LAB DIAGNOSIS OF CTD
BY :DR. ARIF IQBAL
MODERATOR: DR SURUCHI VOHRA
Q1. Senstivity of anti-histone Ab in drug induce
SLE and ideopathic SLE is:
1 1% and 30%
2 30% and 905
3 90% and 30%
Q2. An apparently healthy mother delivers a baby
with annular erythematous skin lesions
predominantly over the face and congenital
A)Which serological marker or markers you
would like to evaluate in the baby and mother?
B) Which ANA pattern will be found on IF?
Q3. Which substrate is used for ANA detection
Q4. Which one of the following is not an indirect
marker of disease activity?
2. Serum factor XIII
4. Serum factor VIII
A. Systemic LE
B. Discoid LE
C. Subacute cutaneous LE
D. Neonatal LE
E. Overlap of two or more LE sub.
F. Overlap of LE with other CTDs
4. Sjögren’s syndrome
6. Overlap and undifferentiated CTD
A.Cutaneous scleroderma (morphea)
B. Systemic scleroderma
1. Limited disease
(acrosclerosis, CREST syndrome)
2. Diffuse disease (SSc)
Mechanism for development of
What are ANA ?
• Specific class of antibodies, capability of binding and destroying certain
structures within the cells.
• Directed to plasma membrane, cytoplasmic and nuclear antigens.
• Predominant reactivity with nuclear antigens, therefore called ANA.
What is there importance ?
• Importance lies in the characterization of connective tissue
• CTD are characterized by presence of high titres of these Ab.
• Patients with CTD have an autoimmune phenomenon.
• Accurate diagnosis depends on evaluation of 4 parameters: clinical
findings, histopathology, tissue immunoflorescence and serologic testing.
• Serologic testing involves identification, characterisation and
measurement of these Ab.
• Serologic testing does not substitute for evaluation of the other criteria.
• Help to confirm a clinical diagnosis, classify subsets of a CTD and thus help
• Not unique to patients with CTD and may be present in the sera of normal
persons or persons with other conditions.
• Mere detection does not always indicate a CTD, however, the total amount
(titre) to a certain antigen is much larger in patients with CTD.
• Specificity of each for the various CTD varies. ( anti Sm and anti ds-DNA for
SLE, anti ssDNA have low specificity).
• The type of antibodies present and the frequency of their occurrence vary
among the various CTDs.
• Each CTD has a unique profile of antibodies.
Indications for fluorescent ANA testing in
• Work-up for photosensitivity.
• Clinical suspicion of CTD
• Baseline for patients undergoing phototherapy
• Work-up of chronic vasculitis
ANAs IN CTD
1. Antibodies to DNA
A. Antibodies to nDNA (dsDNA)
B. Antibodies to ssDNA
2. Antibodies to small ribonucleoproteins
A. Antibodies to Ro(SS-A)
B. Antibodies to La(SS-B)
C. Antibodies to U1RNP
D. Antibodies to Sm
3. Antibodies to histones
4. Antibodies to centromere
5. Antibodies to phospholipid (cardiolipin)
6. Antibodies to other cellular components
dsDNA, Double-stranded DNA; nDNA, native DNA; ssDNA, singlestranded
DNA; U1RNP, uridine-rich ribonucleoprotein.
ANA- the two broad types
• Antibodies to DNA and Histones.
• Antibodies to extractable nuclear antigens(ENA).
Antibodies to DNA and Histones
• These include antibodies against single and double stranded DNA (dsDNA).
• Significant levels of anti-dsDNA antibodies are considered to be
confirmatory in diagnosis of SLE.
• Anti-histone antibodies, indicative of drug-induced SLE.
Ab to extractable nuclear antigens(ENA)
• Named ENA as originally extracted from nuclei with saline.
• Autoantibody to Smith antigen (Sm), considered to be specific for SLE was
the first anti-ENA detected.
• Ribonucleoproteins (RNP), SSA/Ro, or SSB/La, Scl-70, Jo-1 and PM1.
• Topoisomerase-I (Topo-I), centromere protein (CENP)-B, RNApolymerase I-
III (RNA-pol I-III), MU, TM, Ku, Mi-2, RA33 etc.
• Although ENA are disease specific, still a significant overlap exists.
• The sensitivity and specificity also varies depending upon the type of
• Auto-antibodies against cytoplasmic and cell membrane components
though present are less relevant in diagnostics.
Sensitivity and specificity of ANA and its clinically
Autoantibodies Associated CTD Sensitivity Specificity
SLE 93 57
Sjogren's syndrome 48 52
SS 85 54
PM/dermatomyositis 61 63
Raynaud phenomena 64 41
Anti-dsDNA SLE 57 97
Anti-Sm SLE 25–30 High*
Anti-SSA/Ro Sjogren's syndrome, 8-70 87
subacute cutaneous SLE
Neonatal lupus syndrome
Anti-SSB/La Sjogren's syndrome, 16-40 94
subacute cutaneous SLE,
Neonatal lupus syndrome
Anti-U3-RNP SS 12 96
Anticentromere Limited cutaneous SS 65 99.9
Scl-70 SS 20 100
Jo-1 PM 30 95
Techniques of ANA detection
• Presence of autoantibodies in the sera of the patient constitutes one of
the criteria used for diagnosis of CTD.
• Indirect immunofluorescence antinuclear antibody test (IF-ANA) and
enzyme immunoassay (EIA)/enzyme linked immunosorbent assay (ELISA)
are commonly used.
IF-ANA: The standard technique
• Most widely used test for diagnosis of CTD: inexpensive, easy to perform,
with high sensitivity and specificity.
• Detects the presence of ANA in the blood of the patient.
• Ab adhere to reagent test cells (substrate), forming distinct fluorescence
patterns, associated with certain autoimmune diseases.
• Hep-2 cells, cultured laryngeal squamous ca cells, available commercially
prefixed on glass slides are used as substrates ( high senstivity ).
Reporting of IF-ANA results
• Three parameters:
1) Pattern of fluorescence
2) Substrate used
3) Titre of positive test
1. Fluorescence pattern and intensity
• Different staining patterns gives clue to the type of ANA and CTD .
• Nuclear patterns: homogeneous, speckled (fine and
coarse), peripheral/rim, nucleolar, centromeric, PCNA (proliferating cell
nuclear antigen), nuclear dots, nuclear membrane, diffuse grainy.
• Cytoplasmic patterns: speckled, mitochondrial-like, ribosomal-like, Golgi
apparatus, lysosomal-like, cytoskeletal filaments
(actin, vimentin, cytokeratin).
• Mitotic patterns: mitotic spindle, centrosomes, NuMA (nuclear mitotic
apparatus), CENP-F (centromere protein)
Common nuclear patterns
• Homogenous, speckled,
peripheral and nucleolar
patterns are more commonly
• Fluorescence patterns intensity of
staining (qualitative scale) : + to
• Intensity is generally proportional
to antibody concentration and
predicts the severity of the CTD.
Common IF-ANA patterns associated with specific
• ANA pattern Antigen Associated diseases
Speckled ENA, RNP, Sm, SSA/Ro, SLE, MCTD, SS, PM
SSB/La, Scl-70, Jo-1,
Homogenous dsDNA, Histones SLE, drug induced SLE
Peripheral RNP, Sm, SSA/Ro SLE, SS
Nucleolar Anti PM-Scl, anti-RNA pol 1-3 SS, PM
anti U3 RNP, To RNP
Centromeric CENP A-E Limited SS
• Sera of some patients with SLE may be negative on animal substrates i.e.
mouse kidney or rat liver but are positive on human substrate i.e. Hep-2
• Due to variable sensitivity with the substrate used it is essential to report
the type of substrate being used by the lab.
• Directly proportional to antibody concentration.
• Crucial, low titer is less significant than a high titre.
• Low titers (1:40-1:80; 5-10 IU) of ANA:
In healthy subjects (in particular, pregnant women, women older than 40
years, and elderly persons)
As a phenomenon associated with viral infections paraneoplastic
neurologic syndromes, liver disease, chronic fatigue syndrome, and
cancers of various types.
• A titer of 1:160 is taken as significant for the diagnosis of CTDs in majority
Limitations of IF-ANA
• In up to 3% of the normal population it can give false positive result.
• Levels tend to rise when symptoms flare and fall, often being
undetectable, when symptoms are mild or patient is in remission.
• Difficult to specify or categorize an autoantibody.
• Certain patterns i.e. nucleolar and centromeric are less well defined by IF-
• The test therefore is mainly used for screening rather than to diagnose a
• Two types of EIA or ELISA methods currently used for ANA testing:
1) Generic assay which detects ANA of broad specificity similar to IF-ANA.
2) Antigen specific assay that detects ANA and reacts with a single
autoantigen, multiple antigens are coated on to microtitre plates, usually a
combination of SSA/Ro, SSB/La, Sm, and U1-RNP.
• Both highly specific and sensitive and substantially decreases time while
screening large samples.
• Simple to perform, can be automated &does not require highly trained
operators who can recognize microscopic patterns.
• Becoming most widely used method for routine screening as well as for
detection of specific ANA.
Limitations of ELISA
• Performance of ELISA test was compared with the "gold standard" IF-ANA
test: sensitivity of the various ELISAs was 69% to 98% and the specificity
ranged between 81% and 98%(serum with ANA positivity from 87% to
• Figures were arrived at using sera that were positive at 1:160 by the IF-
• The above comparison figures were much lower for sera with IF-ANA titer
• ELISA may miss anti-SSA/Ro, due to vigorous antigen preparation
• Sera that react only with conformational antigens can also miss the
presence of antigen.
• ELISA techniques have also been found to miss a low titer positive ANA as
well as sera with specific ANA.
• Presently, adequate to screen sera only with intermediate to high titers.
1) IF-ANA test using Crithidia luciliae as the substrate(CLIF)
2) The Farr assay
3) ELISA ds-DNA
• CLIF – hemo-flagellate C. luciliae as the substrate. Kinetoplast contains
only dsDNA ( no ssDNA). Sensitivity is comparable to Farr assay.
• Farr assay: radio-labeled assay, precipitation of antibody-antigen
complexes on addition of ammonium sulfate at high concentration, quick,
quite specific and advocated as most reliable assay.
• Disease association:
Highly characteristic of SLE, presence usually associated with positive
Lupus band test, low circulating complement levels, renal disease, and
generally poor prognosis.
• Interpretation of results:
Positive IF test or ELISA value higher than 2-3 S.D. above the mean confirm
a clinical diagnosis of SLE. Low levels of nDNA may be detected in RA,
Hashimoto’s disease, Graves’ disease, Waldenström’s macroglobulinemia,
MCTD, SSc, autoimmune liver disease, & SS.
Negative test does not exclude SLE because nDNA antibodies are positive
in only 50% to 83% of patients with SLE
• Testing technique: ELISA.
• Disease association:
Very low diagnostic value, various forms of LE as well as other CTDs,
including DM, morphea( esp linear morphea in children) and SS.
• Interpretation of results:
Should be much higher than the normal range (>3 standard deviations
above the mean) to be of value in the diagnosis of CTD.
Nonspecific, diagnostic value in workup of CTD is low.
3. Histone antibodies
• Histone antibodies are characteristic of drug-induced SLE.
• Testing technique:
ELISA( commercially available histones), RIA, Complement fixation, IF.
• Disease association:
Majority (approximately90%) of patients with drug-induced SLE have
antihistone antibodies to the exclusion of other antibodies.
Approximately 30% of patients with idiopathic SLE also have antihistone
• Indication and interpretation of results:
Patients suspected of having drug induced lupus, Idiopathic SLE cannot be
ruled out by their presence.
4. anti-RNP Antibodies
• Directed to snRNP ( <1% of total RNA).
• snRNP: RNA and associated protein, antibodies are directed against
epitopes present within protein components.
• Diagnostic specificity variable: Sm specific for SLE, antiRo seen in various
subsets of LE and other CTD.
• Two major techniques for detection:
1) Radial immunodiffusion: high specificity, low senstivity, + test has high
2) ELISA: low specificity, high senstivity. Low specificity is made up by
5. Anti-Ro(SS-A) and anti-La(SS-B) Ab
• Disease association:
Anti-Ro(SS-A) antibodies are characteristic of two CTDs, LE & SS.
Radial immunodiffusion, anti-Ro(SS-A) detected in 50% patients with SS
and a varying percentage in various subsets of LE.
Strongly associated with photosensitivity esp in SCLE.
May be associated with a higher incidence of vasculitis.
Genetic predisposition- HLA-DR3, DQ2, DRw52.
• Anti-La: >90% positivity in patients with +ve antiRo, disease association
same as antiRo.
Incidence of anti-Ro(SS-A) antibodies in
autoimmune CTDs (by radial immunodiffusion)
Antinuclear antibody negative SLE 70
Subacute cutaneous LE 70
Homozygous C2 or C4 deficiency 70
Late onset SLE 80
Neonatal LE 95
Mothers of infants with neonatal LE 95
Discoid LE 0-20
Sjögren’s syndrome 50
SSc, dermatomyositis Rare
Healthy persons < 1
Indications for ordering anti-Ro(SS-A) and
anti-La(SS-B) antibody testing
• Work-up for photosensitivity
• Screening for certain patients with LE ( with photosenstivity)
• Suspicion of subacute cutaneous LE
• Suspicion of neonatal LE
• Suspicion of Sjögren’s syndrome
• Work-up for idiopathic chronic vasculitis (underlying undiagnosed SS)
• Patients with systemic or subacute cutaneous LE with negative screening
fluorescent ANA test ( -ve ANA with these Ab)
6. Ab to U1RNP and U3RNP
• Present in the sera of patients with MCTD (100%) and SLE(30%).
• Presence of U1RNP antibodies in MCTD is to the exclusion of other types
of antinuclear antibodies.
• Patients of SLE with U1RNP usually have other ANAs, important when
differentiating MCTD from SLE.
• Usually associated with sclerodactyly, Raynaud’s phenomenon, esophageal
dysmotility, low incidence of renal disease, pulmonary dysfunction,
arthritis, and myositis.
• Ab to U3RNP is present in Sscl.
7. Other autoantibodies
Directed against the enzyme topoisomerase-I.
Characteristic of SSc, differentiate patients with extensive cutaneous and
systemic involvement from those with limited disease.
Incidence by RID: 10-20%.
Directed against the enzyme histidyl tRNA synthetase
Detected in a small number of patients with dermatomyositis (and
The presence of Jo-1 antibodies is often associated with pulmonary
involvement and possibly the mechanic’s hand skin lesions.
8. Antiphospholipid antibodies
• Directed against negatively charged phospholipids, present in cell
• Most commonly used to detect are:
1) Lupus anticoagulant(LAC)
2) Anticardiolipine(ACL) Ab
3) Anti beta2 glycoprotein 1 AB
• Testing techniques:
Lupus anticoagulant: 4 mandatory steps for detection:
1) Prolongation of aPTT, KCT, dRVVT, dPT: two test with different principles
should be used (Screening step)
2) Mixing study: 1:1 proportion of patient plasma and normal pooled
plasma( d/f specific factor deficiency).
3) Confirmation that inhibitory activity is phospholipid dependent by increasin conc. of
phopholipid in step 1.
4) Exclusion of other coagulopathies that may accompany LAC presence or yield similar
• Anticardiolipine Ab:
ELISA is test of choice. Reference range are:
< 15 IgG phospholipid units (GPL): Absent/ not detected
< 12 IgM phospholipid units (MPL): Absent/ not detected
< 12 IgA phospholipid units ( APL): Absent/ not detected.
For diagnosing APL syndrome, only medium to high titres of IgG/IgM(>40 GPL or MPL) are
• Anti beta 2 glycoprotein 1 Ab:
ELISA is test of choice.
IgG/IgM >99 percentile considered +ve.
Disease association of APLA
• Most prevalent in patients with SLE (approximately 50%).
• Low prevalence with other CTD.
• Drugs: cocaine, interferon alfa, procainamide, hydralazine,
phenothiazines, quinine, quinidine, fansidar, and phenytoin.
• Patients with chronic infections (syphilis, infectious mononucleosis,
tuberculosis, leprosy, leptospirosis, malaria, typhus, trypanosomiasis,
schistosomiasis and filariasis, cytomegalovirus infection, HIV
• Primary APA syndrome.
Indications for APLA testing
• Livedo reticularis
• Purpura and necrosis
• Internal organ thrombosis
• Recurrent miscarriages
• Screening in patients with SLE
9. RA factor and anti CCP Ab
• RA factor:
IgM Ab directed against Fc portion of IgG.
Traditionally used as a laboratory rest to support clinical diagnosis of RA.
Wide range of senstivity (50-85%), moderate specificity (80-90%).
May be –ve in 20% cases of RA, early in course of disease(40%).
Though relate to severity of extraarticular manifestation, not useful for
monitoring course of disease.
• Anti-CCP ( IgG anti-cyclic citrullinated peptide):
Antibody directed against the anti-keratin epitope that contains citrulline.
Citrullinated extracellullar fibrin is found within the synovium of patients with
Highly specific tool for the diagnosis of RA at an earlier stage of the disease.
Senstivity (47.1%), specificity ( 97.4%).
Helpful in confirming diagnosis in patients with a negative or equivocal RF.
Marker for the development of more severe erosive disease.
Monitoring disease activity and prognosis.
Guidelines for the Laboratory Use of Autoantibody Tests
in the Diagnosis and Monitoring of CTD
• Tests to detect autoantibodies are performed only when a consistent
clinical suspicion of autoimmune rheumatic disease is present. ANA
determination should not be used to screen subjects without specific
symptoms because weak ANA reactivity is present in many nonrheumatic
patients and even in “healthy” control subjects.
• IgG ANA determination using indirect immunofluorescence should be the
initial step in the laboratory diagnosis of autoimmune rheumatic diseases.
• When performing IIF-ANA, use the epithelial cell line HEp-2 from human
laryngeal carcinoma, Specify the immunohistochemical patterns as
follows, and add a short interpretation of the significance:
1. Nuclear patterns: homogeneous/peripheral; speckled; centromeric;
nucleolar; PCNA; nuclear dots; nuclear membrane; diffuse grainy
2. Cytoplasmic patterns: speckled; mitochondrial-like; ribosomal-like; Golgi
apparatus; lysosomal-like; cytoskeletal filaments (actin, vimentin,
3. Mitotic patterns: mitotic spindle; centrosomes; NuMA; midbody; CENP-F.
• Titers of 1:40 and 1:160 (or concentrations of 5 and 20 IU, respectively) are
considered decision-making levels that require different operative
1. Titers less than 1:40 (5 IU) should be considered as negative.
2. Titers equal to or more than 1:40 (5 IU) and less than 1:160 (20 IU)
should be considered positive at low titer (in the absence of specific
symptoms, further diagnostic study is not advised, but the patient should
be clinically monitored).
3. Titers equal to or higher than 1:160 (20 IU) should be considered
positive, and patients should undergo further diagnostic study because
they are probably affected by an autoimmune disease.
• In general, the use of variations in the ANA titer by IIF to monitor the course
and therapy of autoimmune rheumatic disease is not advised.
• Do not screen for ANAs with immunoenzymatic methods, unless the
following conditions are present:
1. The method used must have at least 90% concordance with IIF.
2. Positive results must be subsequently confirmed by IIF, specifying pattern
and titer or reactivity.
3. Discordant results (ELISA-positive, IIF-negative) should be considered false-
positive unless anti-Ro/SSA or anti–Jo-1 antibodies are found. If it is strongly
suspected that the patient has an autoimmune rheumatic disease, the patient
must be monitored over time.
4. Negative results should specify which autoantibodies were studied and
found negative, and patients with a clinical picture that raises suspicion of
autoimmune rheumatic disease must be evaluated successively over time.
Guidelines for antiDNA Ab
• Determine anti-dsDNA autoantibodies only when clinical symptoms raise
the suspicion of SLE and when ANA is positive by IIF.
• In the diagnostic phase, use the highly specific radioimmunologic method
(Farr technique) or the IIF assays.
• In monitoring the clinical course of SLE, perform a quantitative
determination of anti-dsDNA antibodies every 6 to 12 weeks, using ELISA
or Farr assay, and express results in IU/mL (WHO).
Guidelines for other Ab
• In general, determine antinuclear specific antibodies only when ANA
screening is positive. In the case of a negative ANA, antinuclear specific
antibody testing is indicated only if the patient has clear signs of an
autoimmune rheumatic disease (especially Sjögren syndrome or
• In the diagnostic phase, extend the detection of antinuclear specific
autoantibodies to the following autoantigens:
Ro/SSA, La/SSB, Sm, U1RNP, Scl70 (topoisomerase I), Jo-1 (histidyl-tRNA
synthetase), CENP-B, rRNP, and nucleosome (chromatin).
• In the diagnostic phase, determine antinuclear specific antibodies with
counterimmunoelectrophoresis, immunoenzymatic, immunoblot, or
• The use of one of the aforementioned methods suffices to identify anti-
Ro/SSA, anti-La/SSB, anti-U1RNP, anti-Sm, anti–Jo-1, anti-Scl70, anti-
rRNP, anti–CENP-B, and antinucleosome (chromatin) antibodies;
however, the finding of a low or negative antibody concentration in a
subject for whom clinical suspicion of autoimmune rheumatic disease is
strong imposes the use of 2 of the above methods.
• Diagnosis of SLE based on the proper constellation of clinical findings and
• The Systemic Lupus International Collaborating Clinics (SLICC) group
revised and validated the ACR SLE classification criteria in 2012.
• Patient is classified as having SLE if:
Patient has biopsy-proven lupus nephritis with ANA or anti-dsDNA
If the patient satisfies 4 of the diagnostic criteria, including at least 1
clinical and 1 immunologic criterion.
• Complete blood count (CBC) with differential
• Serum creatinine
• Urinalysis with microscopy
CBC count may help screen for leukopenia, lymphopenia, anemia, and
Urinalysis and creatinine studies may be useful to screen for kidney disease.
• Other laboratory tests:
Erythrocyte sedimentation rate (ESR) or C-reactive protein (CRP)
Liver function tests
Creatine kinase assay
Spot protein/spot creatinine ratio
• Level of ESR may show a discrepancy relative to a normal CRP level in SLE
flares; if both markers are markedly elevated, suspect the presence of an
• CRP levels change more acutely, and the ESR lags behind disease changes.
• C3 and C4 levels, often depressed in active SLE(consumption). Some
patients have congenital complement deficiency predisposing to SLE.
• Liver test results: azathioprine or nonsteroidal anti-inflammatory drugs
• Creatine kinase levels may be elevated in myositis or overlap syndromes.
• Spot protein/spot creatinine ratio for proteinuria, ratio greater than 0.5
g/day can substitute for the 24-hour protein measurement.
• Active urinary sediment (>5 RBCs per hpf; >5 WBCs/hpf in the absence of
infection; or cellular casts limited to RBC or WBC casts) can substitute for
• Anti ribosomal P antibodies associated with CNS involvement.
• Chillblain lupus, SCLE, & neonatal lupus associated with anti Ro/SSA
• End result of either phagocytosis of distorted nuclear material by a
polymorphonuclear leukocyte or autolysis of one or more lobes of the
• Contains within its cytoplasm the LE body, which appears as
homogenous, pale blue to deep-purplish material pushing the nucleus of
the phagocyte to one side of the cell.
• Not usually found in peripheral blood, although the LE cell phenomenon
could form in the buffy coat of peripheral blood after a period of
• Detected in 85% of SLE patients.
• Lupus erythematosus and EM like syndrome.
• Serological abnormalities:
• Speckled pattern of ANA in DLE, homogenous in SLE.
• Antibodies to saline extracts of human tissue, anti-SjT ( SS-B ).
• LBT +ve in discoid lesions & -ve in EM lesions.
• Anti SS-B and RF developed after EM lesions in one case.
• Joint radiography:
Most common radiographs in SLE show periarticular osteopenia and soft-
tissue swelling without erosions.
• Chest imaging:
Radiography and CT scanning to monitor interstitial lung disease and to
assess for pneumonitis, pulmonary emboli, and alveolar hemorrhage.
• Echocardiography :
To assess for pericardial effusion, pulmonary hypertension, or verrucous
• Brain MRI or MRA:
To evaluate for CNS white matter changes, vasculitis, stroke.
Nonspecific, absent in 42% with NS symptoms.
Joint effusion & CSF studies
Joint effusion- inflammatory/non inflammatory, based on PMN count,
viscosity and gross appearance of fluid.
• Lumbar puncture:
To exclude infection with fever or neurologic symptoms.
Nonspecific elevations in cell count and protein level and decrease in
glucose level may be found.
Biopsies & Histologic findings
• Renal biopsy:
2012 ARA guidelines recommend biopsy for all active, previously
untreated LN patients, unless C/I.
Conform presence of LN, aid in classification, guide therapeutic decisions.
To diff LN from RVT ( compication of APLA syndrome, require
• Renal biopsy is indicated in the presence of the following features:
Increasing serum creatinine in the absence of strong evidence for another
etiology (eg, sepsis, hypovolemia, medication).
Proteinuria of more than 1.0 g per 24 hours, as confirmed by 24-hour
urine specimens or spot protein/spot creatinine ratios.
Proteinuria of 0.5 g or more per 24 hours, along with either (1) hematuria
(≥5 RBCs/hpf) or (2) cellular casts, as confirmed by a minimum of 2 tests
within a short period and in the absence of alternative causes
• Changes less impressive as compared to SCLE or DLE lesions.
• Cell poor interface dermatitis.
• Mild focal vacuolar alteration of keratinocytes, in most severe form, can
display keratinocyte necrosis similar to TEN.
• Upper dermis: pronounced mucinosis, differentiates ACLE from other cell
poor interface dermatitis.
• BM thickening, follicular plugging, alteration of epidermal thickness
• Interface dermatitis.
• Foci of vacuolar alteration of basal keratinocytes alternating with areas of
• Diff diagnosis: atrophic LP, lichenoid drug eruptions.
• Dermis: edema, prominent mucinosis, sparse MN cell infiltration.
• Lesser degrees of HK, FP, dermal melanophages differentiates from DLE
• Stratum corneum: HK with FP.
• Epithelium: thinning & flattening of malphigian layer, hydropic
degeneration of basal cells, dyskeratosis.
• BM: thickening & tortuosity.
• Stroma: pred lymphocytiv infil along DEJ, around hair follicles & other
appendages, and in interstial pattern, interstitial mucin deposition,
edema, vasodilation, extravasation of RBC.
• Subcutis: slight extention of inflammation present.
• Absence of characteristic epidermal & dermal changes of LE.
• Subcutis: lobular lymphocytic panniculitis with PV infiltration with
lymphocytes, plasma cells and histiocytes in deep dermis & s/c fat, hyaline
• Dermis: PV & interstial lymphocytic infiltrate, fibrinoid deneration of
• Mucinous degeneration and calcification in old lesions.
• Boosts senstivity & specificity of diagnosis.
• Not uncommon to see –ve IF in acute, subacute & chronic LE and false
positivity in healthy individuals.
• Deposition of IgG, IgA, IgM and complement components ( C3, C4, C1q,
properdin, MAC C5b-C9) in continuous granular or linear band-like array at
dermal epidermal junction k/s Lupus band.
• Both lesional and non lesional skin.
• Debate regarding use of terminology.
Lesional LBT in 60-80% cases.
Sun damaged skin of healthy individual can show similar results.
Lesional LBT +ve in 60% patients.
Dust-like particle pattern of IgG deposition focussed around epidermal
basal keratinicytes more specific for SCLE, reflect in-vivo bound Ro/SSA Ab.
Lesions on head. Neck & arm: 80%; trunk:60%
Older lesions( > 3months) more than younger lesions.
• LE profundus:
Blood vessel wall of deep dermis and subcutis.
Deposition at D-E Junction may or may not be present.
• Performed on totally sun-protected non lesional skin ( e.g. buttocks).
• Presence of three or more immunoreactants at DEJ has high diagnostic
specificity for SLE.
• Presence of non lesional LBT also correlates positively with risk of
developing LE nephritis.
• Limitation: Information gained by non lesional LBT is not greater than
readily available serologic assays such as antibody to dsDNA.
Lupus band test. Microphotograph of a histologic section of human skin prepared for
direct immunofluorescence using an anti-IgG antibody. The skin is from a patient with
systemic lupus erythematosus and shows IgG deposit at 2 different places: the first is a
band-like deposit along the epidermal basement membrane ("lupus band test" is
positive); the second is within the nuclei of the epidermal cells (anti-nuclear antibodies).
• Laboratory findings:
• Increased erythrocyte sedimentation rate.
• Microangiopathic hemolytic anemia
• Increased creatine phosphokinase levels in patients with muscle
• Increased urea and creatinine levels in patients with kidney involvement
• C-reactive protein: 25%, correlate with disease activity, severity, poor
pulmonary function, and shorter survival.
• Serum procollagen 3 peptide: marker of disease activity.
• Chest radiography:
Normal finding (5-10%), basal fibrosis (30-60%), ground glass & honey
comb pattern ( irreversible, response to t/t).
• Bone radiography:
Generalized osteopenia, mostly affects hands. Intra-articular calcifications
often are observed.
• HRCT and scintigraphy:
Thickening of the alveolar walls and intestinal tissue, honeycomb-
• Gastrointestinal tract changes can be detected by CT, barium swallow,
esophageal manometry, BMFT, barium enema.
Cardiac and pulmonary vascular
• Doppler echocardiography:
Cardiac involvement is one of the major problems in systemic sclerosis.
Evaluation of ventricular function using echocardiographic strain imaging
should be considered.
• Heart changes,myocardial disease, pericardial problems, conduction system
disease, and arrhythmias, can be observed with:
Holter 24-hour monitoring
Doppler ultrasonography (US)
• Lung function test:
• Spirometory: restrictive ventilatory defect, reductions in pulmonary
compliance, VC & TLC
• Decreased DLCO, measure of diffusion capacity.
• ANA positivity by IF-ANA is 97%
• 3 main subgroups of Ab seen, usually mutually exclusive:
• Antibodies against topoisomerase I DNA (Scl 70):
Detected in two thirds of patients with dSSc and interstitial lung fibrosis.
• Anticentromere antibodies (ACAs):
Predict limited skin involvement, less chance of diffuse skin and visceral
invovement, longer survival time.
• Anti RNAP 3 antibodies:
Greatest risk of diffuse skin & renal involvement, shortest survival time.
• Other antibodies( ANA ):
• Spectrum differs in patients with and without cutaneous involvement.
• Antibodies against fibrillarin, 34-kd protein of ribonucleoprotein U3 RNP (
• Anti Th/To RNP ( mitochondrial RNA ) antibodies (4%).
• Anti-PM/Scl antibodies, seen in roughly 24% with polymyositis/systemic
sclerosis overlap syndrome. Also found in 3-10% of systemic sclerosis
• Anti-Ro antibodies ( 9%).
• Anti U1RNP: 6% with overlap.
• Active indurative phase:
Loss of rete ridges occurs.
Epidermal skin appendages atrophy,.
Collagen fibers in the reticular dermis appear broad and hyalinized.
Mononuclear cells, mostly T cells, form a variable perivascular infiltrate in
the deep dermis and subcutis.
• Later, sclerotic changes predominate. Significant sclerotic change not
observed. The number of adnexal structures is reduced, and a loss of
periadnexal fat is noted.
• Approach consideration:
• Laboratory tests.
• Diagnostic imaging ( MRI, chest radiography, ultrasonography,
electromyography [EMG], or computed tomography [CT]).
• Muscle and skin biopsy and other tests as appropriate.
• Older patients: the frequency of an associated malignancy increases.
Assessment for malignancy should be performed upon initial diagnosis
and repeated at least annually for 3 years.
Diagnostic criteria of DM/PM
• Diagnostic Criteria
1. Typical skin rash (Gottron's papules, heliotrope rash, mechanic's/machinist's
2. Symmetric proximal muscle weakness with or without dysphagia or
respiratory muscle involvement
3. Abnormal muscle biopsy specimen
4. Elevation of skeletal muscle enzymes (CK-MM)
5. Abnormal electromyogram.
• Confidence Level for Diagnosis of Dermatomyositis*
Definite dermatomyositis — Rash and three of the other four diagnostic
Probable dermatomyositis — Rash and two of the other four diagnostic
Possible dermatomyositis — Rash and one of the other four diagnostic criteria
• Muscle enzymes:
• Levels are elevated except in CADM.
• Most sensitive/specific enzyme is elevated CK.
• Aldolase and other tests like AST or LDH may also yield abnormal results.
• Elevated levels of CK: hypothyroidism, physical exertion, needle trauma.
• At times, elevation of the CK precedes the appearance of clinical evidence
of myositis, predicting flare in asymptomatic patients.
• Levels may be normal in early or late disease( as atrophy sets in).
• Patients with normal CK have worse prognosis w.r.t. pulmonary disease &
• Aldolase less sensitive, occasionally elevated in presence of normal CK
levels esp Juvenile DM.
• Urinary creatine excretion can be used to monitor the status of myositis.
• Serum myoglobin raised in severe IDM.
• May be helpful in the classification of subtypes for prognosis, but not used
for routine diagnosis.
• Positive ANA: 60-80% of both IIDM & CADM but not diagnostic or
• Myositis-specific antibodies (MSAs) & Myositis associated antibodies.
• MSA (30% with DM or PM), includes antisynthetases ( Jo-1, Mi-2, SRP, PL-
7, PL-12, OJ).
• Myositis associated includes PM/Scl, Ro/SSA, U1RNP.
• Prevalence of MSA: 1 out of 5 patients, routine test is of less clinical use.
• Anti-Mi-2: highly specific for DM, senstivity 25%, associated with acute-onset classic
DM with the V-shaped and shawl rash (poikiloderma), relatively good prognosis.
• Anti–Jo-1 (antihistidyl transfer RNA [t-RNA] synthetase) antibodies: senstivity 20%,
PM>DM, interstitial lung disease, Raynaud phenomenon, arthritis, and mechanic’s
• 5%, severe PM, t/t resistant form with severe cardiac and rare skin involvement.
• Anti–PM-Scl and anti-Ku:
Associated with overlapping features of myositis and scleroderma.
• Autoantibody against p155:
Highly related to cancer-associated myositis and could be a reliable marker of cancer in
patients with dermatomyositis
Antibodies in CADM
• 2/3rd positive for ANA.
• Do not produce either MSA or myositis associated Ab.
• Several new Ab ( 140kD, 155kD and Se) may serve as serologicak markers
• Serum levels of KL-6, expressed by type 2 pneumocytes correlates with
• Ab to 140kD Ag marker for ILD in Japenese CADM patients.
Imaging method of choice for muscle abnormsalities.
Assessing inflammatory myopathy in patients without weakness.
MRI & MRS for status during t/t.
MRS can detect metabolic changes in CADM ( hypomyopathic DM).
As a guide in selecting a muscle biopsy site( obviating need for open ms
Detecting muscle inflammation and damage.
Motor unit AP, recruitment pattern, inc. insertional activity, inc. spon. activity.
Inferior to MRI.
May be normal in 10% of biopsy documented myositis.
• Ultrasonography (doppler & grey-scale USG) of the muscles has been
suggested for evaluation but has not been widely accepted.
• CT scanning: evaluation of potential malignancy associated with myopathy.
• Other tests: PFT, ECG & esophageal manometry.
• Skin biopsy:
Interface dermatitis that is difficult to differentiate from lupus
erythematosus.Vacuolar changes of the columnar epithelium and lymphocytic
inflammatory infiltrates at the dermal-epidermal junction basement membrane
• Muscle biopsy:
• Inflammation is the histologic hallmark of DM, PM & IBM.
• Only DM shows perifascicular atrophy.
• Muscle fiber degeneration/regeneration, perifascicular atrophy & capillary injury
characteristic of DM
• Fibers undergo degeneration and necrosis causing them to lose their staining
ability; termed ghost fibers.
• Collections of inflammatory cells around the blood vessels.
• PM & IBM: infiltration into muscles of infl cells typically surrounding &
displacing, sometimes invading myofibres.
Hematoxylin and eosin paraffin shows dermatomyositis. In dermatomyositis, inflammation is characteristically
perivascular and perimysial. Vessel oriented approximately vertically in center has mild perivascular chronic
inflammatory infiltrate. Endothelium is plump; wall is not necrotic. A few lymphocytes in wall of vessel are
probably in transit from lumen to external aspect of vessel. Some observers may interpret this finding as
vasculitis, but it is certainly neither necrotizing vasculitis nor arteritis.
Hematoxylin and eosin frozen section shows perifascicular atrophy in
dermatomyositis. Fascicles in this sample show atrophy, predominantly at
periphery, along connective-tissue border. Ischemia is considered to cause
perifascicular atrophy. This finding is characteristic of dermatomyositis,
mostly associated with juvenile form but also observed in adult form
• Overlapping clinical features of SLE, scleroderma, and myositis.
• Presence of a distinctive antibody against U1-ribonucleoprotein (RNP).
• Most patients experience Raynaud phenomenon, arthralgia/arthritis,
swollen hands, sclerodactyly or acrosclerosis, and mild myositis.
• 3 clinical subclusters of MCTD manifestations may exist :
Patients with predominantly vascular manifestations, including Raynaud
phenomenon, pulmonary hypertension, and antiphospholipid syndrome
with thromboses ( greatest risk of mortality).
• Patients with a polymyositislike picture, including interstitial lung disease,
esophageal dysmotility, and myositis.
• Patients with erosive polyarthritis with CCP antibodies and sclerodactyly.
• Arthralgia/arthritis (96% cumulatively, 68% at presentation)
• Esophageal hypomotility (66% cumulatively, 9% at presentation)
• Pulmonary dysfunction (66% cumulatively, rare at presentation)
• Swollen hands (66% cumulatively, 45% at presentation)
• Myositis (51% cumulatively, 2% at presentation)
• Rash (53% cumulatively, 13% at presentation)
• Leukopenia (53% cumulatively, 9% at presentation)
• Sclerodactyly (49% cumulatively, 11% at presentation)
• Pleuritis/pericarditis (43% cumulatively, 19% at presentation)
• Pulmonary hypertension (23% cumulatively, rare at presentation)
• CBC count
• Routine blood chemistry
• Muscle enzymes if myositis is suspected clinically
• Antinuclear antibodies:
• High-titer speckled pattern fluorescent antinuclear antibody (FANA) is
typical of mixed connective-tissue disease (MCTD).
• FANA is not specific to MCTD.
• High titers of anti–U1-RNP antibodie are required for diagnosis of MCTD.
• The presence of anti–U1-70 kd is characteristic of MCTD.
• Other immune studies:
• Antiphospholipid antibodies (including anticardiolipin antibodies and
lupus anticoagulant) may be associated with pulmonary hypertension.
• Rheumatoid factor is frequently detected.
• Other lupus-specific antibodies (eg, anti–double-stranded DNA
antibodies) are absent.
• Scleroderma-specific antibodies, including anticentromere, anti–Scl-70
(topoisomerase), and anti–PM-1 (Pm-Scl), are absent.
• C3 and C4 complement levels are more likely to be depleted in lupus than
• Amylase and lipase - To assess for pancreatitis if clinically indicated
• Chest radiography - To assess for infiltrates, effusion, or cardiomegaly
• Pulmonary function testing –declining DLCO, possibly indicating
• ECG and/or cardiac enzymes - To assess for myocardial ischemia and
• Echocardiography -effusion, chest pain, pulmonary hypertension, or
valvular disease (An exercise echocardiography may increase the
sensitivity to identify pulmonary hypertension.)
Ultrasonography/CT scanning - Used to evaluate abdominal pain (
serositis, pancreatitis, or visceral perforation related to vasculitis)
MRI - Used to assess neuropsychiatric signs or symptoms.
Cerebral spinal fluid (CFS) analysis - To monitor for infection, stroke, or
MCTD can enter sustained remission later in the clinical course. Anti-RNP
autoantibodies typically become undetectable in patients in remission.
Indirect markers of inflammation/ Acute phase
• C reactive protein
• Alpha 1 acid glycoprotein
• Alpha 1 antitrypsin
• Serum amyloid A
• Fibrinogen ferritin
• Complement factor C3, C4
• Factor 8
• Stimulus for production is likely to be inflammatory cytokines such as
interleukin-1, interleukin-6 and tumour necrosis factor (TNF)
• Rooks Textbook of Dermatology, 8th edition.
• Fitzpatrick Textbook of Dermatology.
• A practical guide for serologic evaluation of autoimmune connective tissue
• Antinuclear antibodies and their detection methods in diagnosis of
connective tissue diseases: a journey revisited, Diagnostic Pathology 2009,
• Guidelines for the Laboratory Use of Autoantibody Tests in the Diagnosis
and Monitoring of Autoimmune Rheumatic Diseases: Immunopathology.