Lab diagnosis of ctd By Dr Arif Iqbal MD Dermatology UCMS & GTBH

LAB DIAGNOSIS OF CTD

BY :DR. ARIF IQBALMODERATOR: DR SURUCHI VOHRA

Q1. Senstivity of anti-histone Ab in drug induce SLE and ideopathic SLE is:

1 1% and 30%2 30% and 9053 90% and 30%4 None.

Q2. An apparently healthy mother delivers a baby with annular erythematous skin lesions predominantly over the face and congenital heart block.

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 by immunofluoresence?

Q4. Which one of the following is not an indirect marker of disease activity?

1. CRP2. Serum factor XIII3. ESR4. Serum factor VIII

AUTOIMMUNE CTD

1. LE 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

3. Dermatomyositis 4. Sjögren’s syndrome 5. MCTD 6. Overlap and undifferentiated CTD

2. Scleroderma A.Cutaneous scleroderma (morphea) B. Systemic scleroderma 1. Limited disease (acrosclerosis,

CREST syndrome) 2. Diffuse disease (SSc)

Mechanism for development of autoimmune diseases

Antinuclear antibodies (ANA)

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 disorders(CTD).

• 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.

Continued…

• 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 predict prognosis.

• 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.

Continued…

• 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 indermatological practice

• 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 ssDNA2. Antibodies to small ribonucleoproteins A. Antibodies to Ro(SS-A) B. Antibodies to La(SS-B) C. Antibodies to U1RNP D. Antibodies to Sm3. Antibodies to histones4. Antibodies to centromere5. 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 underlying CTD.

• Auto-antibodies against cytoplasmic and cell membrane components though present are less relevant in diagnostics.

Sensitivity and specificity of ANA and its clinically important subtypes

Autoantibodies Associated CTD Sensitivity Specificity ANA SLE 93 57 Sjogren's syndrome 48 52 SS 85 54 PM/dermatomyositis 61 63 Raynaud phenomena 64 41 Specific ANA

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 observed.

• 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 diseases

• ANA pattern Antigen Associated diseases Speckled ENA, RNP, Sm, SSA/Ro, SLE, MCTD, SS, PM SSB/La, Scl-70, Jo-1, ribosomal-P

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

ANA substrate

• 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 cell lines.

• Due to variable sensitivity with the substrate used it is essential to report the type of substrate being used by the lab.

ANA titre• 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 of laboratories

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-ANA tests.

• The test therefore is mainly used for screening rather than to diagnose a CTD.

EIA/ELISA

• 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 95%).

• Figures were arrived at using sera that were positive at 1:160 by the IF-ANA test.

• The above comparison figures were much lower for sera with IF-ANA titer of 1:40.

• ELISA may miss anti-SSA/Ro, due to vigorous antigen preparation methods.

• 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.

Specific ANA1. nDNA/dsDNA

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

2. ssDNA• 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.

• Indication: 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

antibodies.

• Indication and interpretation of results: Patients suspected of having drug induced lupus, Idiopathic SLE cannot be

ruled out by their presence.

Drugs reported with drug-induced SLE

• Allopurinol• Captopril• Chlorpromazine• Clonidine• Danazol• Diphenylhydantoin• Ethosuximide• Griseofulvin• Hydralazine• Isoniazid• Lithium• Lovastatin• Mephenytoin• Mesalazine• Methyldopa• Minocycline• Oral contraceptives

• para-Amino salicylic acid• Penicillamine• Penicillin• Phenothiazines• Phenylbutazone• Piroxicam• Practolol• Primidone• Procainamide• Propylthiouracil• Quinidine• Streptomycin• Sulfasalazine• Sulfonamides• Tetracycline• Thiamazole• Trimethadione• Valproate

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

diagnostic value. 2) ELISA: low specificity, high senstivity. Low specificity is made up by

quantitative assesment.

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 inautoimmune CTDs (by radial immunodiffusion)

Diagnosis % 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) andanti-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• Scl-70: 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%.

• Jo-1: Directed against the enzyme histidyl tRNA synthetase Detected in a small number of patients with dermatomyositis (and

polymyositis). 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

membranes.• 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 results.

• 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

considered +ve.

• 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 infection,hepatitisC1)

• Primary APA syndrome.

Indications for APLA testing• Livedo reticularis• Purpura and necrosis• Ulcers• 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%).

• Limitations: 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 RA.

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 Testsin the Diagnosis and Monitoring of CTD

ANA

• 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, cytokeratin)

3. Mitotic patterns: mitotic spindle; centrosomes; NuMA; midbody; CENP-F.

Titre• Titers of 1:40 and 1:160 (or concentrations of 5 and 20 IU, respectively) are

considered decision-making levels that require different operative algorithms:

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 polymyositis).

• 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 immunodot methods.

• 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.

Laboratory evaluation of individual CTD

SLE• Diagnosis of SLE based on the proper constellation of clinical findings and

laboratory evidence.

• 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

antibodies or If the patient satisfies 4 of the diagnostic criteria, including at least 1

clinical and 1 immunologic criterion.

Diagnostic studies• Complete blood count (CBC) with differential• Serum creatinine• Urinalysis with microscopy

CBC count may help screen for leukopenia, lymphopenia, anemia, and thrombocytopenia.

Urinalysis and creatinine studies may be useful to screen for kidney disease.

• Other laboratory tests: Erythrocyte sedimentation rate (ESR) or C-reactive protein (CRP) Complement levels 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 infectious process.

• 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 (NSAIDS).

• 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 cellular casts.

Antibodies

• Anti ribosomal P antibodies associated with CNS involvement.

• Chillblain lupus, SCLE, & neonatal lupus associated with anti Ro/SSA

LE cell• End result of either phagocytosis of distorted nuclear material by a

polymorphonuclear leukocyte or autolysis of one or more lobes of the nucleus

• 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 incubation.

• Detected in 85% of SLE patients.

Rowell’s syndrome• 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.

Radiologic study• 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

Libman-Sacks endocarditis.• Brain MRI or MRA: To evaluate for CNS white matter changes, vasculitis, stroke. Nonspecific, absent in 42% with NS symptoms.

Joint effusion & CSF studies• Arthrocentesis: 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 anticoagulation).

• 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

Skin Biopsies

ACLE• 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 uncommon.

SCLE• Interface dermatitis.

• Foci of vacuolar alteration of basal keratinocytes alternating with areas of lichenoid dermatitis.

• 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 lesions.

CCLE (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.

Lupus panniculitis• 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 fat necrosis.

• Dermis: PV & interstial lymphocytic infiltrate, fibrinoid deneration of collagen.

• Mucinous degeneration and calcification in old lesions.

Immunohistology• 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.

• ACLE: Lesional LBT in 60-80% cases. Sun damaged skin of healthy individual can show similar results.• SCLE: 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.• CCLE/DLE: 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.

Non-Lesional LBT

• 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).

Summary

Systemic Sclerosis• Laboratory findings:

• Increased erythrocyte sedimentation rate.• Hypergammaglobulinemia• Microangiopathic hemolytic anemia• Increased creatine phosphokinase levels in patients with muscle

involvement• 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.

Imaging studies

• 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-

appearing lungs.• Gastrointestinal tract changes can be detected by CT, barium swallow,

esophageal manometry, BMFT, barium enema.

Cardiac and pulmonary vascular involvement

• 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:

Electrocardiography (ECG) 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.

Antibody profile• 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

( 8%)• 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 patients.

• Anti-Ro antibodies ( 9%).• Anti U1RNP: 6% with overlap.

Histologic findings• 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.

Dermatomyositis• 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

hands) 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 criteria Probable dermatomyositis — Rash and two of the other four diagnostic criteria Possible dermatomyositis — Rash and one of the other four diagnostic criteria

Laboratory studies• 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 & associated malignancy.

• 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.

• Autoantibodies:

• 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 prognostic.

• 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 hands.

• Anti-SRP: • 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 for CADM.

• Serum levels of KL-6, expressed by type 2 pneumocytes correlates with interstitial pneumonia.

• Ab to 140kD Ag marker for ILD in Japenese CADM patients.

Imaging studies• MRI: 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 biopsy).

• EMG: 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.

Histological findings• 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 can occur.

• 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

Lab diagnosis of MCTD

Background

• 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.

Clinical features

• 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)

Laboratory studies• CBC count• Urinalysis• 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

in MCTD• Amylase and lipase - To assess for pancreatitis if clinically indicated

Respiratory system:• Chest radiography - To assess for infiltrates, effusion, or cardiomegaly• Pulmonary function testing –declining DLCO, possibly indicating

pulmonary hypertension.

Cardiovascular system:• ECG and/or cardiac enzymes - To assess for myocardial ischemia and

myocarditis• 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 neuropsychiatric manifestations

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 reactants.

• ESR• C reactive protein• Alpha 1 acid glycoprotein• Alpha 1 antitrypsin• Haptoglobin• Ceruloplasmin• 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)

Bibliography.• Rooks Textbook of Dermatology, 8th edition.

• Fitzpatrick Textbook of Dermatology.

• A practical guide for serologic evaluation of autoimmune connective tissue diseases: JAAD.

• Antinuclear antibodies and their detection methods in diagnosis of connective tissue diseases: a journey revisited, Diagnostic Pathology 2009, 4:1 doi:10.1186/1746-1596-4-1.

• Guidelines for the Laboratory Use of Autoantibody Tests in the Diagnosis and Monitoring of Autoimmune Rheumatic Diseases: Immunopathology.