosis revised to “pulmonary fibrosis”• treated with prednisone for 1 year with clinical,
functional, and radiologic improvement
Pulmonary evaluation: • restrictive PFTs with reduced FVC, TLC, DLCO• HRCT with basilar ground glass, minimal
honeycombing• Serology: -ANA, +SS-A, -Jo-1
Bilateral lower lobe air space disease with ground glass opacities consistent with active alveolitis; minimal fibrosis in lung bases peripherally
Case #3Review of Systems:
• no constitutional symptoms (F/C, NS, weight loss)• denies xerophthalmia/xerostomia, Raynaud’s• denies skin thickening or rashes beyond patchy
hyperpigmentation of palms• currently no cough, significant dyspnea• denies dysphagia, reflux, abdominal pain, change in
bowel habits• no joint pain/swelling/stiffness• mild proximal upper extremity aching without proximal
weakness
Case PresentationPhysical Examination:
• afebrile, normal blood pressure, respiratory rate• grossly adequate tear, salivary pools• without palpable LAD• lungs with good air movement, no use of accessory
muscles; CTA without rales/rhonchi/wheezing• normal radial, posterior tibial pulses• musculoskeletal exam without synovitis• normal proximal, distal muscle strength• skin without Gottron’s rash/papules
Case #3Summary
mechanic’s hands, no muscle weakness
steroid-responsive ILD
-ANA, -Jo-1, +SS-A cytoplasmic staining
What to do next (besides call a rheumatologist)?
Case #4 53 year old male presenting with polyarthritis,
intermittent fever, and weight loss
associated fatigue, sinus congestion, epistaxis
ROS otherwise negative except subjective hearing loss
Physical examination:• Gen: fatigued appearing• T=38.3• slight proptosis (right)• purpuric skin lesions distal lower extremities• mild synovitis PIPs, knees
Case #4
Labs: Hgb=9.7, Cr=2.4, U/A: 1+ protein, 43 RBCs; ESR=83
Imaging studies?
Additional serology?
Overview
1) Mechanisms of autoantibody formation
3) Disease associations
2) Methods of autoantibody detection
4) Autoantibodies as markers of clinical phenotype
Autoimmunity
“Horror Autoxicus”--Paul Ehrlich
Self-antigens targeted
Price of adaptive immunity
Question: why isn’t autoimmunity even more common?
Tolerance
Regulatory mechanism(s) to prevent uncontrolled autoreactivity
B vs. T cell tolerance
Central vs. peripheral tolerance
Autoimmunity: Mechanisms
1) Molecular mimicry•Rheumatic fever, HSK, Guillain-Barre
2) Release of sequestered antigen•Trauma, infection
4) Upregulation of MHC Class II•Release of IFN-γ
3) Generation of cryptic/neo-epitopes•Apoptosis--Granzyme B cleavage•?tissue-specific•Defective clearance--C1q deficiency (SLE)
5) Polyclonal B cell activation•EBV infection
6) Immune system defects, alterations•cytokine milieu--IBD
Antigen Processing/Presentation
Generation and Presentation of Autoantigens:Post-translational modifications
influence Ag structure, immunogenicity–examples include deimination of arginine to citrulline (anti-CCP), deamidaiton of aspartic acid to isoaspartic acid, glycosylation, transglutamination, oxidative damage
modifications can be triggered by aging, cellular stress induced by infection, trauma, apoptosis
modifications may not occur in thymus--escape central tolerance
post-translational changes could also influence subsequent processing, generation of epitopes
may promote epitope spreading--B cell cross reactivity to modified, native version
Antibody Detection Immunofluorescence
• ANA, ANCA
ELISA• CCP• U1RNP• SS-A/SS-B• Scl70
Immunoprecipitation• tRNA synthetases (e.g., Jo-1)• myositis- and scleroderma-specific autoantibodies
Immunodiffusion—tRNA synthetases
Nephelometry—RF
MethodologyImmunofluorescence
MethodologyImmunofluorescence
ANA--Patterns
PatternsA) Rim (dsDNA/chromatin) B) HomogenousC) Speckled (Ro/La, Sm/RNP) D) Nucleolar (Scl-70)
A) B)
C) D)
ACR slide collection
ANA Interpretation
ANCA
C-ANCA (PR3) P-ANCA (MPO)
Limitations- IIF antigen must be present in sufficient copy
number in the cell substrate • fixation solvent may strip antigen• cell may not produce sufficient quantity of Ag
somewhat subjective
limited standardization of pattern reporting
MethodologyELISA
MethodologyELISA
Limitations- ELISApurity of Ag often a problem
• recombinant Ag often expressed in bacteria• Ag purified from animal tissues may include other
antigenic proteins
some assays use only fragments of Ag
patients may have Ab to blocking agent
small operator inconsistencies can result in large errors
Ag coating on plates can be damaged by shipping and handling conditions
MethodologyImmunoprecipitation
Principlepatient’s Ab is bound to Protein
A Sepharose-coated beads and then incubated with an extract derived from human cells
target antigen binds to the Ab
immunprecipitate is eluted, bound Ag is separated by electrophoresis and visualized
190
150
138
126
80
70
62
43
34
32
27
PL-12 110 kd Band
NormalRNA
PolymerasesKuSRP SclerodermaAbs
RNA Pol
KuBands
SRP 54 kd Band
SRPPL-12
Immunoprecipitation
ImmunoprecipitationLimitations
proteins must be soluble, present in sufficient amount, and contain sufficient methionine for labeling
very large and very small antigens cannot be visualized
some antigens cannot be distinguished because they are of similar sizes
takes 1-2 weeks (batching)
MethodologyImmunodiffusion
patient sera and crude Ag mix are placed in wells in a gel an allowed to diffuse
at equivalence point, a visible preciptin line be formed
Autoantibody AssessmentOverall Limitations
RF, ANA lack specificity
SLE Present + -
+ 3 300
AN
A
Result - 1 9696
Lupus prevalence : ~ 4 in 10,000 in general populationProb of ANA >= 1:320 in SLE: ~ 75%
Prob of ANA >= 1:320 in normals: ~ 3%
Positive predictive value = 3/303 = ~1%Negative predictive value = 9696/9697 = >99.98%
Role of ANA TestingANA is useful to exclude lupus, not to
diagnose it
follow-up clinical assessment of ANA+ patients can be beneficial in high-risk populations
ANA has no value in assessing prognosis or disease activity (i.e., do not follow titers)
Autoantibodies:Markers of Clinical Phenotype
Myositis--Autoantibodies
target nuclear and cytoplasmic antigens: Mi-2, PM/Scl, SRP, Ku, tRNA synthetases
Antibody Target Subset PhenotypeMi-2 NuRD DM Shawl, V-neck, Gottron’sCADM-140 MDA-5 DM Amyopathic, ILDSAE SUMO DM ILD, dysphagiaMJ NXP-2 JDM Calcinosis, Ulcerationp155/140 TIF1-γ DM, JDM Severe skin, malignancySRP 72, 54 kDa PM Severe/refractory myositisp200/100 HMGCR IMNM Necrotizing myopathyJo-1 ARS PM/DM Anti-synthetase syndrome
Myositis--AutoantibodiesJo-1 (histidyl-tRNA synthetase): 25%
defines clinically homogeneous patient population: anti-synthetase syndrome (fever, myositis, arthritis, Raynaud’s, mechanic’s hands, ILD)
range of clinical manifestations varies depending upon targeted sytnthetase autoantigen
Systemic Sclerosis:Antibody subsets and Clinical Phenotype
1) Anti-centromere: limited cutaneous, Raynaud’s, pulmonary HTN
2) Anti-Th/To: limited cutaneous, pulmonary HTN and/or ILD
3) Anti-toposisomerase I (Scl-70): diffuse cutaneous, GI, ILD, renal crisis
4) Anti-RNA polymerase III: diffuse cutaneous (rapid), renal crisis
5) Anti-PM/Scl: myositis/scleroderma overlap, ILD
Case #1
Any additional physical exam maneuvers?
Should we order autoantibodies?
19 year old female with mild arthralgia, +Raynaud’s
1. Nailfold Capillaroscopy2. ANA
Case #2--Summary
fatigue, xerophthalmia, polyarthralgia, weakness, Raynaud’s
ANA positive 1:640 in speckled pattern
leukopenia, anemia, elevated ESR
ENA (SS-A, SS-B, Smith, RNP)--MCTD
Additional serology?
Case #3--Summary mechanic’s hands, no muscle weakness
steroid-responsive ILD
-ANA, -Jo-1, +SS-A cytoplasmic staining
Anti-Synthetase Syndrome (incomplete)—anti-EJ
Case #4--Summary
Serology?
53 year old male with:• fever• weight loss, fatigue• epistaxis, hearing loss• polyarthritis
anemia, Cr=2.3, hematuria, elevated ESR
ANCA (c-ANCA/PR3)—Granulomatosis with Polyangiitis
Imaging?
CXR: pulmonary nodules
ConclusionAutoantibody formation reflects breakdown of tolerance
•combination of T and B cell dysregulation
Clinical picture should determine serological profiling
•RF, CCP—rheumatoid arthritis
Autoantibodies serve as biomarkers of disease subsets
•ANA—SLE (dsDNA, Sm), Sjogren’s (SS-A, SS-B, RF), MCTD (RNP), systemic sclerosis (Scl-70, RNApol III, centromere)
•cytoplasmic ANA/ANA negative--myositis
•ANCA—GPA (c-ANCA/PR3), mPAN, EGPA (p-ANCA/MPO)
Caveat: RF, ANA lack specificity