E Toxo IgM 9K09-20 B9K090 49-3257/R4 Toxo IgM This package insert must be read carefully prior to use. Package insert instructions must be followed precisely. Reliability of assay results cannot be guaranteed if there are any deviations from the instructions in this package insert. Read Highlighted Changes Revised March, 2010 2004, 2010 Abbott / Printed in Germany / AxSYM Toxo IgM March 2010 Key to symbols used List Number Lot Number For In Vitro Diagnostic Use Expiration Date Store at 2-8 C Index Calibrator Store at 15-30 C Control Negative, Positive (NEG, POS) Reagent Pack CAUTION: Handle human sourced materials as potentially infectious. Consult instructions for use. (Infection Risk) Reaction Vessels Matrix Cells 0088 Legal Manufacturer Sample Cups Consult instructions for use See REAGENTS section for a full explanation of symbols used in reagent component naming. ABBOTT Diagnostics Division 1
NAME Toxoplasma IgM Antibody INTENDED USE The AxSYM Toxo IgM assay is a Microparticle Enzyme Immunoassay (MEIA) for the measurement of IgM antibodies to Toxoplasma gondii in human serum or plasma (EDTA, heparin or sodium citrate) to aid in the diagnosis of primary infection. The AxSYM Toxo IgM assay is not for use with cord blood or neonatal specimens. SUMMARY AND EXPLANATION OF THE TEST Toxoplasma gondii is an obligate intracellular parasite capable of infecting a wide variety of intermediate hosts including man (see reference 1 for a comprehensive review of toxoplasmosis). Infected definitive hosts (cats) shed oocysts in feces which rapidly mature in soil and become infectious. When ingested by intermediate hosts, tachyzoites form and multiply rapidly with eventual development of cysts containing the slower growing, but infectious bradyzoites. Thus, toxoplasmosis is acquired by man via ingestion of cat feces or undercooked meats infested with cysts. 2,3 Infection of the normal adult is commonly asymptomatic. In those cases with clinical manifestations, the most common of the symptoms is lymphadenopathy which may be accompanied by an array of other symptoms making differential diagnosis difficult. 4,5 On the other hand, severe to fatal infections do occur in adults immunocompromised by cancer chemotherapy or immunosuppressive treatment and in patients with AIDS. Infections in the immunocompromised adults are thought to be reactivation of latent acquired infections and usually involve the central nervous system although involvement of other sites has been reported. 6-9 Transplacental transmission of the parasite resulting in congenital toxoplasmosis can occur during the acute acquired maternal infection. The risk of fetal infection is a function of time at which acute maternal infection occurs during gestation. Maternal infections acquired before conception present very little, if any, risk to the fetus. However, the incidence of congenital toxoplasmosis increases as pregnancy progresses; conversely, the severity of congenital toxoplasmosis is greatest when maternal infection is acquired early during pregnancy. A majority of infants infected in utero are asymptomatic at birth, particularly if maternal infection occurs during the third trimester, with sequelae appearing later in life. 10,11 Congenital toxoplasmosis results in severe generalized or neurologic disease in about 20-30% of the infants infected in utero; approximately 10% exhibit ocular involvement only, and the remainder (about 70%) are asymptomatic at birth. 11,12 Subclinical infection may result in premature delivery and subsequent neurologic, intellectual, and audiologic defects. 10,13 Prospective studies of pregnancies have shown that prenatal diagnosis of infection followed by prenatal therapy reduces the frequency and the severity of congenital toxoplasmosis. 10,14 Serologic tests can be used to identify those pregnancies at risk; women who are seronegative at the time of diagnosis of pregnancy can be monitored during pregnancy. Seroconversion is indicative of T. gondii infection and establishes gestational age when maternal infection occurred. 10,14,15 Serologic tests specific for T. gondii IgM antibodies are useful aids in the diagnosis of both congenital and acute acquired toxoplasmosis. 16-18 Levels of IgM antibody increase rapidly following acute acquired infections and begin to decline after several months but can persist at detectable levels for a year or more. 19-21 Since persisting IgM levels may be detected long after the onset of acquired infection, the use of a single serological test result must be used with caution in those cases when it is critical to establish the time of infection. This applies to the diagnosis of acute T. gondii infection acquired during pregnancy. Determination of the date of infection based solely on the results of detectable IgM antibody to T. gondii is not recommended. That determination should include clinical history and previous serology, since low levels of IgM antibody may persist for a year or more. 19-21 The use of a test to determine a rise in IgG antibody to T. gondii may provide additional information as to the date of infection. The AxSYM Toxo IgM assay is a method for the measurement of T. gondii specific antibodies in human serum and plasma (EDTA, heparin or sodium citrate). BIOLOGICAL PRINCIPLES OF THE PROCEDURE The AxSYM Toxo IgM assay is based on the Microparticle Enzyme Immunoassay (MEIA) technology. Sample and all AxSYM Toxo IgM reagents required for one test are pipetted by the Sampling Probe into various wells of a reaction vessel (RV) in the Sampling Center. The RV is immediately transferred into the Processing Center. Further pipetting is done in the Processing Center by the Processing Probe. The reactions occur in the following sequence: SAMPLING CENTER The Sampling Probe dilutes the sample in Solution 4 (Line Diluent) and delivers an aliquot of diluted sample and T. gondii Coated Microparticles to an incubation well of the RV. The T. gondii antibody binds to the T. gondii Coated Microparticles forming an antigen-antibody complex. PROCESSING CENTER Assay Diluent is added to the reaction mixture and an aliquot of the antigen-antibody complex is transferred to the matrix cell. The microparticles bind irreversibly to the glass fiber matrix. The matrix cell is washed with RF Neutralization Buffer to remove RF interference antibodies (if present) from the antigen-antibody complex. The matrix cell is washed to remove unbound materials. The Anti-Human IgM: Alkaline Phosphatase Conjugate is dispensed onto the matrix cell and binds to the antigen-antibody complex. The matrix cell is washed to remove unbound materials. The substrate, 4-Methylumbelliferyl Phosphate, is added to the matrix cell and the fluorescent product is measured by the MEIA optical assembly. For further information regarding MEIA technology, refer to the AxSYM System Operations Manual, Section 3. REAGENTS REAGENT PACK, 100 Tests AxSYM Toxo IgM Reagent Pack (9K09-20) 1 Bottle (7.8 ml) T. gondii Coated Microparticles in Tris buffer with protein stabilizers. Preservatives: Antimicrobial Agents. (Reagent Bottle 1) 1 Bottle (9.5 ml) Anti-Human IgM (goat): Alkaline Phosphatase Conjugate in Tris buffer with protein stabilizers. Minimum concentration: 0.3 μg/ml. Preservative: Sodium Azide. (Reagent Bottle 2) 1 Bottle (25.7 ml) Assay Diluent in Tris buffer with protein stabilizers. Preservatives: Antimicrobial Agents. (Reagent Bottle 3) 1 Bottle (26.0 ml) RF Neutralization Buffer, Citrate. Preservatives: Antimicrobial Agents. (Reagent Bottle 4) CALIBRATOR AxSYM Toxo IgM Index Calibrator (9K09-40) 1 Bottle (3 ml) AxSYM Toxo IgM Index Calibrator prepared with recalcified human plasma reactive for T. gondii IgM Ab and nonreactive for HBsAg, HIV-1 Ag or HIV-1 NAT, anti-hcv and anti-hiv-1/hiv-2. Preservative: Sodium Azide CONTROLS AxSYM Toxo IgM Controls (9K09-10) 1 Bottle (5 ml) AxSYM Toxo IgM Negative Control prepared with recalcified human plasma nonreactive for HBsAg, HIV-1 Ag or HIV-1 NAT, anti-hcv, anti-hiv-1/hiv-2 and T. gondii IgM Ab. 1 Bottle (5 ml) AxSYM Toxo IgM Positive Control prepared with recalcified human plasma reactive for T. gondii IgM Ab and nonreactive for HBsAg, HIV-1 Ag or HIV-1 NAT, anti-hcv and anti-hiv-1/hiv-2. Bottle Anti-Toxo IgM Index Value Ranges (Index) 0 0-0.399 1.5 1.000-2.000 Preservative: Sodium Azide. 2
OTHER REAGENTS Solution 1 (MUP) (8A47-04) 4 Bottles (230 ml each) Solution 1 (MUP) containing 4-Methylumbelliferyl Phosphate, 1.2 mm, in AMP buffer. Preservative: Sodium Azide. Solution 3 (Matrix Cell Wash) (8A81-04) 4 Bottles (1000 ml each) Solution 3 (Matrix Cell Wash) containing 0.3 M Sodium Chloride in 0.05 M Tris buffer. Preservatives: Sodium Azide and Antimicrobial Agents. Solution 4 (Line Diluent) (8A46) 1 Bottle (10 L) Solution 4 (Line Diluent) containing 0.1 M Phosphate buffer. Preservatives: Sodium Azide and Antimicrobial Agents. AxSYM Probe Cleaning Solution (9A35-05/9A35-04) 2 Bottles (220 ml each) AxSYM Probe Cleaning Solution. Contains 2% Tetraethylammoniumhydroxide (TEAH). WARNINGS AND PRECAUTIONS For In Vitro Diagnostic Use. SAFETY PRECAUTIONS CAUTION: This product contains human sourced and/or potentially infectious components. For a specific listing, refer to the REAGENTS section of this package insert. Components sourced from human blood have been tested and found to be nonreactive for HBsAg, HIV-1 Ag or HIV-1 NAT, anti-hcv and anti-hiv-1/hiv-2. No known test method can offer complete assurance that products derived from human sources or inactivated microorganisms will not transmit infection. Therefore, all human sourced materials should be considered potentially infectious. It is recommended that these reagents and human specimens be handled in accordance with the OSHA Standard on Bloodborne Pathogens. 22 Biosafety Level 2 23 or other appropriate biosafety practices 24, 25 should be used for materials that contain or are suspected of containing infectious agents. The AxSYM Probe Cleaning Solution (2% TEAH) may cause mild eye irritation. If this solution comes in contact with eyes, rinse immediately with water. If irritation persists, seek medical advice. Some components of this product contain Sodium Azide. For a specific listing, refer to the REAGENTS section of this package insert. The components containing Sodium Azide are classified per applicable European Community (EC) Directives as: Harmful (Xn). The following are the appropriate Risk (R) and Safety (S) phrases. R22 Harmful if swallowed. R32 Contact with acids liberates very toxic gas. S35 This material and its container must be disposed of in a safe way. S36 Wear suitable protective clothing. S46 If swallowed, seek medical advice immediately and show this container or label. Information for European Customers: For product not classified as dangerous per European Directive 1999/45/EC - safety data sheet available for professional users on request. HANDLING PRECAUTIONS Do not use Solution 1 (MUP) beyond expiration date or a maximum of 14 days on-board the AxSYM System. When loading new Solution 1 (MUP) it is important to immediately tighten the instrument cap for MUP to minimize exposure to air. Prolonged exposure of MUP to air may compromise performance. Do not use Reagent Pack beyond the expiration date or a maximum of 336 cumulative hours on-board the AxSYM System. Do not mix reagents from different reagent packs. Additional safety and handling precautions and limitations for the Reagent Packs, Index Calibrator, Controls, patient samples and other reagents are described in the AxSYM System Operations Manual, Sections 7 and 8. STORAGE INSTRUCTIONS The AxSYM Toxo IgM Reagent Pack, Index Calibrator and Controls must be stored at 2-8 C. The AxSYM Toxo IgM Reagent Pack, Index Calibrator, and Controls may be used immediately after removing them from the refrigerator. Index Calibrator and Controls should be returned to 2-8 C storage immediately after use. Do not freeze the AxSYM Toxo IgM Reagents. The AxSYM Toxo IgM Reagent Pack may be on-board the AxSYM System for a maximum of 336 cumulative hours; for example 42 eight hour shifts. Recalibration may be required to obtain maximum on-board reagent stability. More frequent use of controls may be required to monitor reagent performance within the same lot. Refer to the AxSYM System Operations Manual, Sections 2, 5 and Appendices, for further information on tracking on-board time. Solution 1 (MUP) must be stored at 2-8 C. It may be on-board the AxSYM System for a maximum of 14 days. After 14 days, it must be discarded. It may be used immediately after removing it from the refrigerator. Do not freeze MUP. The AxSYM Probe Cleaning Solution, Solution 3 (Matrix Cell Wash) and Solution 4 (Line Diluent) must be stored at 15-30 C. INSTRUMENT PROCEDURE Assay File Installation The AxSYM Toxo IgM Assay File must be installed on the AxSYM System from one of the following software disks, prior to performing Toxo IgM assays: 8B96-02 (112 hours on-board Stability) 7D16-01, or higher (336 hours on-board Stability) Refer to the AxSYM System Operations Manual, Section 2 for proper installation procedures. AxSYM Toxo IgM Assay Parameters The default values for the assay parameters used for the AxSYM Toxo IgM assay are listed below. Assay parameters that can be edited contain a (>) symbol. These parameters can be displayed and edited according to the procedure in the AxSYM System Operations Manual, Section 2. In order to obtain values for the parameters with an asterisk (*), review the specific Assay Parameter screen. Press PRINT to print the assay parameters. Assay Parameters 1 Long assay name (English): TOXO_M 6 Abbrev Assay Name (English): TOXO_M 11 Assay Number: 734 12 Assay Version: * 13 Calibration Version: * 14 Assay File Revision: * 15 Assay Enabled > ON 17 Assay Type: MEIA 18 Standard Cal Reps > 2 19 Master Cal Reps: 0 20 Cal Adjust Reps: 0 21 Cal A Concentration: 0.000 22 Cal B Concentration: 0.000 23 Cal C Concentration: 0.000 24 Cal D Concentration: 0.000 25 Cal E Concentration: 0.000 26 Cal F Concentration: 0.000 27 Master Calibrator 1 Concentration: 0.000 28 Master Calibrator 2 Concentration: 0.000 29 Cal Adjustment Concentration: 0.000 43 Default Dilution Protocol > UNDILUTED 44 Default Calibration Method > Index Cal 45 Selected Result Concentration Units > Index 46 Selected Results Decimal Places > 3 62 Blank I - Max background intensity: 0.0000 63 Min Tracer-Min net intensity: 0.0000 64 Max Intercept-Max MUP intercept: 16000.0000 3
Assay Parameters 65 Min Intercept-Min MUP intercept: 2500.0000 66 Upper limit for NRMSE for low rates: 9999.9900 67 Upper limit for NRMSE for high rates: 0.3000 68 Max Rate-Max rate used to check Min MUP Intercept: 1500.0000 69 Min Rate-Rate cutoff for NRMSE and Corr. Coef.: 100.0000 70 Min correlation coefficient for low rates: 0.9000 71 Min correlation coefficient for high rates: 0.9700 72 MUP T Delay-Time delay following MUP: 1.7500 73 Low Limit-Normal/Therapeutic Range lower limit > 0.000 74 High Limit-Normal/Therapeutic Range upper limit > 0.000 75 Low Extreme Value > 0.000 76 High Extreme Value > 0.000 77 Lo Norm-% Uptake Normal Range Low > 0.0000 78 Hi Norm-% Uptake Normal Range High > 0.0000 80 Interpretation Option to use > 1 84 Hold results with POS interpretation > OFF 85 Hold results with NEG interpretation > OFF 86 Hold results with GRY interpretation > OFF 91 Low range Undiluted: 0.000 92 High range Undiluted: 99999.000 96 Low Range Dil1: 0.000 97 High Range Dil1: 0.000 101 Low Range Dil2: 0.000 102 High Range Dil2: 0.000 106 Low Range Dil3: 0.000 107 High Range Dil3: 0.000 112 Max End-Point Deviation: 0.0000 113 Max Baseline Intensity: 0.0000 114 Min Baseline Intensity: 0.0000 115 Max Percent Transmission: 0.0000 NOTE: Parameters 43, 44, 45, and 46 cannot be edited. Refer to the AxSYM System Operations Manual for a detailed description of Instrument Procedures. For details on Automatic Sample Retest Configuration, refer to the AxSYM System Operations Manual, Section 2. SAMPLE COLLECTION AND PREPARATION FOR ANALYSIS Serum or plasma (EDTA, heparin or sodium citrate) may be used in the AxSYM Toxo IgM assay. Follow the manufacturer s processing instructions for serum or plasma collection tubes. Samples may stored for up to 7 days at 2-8 C after the date of collection. If testing will be delayed more than 7 days, the specimens should be stored at -10 C or colder. Mix after thawing to insure consistency in results and centrifuge at > 10,000 x g for 10 minutes before testing. This assay has not been validated for specimens which have undergone more than five freeze/thaw cycles. Specimens should be free of fibrin, red blood cells, or other particulate matter. Specimens showing particulate matter, erythrocytes or turbidity must be clarified by centrifugation at > 10,000 x g for 10 minutes before testing. Heat treated specimens, lipemic specimens, grossly hemolyzed specimens, or specimens with obvious microbial contamination should not be tested with this procedure. When shipped, samples must be packaged and labeled in compliance with applicable federal and international regulations covering the transport of clinical samples and etiologic agents. The AxSYM System does not provide the capability of verifying sample type. It is the responsibility of the operator to verify the correct sample type(s) is (are) used in the AxSYM Toxo IgM assay. All samples (patient samples, controls and calibrators) should be tested within 3 hours of being placed on-board the AxSYM System. Refer to the AxSYM System Operations Manual, Section 5, for more detailed discussion of on-board sample storage constraints. Inspect all samples for bubbles. Remove all bubbles prior to analysis. SAMPLE VOLUME The sample volume required to perform a single Toxo IgM test on the AxSYM System varies depending on the type of sample container used. For sample cups, a ROUTINE test requires 150 μl and a STAT test requires 83 μl. For every additional Toxo IgM test performed (ROUTINE or STAT) from the same container, an additional 33 μl of sample is required. The sample cup minimum volumes for both STAT and ROUTINE tests are calculated by the AxSYM System. They are displayed on the Order screen at the time the test(s) is (are) ordered. When using Host Order Query, the Order screen information and Orderlist Report are not available. Refer to the AxSYM System Operations Manual, Section 5: Ordering Patient Samples, for a description of the Host Order Query option. If the assay is configured for Auto Retest the additional sample volume needed for the retest will not be displayed on the Order screen at the time the test(s) is (are) ordered. Therefore the total sample volume should include the additional 33 μl of sample. Refer to the AxSYM System Operations Manual, Section 2, for details on Automatic Sample Retest Configuration. To obtain the recommended volume requirements for AxSYM Toxo IgM Index Calibrator and Controls, hold the bottles vertically and dispense 4 drops of Index Calibrator or 4 drops of Positive or Negative Control into each respective sample cup. For sample volume requirements in primary or aliquot tubes and Control volume requirements for multiple AxSYM Toxo IgM reagent lots, refer to the AxSYM System Operations Manual, Section 5. AxSYM Toxo IgM PROCEDURE Materials Required 9K09-20 AxSYM Toxo IgM 8A75-02 100 8A73-02 100 AxSYM System 9K09-40 AxSYM Toxo IgM Index Calibrator 9K09-10 AxSYM Toxo IgM Controls 8A47-04 8A81-04 8A46 9A35-05/ AxSYM 9A35-04 8A76-01 Materials Required But Not Provided Pipettes and pipette tips CAUTION: When manually dispensing samples into sample cups, verify that dispensing equipment does not introduce cross contamination and delivers the specified sample volume. For optimal performance, it is important to follow the routine maintenance procedures defined in the AxSYM System Operations Manual, Section 9. If your laboratory requires more frequent maintenance, follow those procedures. Assay Procedure Sections 5 and 6 of the AxSYM System Operations Manual contain detailed steps for performing assay calibration and sample testing procedures. Prior to ordering tests, confirm that System Inventory of matrix cells, bulk solutions and waste levels are acceptable. The Orderlist Report contains sample placement information and sample cup volume requirements for all ordered tests. It is recommended that this report be referenced when loading samples into sample segments. When using Host Order Query, the Order screen information and Orderlist Report are not available. Refer to the AxSYM System Operations Manual, Section 5: Ordering Patient Samples, for a description of the Host Order Query option. 4
CAUTION: When operating the AxSYM System, always observe the following: The System status must be WARMING, PAUSED, READY, or STOPPED before adding or removing sample segments, reagent packs, or reaction vessels. Do not open the Interior Waste Door or the Processing Center Cover while any test is in progress. If opened, all processing will stop. All tests will be terminated and must be repeated. When testing is completed, it is recommended that samples and the AxSYM Toxo IgM Reagent Pack are removed from the Sampling Center to maximize the on-board reagent pack use. Store at 2-8 C. QUALITY CONTROL PROCEDURES CALIBRATION The AxSYM Toxo IgM Assay must be calibrated by testing 2 replicates of the Index Calibrator. Dispense 4 drops of the Index Calibrator into a sample cup. A single sample of the AxSYM Toxo IgM controls must be tested as a means of evaluating the assay calibration. Once an AxSYM Toxo IgM Calibration is accepted and stored, all subsequent samples may be tested without further calibration unless: A reagent pack with a new lot number is used Control values are out of their specified range Refer to the AxSYM System Operations Manual, Section 6, for: Setting up an assay calibration When recalibration may be necessary Calibration verification The AxSYM System verifies that the results of an assay calibration meet the specifications assigned to selected validity parameters. An error message occurs when the calibration fails to meet a specification. Refer to the AxSYM System Operations Manual, Section 10 for an explanation of the corrective actions for the error code. Refer to the AxSYM System Operations Manual, Appendices, for an explanation of the calibration validity parameters that may be used by the AxSYM System. QUALITY CONTROL The recommended control requirement for the AxSYM Toxo IgM assay is a single sample of each of the Positive and Negative Controls tested once every 8 hours, each day of use. Controls may be placed in any position in the sample carousel. If quality control procedures in your laboratory require more frequent use of controls to verify test results, follow those procedures. To achieve maximum on-board reagent stability, more frequent use of controls may be required to monitor reagent performance within the same lot. Ensure that AxSYM Toxo IgM Control values are within the acceptable ranges specified in the package insert. Refer to REAGENTS, CONTROLS section of this package insert. INDICATIONS OF INSTABILITY OR DETERIORATION OF REAGENTS When an AxSYM Toxo IgM Positive or Negative Control value is out of the expected range, it may indicate deterioration of the reagents or errors in technique. Associated test results should be considered invalid and require retesting. Assay recalibration may be indicated. Refer to the AxSYM System Operations Manual, Section 10, for further troubleshooting information. The AxSYM System has a capability to generate a Levey-Jennings plot of each assay s quality control performance. Refer to the AxSYM System Operations Manual, Section 5. At the discretion of the laboratory, selected quality control rules may be applied to the quality control data. Fluorescence Background Acceptance Criteria Quality control of the MUP substrate blank is automatically determined by the instrument and checked under Assay Parameter 64 (Max Intercept- Max MUP Intercept) each time a test result is calculated. If the MUP Intercept value is greater than the maximum allowable value, the result is invalid. The test request will be moved to the Exceptions List where it will appear with the message 1064 Invalid test result, intercept too high and the calculated intercept value. Refer to the AxSYM System Operations Manual, Section 10, when this error message is obtained. Refer to the AxSYM System Operations Manual, Section 2, for further information on this parameter. RESULTS CALCULATION The AxSYM System calculates a Toxo IgM Index Value based on the ratio of the sample (patient/control) rate to the Index Calibrator Mean Rate. Sample Rate Index Value = Index Calibrator Mean Rate FLAGS Some results may contain information in the Flags field. For a description of the flags that may appear in this field, refer to the AxSYM System Operations Manual, Sections 1 and 2. INTERPRETATION OF THE RESULTS Samples with Index Values less than or equal to 0.499 are considered nonreactive for IgM antibody to T. gondii by the criteria of AxSYM Toxo IgM. Samples with Index Values in the range of 0.500 to 0.599 are considered equivocal (grayzone). Samples interpreted as equivocal (grayzone) may contain very low levels of IgM. A second specimen should be obtained and tested. Samples with Index Values equal to or greater than 0.600 are considered reactive for IgM antibody to T. gondii by the criteria of AxSYM Toxo IgM. LIMITATIONS OF THE PROCEDURE The use of the AxSYM Toxo IgM Antibody assay to diagnose recent infection by testing paired sera has not been validated. Performance has not been established for cord blood, neonatal samples, or body fluids such as urine, saliva, semen, amniotic fluid or cerebrospinal fluid. Negative results by this test do not preclude recent primary T. gondii infection. IgM type rheumatoid factor in the presence of T. gondii specific IgG can interfere in IgM class assays, causing false positives results. The RF neutralization buffer is provided to minimize such interference, however, high levels of IgG and RF could cause false positive results. The level of anti-t. gondii IgG inhibited by the RF neutralizing buffer has not been determined. If the AxSYM Toxo IgM assay is used as a general screen in the absence of clinical symptoms or known exposure, a diagnosis of an unsuspected primary T. gondii infection should not be based solely upon a positive result. Another method should be used for confirmation before making that diagnosis. Epstein-Barr Virus (EBV) is known to be a potent B-Cell stimulator. 26 Infections with EBV have been suspected to elicit antigen-specific IgM responses in individuals previously sensitized to a variety of non- EBV infectious agents. 27,28 Specimens showing particulate matter, erythrocytes, or turbidity must be clarified by centrifugation at > 10,000 x g for 10 minutes before testing. Heat-treated serum, lipemic serum, grossly hemolyzed serum, or serum with obvious microbial contamination should not be tested with this procedure. EXPECTED RESULTS The prevalence of IgM antibody to T. gondii in a population will vary with age and geographic location. In this study 800 specimens from pregnant women and 500 specimens from random low risk individuals in Europe and the U.S. were tested. Of these specimens, 88 (6.8%) were positive, 15 (1.1%) were equivocal, and 1,197 (92.1%) were negative by the AxSYM Toxo IgM Antibody assay. The distribution for this population is shown in Figure 1. 5
SPECIFIC PERFORMANCE CHARACTERISTICS PRECISION Assay reproducibility was determined as described in National Committee for Clinical Laboratory Standards (NCCLS) Protocol EP5-T2. 29 A twelve member panel of serum and plasma, as well as a Negative Control and Positive Control were assayed at 3 laboratories (Site I, II and III) in replicates of 2 at two separate times per day for twenty days (N= 80 for each panel and Control) using a single lot of reagents and a single calibration. The two runs assayed per day were separated by a minimum of two hours. The within run and total reproducibilities were evaluated by calculating the standard deviation (SD) and coefficient of variation (%CV) of each panel member and Control. The results of the reproducibility study are summarized in Table 1 of this section and show that acceptable reproducibility results were obtained for Negative and Positive Controls and all panel members. Table 1 REPRODUCIBILITY FOR AxSYM TOXO IgM ANTIBODY ASSAY Negative Control I 0.2 80 0.013 7.4 0.024 13.5 II 0.2 80 0.011 5.9 0.015 8.0 III 0.2 80 0.008 5.0 0.012 7.7 Positive Control I 1.2 80 0.081 6.5 0.096 7.7 II 1.3 80 0.054 4.2 0.087 6.7 III 1.3 80 0.082 6.4 0.107 8.3 Panel Member 1 (serum) I 0.1 80 0.008 6.7 0.009 7.9 II 0.1 80 0.007 5.7 0.009 6.5 III 0.1 80 0.007 5.0 0.009 6.9 Panel Member 2 (serum) I 0.6 80 0.039 6.2 0.061 9.7 II 0.6 80 0.034 5.4 0.053 8.3 III 0.7 80 0.041 6.1 0.053 7.9 Panel Member 3 (serum) I 1.0 80 0.061 6.0 0.097 9.7 II 1.0 80 0.044 4.4 0.076 7.6 III 1.0 80 0.055 5.3 0.075 7.2 Panel Member 4 (EDTA) I 0.1 80 0.007 6.3 0.010 9.2 II 0.1 80 0.007 5.1 0.009 6.8 III 0.1 80 0.006 5.0 0.010 7.8 Panel Member 5 (EDTA) I 0.6 80 0.046 7.2 0.052 8.2 II 0.6 80 0.034 5.3 0.048 7.5 III 0.7 80 0.028 4.3 0.054 8.0 Panel Member 6 (EDTA) I 1.0 80 0.058 5.8 0.080 8.1 II 1.0 80 0.050 5.0 0.075 7.4 III 1.0 80 0.053 5.1 0.075 7.1 Panel Member 7 (sodium citrate) I 0.1 80 0.007 5.9 0.009 7.8 II 0.1 80 0.007 5.1 0.008 6.2 III 0.1 80 0.007 4.8 0.009 6.8 Panel Member 8 (sodium citrate) I 0.6 80 0.040 6.3 0.051 8.0 II 0.6 80 0.039 6.0 0.048 7.5 III 0.7 80 0.033 4.9 0.057 8.5 Panel Member 9 (sodium citrate) I 1.0 80 0.059 6.1 0.088 9.0 II 1.0 80 0.046 4.6 0.060 6.0 III 1.1 80 0.058 5.5 0.082 7.8 Panel Member 10 (heparin) I 0.1 80 0.006 5.3 0.009 7.5 II 0.1 80 0.009 6.6 0.011 7.9 III 0.1 80 0.008 5.5 0.011 7.5 Panel Member 11 (heparin) I 0.6 80 0.043 6.8 0.053 8.5 II 0.6 80 0.035 5.4 0.047 7.3 III 0.7 80 0.039 5.7 0.065 9.5 Panel Member 12 (heparin) I 1.0 80 0.062 6.3 0.098 10.1 II 1.0 80 0.058 5.7 0.079 7.8 III 1.1 80 0.062 5.9 0.098 9.3 6
RELATIVE AGREEMENT The presence of IgM antibody to Toxoplasma gondii in 1,363 specimens was determined by 2 U.S. (Site I and II) and 1 European laboratory (Site IV) using the AxSYM Toxo IgM Antibody assay. In addition, each specimen was tested using a legally marketed EIA. The relative agreement was calculated to be 99.3% (Exact 95% confidence interval: 98.8% to 99.7%) (1354/1363). RELATIVE SENSITIVITY Of the 1,363 specimens tested, 182 were found to be positive by EIA. Of these 182, 156 were positive, 20 were equivocal, and 6 were negative by AxSYM Toxo IgM Antibody assay for a relative sensitivity of 96.3% (Exact 95% confidence interval: 92.1% to 98.6%) (156/162). RELATIVE SPECIFICITY Of the 1,363 specimens tested, 1,209 were found to be negative by EIA. Of these 1,209, 1,198 were negative, 8 were equivocal, and 3 were positive by AxSYM Toxo IgM Antibody assay for a relative specificity of 99.8% (Exact 95% confidence interval: 99.3% to 100%) (1198/1201). NOTE: Specimens giving equivocal results using AxSYM and/or EIA were not included in the calculation of relative agreement, relative sensitivity and relative specificity. Table 2 Comparison of AxSYM Toxo IgM with EIA Site I Site II Site IV Total Relative 99.4% 98.5% 99.7% 99.3% Agreement (338/340) (333/338) (683/685) (1354/1363) Relative 94.4% 94.6% 97.8% 96.3% Sensitivity (34/36) (35/37) (87/89) (156/162) Relative 100% 99.0% 100% 99.8% Specificity (304/304) (298/301) (596/596) (1198/1201) AxSYM 2.3% 3.1% 1.9% 2.3% Equivocal (8/350) (11/350) (13/700) (32/1400) Site IV tested multiple bleeds from 30 patients demonstrating seroconversion to T. gondii. For 21 seroconversion patients whose series contained a positive IgG bleed drawn no longer than 8 weeks following the last negative bleed, evaluation of IgG and IgM antibody response was performed. The first specimen in the series which gave a positive result by the Sabin-Feldman Dye test was also positive by AxSYM Toxo IgM Antibody assay in all 21 cases. The specificity of the AxSYM Toxo IgM assay was further evaluated by testing 201 specimens positive for anti-nuclear antibody, systemic lupus erythematosus, rheumatoid factor, herpes simplex virus, Epstein-Barr virus, measles, parvovirus, varicella zoster virus, hyper IgG, hyper IgM, and influenza vaccine recipients. With these specimens, AxSYM Toxo IgM and the EIA showed 98% agreement (197/201). Additional studies were performed to evaluate the effect of endogenous substances that may be present in clinical specimens. The following compounds did not show interference in the AxSYM Toxo IgM assay at the levels indicated: 1000 mg/dl hemoglobin, 20 mg/dl bilirubin, 2000 mg/dl triglycerides, 10 g/dl protein, or 0.4% red blood cells. BIBLIOGRAPHY 1. Remington JS, Desmonts G. Toxoplasmosis. ln: Remington JS, Klein JO, editors. Infectious Diseases of the Fetus and Newborn Infant. Philadelphia: WB Sanders 1983:143 2. Wallace GD, Marshall L, Marshal M. Cats, Rats and Toxoplasmosis on a Small Pacific Island. AM J Epidemiol 1972;95:475. 3. Frenkel JK. Toxoplasma in and Around Us. Bioscience 1973;23:343. 4. Remington JS. Toxoplasmosis in the Adult. Bull N Y Acad Med 1974;50:211. 5. Krick JA, Remington JS. Toxoplasmosis In the Adult- An Overview. N Eng J Med 1978;298:550. 6. Ruskin J, Remington JS Toxoplasmosis in the Compromised Host. Ann Intern Med 1976;84:193. 7. Mendelson MH, Finkel LJ, Meyers BR, Lieberman JP, Hirschman SZ. Pulmonary Toxoplasmosis in AIDS. Scand J Infect Dis 1987;19:703. 8. Luft BJ, Remington JS. Toxoplasmosis of the Central Nervous System. Curr Clin Top Infect Dis 1985;6:316. 9. Luft BJ, Remington JS. Toxoplasmic Encephalitis. J Infect Dis 1988; 157:1. 10. Desmonts G, Couvreur J. Congenital Toxoplasmosis. A Prospective Study of 378 Pregnancies. N Engl J Med 1974; 290:1110. 11. Desmonts G, Couvreur J. Toxoplasmosis: Epidemiologic and Serologic Aspects of Prenatal Infection. In: Krugmann S.,Gershon AE, editors. Infections of the Fetus and Newborn Infant. New York: Alan R. Liss, Inc. 1975;115. 12. Alford CA Jr, Stagno S, Reynolds DW. Toxoplasmosis: Silent Congenital Infect. In: Krugmann S, Gershon AE, editors. Infections of the Fetus and Newborn Infant. New York: Alan R. Liss, Inc. 1975;133. 13. Wilson BD, Remington JS, Stagno S, Reynolds DW. Development of Adverse Sequelae in Children Born With Subclinical Toxoplasma Infection. Pediatrics 1980;66:767. 14. Daffos F, Foestier F, Capella-Pavalovsky M Thulliez P, Aufrant C, Valenti D, Cox WL. et al. Prenatal Management of 746 Pregnancies At Risk For Congenital Toxoplasmosis. N Engl J Med 1988;318:271. 15. McCabe R, Remington JS. Toxoplasmosis: The Time Has Come. N Engl J Med 1988;318:313. 16. Remington JS, Miller MJ, Brownlee I. IgM Antibodies In Acute Toxoplasmosis: I. Diagnostic Significance In Congenital Cases and a Method for Their Rapid Demonstration. Pediatrics 1968;41:1082. 17. Remington JS, Miller MJ, Brownlee I. IgM Antibodies In Acute Toxoplasmosis: II. Prevalence and Significance In Acquired Cases. J Lab Clin Med 1968;71:855. 18. Naot Y, Remington JS. An Enzyme-Linked Immunosorbent Assay For The Detection of IgM Antibodies to Toxoplasma gondii: Use for Diagnosis of Acute Acquired Toxoplasmosis. J Infect Dis 1980;142:757. 19. Naot Y, Guptill DR, Remington JS. Duration of IgM Antibodies to Toxoplasma gondii After Acute Acquired Toxoplasmosis. J Infect Dis 1982;145:770. 20. Sulzer AJ, Franco EL, Takafuji E, Benenson M, Walls K, Greenup RL. An Oocyst-Transmitted Outbreak of Toxoplasmosis: Patterns of Immunoglobulin G and M Over One Year. Am J Trop Med Hyg 1986;35:290. 21. Brooks RG, McCabe RE, Remington JS. Role of Serology In the Diagnosis of Toxoplasmic Lymphadenopathy. Rev Infect Dis 1987;9:1055. 22. U.S. Department of Labor, Occupational Safety and Health Administration, 29 CFR Part 1910.1030, Occupational Exposure to Bloodborne Pathogens. 23. U.S. Department of Health and Human Services. Biosafety in Microbiological and Biomedical Laboratories. HHS Publication (CDC) 93-8395. Washington: U.S. Government Printing Office, May 1999. 24. World Health Organization. Laboratory Biosafety Manual. Geneva. World Health Organization, 1993. 25. National Committee for Clinical Laboratory Standards. Protection of Laboratory Workers from Instrument Biohazards and Infectious Disease Transmitted by Blood, Body Fluids, and Tissue. Approved Guideline. NCCLS Document M29-A2. Wayne, PA: NCCLS, 2001. 26. Moller E, Strom H, Al-Balaghi S. Role of Polyclonal Activation in Specific Immune Responses. Relevance for Findings of Antibody Activity in Various Diseases. Scand J Immunol, 1980;12:177-82. 27. Rasmussen L, Keisall D, Nelson R, Carney W, Hirsh M, Winston D, et al. Virus-Specific IgG and IgM Antibodies in Normal and Immunocompromised Subjects Infected with Cytomegalovirus. J Infect Dis 1982:145:191-9. 28. Morgan-Capner P, Tedder RS, Mace JE. Rubella-Specific IgM Reactivity in Sera from cases of Infectious Mononucleosis. J Hyg (Lond) 1983;90:407-13. 29. National Committee for Clinical Laboratory Standards. Evaluation of Precision Performance of Clinical Chemistry Devices - Second Edition; Tentative Guideline. NCCLS Document EP5-T2, March 1992. AxSYM is a registered trademark of Abbott Laboratories, Abbott Park, IL, USA. For additional product information, please contact your local customer service organization. 7