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EliA Fibrillarin

EliA Fibrillarin. Launch Presentation 2011-12-19 EM. Content. Indications for testing Clinical background The antigen Methods to detect anti-Fibrillarin Product Details – Technical Performance Clinical Performance Competition Sales argumentation. Indications for testing.

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EliA Fibrillarin

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  1. EliA Fibrillarin Launch Presentation 2011-12-19 EM

  2. Content • Indications for testing • Clinical background • The antigen • Methods to detect anti-Fibrillarin • Product Details – Technical Performance • Clinical Performance • Competition • Sales argumentation

  3. Indications for testing • Suspicion of scleroderma1 • Differential diagnosis of diseases characterized by Raynaud‘s phenomenon1 • Assessment of risk group members1 (e.g., persons with exposure to silica or mercury) • Prognostic use: immunological differential diagnosis to predict potential development of scleroderma1 • Follow-up of a positive IIF with fibrillarin-typical pattern 1 Conrad K et al (2002)

  4. Clinical background Synonyms forFibrillarin : • Scl-34 • U3-RNP • rRNA 2'-O-methyltransferase Associated diseases: • Anti-fibrillarin occurs in lessthan 15% ofpatientswithsystemicsclerosis (SSc)2, but ishighlyassociatedwith diffuse cutaneousSSc (upto 64%)4 2 Pollard KM and Hultman P (2007) 4 Steen VD (2008)

  5. Systemic sclerosis • Definition: Fibrosing systemic disease with lesions of the vessels, leading to atrophy and fibrosis of almost all organs.(Fibrosis = proliferation of connective tissue) • Two major forms: limited and diffuse systemic sclerosis • Prevalence app. 10 in 100.000, depending on the population tested6 • Sex Ratio: male:female = 1:2 • Age: mostly adults, peak at 40-50 years • Antibodies against Scl-70, CENP, Fibrillarin, RNA Polymerase III and others (nucleolar pattern in IIF) • Symptoms: • Raynaud‘s phenomenon • Swollen face and hands in the morning • Calcium deposits in the skin • Ulcerations of the fingers • Telangiectasia • Involvement of the lung in >60%

  6. Limited systemic sclerosis (formerly called CREST) Calcinosis Raynaud’s phenomenon Esophageal dysmotility Sclerodactyly Telangiectasia • restricted to the fingers or distal limbs (not truncal) • more benign and indolent disease course • Internal organ involvement restricted to gastrointestinal tract. • CENP antibodies

  7. Diffuse cutaneous systemic sclerosis (dcSSc) • Diffuse cutaneous systemic sclerosis (dcSSc) is in contrast to the limited form of systemic sclerosis (CREST) not restricted to the fingers or distal limbs but affects also proximal extremities, trunk, and face. • Antibodies in dcSSc: anti-fibrillarin (up to 64%), anti-RNA-Pol III (up to 85%), anti-Scl70 (up to 71%)4 • Additional symptoms occuring in anti-fibrillarin positive patients: • Multiorgan involvement including • small intestinal involvement2 • renal crisis2 • pulmonary fibrosis/pulmonary hypertension2 • muscle inflammation3 • dcSSc patients positive for anti-fibrillarin have the worst prognosis.4 • Treatment approaches depend on antibody reactivity and diagnosis.4 2 Pollard KM andHultman P (2007) 3 Steen VD (2005) 4Steen VD (2008)

  8. Prevalence of Fibrillarin antibodies Prevalence in patients (sensitivity) • 7-14% of patients with scleroderma2 • Occur more frequently in a subset of SSc patients who are often of African descent (>50%), male, and with serious cutaneous and visceral disease2 • It is the second most ANA antibody in African American with SSc Prevalence in controls (specificity) • Anti-fibrillarin is a specific marker for dcSSc. However, the antibodies may occur in several systemic autoimmune diseases like SLE, Raynaud‘s phenomenon, and scleroderma.2 • Clinical features and the frequency of scleroderma subsets and antibodies are different in different countries and ethnicities4 2 Pollard KM and Hultman P (2007) 4 Steen VD (2008)

  9. Fibrillarin is a prognostic marker • For scleroderma patients, especially young patients5 (mean age of onset 35 years) to develop internal organ involvement, such as • Small intestinal involvement1 • Skeletal muscle involvement1 • Pulmonary hypertension1 • Associated with severe SSc and worst prognosis because of severe organ involvement.4 • Anti-fibrillarinpositive patients are likely to develop alveolitis, pulmonaryfibrosis and later on severe vascular pulmonary hypertension and need to be monitored.4 1 Conrad K et al (2002) 4Steen VD (2008) 5TormeyVJ et al. (2001)

  10. The antigen • Fibrillarin (34 kDa) is a componentofseveral ribonucleoproteinsincluding a nucleolarsmall nuclear ribonucleoprotein(snRNP) and one of the two classes of small nucleolarribonucleoproteins (snoRNPs). • It is localized exclusively in the fibrillar region of the nucleolus, including both the dense fibrillar and the fibrillar center regions. • Function: Fibrillarincatalyzes site-specificribosemethylationofribosomalRNA occuring in RNA-splicingandribosomal RNA processing. • Fibrillarinispresent in all nucleatedcelltypes.

  11. Methods to detect anti-Fibrillarin • IFA on HEp-2 cells: nucleolar („clumpy“) pattern (distinct chromosomal staining in the metaphase cells, but no staining of the nucleoplasm in interphase cells)2 • NOTE: differentiationof different nucleolarpatternsrequiresexperienceandisfacilitatedbyviewingthenucleolarstaining in variousfocal planes: difficult! • Immunoprecipitation(IP) of radiolabeled cell culture extracts or recombinant proteins2 • Western Blot (WB) using nucleolar extracts or nucleolar protein preparations2 • ELISA: currently no ELISA assay available • Methods using native proteins in cell (WB) or from extracts (IP, WB) do not unequivocally identify anti-fibrillarin because a positive reaction could be due to interaction with snoRNPcomponentsotherthanfibrillarin.2 • Only assays using recombinant proteins have the specificity to show the association of anti-fibrillarin antibodies with the severe form of systemic sclerosis.The available assays are not often used in clinical routine.2 2 Pollard KM and Hultman P (2007)

  12. Product Details

  13. EliA Controls • Fibrillarin activity is not included in the EliA ANA Positive Control. • Due to expected low volume and difficult serum supply we are not able to offer EliA Controls for EliA Fibrillarin. • This means that both the EliA ANA Positive Control and the EliA IgG/IgM/IgA Negative Control must not be used with EliA Fibrillarin.

  14. EliA Fibrillarin – Precision Excellent precision on all Phadia instruments

  15. EliA Fibrillarin – Serum/Plasma Correlation Both serum and all types of plasma can be used

  16. EliA Fibrillarin – Blood Donors and Normal Range 400 blood donors were measured on Phadia 250. A substudy using 70 blood donors on Phadia 100 and Phadia 2500 showed good comparison. Equivocal Most blood donors far below cut-off, good discrimination

  17. Clinical Performance • Sample panels used in the evaluation:300 clinically defined samples Patients: • 100 scleroderma Controls: • 70 CTD (SLE, Sjögren, MCTD, PM/DM) • 40 RA • 70 infections (HCV, HBV, EBV, bacterial) • 20 tumors

  18. Clinical Performance Cut-off Equivocal range Sensitivity in therangereported in theliterature, but depending on the sample panel. Optimal specificitygivesthistest an excellentclinicalvalue.

  19. Competition • No ELISA based tests available • Some Euroimmun Line Blots include fibrillarin, but no single fibrillarin test available • IIF shows a typical pattern, however it is not easy to identify and requires a lot of experience • Immunoprecipitation

  20. Comparison to IFA on HEp-2 cells • Fibrillarin antibodies give a nucleolar pattern in IFA. However, since there are several other unkown antibodies giving a similar pattern, you cannot be sure that a nucleolar pattern is due to fibrillarin antibodies. • In our study 17 samples of scleroderma patients gave a nucleolar pattern in IFA. • 5 samples out of the 17 IFA positives showing a nucleolar pattern were positive in EliA Fibrillarin. • 3 more samples were positive in EliA RNA Pol III. • Since all EliA Fibrillarin positive samples were also IFA positive showing a nucleolar pattern, we are sure that EliA Fibrillarin is well in-line with IFA, but more specific.

  21. Sales Arguments • First single anti-fibrillarin test for routine use, saving costs for additional tests • First automated anti-fibrillarin test, reducing the workload for your lab personnel • Clear differentiation between systemic sclerosis and other connective tissue diseases • Reliable follow-up of a positive EliA CTD Screen • Reliable follow-up of a positive IIF result

  22. Sales Arguments • First single anti-fibrillarin test for routine use, saving costs for additional tests • To date no single tests are available for anti-fibrillarin, but only Line Blots including fibrillarin among many other antigens, making anti-fibrillarin testing expensive. • EliA Fibrillarin brings single anti-fibrillarin testing to the routine lab, making it flexible and cost-efficient.

  23. Sales Arguments • First automated anti-fibrillarin test, reducing the workload for your lab personnel • The possibility of single testing combined with automation makes anti-fibrillarin testing cost-efficient, saving both labour time and costs for other tests which are not needed. Phadia 100 Phadia 250 Phadia 2500 Phadia 5000

  24. Sales Arguments • Clear differentiation between systemic sclerosis and other connective tissue diseases • EliA Fibrillarin pinpoints systemic sclerosis without generating false positives. This results in an optimal positive predictive value and provides reliable early guidance for the physicians. Cut-off Equivocal range

  25. Sales Arguments • Reliable follow-up of a positive EliA CTD Screen • If EliA CTD Screen is positive but the most frequent antibodies are negative, this may be due to anti-fibrillarin. Both tests are perfectly aligned. • If systemic sclerosis is suspected and EliA CTD Screen is positive, EliA Fibrillarin is the follow-up test of choice, besides EliA Scl-70, CENP and PM-Scl, providing clear, quantitative results of high clinical value

  26. Sales Arguments • Reliable follow-up of a positive IIF result • If IIF shows a pattern (nucleolar („clumpy“) pattern; distinct chromosomal staining in the metaphase cells, but no staining of the nucleoplasm in interphase cells) which is typical for anti-fibrillarin, but very difficult to differentiate from other nucleolar patterns. EliA Fibrillarin can unequivocally confirm or rule out the presence of anti-fibrillarin due to its optimal specificity

  27. References 1 Conrad K et al (2002). Fibrillarin Antibodies. In: Autoantigens, Autoantibodies, Autoimmunity, Vol 2, Conrad, Sack (eds), Pabst, Lengerich: pp 78-79 2 Pollard KM and Hultman P (2007). Fibrillarin Autoantibodies, Vol 2, Shoenfeld, Gershwin, Meroni (eds), Elsevier, Amsterdam: pp 317-323 3 Steen VD (2005). Autoantibodies in Systemic Sclerosis. Semin Arthritis Rheum 35: 35-42 4 Steen VD (2008). The Many Faces of Scleroderma. Rheum Dis Clin N Am 34: 1-15 5 Tormey VJ et al. (2001). Anti-fibrillarin antibodies in systemic sclerosis. Rheumatology (Oxford) 40: 1157-1162 6 Chifflot H et al. (2008). Incidence and prevalence of systemic sclerosis: a systematic literature review. Semin Arthritis Rheum37: 223-235

  28. EliA FibrillarinPinpoints systemic sclerosis

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