NOVA Lite HEp-2 External EB Kits For In Vitro Diagnostic Use

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1 NOVA Lite HEp-2 External EB Kits For In Vitro Diagnostic Use Product Code: , CLIA Complexity: High Intended Use This product (kit or substrate slides) is intended for use in the screening and titration of circulating antinuclear antibodies by means of an immunofluorescence test. It is to be used as an aid in the diagnosis and treatment of systemic lupus erythematosus (SLE), Sjögren s Syndrome, rheumatoid arthritis and other connective tissue disorders. Summary and Explanation of the test Antinuclear antibody (ANA) is a term which describes a variety of autoantibodies against constituents of cell nuclei including DNA, RNA and various nuclear proteins. 1 These ANAs are found with high frequency in patients with connective tissue or rheumatic diseases, in particular, SLE. There is a high correlation of positive ANA with SLE, and a negative ANA essentially rules out the disease. 2 However, patients with other connective tissue diseases such as rheumatoid arthritis, scleroderma and dermatomyositis are often ANA positive and low ANA titres are seen in other disease states. Positive ANA results have also been reported in older, healthy people. Although indirect immunofluorescence is the method of choice for screening circulating ANAs, it is recommended all ANA positive samples are titred to end point and further investigated by more specific testing for autoantibodies to double stranded DNA (dsdna) and extractable nuclear antigens (ENAs). Frozen sections of rat liver were historically the most popular substrate for demonstration of ANAs but these have now been replaced by various types of cell lines. HEp-2 cells 3 are an epithelial cell line characterised by extremely large nuclei compared to other cell lines. When used in ANA screening the advantages of HEp-2 cells are firstly, they are a standardised antigen substrate with less batch to batch variability than with rodent tissue; secondly, HEp-2 cells are larger enabling easier visualisation of cell morphology with a consequent increase in assay sensitivity over rat liver sections and finally, many HEp- 2 cells are actively dividing, exposing antigens not normally expressed in the resting cells of rat liver sections. 3 Principles of the Procedure An indirect immunofluorescence technique 4 is utilised where patient samples and appropriate controls are incubated with the HEp-2 substrate. The unreacted antibodies are washed off and then an appropriate fluorescein labelled conjugate is applied. Unbound conjugate is washed off as before. Slides are viewed with a fluorescence microscope and positive samples give rise to apple-green fluorescence which corresponds to areas of the HEp-2 cell where autoantibody has bound. Reagents 1. HEp-2 ANA substrate slides on 6 or 12 well slides 2. Positive control serum (homogeneous pattern, 3+ staining intensity), containing 0.09% sodium 3. Positive control serum (centromere pattern, 3+ staining intensity), containing 0.09% sodium 4. Negative control serum (universally negative for all autoantibodies), containing 0.09% sodium 5. FITC IgG (H&L) fluorescein, containing 0.09% sodium 6. 1% Evans Blue, as an optional counterstain. 7. Phosphate buffered saline (PBS II), provided as a 40-fold concentrate in liquid form. 8. Mounting medium, containing an anti-fading agent 9. Coverslips Warnings/Precautions This product is for In Vitro Diagnostic Use. This product should only be used by suitably trained persons for the purposes stated. Adherence to the given procedure is recommended. All donors of human serum supplied (kits only) have been serum tested and found to be negative for Hepatitis B surface antigen and antibodies to Hepatitis C virus and Human Immunodeficiency Virus (HIV 1 & 2), however, these tests cannot guarantee the absence of infective agents. Proper handling and disposal methods should be established as for all potentially infective material and only personnel adequately trained in such methods should be permitted to perform the procedures. Some of the kit components contain 0.09% sodium azide as a preservative and must be handled with caution do not ingest or allow contact with skin or mucous membranes. If contact does occur wash with large volume of water and seek medical advice. Explosive metal azides may be formed with lead and copper plumbing; on disposal of reagent, flush with a large volume of water to prevent azide build up. 1

2 Storage Conditions Unopened kits or slides should be stored at 2-8 C and can be used until the given expiry date. DO NOT FREEZE. Once slides are removed from a foil bag, they should be used immediately. Diluted PBS II buffer can be stored for up to one month at 2-8 C. The fluorescein conjugate should be kept out of sunlight, fluorescent or U.V. light whenever possible. All reagents should be stored at 2-8 C. Specimen Collection Blood samples should be collected by venepuncture, allowed to clot naturally and the serum seperated as soon as possible to prevent haemolysis. The serum may be stored at 2-8 C for up to 7 days prior to assay, or for prolonged storage, aliquoted and stored at -20 C or below 11. Repeated freezing and thawing should be avoided. Avoid using lipaemic, haemolysed or microbially contaminated sera as decreased titres or unclear staining patterns may occur. Procedure Materials Provided well HEp-2 ANA Substrate Slides 1 1mL Homo. Ptn. (use w/conj or ) 1 1 ml Centromere Positive Control 1 1mL IFA System Negative Control 1 7mL FITC IgG (H&L) HEp Conjugate 1 3mL PVA Mounting Medium 2 25mL PBS II Concentrate (40x) 1 3mL 1% Evans Blue 1 10 Coverslips well HEp-2 ANA Substrate Slides 1 1mL Homo. Ptn. (use w/conj or ) 1 1 ml Centromere Positive Control 1 1mL IFA System Negative Control 1 15mL FITC IgG (H&L) HEp Conjugate 1 10mL PVA Mounting Medium 2 25mL PBS II Concentrate (40x) 1 3mL 1% Evans Blue 1 20 Coverslips Additional Materials Required But Not Provided 1. Distilled or deionized water to dilute PBS II concentrate. 2. Container for PBS II buffer and plastic squeeze bottle for initial wash in PBS II. 3. Micropipettes and disposable tips to apply patient samples. 4. Humid chamber for incubation steps 5. Fluorescence microscope with 495nm exciter filter and 515nm barrier filter. If using substrate slides only, the following materials may be obtained from INOVA: positive controls eg. Homo. Ptn. (use w/conj or ) (504090), centromere (508184), negative control (508186), anti-human IgG (H+L) AFF FITC conjugate (504088, ), prediluted ready for use, or , (dilute 1/50 1/200 for use), PBS II (508998), Evans Blue (504049), mounting medium (504046, ), coverslips. Test Procedure Quality Control Positive and negative controls should be used every time samples are tested. 1. Mounting medium: Remove the mounting medium from the fridge to allow it to reach room temperature (18-28ºC) before it is needed. 2. Dilute PBS II concentrate. Dilute PBS II concentrate with distilled or deionized water (1 part PBS II concentrate + 39 parts distilled or deionized water) and mix. NB: only make up the entire amount of kit PBS II if the entire kit is to be used within one month. The PBS II is used for diluting patient samples and as a wash buffer. 3. Dilute patient samples. Screening: Dilute patient samples 1/40 by adding 25µL of serum to 975µL of PBS II buffer. Titration: Make serial dilutions of positive screened samples with PBS II buffer (e.g. 1/40, 1/80, 1/160, 1/320 and 1/640 etc.). For example: Take 500µL of the 1/40 patient sample dilution, mix with 500µL of PBS II to give a 1/80 dilution. Repeat this procedure for further doubling dilutions. 4. Substrate slides. Allow substrate slide(s) to reach room temperature (18-28ºC) for approximately 30 minutes prior to removal from pouch(es). Label slides appropriately, place in the humid chamber and add one drop of each kits two positives and one negative control to wells 1, 2 and 3 respectively. Add 25µL of diluted patient samples to the remaining wells. 5. Slide incubation. Incubate slides for 30 minutes in a humid chamber at room temperature (18-28ºC). (Moistened paper towels at the bottom of the chamber will maintain humidity.) 2

3 6. PBS II wash. Remove slides from humid chamber and rinse thoroughly but gently with PBS II in a squeeze bottle. Do not squirt directly on the wells. Place the slides in a coplin jar of diluted PBS II or rack immersed in PBS II for up to 5 minutes. 7. Addition of fluorescent conjugate. Shake off excess PBS II. Return slides to humid chamber and immediately cover each well with a drop of fluorescent conjugate. DO NOT LEAVE WELLS UNCOVERED FOR LONGER THAN 15 SECONDS. DRYING OUT OF THE SUBSTRATE SERIOUSLY AFFECTS THE RESULTS. 8. Slide incubation. Incubate slides for 30 minutes in humid chamber at room temperature (18ºC- 28ºC) in the dark. 9. PBS II wash. Wash again as described in step 6. Optional counterstain: add 2-3 drops of 1% Evans Blue per 100mL of PBS II prior to slide immersion. 10. Mounting with coverslip. Remove one slide at a time from PBS II wash. Quickly dry around the wells and add a drop of mounting medium to each well. Carefully lower the slide onto the coverslip, avoiding air bubbles, but if present do not attempt to remove. 11. View slides under fluorescence microscope. Finished slides should be viewed as soon as possible but may be stored at 2-8 C in the dark for up to 1 week, without significant loss of fluorescence. Quality Control The kit homogeneous positive control should give a bright 3+ apple-green homogeneous staining pattern in cell nuclei. The kit centromere positive control should give a 3+ discrete speckled pattern (usually in multiples of 46). The kit negative control should show dull green staining in all HEp-2 cells, with no discernible fluorescence. If the controls do not appear as described, the test is invalid and should be repeated. NB: If Evans Blue is used as a counterstain, a negative sample will give a dull orange-red appearance. If positive, the nuclei will stain apple-green according to the ANA pattern being demonstrated and the unstained areas of the cell will be dull red in colour. Interpretation of Results Negative A sample is considered negative if specific nuclear staining is equivalent to or less than the negative control well. Positive A sample is positive if nuclear staining is observed to be greater than the negative control well, and a clearly discernible pattern can be seen in most of the HEp-2 cells. Kit controls are usually 3+ in intensity. The following table summarises a variety of nuclear staining patterns which may be observed depending on the types and relative amounts of autoantibodies present in the sample: PATTERN Homogeneous Speckled Nucleolar Centromere Mitochondrial Jo-1 APPEARANCE Solid nucleus staining (Scl-70 may have a speckled appearance) Fine or granular (coarse) speckles, usually without staining of the nucleoli Large coarse speckles (usually less than 6 speckles per nucleus, with or without fine speckles) Discrete speckles (usually a multiple of 46) Coarse granular filamentous cytoplasmic speckled pattern extending around the nucleus and throughout the cytoplasm Fine speckled staining concentrated around the nucleus ANTIGENS INVOLVED dsdna ssdna Histones Scl-70 SS-A SS-B Sm RNP + others Pm-Scl Nucleolin Ku and other unknown nuclear antigens Chromosomal Centromere M2 9,10 Jo-1 (histyl-trna synthetase) MAIN DISEASE ASSOCIATIONS High titres : SLE Low titres : SLE or other connective tissue disorders 5 High titres: Sjögrens syndrome -sicca complex SLE Mixed connective tissue disease Scleroderma Low titres: other connective tissue diseases High titres: Scleroderma & Sjögren s syndrome 6 CREST Syndrome 7 Primary biliary cirrhosis (common) Scleroderma (40%) and occasionally in overlap syndromes Polymyositis, dermatomyositis associated with interstitial pulmonary disease 3

4 Limitations of the Procedure 1. High-titre ANA is highly suggestive of connective tissue disease but the patient s clinical history and other serological results must be considered before reaching a diagnosis. 2. A small percentage of SLE patients may be ANA negative by indirect immunofluorescence but have ANAs by other methods Patients with drug-induced SLE may have positive homogeneous or peripheral ANAs. Conversely, patients undergoing steroid therapy may give negative ANAs by other methods More than one autoantibody may be present in a patient sample. By diluting the serum, the various patterns may be more easily discerned. Similarly, with very high titres, a prozone effect may mask the true strength. All suspicious or apparently low titre specimens should be further diluted. 5. The light source, filters and optics of different makes of fluorescence microscope will influence test sensitivity. The performance of the microscope is significantly influenced by correct maintenance especially centring of the mercury vapour lamp and changing of the lamp after the recommended period of time. 6. Low level ANA may be detected in patients who have clinical conditions other than rheumatoid and connective tissue diseases. The incidence of posititve ANA results increases with age in normal individuals. 7. When interpreting staining patterns, the possible presence of more than one autoantibody specificity should be considered. Combinations of autoantibodies can induce homogeneous or speckled patterns and it is recommended that specific testing for dsdna and ENAs be performed on all homogeneous samples. 8. Although a 1/40 dilution of patient serum is used routinely for screening, it is possible that low level non-specific staining of the nuclei may occur with some samples at low sample dilutions. 9. The ANA test is a diagnostic aid only, and is not in itself diagnostic. Test results must be interpreted as part of a complete clinical picture. Slides sold separately are classified as Analyte specific reagents. Except as a component of the kit, analytical and performance characteristics are not established. Performance Characteristics Precision The quality control procedure for each batch involves testing multiple slides with a known control diluted to end-point. Each batch meets a predetermined end-point titre. This demonstrates that there is no significant difference in performance between slides in the same batch or in different batches. Sensitivity The sensitivity of the tests and the limit of detection are dependent on the microscope used. There is no international calibrator available to standardise this. Specificity A panel of more than 50 clinical samples of various specificities, including at least 10 negatives, was tested on The Binding Site s HEp-2 kit and on an equivalent commercially available HEp-2 kit. The Binding Site s kit performance was comparable to the equivalent kit in terms of specificity and intensity. Summary of Procedure 1. Equilibrate mounting medium to room temperature. 2. Dilute PBS II 1/40 with distilled or deionized water. 3. Dilute patient sera 1/40 with PBS II. 4. Remove slides from refrigerator and equilibrate to room temperature (18-28ºC). 5. Remove slide from foil bag and place in a humid chamber. Add 25µL of controls and diluted patient sera. Incubate for 30 minutes at room temperature (18-28ºC). 6. Rinse slides with a stream of PBS II. Wash slides for 5 minutes in a coplin jar or rack. 7. Return slide to humid chamber and immediately add a drop of fluorescent conjugate. 8. Incubate slides for 30 minutes. 9. Wash again as described in step Add mounting media to each well and coverslip. 11. View slides under a fluorescence microscope. 4

5 References 1. Tan, E.M. (1982): Autoantibodies to nuclear antigens: Their immunobiology and medicine. Advances in Immunology. 33: Tan, E.M. et al (1982): The 1982 revised criteria for classification of systemic lupus erythematosus. Arth and rheum. 25: Miyachi, K., Fritzler, M.J., Tan, E.M. (1978): Autoantibody to nuclear antigen in proliferating cells. J. Immunol. 121: Weller, T.H., Coons, A.H. (1954): Fluorescent antibody studies with agent of Varicella and Herpes Zoster propagated in vitro: Prov. Soc. Exp. Biol. Med. 86: Notman, D.D., Kurata, N., Tan, E.M. (1975): Profiles of antinuclear antibodies in systemic rheumatic diseases. Ann. Int. Med. 83: Ritchie, R.F. (1970): Antinuclear antibodies. Their frequency and diagnostic application. N. Enr. J. Med. 282: Tan, E.M., Rodman, G.P., Garcia, I. (1980) et al: Diversity of antinuclear antibodies in progressive systemic sclerosis. Arthritis Rheum. 23: Gladman, D.D., Chalmers, A., Urowitz, M.B. (1978): Systemic lupus erythematosus with negative LE cells and anitnuclear factors. J. Rheum.: MacKay, I.R., Gershwin, M.E. (1989): Molecular basis of mitochondrial autoreactivity in primary biliary cirrhosis. Immunol.Today: 10: 9: Laung, P.S.C., MacKay, I., Gershwin, M.E. (1994): Mitochondrial autoantigens. Man Biol. Mark; B8.1,1-14. Eds W.J.Van Venrooij and R.N. Maini. Kluwer Academic Pubs. Netherlands. 11. Protein Refence Unit Handbook of Autoimmunity (3 rd Edition) Ed A Milford Ward. J Sheldon, GD Wild. Publ. PRU Publications, Sheffield. 14. Manufactured By: INOVA Diagnostics, Inc Old Grove Road San Diego, CA United States of America Technical Service (U.S. & Canada Only) : Technical Service (Outside the U.S.) : support@inovadx.com Authorized Representative in the EU: Medical Technology Promedt Consulting GmbH Altenhofstrasse 80 D St. Ingbert, Germany Tel.: Fax.: Rev. 5 January 2014 NOVA Lite and INOVA Diagnostics, Inc. are registered trademarks. Copyright 2013 All Rights Reserved 5