golgi, ribosmomes, lysosomes, mitochondria and cytoskeletal elements (smooth muscle antibodies) should be followed up with appropriate frozen tissue sections. HEp-2 substrates are not equally sensitive for mitochondrial and smooth muscle testing when compared with tissue section antigens. ANTINUCLEAR ANTIBODY TEST SYSTEM HEp-2 REF 10-1120 120 Tests Store kit at +2 to +8°C Pour d'autres langues Für andere Sprachen Para otras lenguas Per le altre lingue Dla innych języków Para outras línguas Για τις άλλες λώσσες För andra språk For andre språk wwww.trinitybiotech.com INTENDED USE MarDx Antinuclear Antibody Test System is intended for testing human serum for the presence of human antinuclear antibodies (ANA) as an aid in the diagnosis of chronic autoimmune disorders. For in vitro diagnostic use. High complexity test. SUMMARY AND PRINCIPLES Indirect fluorescent antibody (IFA) Techniques have been used extensively for the detection of antinuclear antibodies (ANA) in patient sera for diagnostic evidence, prognostic significance and therapeutic management. Screening for these antibodies is routinely done on patients with various connective tissue diseases, particularly in Systemic Lupus Erythematosis (SLE) which may be autoimmune or drug induced (see Table I). Drug induced lupus is differentiated from classic lupus by the absence of anti- DNA antibodies and the presence of anti-histone antibodies. TABLE I SLE INDUCING DRUGS Group I: Inducted by Pharmacological Action Hydralazine Procainamide Anti-convulsants: Mephenytoin Phenytoin Trimethadione Ethosuximide Carbamazepine Pheneturide Isoniazid Chlorpromazine 1. HOMOGENEOUS PATTERN: Fluorescence is uniform and diffuse in the nucleus of the interphasic cells. Metaphasic cells demonstrate homogeneous fluorescence of the condensed chromosomal region Specificity: The homogeneous pattern is obtained with antibodies to DNA, DNP, and histones. 2. PERIPHERAL PATTERN: Fluorescence is uniform and diffuse with staining of a greater intensity at the outer region of the nucleus. Metaphasic cells demonstrate strong staining of the condensed chromosomal submembranal region. Specificity: The peripheral pattern is obtained with antibodies to DNA and histone. 3. COARSE SPECKLED WITHOUT NUCLEOLUS PATTERN: Fluorescence is uniform with coarse specks in the nuclear matrix with no nucleolar staining. Metaphasic cells demonstrate no staining of the condensed chromosomal region. Specificity: The coarse speckled pattern without nucleolus staining is obtained with anti- SM and anti-RNP antibodies. 4. COARSE SPECKLED WITH OCCASIONAL NUCLEOUS PATTERN: Fluorescence is uniform with coarse flat specks in the nuclear matrix with occasional nucleolar staining. Metaphasic cells demonstrate no staining of the condensed chromosomal region. Specificity: The coarse speckled pattern with occasional nucleolar staining is obtained with anti-SS-B antibodies. 5. FINE SPECKLED WITHOUT NUCLEOLUS PATTERN: Fluorescence is uniform with fine specks in the nuclear matrix without nucleolar staining. Metaphasic cells demonstrate no staining of the condensed chromosomal region. Specificity: The fine speckled pattern without nucleolus staining can be found with several antibodies including SS-A, Mi-1, Mi-2, and SL. Group II: Induced by Allergic Action Aminosalysilic Acid Chlorthalidone D-Penicillamine Griseofulvin Isoquinazepon Levodope Methyldopa Methysergide Methylthiouracil Oral Contraceptives Oxyphenisatin TABLE II CLASSIFICATION OF CLINICALLY SIGNIFICANT SINGLE PATTERN ANA REACTIONS USING HEP-2 CELLS. Pencilillin Phenyibutazone Practolol Propylthiouracil Quinidine Reserpine Streptomycin Sulfonamides Tetracycline Tolazamide The detection of positive antinuclear antibodies depends in part on the antigenic substrate and the fixation method used in the preparation of the substrate. ANA testing can utilize many substrate sources such as liver or kidney tissue sections which can be derived from rats or mice. The use of human tissue culture cells (specifically HEp-2) has provided an alternative substrate to tissue sections. HEp-2 substrate allows for differentiation of pattern recognition on cells in all phases of mitosis which is not necessarily possible using tissue sections as substrate. The high level of mitotic cells in tissue culture HEp-2 cells allows for the definitive differentiation of "centromere" antigen staining of the condensed chromosomal region of metaphasic cells. Centromere positive sera are generally reported as "speckled" staining patterns when tissue sections are used. The high level of mitotic cells found in HEp-2 cells also aids in the determination of mixed homogeneous and speckled antigenic patterns. Most speckled patterns stain the nucleoplasm surrounding the mitotic cell but do not stain the condensed chromosomal region of the metaphasic HEp-2 cells. Homogeneous patterns will stain the condensed chromosomal region with an intense homogeneous or peripheral stain. Therefore, staining in the same mitotic HEp-2 cell of both homogeneous condensed chromatin of metaphasic cells and a speckled surrounding nucleoplasm of metaphasic cells indicates the presence of two types of nuclear specificities in the same sample. Mixed homogeneous/speckled specificities using tissue sections are generally observed upon titration of the sample which reveals patterns present in conjunction with the homogeneous pattern. SS-A antigens are specifically fixed in the HEp-2 substrate preparation to assure its reproducible abundance in testing for this clinically important antigenic determinant. Tissue sections may vary in the amount of SS-A present from manufacturer to manufacturer. Anti-SS-A is found in neonatal lupus syndrome, Sjogren's Syndrome and SLE. Anti-SS-A antibodies bind to skin and heart tissue. Study of expectant mothers with rheumatic disease symptoms for the presence of anti-SS-A is recommended to prevent untoward neonatal death. Postpartum clinical monitoring of mothers of children with heart block and rashes for the development of clinical and serologic SLE is clinically indicated. Both ANA testing with immunofluorescence and double gel diffusion testing with extractable nuclear antigens (ENA) are recommended for detecting and typing the Anti-SS-A response. Proliferating cell nuclear antigen is also detected in HEp-2 cells which may vary in tissue section substrates. Other nuclear patterns may be observed in HEp-2 cells which are not yet clinically defined. They include staining of the mitotic cell spindle apparatus including mitotic spindle fibers, centriole, chromosomes, midbody of telophase cells and nuclear cell membrane. Additionally, non-nuclear cytoplasmic organelles can stain with various staining patterns. Generally cytoplasmic staining of 6. FINE SPECKLED WITH NUCLEOLUS PATTERN: Fluorescence is uniform with fine specks in the nuclear matrix with nucleolar staining. Metaphasic cells demonstrate staining of the condensed chromosomal region. Specificity: The fine speckled with nucleolus pattern is demonstrated by antibodies to Scl-70. This pattern is considered a marker antibody specific for proteinkinase (70K) and is clinically diagnostic of scleroderma. 7. DIFFUSE SPECKLED (CENTROMERE) WITHOUT NUCLEOLUS PATTERN: Fluorescence is uniform with spherical specks approximately 40-60 per cell nucleus. Metaphasic cells demonstrate intense speckled staining of the condensed chromosomal region corresponding to the centromere region. Telophase cells do not demonstrate speckles. Specificity: Diffuse speckled with centromeric staining of metaphasic cells is demonstrated by antibodies to the kinetocore antigens of the centromere. This pattern is considered a marker antibody and is diagnostically specific for the CREST syndrome. 8. ATYPICAL DISCRETE SPECKLED (PSEUDOCENTROMERE, NUCLEAR DOTS): Occasional speckled fluorescence of less than 10 bright dots in the nuclei of interphasic cells only. Metaphasic cells demonstrate no speckling of condensed chromosomal region. Specificity: The chemical nature of the pseudocentromeric pattern is not yet known. The antibody has been defined as an anti Nsp-1. This antibody is associated with PBC and Chronic Hepatic Disease. 9. PLEOMORPHIC SPECKLED OF VARIOUS INTENSITIES: Varying fluorescence of fine to coarse speckling in approximately 30-60% of the cells. Both the number of cells staining and the intensity of the speckles vary. Metaphasic cells may demonstrate positive or negative staining of the condensed chromosomal regions. Specificity: The antibody responsible for pleomorphic speckled fluorescence is proliferating cell nuclear antibody (PCNA) and is active against cyclin. 10. NUCLEOLAR ONLY: The staining of the nucleolus may be either homogeneous, clumpy, speckled or perinuclear. Metaphasic cells generally do not show staining of the condensed chromosomal regions. Positive staining can be seen in the nucleolus of cells at the end of anaphase in telophase. Specificity: The antibodies responsible for staining of the nucleolus include: Anti-4-6 RNA, Anti-RNA and RNP, and Anti-DNA Nucleolar Organizer. Page 1 of 4-EN 10-1120-29-Rev. 2 SUMMARY AND PRINCIPLES (continued) CYTOPLASMIC ORGANELLE PATTERNS: 1. FINE CYTOPLASMIC: (Jo-1) Fine perinuclear cytoplasmic fluorescence. Specificity: The antibodies responsible for staining of the perinuclear bodies are designated as Jo-1 and are active against a histidyl-modified t-RNA synthetase moiety. The Jo-1 antibody is considered a diagnostic marker antibody for polymyositis where it has a 31% disease association. The use of gel diffusion tests to identify this antigen is recommended, as its characterization from other HEp-2 cytoplamic patterns is difficult. 2. COARSE CYTOPLAMIC: (Anti-mitochondrial) Coarse cytoplasmic fluorescence extending around the nucleous along the cytoskeletal filaments. (anti-mitochondrial) Specificity: The antibodies responsible for mitochondrial fluorescence include antibodies against various mitochondrial specificites including M1, M2, M3, M5, and M6. HEp-2 cells detect the M2 type antigen. However, the use of kidney tissue sections is recommended since the kidney section has greater sensitivity in the detection of all mitochondrial antibody specificities for mitochondrial antibody testing. Nonspecific patterns resembling mitochondrial reactions on HEp-2 cells may occur and nonspecific mitochondrial-like HEp-2 patterns will be negative using tissue sections. Therefore, HEp-2 mitochondrial patterns should be confirmed on tissue sections. 3. FILAMENTOUS OR FIBROUS CYTOPLASMIC: (Anti-smooth muscle) Cytoskeletal fluorescence surrounding the nucleus. The fluorescence may occasionally appear as fine perinuclear radial filaments with coils due to vimentin antibodies or they may occasionally appear as fine fibrous staining seen with antibodies to actin. Smooth muscle staining of tissue sections due to desmin will not stain the cytoskeleton of HEp-2 cells. Specificity: As with mitochondrial antibody testing tissue section substrates are more specific and sensitive than HEp-2 cells for routine testing of all three types of anti-smooth muscle antibodies. The primary reaction occurs during the first incubation period while the patient's serum covers the substrate. The secondary reaction follows a PBS rinsing to remove any unbound human antibody. The reagent used in the secondary reaction is a fluorescein labeled anti-human conjugate which has been affinity purified for use with Hep-2 cell culture substrates and has been adjusted for the optimum use dilution and is free of most nonspecific staining of the HEp- 2 cell. After a second PBS rinse to remove any unbound anti-human globulin conjugate, the specific type of nuclear fluorescence observed and its fluorescence intensity is reported visually using a fluorescence microscope at a magnification of 400X. (See Table II). 1. 2. 3. 4. 5. 6. 7. The disease association of various ANA antibodies in Systemic Rheumatic Diseases is summarized in Table III. 8. 9. TABLE III ASSOCIATION OF ANA WITH SRD ANTIBODIES TO: DS-DNA only DA and ssDNA SS-DNA only (exposed purine and pyrimidines) Histones Non-Histone Antigens Sma nuclear RNP (u! RNP) SS-B/La PCNA Ma Scl-70a Centromere/kinetochorea RANA PM-1 (PM/Sol) Mi-1 J0-1a Ku Nucleoli 4-6s RNA RNA and RNP DNA:nucleolar organizer DISEASE ASSOCIATION SLE:rare cases SLE:60-70%, in other diseases may occur in low titer SLE:other rheumatic diseases, some nonrheumatic diseases SLE:60-70% Drug LE (procainamide): 95% Other drug LE: 30-90% RA:30% low titer 10. 11. 12. 13. PRECAUTIONS Always wear suitable protective clothing, gloves and eye/face protection when working with this product. Each donor unit used in the preparation of this material was tested by an FDA approved method for the presence of the antibody to HIV as well as for HBsAg and found to be negative (were not repeatedly reactive). WARNING - POTENTIAL BIOHAZARDOUS MATERIAL Because no test method can offer complete assurance that human immunodeficiency virus (HIV), hepatitis B virus, or other infectious agents are absent, these human control reagents should be handled at the Biosafety Level 2 as recommended for any potentially infectious human serum or blood specimen in the Centers for Disease Control/National Institutes of Health Manual "Biosafety in Microbiological and Biomedical Laboratories", 1999. (23) The phosphate buffered saline and mounting medium found in this kit are irritating to the eyes, respiratory system and skin. Some components in this kit contain 0.1% Proclin 300. At full strength Proclin 300 is corrosive and will cause burns and possibly sensitisation by skin contact. The conjugate in this kit contains 0.015% Evan’s Blue. Evan’s Blue is a possible carcinogen and may cause reproductive harm. Some components in this kit contain 0.02% Thimerosal. Thimerosal is toxic by inhalation, in contact with skin, and if swallowed, and is a reproductive hazard. The conjugate and controls in this kit contain sodium azide at a concentration of less than 0.1 %. Sodium azide is toxic if ingested and forms potentially explosive copper and lead azide compounds in waste plumbing lines. Should the reagents come in contact with copper or lead waste plumbing, flush the waste line with large quantities of water to prevent the formation of potentially explosive compounds. Do not use components beyond their expiration date. Follow the procedural instructions exactly as they appear in this insert to insure valid results. For in vitro diagnostic use. Handle slides by the edges since direct pressure on the antigen wells may damage the antigen. All reagents must be brought to 20 to 25°C before performing the test procedure. Once the procedure has been started do not allow antigen in the wells to dry out. This may result in false negative test results, or unnecessary artifacts. R43: S28-37: SLE:25-40%, marker antibody MCTD: 95-100%, lower frequency in SLE, discoid LE, PSS SS-A/Ro Sjogren's Syndrome: 60-70% SLE: 30-40% Neonatal Lupus Syndrome: 100% Sjogren's Syndrome: 50-60% SLE:10-20% SLE:<10% SLE:20% SS: 15-20% Prod# 10-1012 10-1202 10-1206 10-1201 10-1513 90-1612 90-1607 90-1700 90-1712 CREST syndrome: 70-90% RA: 90-95% Polymyositis/scleroderma overlap: 64% Dermatomyositis: 17% Dermatomyositis: 11% Polymyositis: 31% Polymyositis/SS overlap: (55%) SS and certain overlap diseases Sjogren's Syndrome Sjogren's Syndrome 1. 2. 3. 4. 5. 6. 1. 2. a Marker Antibody = Presence of antibody is virtually diagnostic. The HEp-2 substrate utilized in this IFA test system contains a mixture of cells in various mitotic stages. The majority of the cells are in a resting phase (interphase), the ideal phase for routine ANA screening. The remainder are a mixture of the four phases of mitosis: 1. prophase 2. metaphase 3. anaphase 4. telophase. When interpreting the patient sample, both resting and mitotic cells should be evaluated. ANA antibodies are not organ or species specific. The primary test reaction involves circulating antinuclear antibodies present in the patient's serum which attach to their homologous nuclear antigens. 3. 4. 5. 6. Xi - Irritant May cause sensitization by skin contact After contact with skin, wash immediately with plenty of water and soap. Wear suitable gloves MATERIALS PROVIDED Description HEp-2, 12 well slide ANA (4+) Homogeneous Positive Control ANA (1+) Homogeneous Positive Control Autoimmune Negative Control FITC Conj., HEp-2 with Evans’ Blue (Goat) FITC HEp-2 Mounting Medium (pH 7.5) Phosphate Buffered Saline (pH 7.5) Coverslips, 70x22 mm Blotters, 12 well Quantity 10 ea 0.5mL 0.5mL 0.5mL 7.0mL 3.0mL 2x10g 12 ea 10 ea ADDITIONAL MATERIALS REQUIRED BUT NOT PROVIDED Test tubes, test tube rack, pipettes. Volumetric flask (1 liter) Staining dish. Epifluorescence microscope Microscope Slide Roller Humid Chamber STORAGE AND STABILITY Antigen slides Prod# 10-1012 should be stored at +2 to +8°C. Slides are stable until their expiration date on the product label. Positive control Prod# 10-1202 and Prod# 10-1206 should be stored at +2 to +8°C. Refer to expiration date on label. Negative control Prod# 10-1201 should be stored at +2 to +8°C. Refer to expiration date on label. FITC labeled anti-human conjugate Prod# 10-1513 should be stored at +2 to +8°C. Refer to expiration date on label. Mounting Medium Prod# 90-1612 should be stored at +2 to +8°C. Refer to the expiration date on label. Phosphate buffered saline pH 7.5 Prod# 90-1607 are stable at room temperature. Reconstitute each vial of PBS buffer salts with 1.0L of distilled water. The PBS contains no preservative and should be stored at +2 to +8°C. Discard if turbidity develops. Page 2 of 4-EN 10-1120-29-Rev. 2 TEST PROCEDURE Dilute test serums 1:40 in PBS if testing is being performed for screening purposes. For titrations set up doubling dilutions of serum starting at 1:40, (i.e. 1:40, 1:80, 1:160, 1:320, etc.). The slide, controls and conjugate are ready to use. 1. Tear envelope at notch. Carefully remove the slide and avoid touching the antigen areas. The slide is now ready to use. 2. Place a drop of diluted serum (15 to 20 l) over the antigen wells. 3. Place slide with patient's serum and controls in a moist chamber for 30 minutes at room temperature. (approximately 20°C) 4. Remove slide from moisture chamber. Using a wash bottle, gently rinse remaining sera from slides being careful not to aim the stream directly on the well. 5. Wash in PBS for two separate five minute changes 6. Remove the slides from PBS and place slide antigen side facing up on a dry paper towel. Carefully place the blotter over the slide so that the blotter is indexed to the surface of the microscope slide. Hold one edge of the blotter with one hand to keep the blotter in place and apply sufficient gentle pressure with the microscope slide roller to remove the moisture between the antigen wells. DO NOT ALLOW THE ANTIGEN WELLS TO DRY. 7. Using dispenser provided, deliver 1 drop (25 l) of conjugate per antigen well. Repeat steps 3-6. 8. Place 4-5 drops of mounting medium on slide. 9. Apply a 22 x 70mm cover glass. Examine the slide under a fluorescent microscope. Note: To maintain fluorescence, store mounted slide in a humid chamber placed in a dark refrigerator. * The conjugate dispenser is provided with a calibrated tip and allows quantitative delivery of reagents from the storage battle. To use, wipe the tip with a paper towel, invert the bottle and squeeze gently to release one drop. If the tip contains an air bubble, tap the battle gently to remove air bubble which will ensure precise drop delivery. SPECIMEN COLLECTION AND STORAGE Serological specimens should be collected under aseptic conditions. Hemolysis is avoided through prompt separation of the serum from the clot. Serum should be stored at 2°C to 8°C if it is to be analyzed within 4-7 days. Serum may be held for 3 to 6 months by storage at -20°C or lower. Lipemic and strongly hemolytic serum should be avoided. When specimens are shipped at ambient temperatures, additions of a preservative such as 0.01% thimerosal (merthiolate) or 0.1% sodium azide is strongly recommended. The CLSI provides recommendations for storing blood specimens (Approved Standard Procedure for the Handling and Processing of Blood Specimens, H18-A2 2005). (24) TITER INTERPRETATION The titer is the highest dilution of patient's serum showing weak (1+) fluorescence. Less than 1:40 Normal; virtually rules out active SLE provided patient is not on immunosupressive therapy or in remission. 1:40-1:80 1:160 or greater Positive; test often in RA and other connective tissue diseases. A fresh sample should be tested in two weeks. If the titer increases active SLE is suggested. No change in titer indicates other possible autoimmune disease in a static condition or a treated controlled SLE patient. A decrease in titer indicates an SLE case in remission or a treated controlled SLE case or another autoimmune process. Strongly suggests SLE although other autoimmune diseases and drugs may induce these high titers. PATTERN INTERPRETATION ANA patterns are generally reported as: Homogeneous, Peripheral, Speckled, Centromere, Nucleolar and in multiple combinations. The nuclear ANA patterns found in IFA may be of diagnostic and/or prognostic significance. (SEE TABLE II & TABLE III). 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. LIMITATIONS OF PROCEDURE No diagnosis should be based upon a single ANA test result, since various host factors must be taken into consideration. Among these host factors are sex and age. There is an increasing incidence in positive ANA results in both males females as age increases (10). Normal females between 20-60 have 7% incidence of ANA: normal males, a 3% incidence. Normal males and females over 80 years of age have a 50% incidence of ANA. Various medications including antibiotics, tranquilizers, aspirin and birth control pills can induce a Lupus-Iike condition resulting in high ANA titers (11). (See table I) Drug- induced Lupus generally goes into sustained clinical remission following removal of the triggering medication. Various autoimmune processes induce positive ANA tests. Further evidence for a diagnosis of SLE is provided by low complement levels, particularly C1Q, C3 and C4. (12). ANA tests may not agree with LE Prep tests or with latex tests. Management of therapy should be based not only on positive serologic tests for SLE, but should include the presence of active clinical disease. (13). Elderly patients with SLE have a better prognosis and their clinical symptoms differ substantially from those seen in younger patients. (14). Although the predominant class of antinuclear antibodies (ANA) is immunoglobulin G, the presence of immunoglobulin E maybe of pathogenic importance in SLE (15). Staining patterns often change with titration of the sera revealing multiple patterns not seen in the lower dilutions. Identification of antibodies based only upon patterns could be misleading and should be confirmed using other serological tests such as ENA double gel diffusion tests, specific nDNA tests and histone tests. 1. 2. 3. 4. 5. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. QUALITY CONTROL Positive 4+, 1+, and negative serum controls must be included in each day's testing to confirm reproducibility, sensitivity and specificity of the test procedure. The negative serum control should result in little(+) or no fluorescence of the nuclei. If this control shows bright fluorescence either the control or the antigen may be at fault. The positive 4+ serum control should result in 3+ to 4+ fluorescence of the type specified on the label. If this control shows little or no fluorescence either the control, antigen, conjugate or technique may be at fault. The positive 1+ serum control should result in 1+ fluorescence of the type specified on the label. If this control shows little or no fluorescence either the control, antigen, conjugate or technique may be at fault. In addition to positive and negative serum controls, a PBS control should be run to establish that the conjugate is free from nonspecific staining of the antigen substrate. If the antigen shows bright fluorescence in the PBS control repeat using fresh conjugate. If the antigen still fluoresces either the conjugate or the antigen may be at fault. REFERENCES Feely RH, Systemic Lupus Erythematosus: A Review. Rheum. and Rehab., 17:79, 1978. Burnham TK, Antinuclear Antibodies II. The Prognostic Significance of Nuclear Immunofluorescent Patterns in Lupus Erythematosus. Arch. of Derm., 11.203, 1975. Greenwald CA, Peebles CI, Nakamura RM, Laboratory Tests for Antinuclear Antibody (ANA) in Rheumatic Disease. Lab. Med., 9., 1978. Nisengard RJ, Antinuclear Antibodies: Significance of Titers. Immunology of the Skin by EH Beutner, TP Chorzelski, SE Bean. 2nd Edition, John Wiley and Sons, p.287. 1979 Barrett EV, Immunofluorescence Tests in Immune Techniques and Applications Amer. Jour. Clin. Path 68:662, 1977 Lowenstein MB, Rothfield NF, Family Study of Systemic Lupus Erythematosus, Arth. and Rheum. 20:1293, 1977 Hahon N, Eckert HL, Stewart J, Evaluation of Cellular Substrates for Antinuclear Antibody Determinations. J. Clin. Microbiol. 2:42, 1975. Ritchie RH, The Clinical Significance of Titered Antinuclear Antibodies. Arthritis and Rheumatism. 10.6,1967 Tan EM, Rodnan GP, Garcia I, Moroi Y, Fritzler MJ and Peebles C, Anti Centromere Antibody and its relationship to Crest Syndrome. Arth and Rheum. 23:6, 1980. Castanedo JP, White JG, Williams RC Jr. Antinuclear Antibodies in Normal Human Subjects. Arth and Rheum. 10:5, 1967. Holborow EJ, Weir DM, Johnson GD. A Serum Factor in Lupus Erythematosus with Affinity for Tissue Nuclei. British Med Jour Sept 28, 1957. Clark G, Reichlin M, Tomasi TB Jr. Characterization of a Soluble Cytoplasmic Antigen Reactive with Sera from Patients with systemic Lupus Erythmatosus. J Immunology 103:1, 1969. Solomon SD, The PM-1 Antibody Test in a Patient with Rheumatic Complaints. J.Rheum 1982. Sharp GC, Irvin WS, Tan EM, Gould RG, Holman HR, Mixed Connective Tissue DiseaseAn apparently Distinct Rheumatic Disease Syndrome Associated with a Specific Antibody to an Extractable Nuclear Antigen (ENA). Am J Med Vol 52, 1972. Alspaugh M, Maddison, P, Resolution of the Identity of Certain Antigen-Antibody systems in Systemic Lupus Erythematosus and Sjogren's Syndrome: An Interlaboratory Collaboration, Frief Reports. p. 796-798. Feb 21, 1979 Cox JV, Schenk EA, Olmstead JB, Human anticentromere Antibodies: Distribution, Characterization of Antigens, and Effect on Microtubule Organization. Cell Vol 35, p331339, 1983. Moroi Y, Peebles C, Fritzler M, Steigerwald J, Tan EM. Autoantibody to Centromere (Kinetochore) in Scleroderma Sera. Proc Natl Acad Sci 77:1627- 1631, 1980 Sluder G, Begg DA, More on the Spatial Arrangement of Spindle Components in the Timing of Mitotic Events. J Cell Bio Vol 97, 1983. Nakamura R, Peebles CL, Rubin RL, Molden DP, Tan E, Autoantibodies to Nuclear Antigens (ANA), Advances in Laboratory Tests and Significance in Systemic Rheumatic Diseases. ASCP Second Edition, 1985. McCarthy GA, Autoantibodies and Their Relation to Rheumatic Diseases. Med. Clin. of N.A. 70:237-261 1986. Fritzler MJ, Tan EM, Antinuclear Antibodies and the Connective Tissue Diseases. Chapter 8. Laboratory Diagnostic Procedures in the Rheumatic Diseases. Grune & Stratton pp207243 1985. Walravens M, Maini RN, First European ANA Symposium Leuven, Belgium May 1984. CI. Rheu. Acta Medica Belgica Vol6 Suppl N1, pp 1-108 June 87. Centers for Disease Control/National Institutes of Health (CDC-NIH) Manual. 1999. In: Biosafety in Microbiological and Biomedical Laboratories, 4th Edition, U.S. Dept. of Health and Human Services, Public Health Service. Clinical Laboratory Standards Institute (CLSI). 2005. Procedures for the Handling and Processing of Blood Specimens; Approved Guideline – Second Edition. CLSI Publication H18-A2. Page 3 of 4-EN 10-1120-29-Rev. 2 Consult Instructions for Use REF Product Number LOT Lot Number IVD In Vitro Diagnostic Medical Device EC Authorized Representative in the European Community REP Use By Caution, consult accompanying documents Temperature limitation Manufacturer Irritant- Precaution CONTROL - Negative Control CONTROL ANA 4+ ANA 4+ Homogeneous Positive Control CONTROL ANA 1+ ANA 1+ Homogeneous Positive Control CONJ Conjugate PBS Phosphate Buffered Saline MTMED Mounting Medium CVRSLP BLTRS SLD Coverslips 12W 12W EC REP-Trinity Biotech plc. IDA Business Park Bray, County Wicklow, Ireland Phone: +353-1-276-9800 Fax: +353-1-276-9888 Web:www.trinitybiotech.com Blotters, 12 Well Slide, 12 Well Manufactured By MarDx Diagnostics,Inc. A Trinity Biotech Company 5919 Farnsworth Court Carlsbad, CA 92008 Phone: 800-325-3424 Fax: 760-929-0124 10-1120-29-Rev. 2 10/2010 Page 4 of 4-EN 10-1120-29-Rev. 2
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