This guidance was written prior to the February 27, 1997 implementation of FDA’s Good Guidance Practices, GGP’s. It does not create or confer rights for or on any person and does not operate to bind FDA or the public. An alternative approach may be used if such approach satisfies the requirements of the applicable statute, regulations, or both. This guidance will be updated in the next revision to include the standard elements of GGP’s.
REVIEW CRITERIA FOR IN VITRO DIAGNOSTIC DEVICES FOR THE ASSESSMENT OF THYROID AUTOANTIBODIES USING INDIRECT IMMUNOFLUORESCENCE ASSAY (IFA), INDIRECT HEMAGGLUTINATION ASSAY (IHA), RADIOIMMUNOASSAY (RIA), AND ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA). This is a flexible document representing the current concerns and suggestions regarding thyroid autoantibodies in vitro diagnostic devices employing IFA, IHA, RIA, and ELISA methodologies. It is based on (1) current basic science, (2) clinical experience, (3) the Safe Medical Devices Act of 1990 (SMDA) and (4) FDA regulations in the Code of Federal Regulations (CFR). As advances are made in science and medicine, these review criteria will be re-evaluated and revised as necessary. PURPOSE OF THE GUIDANCE DRAFT The purpose of this document is to provide guidance and clarification on information to present to the Food and Drug Administration (FDA) before a device to detect, quantitate and/or semi-quantitate thyroid autoantibodies in clinical specimens can be cleared for marketing. A premarket notification 510(k) submission provides evidence that the device is accurate, safe, effective and substantially equivalent to a predicate device legally marketed in the United States. DEFINITION This generic type of device is intended for use in clinical laboratories or physician's office laboratories* as an in vitro diagnostic test for the qualitative, quantitative and/or semi-quantitative measurement of thyroid autoantibodies by IFA, IHA, RIA or ELISA. *Devices may be cleared for use in physician's office laboratories when additional data are submitted to demonstrate performance in these settings. PRODUCT CODES: JNL, DDC, DDJ, JZO REGULATION NUMBER: 21 CFR 866.5870 Thyroid autoantibody immunological test system. Identification. A thyroid autoantibody test system is a device that consists of the reagents used to measure, by immunochemical techniques, thyroid autoantibodies (antibodies produced against the bodies own tissues). Measurement of thyroid autoantibodies may aid in the diagnosis of certain thyroid disorders, such as Hashimoto's disease (chronic lymphocytic thyroiditis), nontoxic goiter (enlargement of the thyroid gland), and Graves' disease (enlargement of the thyroid gland with protrusion of the eyeballs). CLASSIFICATION: CLASS II (Performance Standards) PANEL: Immunology (82) REVIEW REQUIRED: Premarket notification (510(k)) I. CLINICAL INDICATIONS/SIGNIFICANCE/INTENDED USE A. INTRODUCTION The principle role of the thyroid gland is the storage and synthesis of thyroid hormones. The normal thyroid gland is composed of numerous follicles, each of which consists of a single layer of epithelial cells surrounding a central lumen containing colloid. Thyroglobulin is the major component of the thyroid follicular colloid. It is produced by the thyroid epithelial cells and is a water soluble glycoprotein with a molecular weight of 670,000 daltons. Small amounts of thyroglobulin are present in serum. Microsomal antigen is a 110,000 dalton glycoprotein present in the cytoplasm and on the apical membrane of the thyroid cell. Reports have shown that thyroid peroxidase (TPO) is the major component of the thyroid microsomal antigen and is the antigenic component recognized by autoantibodies directed against the microsomal glycoprotein. 1 Autoimmune thyroid disease is organ specific and is defined by the presence of circulating antibodies. The most common antibodies seen are antibodies to thyroglobulin and microsomal or thyroid peroxidase (TPO) of the thyroid epithelial cell lining. Autoantibodies to these thyroid antigens are detected in Hashimoto's disease and its variants, Graves' disease, myxedema, nontoxic goiter, and thyroid carcinoma. Antithyroglobulin and antimicrosomal (TPO) antibodies are seen most often and are in the highest titers in Hashimoto's disease. Patients with Graves' disease can also demonstrate thyroid antibodies of relatively high titer. These autoantibodies are also seen in low titers in other thyroid diseases and in individuals without clinical evidence of disease. 2 Two other thyroid antibodies less commonly seen are antibodies to the second antigen of the colloid or CA-2 and human thyroid-stimulating immunoglobulin (TSI). CA-2 is a minor protein of colloid distinct from thyroglobulin and antibodies to it are of uncertain significance. Positive CA-2 reactions are seen in a low percentage of thyroiditis patients in the absence of other antithyroid antibodies and in 5 to 10 per cent of patients with Graves' disease and thyroid cancer. 2 CA-2 antibodies can be detected by IFA. TSI, previously referred to as long-acting thyroid stimulator (LATS), is a polyclonal gamma globulin which appears to bind to a receptor on thyroid cells and stimulates thyroid activity. It is present in the sera of about 50 per cent of those with Graves' disease and is absent or present only in a small number of patients with nodular toxic goiter or other thyroid disorders. TSI can be measured by a competitive inhibition assay using 125I labeled TSH and thyrotropin (TSH) receptors. Hashimoto's thyroiditis is an inflammatory condition occurring in about 1 to 2 per cent of the population, mainly in middle-aged women, and is characterized by gland enlargement as a result of marked lymphocytic inflammatory changes. The latter may consist of lymphoid follicles with active germinal centers in which much of the antithyroglobulin antibody appears to be synthesized. Normal thyroid glandular structures are adversely altered, and in prominent cases progressive disease may lead to thyroid atrophy and myxedema (dry, waxy type of swelling, with abnormal deposits of mucin in the skin and other tissues associated with hypothyroidism). In thyrotoxicosis, the thyroid may contain small areas of lymphoid infiltration as well as evidence of the typical glandular hyperactivity. Graves' disease is a multi-systemic disorder, particularly in young to middle-aged females, consisting of varying degrees of (1) hyperthyroidism with diffuse hyperplasia of the thyroid (the most common pattern seen with diffuse toxic goiter); (2) a myopathy; and (3) an infiltrative ophthalmopathy, frequently leading to exophthalmos (protrusion of the eye ball). 2 As with all the organ-oriented diseases associated with autoantibody production, it is important to determine when the antibodies under discussion are pathogenic or are reacting to antigens liberated as a result of tissue damage due to non-immune causes. Immune reactivity may not be the primary pathogenic event but, once present, causes further tissue damage. Evidence against a primary pathogenic role for thyroid autoantibodies in Hashimoto's thyroiditis and Graves' disease is (1) the lack of correlation between the level of autoantibody and the severity of disease in individual cases, and (2) the lack of development of thyroid disease in infants with high levels of antithyroid antibodies because of placental transfer. B. INSTRUCTIONS: Provide a concise discussion to include the following as appropriate. Support the discussion with key literature citations. 1. Clinical indications, significance and intended use. 2. Background description of the thyroid disease involved including the type of population affected (sex, age, etc.) 3. Significance of a positive result (disease indication and follow up testing). 4. Significance of false positive and false negative results. 5. Salient concerns of the medical community including relevant medical issues that may impact the review process or possibly the development of public policy. 6. A brief historical summary of all test methodologies used to detect the antibody(ies). 7. Merits/advantages and limitations/disadvantages of the device methodology(ies) compared to other available methodologies. 8. Matrices. II. DEVICE DESCRIPTION: The determination of substantial equivalence is based on the specific intended use (what analyte is detected and the indications for use) and the technology/methodology utilized in the device. Discuss the principles of the device methodology and whether it is well-established or new and unproven. III. CLINICAL AND NONCLINICAL LABORATORY STUDIES: SPECIFIC PERFORMANCE CHARACTERISTICS: FDA requests different types and amounts of data and statistical analyses in pre-market notification applications to market in vitro diagnostic devices. The amount and type of data requested depends on the intended use, technological characteristics of the new device, whether the test is qualitative, quantitative or semi-quantitative and on certain claims made by the manufacturer. The performance of the device can be established by comparison to any legally marketed medical device (the predicate) with the same intended use. Prove all claims for substantial equivalence and specific performance characteristics for using the device. Clearly document all protocols for in vitro testing. Present test data results with analyses and conclusions. Summarize results and include explanations for unexpected results and any additional testing performed. Charts (scattergrams, histograms, etc.) may be used as part of the analyses and conclusions when appropriate. Actual, unprocessed laboratory data may be requested. A. ANALYTICAL/LABORATORY/IN VITRO STUDIES 1. Validation of the Cut-off Describe the rationale for determination of the assay cut-off(s). Furnish descriptive information and laboratory data to show how the cut-off point (distinction between positivity and negativity or medical decision limit) was determined by the assay. a. Define the population(s) used, including the following information: i. Number of samples in the normal population (used to determine initial screening dilution) with samples summarized according to gender and age groups. 4 ii. Number of specimens included in each disease group summarized according to gender and age groups. iii. Geographical area(s) from which the population was derived. iv. Graphical (e.g., scattergrams, histograms, etc.) representation of population characteristics. b. Define the statistical method used to determine the cut-off point(s). c. Present a Receiver Operator Curve (ROC) analysis of cut-off point selection and other graphical representations as appropriate. d. Define the basis for the equivocal zone (if applicable). 2. Reagent characterization a. Give a brief description of the antigen(s) and antibody(ies) used in the assay. b. If any recombinant technology was used in the preparation of the antigen(s), describe method used. 3. Assay Specificity/Interfering Substances Any potentially cross reacting or interfering substances encountered in specific specimen types or conditions should be tested using the assay system, e.g., hemolysis, lipemia, microbial contamination, additional analytes or other autoantibodies present, and storage or freeze-thawing. a. Verify that recommended storage conditions are compatible with the assay. State the optimal conditions based on specimen storage stability studies. Both false positivity and negativity should be evaluated (if applicable). b. If the use of plasma is claimed, a study with each anticoagulant must be performed to show that each anticoagulant does not interfere with the assay. i. For each anticoagulant, test 10 matched serum and plasma specimens which are positive at the cut-off point. ii. For each anticoagulant, test 10 matched negative serum and plasma specimens. Provide an explanation if interference from the anticoagulant is not anticipated (e.g., high dilution factor). 4. Performance Characteristics Include the following performance characteristics: a. Analytical Sensitivity (if applicable) The analytical sensitivity or detection limit is defined as the lowest quantity differentiated from zero (95% confidence intervals or 2 standard deviations (SD) above the mean of the Zero control are commonly used). 4,5 Run the Zero standard (Zero diluent) at least 20-25 times in the same run and calculate the mean of the Zero standard and 2 SD of the mean (counts, OD's, etc.). If levels of the analyte are not clinically significant, determination of the detection limit may be irrelevant. b. Relative Sensitivity and Specificity The relative sensitivity and specificity as determined by comparison to a legally marketed device or to a reference method should be determined and reported in the Performance Characteristics section of the package insert. c. Linear Range Validate the linear range of the assay with normal and abnormal specimens covering the entire reportable range of the assay. 6 d. Reproducibility and Repeatability Studies 4,5,6,7,8 The National Committee for Clinical Laboratory Standards (NCCLS) recommends an analysis of variance experiment testing two clinically significant levels near medical decision limits (normal or elevated) of an analyte, in this case thyroid autoantibodies. 8 Use controls simulating patient samples or actual patient specimens 3 times in the same run and in two different runs each day for 20 days. This permits separate estimation of between-day, between-run and within-day standard deviations (SDs), as well as within-run and total SDs. i. Qualitative/Quantitative Tests: Calculate total, between- and within-day and between- and within-run means and coefficients of variation of imprecision for each set of values. ii. Semi-quantitative Tests: In devices with a titration format, e.g., immunofluorescence assays, demonstrate that intra-run reproducibility is within the commonly accepted limits of plus or minus one two-fold dilution. iii. Means, SDs, and Coefficients of Variation: Report in the Performance Characteristics section of the package insert the appropriate means, SDs, and/or coefficients of variation with confidence levels according to number of times the sample is repeated. Report the number of runs per day. e. Prozone or High-Dose Hook Effect Studies Test a sample with the highest titer available, serially diluted and undiluted. If prozone problems are encountered, state in the Performance Characteristics section of the package insert the titer at which prozone problems were detected and a procedure for the user to follow to correct the problem. Where appropriate, describe the appearance of a prozone reaction for subjective tests. f. Alternative Testing Sites Include reproducibility studies performed in these settings. In-site testing for new technologies should include at least three independent alternative testing sites. At each of the three sites, the precision and accuracy of the device should be evaluated. A statistically valid number of samples should be tested by the site personnel and by professional laboratory personnel, and the results compared, to show how the device performs in the hands of the lesser trained user. 5. Comparison Studies Compare the new device to a legally marketed device. Include the package insert for the legally marketed device. It is recommended that a recognized reference method (if available) also be employed for comparison to enable a fair evaluation of the proposed device's performance characteristics, particularly if there are broad differences in methodology/ technology between the new device and the legally marketed device. a. Qualitative Tests: The studies should be performed on an adequate number of positive and negative specimens to support statistical significance. (An appropriate number may be suggested by a statistician.) b. Quantitative/Semi-quantitative Tests: An assay is considered quantitative only if a recognized reference material of known concentration is available for standardization of a calibrator or standard used in the assay to calculate results. If the same reference material is used in the new device as in the predicate device to substantiate the quantitative claim, comparison data should be presented to show correlation between the two assays when running the reference material as samples. Run the serially diluted reference material by the new device and the predicate device. The assays should show similar results. Compare results obtained using positive thyroid autoantibody samples free from interfering substances from 40-100 persons covering the whole assay range (from low to high levels of antibodies). 7,9 Perform a linear regression analysis and report the slope, intercept, correlation coefficient, the assay range, and the nature and number of samples tested. c. Comparison Discrepancies: Equivocal results or discrepancies between the new device and the comparison method should be resolved using another method or clinical diagnosis. 6. Specimen Collection and Handling Conditions State specimen collection, storage and handling conditions in the package insert and provide data or appropriate literature references in the submission to substantiate claims. 7. Computer Controlled Medical Devices For information regarding computer assisted clinical laboratory devices, refer to "Review Guidance for Computer Controlled Medical Devices Undergoing 510(k) Review" available from the Division of Small Manufacturers Assistance (DSMA), 1-800-638-2041. B. CLINICAL INVESTIGATIONS In certain instances it is necessary to require comparative clinical data to establish substantial equivalence, e.g., a new or unfamiliar methodology or technological feature is introduced in a device category in which clinical performance is claimed to be equivalent to a legally marketed device using "conventional" technology. For 510(k) submissions, perform a comparison of the device to a legally marketed device. Ideally this study should be done at independent clinical laboratory site(s). A minimum of two additional independent investigators at separate outside locations is recommended. The investigators should be identified by institutional name and address. 1. Adequate Clinical Investigations a. Prove all claims for substantial equivalence and specific parameters for using the device. b. Describe all protocols for clinical studies and consistently adhere to the protocols. c. Determine the sample size, prior to beginning the study, that will be statistically sufficient to determine whether or not the device is safe and effective. d. Sampling Method: Describe sampling method used in the selection and exclusion of patients. i. Patient selection: Include samples from individuals with diseases or conditions that may cause false positive or false negative results with the device. Ideally, a prospective study is preferred. However, if a retrospective study is used, include all eligible patients who meet the patient selection criteria as specified in the protocol. ii. Account for all patients and samples. Insure that data points are included for every sample for every patient. 2. Establishing Reference Ranges a. Normal individuals: Establish a normal reference range with a statistically sufficient number of samples from normal persons characterized by age, sex, geographical location and any other factors that would influence the values obtained. 3,10 b. Patient groups: i. Confirm that the new device detects the percentage of positives generally expected for each disease for which the device is intended. Use a statistically sufficient number of patients characterized by age, sex, geographical location, any symptoms of disease, clinical presentation, and any other factors that would influence the values obtained. 3,10 ii. False results: Patients positive for antinuclear antibodies (ANA) could give a false positive result in an IFA assay for the detection of thyroid autoantibodies. Radioisotopes administered to the patient for diagnostic or therapeutic purposes may interfere in some RIA assays. Provide reports, if any, of false positive and false negative results for each disease as appropriate. c. Sample Types Claimed: Investigate all sample type(s) claimed in the intended use statement unless other data proves that there is no difference between them. IV. LABELING CONSIDERATIONS The following are additional details for some of the points in the statute [502(f)(1)] and regulations [21 CFR 809.10(b)]. Package Insert Include the package insert for the new thyroid autoantibody device. Support the statements throughout the document with key literature citations. A. INTENDED USE Briefly describe the intended use based on the technology/methodology used in the device. Include the following information: 1. Whether the assay is qualitative, quantitative, or semi-quantitative. In order to claim to provide a quantitative result, the calibrators or standards must be calibrated to a reference material of known, established value. 2. Test methodology. 3. Specimen type(s). 4. Indicate if the device is for use in clinical laboratories and/or alternative care sites. The Limitations section should include any specific training required for test performance. A typical Intended Use statement would be: "ABC's *** test system is a device for the semi-quantitative measurement of anti-thyroglobulin antibodies by indirect immunofluorescence in human serum to aid in the diagnosis of certain thyroid disorders, such as Hashimoto's disease, nontoxic goiter, and Graves' disease." B. QUALITY CONTROL (QC) The package insert should recommend levels of quality control samples and their number, matrix type, placement and interpretation to ensure that the system meets its performance claims. Include a statement that if controls do not behave as expected, assay results are considered invalid and should be repeated. Controls should be handled in the same manner as patient samples. For example, if the patient sample is diluted or titered the control material should also be diluted or titered using the same diluent. C. RESULTS Give an adequate description of expected results and interpretation. 1. IFA a. Give a description of the fluorescence for a positive and negative result. b. List possible staining patterns which may be found. Photographs or diagrams may be helpful. c. Give pattern description and interpretation as it relates to the particular antibodies. d. Give instructions for titering positive samples to end point. 2. IHA a. Describe in detail the appearance of a positive and negative result. b. Give a definition and description of the end point for a positive result. c. Describe the appearance of a prozone reaction (antibody excess) and instruct the user what to do if prozoning is suspected. d. Explain the procedure for repeating samples which have results above the linearity of the assay. 3. RIA a. Explain the procedure for manual (if applicable) calculation of percent bound for each sample and include a sample calculation. b. Give instructions for plotting percent bound versus concentration of the standard showing examples of typical results (numerical and graphical). c. Give a brief explanation of how automated calculations are performed, e.g., the type of data reduction program used. 4. ELISA a. Explain the procedure for calculating the value of the unknown including a sample calculation. b. Explain the procedure for repeating samples which are above the linearity of the assay. Give instructions for dilution of samples including the dilution factor and type of diluent to be used. D. LIMITATIONS OF THE PROCEDURE Include a statement of limitations of the procedure to include the following: 1. A statement that the test result in and of itself is not diagnostic for thyroid disease and should be considered in conjunction with iodine uptake and other standard thyroid tests and the clinical presentation of the patient. 2. IFA a. Explain possible variations between different types of fluorescent microscopes. b. Give warnings concerning distinguishing the thyroid specific cytoplasmic fluorescence from that obtained with mitochondrial antibody in primary biliary cirrhosis. If mitochondrial antibody is suspected, the distinction can be made by running more specific tests for antinuclear antibodies. c. Multiple antibodies may be present and complicate the staining interpretation. Serially diluting the patient sample will often aid in distinguishing multiple patterns. d. A prozone reaction can appear as a doubtful positive or negative because of a small amount of antigen in relation to the large amount of antibody present. If prozone is suspected, the patient sample should be serially diluted. 3. IHA Give warnings concerning heterophile antibodies and a possible prozone reaction 4. RIA a. Give warnings concerning possible interference from radioisotopes administered to the patient for diagnostic or therapeutic purposes. b. Supply instructions for proper disposal of radioactive materials. E. EXPECTED VALUES 1. The expected value in the normal population is negative. However, apparently healthy, asymptomatic individuals (5-10%) may test positive for thyroid autoantibodies. The incidence of these antibodies increases with increasing age beginning in the seventh decade for women and the eighth decade for men. 2. Thyroid autoantibodies may be present in non-thyroid disorders such as pernicious anemia, diabetes mellitus, Addison's disease, and Sjogren's syndrome. 3. Present information showing the incidence or prevalence of each type of thyroid autoantibody for each disease state. From: Division of Clinical Laboratory Devices Immunology Branch Prepared by: Deborah M. Moore, Scientific Reviewer February, 1994 V. BIBLIOGRAPHY 1. Czarnocka B,et al. Purification of the Human Thyroid Peroxidase and its Identification as the Microsomal Antigen involved in Autoimmune Thyroid Disease. FEBS 190:147 (1985). 2. Henry J, Immunology and Immunopathology. Clinical Diagnosis and Management by Laboratory Methods, 18th ed 1985; WB Saunders Co., Philadelphia, PA. 3. National Committee for Clinical Laboratory Standards. How to define, determine, and utilize reference intervals in the clinical laboratory; proposed guideline. Villanova, PA 1991. Order code C28-P. 4. Vadlamudi SK, Stewart WD, Fugate KJ, Tsakeris TM. Performance characteristics for an immunoassay. Scand J Clin Lab Invest 1991;51:134-138. 5. Peters T, Westgard JO. Evaluation of methods, Chapter 7 in: Tiets NW, editor. Fundamentals of Clinical Chemistry, 3rd ed, 1987: 225-37 Philadelphia, PA; WB Saunders Co. 6. National Committee for Clinical Laboratory Standards. Evaluation of the linearity of quantitative methods; proposed guideline. 1986 Order code EP6-P. 7. Information for authors. Clin Chem 1991; 37:1-3. 8. National Committee for Clinical Laboratory Standards. Evaluation of precision performance of clinical chemistry devices - 2nd ed; tentative guideline. 1991:1-56. Order code EP5-T2. 9. National Committee for Clinical Laboratory Standards. User comparison of quantitative clinical laboratory methods using patient samples; proposed guideline. 1985; 6(1). Order code EP9-P. 10. Ash KO. Reference Intervals (Normal Ranges): A Challenge to Laboratories. Am J. Med Tech 1980; 46:504-11.