Immunoserological Diagnosis in Rheumatologic Diseases

2. Immunoserological Diagnosis in Rheumatologic Diseases M. Mahdi Mohammadi, LMD, PhD,MPH Kerman University of Medical Sciences m3mahdi@hotmail.com

3. حداقل های آموزشی هر آزمایش • نام تست • اساس تست • کاربردها • جایگاه در بین اقران • ملزومات وشیوه کار • ارزشیابی و تفسیر نتایج

4. My Definition 4:Lab TEST به کلیه فعالیتهای منظم و روشمندانه ای اطلاق میشود که بر روی یک نمونه بیولوژیک (خواه مایع یا غیرمایع) انجام میگیرد تا کمیت یا کیفیت چیزی (یک آنالیت یا یک پدیده ) را با دقت و صحت مطلوب اندازه گیری کرده و در زمان مناسب کزارش نماید.

5. Lab Definition? سازمانی است که بر روی نمونه های انسانی (بافتها - توپر یا روان – و مایعات بدن ) ویا نمونه های مرتبط (آب و غذا و دارو و . . .) کار میکند. Practical Objectives of a Lab: • Screening (eg Employment) • Dx Verification • R/O (eg in Emergency Room) • Monitoring (eg TDM) • Prognosis And so, WHAT IS A MED Lab TEST?

6. Tests Classification By Techniques: Microscopy Photometry ELISA Culture Agglutination Chromatography Electrophoresis PCR etc • By Disease: • Endocrinology • OB & GYN • Cardiovascular • GI & Hepatobil. • Rheumatology • Nephrology • Hemato-oncology • etc By Methods: • Biochemistry • Hematology • Immunology • Microbiology Bacteriology Parasitology Mycology Virology • Forensic • etc

7. معیارهای یک آزمایش خوبو یک مارک خوب سادگی سرعت حساسیت ویژگی دقت صحت پذیرش مردمی ارزش اخباری • Equipments, Staff, Steps • Short T.A.T. (Clinical or Analytical) • Inherent Characteristics!/cutoff point? • Interferences, mAb Cross Reactivity • Precision, پایایی, Rel, Repro, Repea. • Accuracy, روایی, Validity, Trueness • Sampling; Price; Preanalytical Errors • Where from do you order a test?

8. Types of the Lab Results • Quantitative or Qualitative? • Even Semi-ones! • Objective or Subjective?

9. Specimen, Sample, SamplingDevices,Containers & VesselsBlood (a tissue) vs Urine (a fluid) • راحتی و صرفه - سهولت در نمونه گیری (غیرتهاجمی) - سهولت در تکنیک آزمایش واجرا - ارزانتر در آماده سازی و استخراج • غلظت - مقدار نمونه ی بیشتر - مقدار دارو و متابولیتهای بیشتر - آشغال کمتر • پایداری - دوام حضور متابولیتها - ماندگاری بیشتر نمونه

10. Normal Factors Affecting The Test Result Biological: Others: Dose Drug Impurity Presence of additves • Weight • Underlying Diseases • Route of Drug Entrance • Last Consumption Time • Duration

11. Immunoserological Diagnosis in Rheumatologic Diseases

12. Historical Milestones 1937 (Waaler)-Rheumatoid Factor: Agglutination of sensitized sheep RBCs by antibodies of rabbit origin, when they were exposed to Rheumatoid sera 1948 (Hargraves)-LE cell phenomenon: Antinuclear antibodies opsonize damaged lymphocytic nuclei which then become phagocytosed by neutrophils 1957 (Laurell & Nilsson)-Lupus Anticoagulant: Antiphospholipid antibodies found to be responsible for both anticoagulant effect as well as false-positive VDRL test in patients with SLE. 1982 (Davis)-Antineutropil cytoplasmic antibodies: Discovery of antibodies against neutrophils in the sera of patients with small vessel necrotizing glomerulonephritis

13. Rheumatoid Factor&Anti-cyclic citrullinated Peptides

14. Rheumatoid Factors • Antibodies (IgM, IgG, IgA) against Fc portion of IgG • Frequency in healthy population: 1.3-4% in Caucasions 30% in N. American Indian tribes • The frequency of IgM-RF increases with age while that of IgG-RF declines in elderly. • Techniques of detection: 1) Latex or sheep RBC agglutination 2) Enzyme immunoassay 3) Nephelometry IgM-RF predates development of RA. Those with persistent high titer RF and those who have both IgA-RF and IgM-RF are at increased risk of RA.

15. Rheumatoid Factor in RA • Frequency: up to 70% • Correlated with: Nodulosis, Vasculitis, Radiological erosions and Sicca. • IgA-RF is particularly associated with early development of erosions and a persistent disease course. Sensitivity Specificity RF 66-85% 72-82% Anti-CCP 56-74% 90-92% RF+Anti-CCP 48% 96%

16. Anti-Cyclic Citrullinated Peptide 295 (clinically proven RA) + 163 (DJD and non-RA inflammatory arthropathies) + 103 (other CTD/Vasculitis) + 154 (healthy controls) N=715 AssaySensitivitySpecificity In RA patients with highdisease activity, anti-CCP showed higher IgG-RF 43.7% 91/0% sensitivity than all RF isotypes. IgA-RF 50.9% 88.3% Of all tests done, anti-CCPshowed the best correlation IgM-RF 66.4% 82.1% with joint damage. Anti-CCP-2 64.4% 97.1% Ann Rhem Dis 2004;63:1079-1084

17. Laboratory Diagnosis in Antiphospholipid Syndrome

18. Clinical Correlations • Among those with recurrent arterial and venous thrombosis, 5-15% have antiphospholipid antibodies. • Antiphospholipid antibodies can develop in response to various infections which usually disappear within 12 weeks. • Drug-induced antiphospholipid antibodies are usually of IgMisotype and they do increase risk of thrombosis.

19. Evolving Laboratory Criteria of APS

20. Lupus Anticoagulants & Anticardiolipin antibodies; Different or the Same? • LA usually is a functional test. It is caused by antibodies against various phospholipids bound to proteins (B2GP-I, Prothrombin, Annexin-V). • ACLA-ELISA is a technique to look for antibodies against cardiolipin in the sample. • LA and ACA occur concurrently in 50%-75% of cases.

21. Lupus Anticoagulant Testing • Blood specimens should be collected in 3.2%sodium citrate tubes. • The specimen should be centrifuged before freezing & with 4 hours of blood collection to obtain platelet-poor plasma (Platelet count less than 10,000/mm3) Step 1: Screening (prolonged Pl-dependent coagulation based on more than two screening tests [dRVVT, dPTT, KCT, …]) Step 2: Mixing (failure to correct screening test by equal volume of pooled normal platelet-poor plasma) Step 3: Phospholipid neutralization (Correction of prolonged screening test by addition of excess phospholipid [lysed Platelet membranes, commertial bilayer, hexagonal phase]) Step4: Exclusion of other coagulopathies

22. Problems with LA testing • No LA-positive standard sample is available. • Lack of universal cut-off point for each step of the test • The impact of between-day imprecision • Weak LAs • Different sensitivities of various reagents in dPTT • Non-LA, factor inhibitors causing false positive screening and mixing steps (VIII & IX) • Contamination of plasma samples with the prescribed anticoagulants (Heparin, Warfarin, IV thrombin inhibitors, direct factor Xa and IIa inhibitors, Fondaparinux)

23. Isotypes of Anticardiolipin Antibodies Isotype distribution of anticardiolipin antibodies in patients with antiphospholipid syndrome: Only IgG…………………………..…36% Only IgM……………………..………17% Only IgA…………………………..….14% Various admixtures……………….…33% All three isotypes of Anti-cardiolipin antibodies IgG, IgA, IgM) should be tested in solid-phase ELISA method.

24. Subtypes of Antiphospholipid Antibodies • The frequency of Lupus Anticoagulant-negative and Anticardiolipin antibody-negative Antiphospholipid syndrome: Type I: (DVT/PE)…………………………………………………. 7% Type II: Coronary/peripheral artery thrombosis……………….15% Type III: Cerebrovascular/retinal thrombosis……….......…15-24% Type IV: Recurrent miscarriage…………………….......….......22% • Antibodies with possible role in the subtypes of APS: Anti-Phosphatidylserine, Anti-Phosphatidylcholine, Anti-Phosphatidylinositol, Anti-Phosphatidylglycerol, Anti-Phosphatidylethanolamine, Anti-Annexin-V

25. Looking For Antinuclear Antibodies

26. LE cell • Direct assay: Patient’s blood mixed with glass beads, incubated an hour in 37 C and buffy coat is stained with Giemsa stain. The nuclei of damaged lymphocytes are opsonized & phagocytosed by PMNs. • In vivo formation of LE cells are considered to be strong evidence of SLE. • Antibodies against Histone H1 are the major drive behind formation of LE cell.

27. LE cell versus “Tart cell” • Unlike LE cell, the ingested nuclei in Tart cell have not reacted with “LE cell factor” so the ingested nuclei retained their chromatin pattern and stain blue. • Tart cells do not correlate with SLE.

30. Antinuclear Antibody Diseases and Related Conditions

31. Antinuclear Antibody Diseases and Related Conditions

32. Antinuclear Antibody Diseases and Related Conditions

33. Antinuclear Antibody Pattern

34. AUTOANTIBODIES IN RHEUMATIC DISEASES.

35. Antinuclear Antibody Diseases and Related Conditions

36. Fluorescent Antinuclear Antibodies(FANA) • Substarte: • Slice of rodent liver • HEp-2 cells fixed with ethanol or acetone • HEp-2000 Addition of patient’s plasma washing Extractable NuclearFluorescently-labeled Antigens (ENA) anti-human globulin washing UV microscopy

37. Extractable Nuclear Antigens • Water soluble nuclear antigens that are washed away during FANA procedure • Responsible for “ANA-negative Lupus” • They are: SSA/Ro, SSB/La, Smith, RNP, Jo-1, … • Anti-Smith has high specificity for SLE and can be detected by gel diffusion, counterimmunoelectrophoresis and enzyme immunoassay. • Anti-RNP is found in high titer in 95-100% of MCTDs. • Anti-SSA is present in 70% of subacute cutaneous lupus erythematosus. • Anti-SSA is the main antibody responsible for congenital heart block in neonatal lupus. • Anti-Jo-1 is an antibody against histidyl-tRNAsynthetase and causes cytoplasmic speckled staining with faint nuclear staining. • Anti-Jo-1 is present in one third of PM/DM patients and is strongly correlated with interstitial lung disease.

38. Patterns of FANA • The most useful pattern of FANA in HEp-2 cells are Homogenous, Rim, Nucleolar, Speckled and Centromere. • Many SLE patients have more than one pattern that can be seen in various dilutions.

39. Antinuclear Membrane v/s Rim Pattern • Antinuclear membrane pattern has been associated with primary biliary cirrhosis. • Unlike homogenous-rim pattern which is caused by anti-dsDNA, anti-ssDNA and anti-Histone, antinuclear membrane pattern is because of antibodies to some nuclear membrane proteins. • Unlike homogenous/rim pattern, anti-nuclear membrane pattern does not stain mitotic figures.

40. Nucleolar FANA Pattern • The nucleolar pattern is the one most closely associated with Systemic sclerosis. • This pattern is caused by antibodies against Topoisomerase (Scl-70), RNAP-I, U3-RNP, PM-Scl,

41. Problems with FANA Testing • Different antigen concentrations in commercially available substrates • If high enough concentration of serum is used, most normal people give a positive FANA screen. Cut off titer? • Feltkamp recommends that about 200 sera from known healthy controls with broadly represented ages and equally distributed by sex should be used to determine the cut off. • Variability of titer from one laboratory to another.

42. ANA Enzyme Immunoassay • The concentration and specific preparation of each antigen is not currently standardized. • By including only a selected menu of antigens, the ANA-EIA can eliminate non-specific false-positives that provide confusing information.

43. Patient Selection and ANA Testing • The prevalence of SLE is roughly 1 in 1000 individuals. • Even in best circumstances, 5% of normal individuals give a positive ANA result. • If one tested all individuals among 100,000 unselected subjects one would detect 5000 positive results. • There is no more than 100 SLE cases among 100,000 unselected individuals. • This means that 4900 positive ANA test results among unselected individuals are false positive results. To avoid false positive ANA results, pretest selection of patients with appropriate symptoms is crucial.

44. Anti-neutrophil Cytoplasmic Antibody (ANCA)Testing

45. ANCA Testing • ANCA is an indirect immunofluorescence test. • ANCA performed with ethanol-fixed PMNs may result in two patterns: 1) Cytoplasmic (c-ANCA) 2) Perinuclear (p-ANCA) • Some antibodies give a positive reaction on ethanol-fixed PMNs but weak and smudgy appearance on formalin-fixed substrate. These are called Atypical ANCA (a-ANCA).

46. Targets of Antibodies Causing ANCA Patterns • c-ANCA pattern is mainly caused by Anti-PR3 and sometimes seen as a result of human leuckocyte elastase (HLE) • p-ANCA is caused by variety of antigens like MPO, Lactoferrin, BPI, Azuridin, lysozyme. The most clinically relevant of all is MPO. • Clinical correlations: Anti-PR3: Wegener’s Granulomatosis Anti-MPO: Microscopic polyangiitis, (WG in China) Anti-HLE: Drug-induced vasculitis/lupus, Cocaine- induced nasal destruction If ANCA phenomenon is found to be directed to more than one ANCA antigen, a drug-induced condition should be suspected.