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2 Laboratory Update Mark A. McQuillan MD FACP SFHM June 15, 2016RHEUM UPDATE II Laboratory Update Mark A. McQuillan MD FACP SFHM June 15, 2016

3 DISCLOSURES None

4 Acknowledgements Blake Roessler MD David A. Fox, Division ChiefW. Joseph McCune Ruba Kado MD Josef Holoshitz MD UMHS Rheumatology William Repaskey MD MFH Magrey and Abelson, CCF

5 Goals Rheumatology Labs: how much is reasonable? What rheum labs are pathognomonic? (audience top 2 choices) Costs How often should the key tests be obtained? Repeated? What tests are helpful before referral? What tests are helpful for monitoring? Local tips for your EMR?

6 Update in LABS

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8 WESR—sick or not sick? Causes of falsely Low WESRVibration Timing (RBC alignment and hydrostatic bonds do not necessarily follow a linear response curve) Anemia (? All types) Hypofibrinogenemia Coagulopathy (esp. DIC) Hemoglobinopathy (Hgb S or S-C) Muscle Diseases (?mechanism) Cryoglobulinemia Falsely HIGH with age, nephritis, hypergammaglobulinemia

9 C-reactive protein Very sensitive in identifying inflammatory changes, as early as 4-6 hours after tissue injury Usually not elevated in SLE pts, so elev CRP in a pt with SLE may raise suspicion of bacterial infection Very useful in inflammatory bowel disorders Turnaround time has improved so often coincides with WESR and is promptly available, though possibly more costly

10 Can you always follow the WESR?Certain diseases classically do not have WESR elevation associated with flares… PM/DM RA OA SD SSA PMR ?

11 “Sedimentation” (historic perspective)ESR described in writings of British Surgeon and Anatomist John Hunter ( ). Famous for experimental self-inoculation with GC which led to his subsequent fatal co-infection by syphilis Whose home was supposedly model for “Dr. Jekyll and Mr. Hyde” Book entitled, “Treatise on the Blood, Inflammation, and Gun-Shot Wounds”

12 Dr. Hunter’s observation on sedimentation:“…The red globules subsided much faster in the inflammatory blood than in the other…” Subsequent distinction between “inflammatory fevers” and “symptomatic fevers”

13 ESR measurement Described by Polish physician Edmund F. Biernacki ( ) Designed glass cylinders Correlated with fibrinogen level 1897 His studies later associated ESR with RA, TB, fevers, nephritis, hepatic disorders

14 ESR early investigators1917 Ludwik Hirszfeld, Polish microbiologist (malaria) 1918 Robert Fahraeus, Swedish hematologist, ESR elevation in pregnancy Alf Westergren, Swedish internist, used sodium citrate as anticoagulant, helpful in prognostication for TB patients 1973, J Clin Pathology established Westergren method as preferred

15 Acute Phase REactants ESR (Westergren or Wintrobe) C-reactive proteinInterleukins IL-6, IL-1, Tumor necrosis factor alpha (TNFa) Interferon gamma (IFN) Transforming growth factor beta (TGF-B) All of the above are proteins whose plasma concentrations rise by 25% or more with acute inflammation, causing a change in hepatic protein synthesis Ref: Magrey and Abelson, CCF

16 ENA Sm, Ro, La, RNP Centromere Histone OthersANA PATTERNS—diffuse, rim, speckled, homogeneous, nucleolar, centromere ?? Significance ??

17 Advances in ENA testingParaneoplastic panel Unusual markers ?significance

18 ENA staining patterns

19 Antinuclear antibody staining patterns on mouse liver by immunofluorescence. (Top left panel) There is homogeneous or diffuse staining of the nuclei. The pattern is strongly associated with antibodies to nucleosomes. It is seen in patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and drug-induced lupus. (Top right panel) The peripheral (also called rim) pattern, in which the periphery of the nucleus reacts with the patient's serum. This pattern correlates with antibodies to double-stranded DNA (dsDNA) and nuclear envelope proteins and is suggestive of SLE. (Bottom left panel) The speckled pattern in which there may be few or many speckles, and small or large speckles. The pattern represents antibodies to the components of extractable nuclear antigens, eg, Smith (SM), ribonucleoprotein (RNP), Ro (SSA), La (SSB) and probably other nuclear antigens. The pattern may be seen in patients with SLE, Sjögren's syndrome, scleroderma, infectious mononucleosis and other conditions, and in normal subjects (especially in low titer). (Bottom right panel) There is a nucleolar pattern, in which the patient's serum only reacts with the nucleoli. This pattern represents antibodies to nucleolar RNA associated proteins or complexes, and is primarily seen in scleroderma. Courtesy of Peter H Schur, MD. Graphic Version 8.0

20 ENA patterns and associationsPossible Disease Associations Homogeneous Rim Speckled Nucleolar RA, SLE, drug-induced SLE dsDNA, SLE Non-specific* Scleroderma, MCTD * Smith, Ro, La, RNP SLE, Sjogren’s, SD, mono, Normals (low titer)

21 ANA testing ANA is important to confirming diagnosis of SLEANA does NOT correlate with disease activity ANA serial exams in SLE is of unknown value Anti-DNA testing is useful in SLE, by either Crithidia Lucida assay or ELISA, and may vary with disease activity in some patients; may also correlate with renal disease in SLE patients

22 Anti-CCP Antibodies to citrullinated cyclic polypeptidesEarly and sensitive indicator of RA Specificity ? Confounders: MCTD Other Overlap syndromes

23 WESR vs CRP Cheap Turnaround time Sensitivity Poor specificityFalse low WESR Lab interference ? Serial values CRP more costly More sensitive w IBD ?Serial values ? Less sensitive vs more sensitive

24 scleroderma tests Anti-SCL-70 is useful for systemic sclerosisAnti-centromere Ab is useful in identifying limited scleroderma

25 “ANCA positive Vasculitis”MPO—a type of p-ANCA which is positive in 40-80% of patients with microscopic polyangiitis PR3—a type of c-ANCA which is present in % of pts with granulomatous polyangiitis Negative ANCA assay does not exclude GPA Value of sequential or serial ANCA testing is unknown

26 Complement deficienciesInherited deficiency of C1, C2, and C4 predispose to SLE

27 OTHERS Anti-Synthetase AbMay be seen in inflammatory muscle disease; (PM/DM) IgG 4 subclass disorder Emerging as a rare cause of pancreatitis and other abdominal findings REF: Ruba Kado MD UMHS

28 Goals Rheumatology Labs: how much is reasonable? What rheum labs are pathognomonic? (audience top 2 choices) Costs How often should the key tests be obtained? Repeated? What tests are helpful before referral? What tests are helpful for monitoring? Local tips for your EMR? (eg, Crithidia, ENA) Referring to algorithm can help to limit testing and help with decision to refer and timing !!

29 Vasculitis Update June 2016William Repaskey MD Mark A. McQuillan MD FACP SFHM

30 OBJECTIVES Update in classification of VasculitisUpdate in nomenclature Update in Diagnostic testing interpretation Case-based Invitation to question

31 Why update classification?Pathophysiology Testing Treatment advantage

32 Nomenclature update Eponymous expungement Examples Reactive arthritisMicro-angiitis others

33 Rheumatology Labs: C3 normal CH50 220(H) P-ANCA positive (1:80)MPO 1.0 (upper limit of normal 0.9); PR3 negative

34 Other Rheum Labs

35 Bronchoscopy

36 BAL Findings

37 …Encore ICU Admit 1 week later: “…87 year old male with a recent admission for hemoptysis found to have pseudomonas on bronch, who is readmitted with worsened SOB, hemoptysis, and interval worsening of b/l airspace disease on CT concerning for DAH…”

38 CXR

39 Tale of the Soup AEC (nl 0-500) 900 on 5/6< in March 2013< in August 2012 ENA11 neg NAB neg ANCA neg<---1:80 MPO + 4.3<--1.0 PR3 neg<--neg Anti-GBM neg CAB neg B2GP1 RF 33<---10 hep C neg anti ccp 5 ck 54 esr 100 <--75 crp 22 <--4.5 c3 116 c4 22 UA no blood, no protein. initial UA 6-10 RBC The ANA screen test (ANA) detects autoantibodies to 11 clinically relevant antibodies (dsDNA, SS-A, SS-B, Sm, RNP, SmRNP, Scl-70, Jo-1, Centromere B, Ribosomal P, and Chromatin). A negative result means that the patient’s serum shows no reactivity for these common antibodies associated with connective tissue diseases. The ANA by IFA (NAB) assay is recommended if the ANA screen test is negative, but patient symptoms are compelling for SLE. Antinuclear Antibody Screen ANA 86038 Antinuclear Antibody by IFA NAB 86038 EXTRACTABLE NUCLEAR ANTIBODY TESTING ENA testing is recommended to follow-up a positive ANA screen. B2GP1 (apolipoprotein H) – binds to cardiolipin - Anti-cardiolipin antibodies are found in both infectious (syphilis) and autoimmune disease (sclerosis, lupus).

40 DAH Not a specific disorder, but a syndromePersistent or recurrent pulmonary hemorrhage. Autoimmune disorders most common. dyspnea, cough, hemoptysis, and new alveolar infiltrates on chest imaging Hemoptysis absent in up to one third of patients (most w/ anemia, dropping hct) Symptoms, signs, and chest-x-ray findings are not specific BAL shows persistent/increasing hemorrhage with sequential lavage samples and hemosiderin-laden macrophages

41 DAH Causes

42 BAL in DAH

43 Clinical Classification of Systemic Vasculitis

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45 Pauci-Immune Small Vessel Vasculitides (name changes)Microscopic polyangiitis (MPA) aka PAN Granulomatosis with polyangiitis (GPA, formerly known as Wegener granulomatosis), as of 2011 ACCP editorial; (in 2000 WG had been renamed “ANCA-associated Granulomatous Vasculitis” when ACCP identified Wegener’s Nazi past and rescinded the “master clinician” award bestowed in 1989 Eosinophilic granulomatosis with polyangiitis (EGPA, formerly known as Churg-Strauss syndrome)

46 What are ANCAs? Anti-neutrophil cytoplasmic antibodiesAntibodies against myeloperoxidase (MPO-ANCA) and Proteinase 3 (PR3-ANCA) MPO & PR3 are found in the azurophilic granules of neutrophils and the lysosomes of monocytes. ANCAs are autoantibodies Therefore ANCA-associated vasculitis is an autoimmune disease MPO: MPO produces hypochlorous acid (HOCl) from hydrogen peroxide (H2O2) and chloride anion (Cl-) deficiency of the enzyme predisposes to immune deficiency. PR3 is a serine protease enzyme expressed mainly in neutrophil granulocytes. Its exact role in the function of the neutrophil is unknown but in human neutrophils proteinase 3 contributes to the proteolytic generation of antimicrobial peptides John H. Stone, MD, MPH

47 ANCA testing Although particular ANCAs are linked with particular diseases, it is possible to be ANCA + without manifesting a disease syndrome, and it is possible to manifest a traditional ANCA-associated vasculitis without + ANCAs Any of the ANCA-associated vasculitides can be associated with either type of ANCA or no ANCAs at all. Other autoimmune disease can be ANCA positive, at least on immunofluorescence

48 Three patterns of ANCA p-ANCA, show a perinuclear staining pattern c-ANCAs, show a diffusely granular, cytoplasmic staining pattern Atypical that develop against antigens other than MPO or PR3 will occasionally result in patchy staining

49 pANCA disease association: cANCA disease : associationPrimary vasculitis Microscopic polyangitis Churg-Strauss syndrome Polyarteritis nodosa Collagenosis Felty's syndrome SLE Rheumatiod arthritis Sjögren's syndrome  Chronic inflammatory bowel disease   Chronic liver disease Primary sclerosing cholangitis cANCA disease : association Primary vasculitis Wegener's granulomatosis Microscopic polyangitis Churg-Strauss syndrome. Atypical Primary sclerosing cholangitis Primary biliary cirrhosis Autoimmune hepatitis SLE RA

50 Applications of ANCA Churg-Strauss Syndrome Wegener's GranulomatosisMicroscopic Polyangiitis ANCA 10% 80% 40 % PR3-ANCA 60% 20% 50% MPO-ANCA 30% 5% Negative

51 Microscopic PolyangiitisMost common ANCA vasculitis (though rare: 13 to 19 cases/million) Median age ~ 50s Vasculitis of small and medium sized arteries No granulomas. 60% of patients are ANCA-positive, usually perinuclear ANCA (p-ANCA) with antibodies against myeloperoxidase but can be PR3-ANCA and, rarely, ANCA negative

52 More MPA “Pauci-immune” (ie, immune globulin deposition is not seen on tissue biopsy) Differentiates from immune complex-mediated small-vessel vasculitides (eg, immunoglobulin A–associated vasculitis—formerly known as Henoch-Schönlein purpura) Older literature (ie, before 1994) did not adequately distinguish between polyarteritis nodosa and MPA―alveolar hemorrhage and glomerulonephritis can occur in MPA but not in polyarteritis nodosa.

53 Churg-Strauss SyndromeAllergic Granulomatosis and angiitis Vasculitis of small and medium sized arteries Necrotizing vasculitis Builds over years until full presentation as it progresses through the following phases: Prodromal Phase- teens-20s Allergic rhinitis Asthma Eosinophilic phase Vasculitic phase as early as 20s-30s, but mean age is 50s Curr Opin Rheumatol. 2010;22(1):21-8

54 References Al-Saadi, A. In: Clinical immunology principles and practice RICH, 2nd edition Methodological update ANCA.mht, 2008 Schrier, P. In: Anti-Neutrophil Cytoplasmic Antibody- Associated Vasculitis and Anti-Glomerular Basement Membrane Antibody Glomerulonephritis, PPT, 2011 Gota, C. In : The Merck Manual, 2012 Stone, J. In: UpToDate, 2013.

55 Development of ANCA Theory of molecular mimicry. Superantigens have the power to stimulate a strong immune response . THEY have regions that resemble self-antigens – this is the theory of molecular mimicry. classical example in post group A streptococcal rheumatic heart disease, where there is similarity between M proteins of Streptococcus pyogenes to cardiac myosin and laminin. Theory of defective apoptosis. ANCA may be developed either via ineffective apoptosis or ineffective removal of apoptotic cell fragments, leading to the exposure of the immune system to molecules normally sequestered inside the cells. This theory solves the paradox of how it could be possible for antibodies to be raised against the intracellular antigenic targets of ANCA.[4] Methodological update ANCA.mht

56 Friedrich Wegener Friedrich Wegener (born 1907 in Varel – July 9, 1990, Lübeck) was a German pathologist who is notable for his description of a rare disease. Although this disease was known before Wegener's description, since the 1950s it has been called by the name Wegener's granulomatosis.[1] Wegener joined the Nazi Party in 1932.[1] As a relatively high-ranking military doctor, he spent some of World War II in a medical office three blocks from the Łódź Ghetto, a Jewish ghetto in Łódź, Poland.[1] There is speculation that he participated in experiments on concentration camp inmates.[1] The American College of Chest Physicians (ACCP) awarded Wegener a “master clinician” prize in 1989.[1] After his Nazi past was discovered in 2000, the ACCP rescinded the prize and, separately, a campaign was begun to rename Wegener's granulomatosis to ANCA-associated granulomatous vasculitis.[1] More recently, several journals proposed the name 'granulomatosis with polyangiitis' in a 2011 editorial.[2]

57 Wegener (cont’d.) "The facts we have uncovered do not prove Dr Friedrich Wegener guilty of war crimes. However, the evidence suggests that Dr Wegener was, at least at some point of his career, a follower of the Nazi regime. Dr Wegener’s mentor, Martin Staemmler, was an ardent supporter of the racial hygiene. In addition, our data indicate that Dr Wegener was wanted by Polish authorities and that his files were forwarded to the United Nations War Crimes Commission. Finally, Dr Wegener worked in close proximity to the genocide machinery in Lodz."[8]

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