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1 BIO-FLASH Toxo IgM tests The BIO-FLASH Toxo IgM is a fully automated chemiluminescent two-step immunoassay for qualitative measurement of IgM antibodies to Toxoplasma gondii in human serum or plasma on the BIO-FLASH instrument. Summary Toxoplasmosis is an infectious disease affecting both animals and humans which is caused by the protozoan parasite Toxoplasma gondii. 1 Acquired Toxoplasmosis is usually asymptomatic and benign. 2 In pregnant women, however, the infection acquires a special significance as the parasite may enter the fetal circulation through the placenta and cause congenital Toxoplasmosis. Several studies have shown that the risk and severity of congenital Toxoplasmosis is greatest when acquired during the first three months of pregnancy. 3-6 The consequences of congenital Toxoplasmosis range from spontaneous abortion and prematurity to generalized and neurological symptoms, which often involve ocular complications. Infants with congenital Toxoplasmosis may also remain asymptomatic for several weeks after birth. Numerous studies have shown that the presence of a certain concentration of maternal IgG against toxoplasma, determined prior to pregnancy, protects the fetus against infection by the parasite. Serologic tests can also be used to identify those pregnancies at risk; women who are seronegative at the time of diagnosis of pregnancy can be monitored during pregnancy. Testing of specific antibody isotypes to Toxoplasma gondii can provide information as to when the infection was acquired. 7,8 This allows a recent infection to be detected and the appropriate prophylactic and therapeutic measures to be taken. The detection of specific IgM allows the follow-up of pregnant women, since toxoplasma infections are often clinically unapparent. The serological follow-up should allow the early detection of a seroconversion and, considering the seriousness of congenital infection in the fetus, the quick implementation of the necessary measures. Toxoplasmosis can be life-threatening in immunocompromised patients (AIDS, organ transplantation). In AIDS patients, acute Toxoplasmosis usually occurs due to re-activation of a latent previously acquired infection, although primary infections are also diagnosed. 9 Principle When BIO-FLASH Toxo IgM paramagnetic microparticles are mixed and incubated with the sample, the specific anti-toxoplasma antibodies if present in the sample will bind to the toxoplasma antigens coated on the microparticles. A magnetic separation followed by a wash step is done to remove residual sample. Immediately after, a tracer consisting of an isoluminol-labeled monoclonal antibody anti-human IgM is added and may bind to the anti-toxoplasma IgM captured by the microparticles. After a second incubation, a magnetic separation and a wash step, reagents that trigger the chemiluminescent reaction are added. The emitted light is measured as relative light units (RLU) by the BIO-FLASH luminometer. The RLUs are directly proportional to the anti-toxoplasma IgM concentration in the sample. The BIO-FLASH utilizes a 4 Parameter Logistic Curve fit data reduction method (4PLC) to generate a Master Calibration Curve (MCC). The MCC is predefined and lot dependent and it is stored in the instrument through the cartridge barcode. With the measurement of calibrators (supplied in a separate kit), the predefined MCC is transformed to a new, instrument specific Working Calibration Curve (WCC). The concentration values of the calibrators are included in the calibrator tube barcodes. Reaction Scheme: READ HIGHLIGHTED CHANGES Triggers RLU Magnetic particle Sample Tracer Light emission

2 Components Reagent cartridge The BIO-FLASH Toxo IgM kit contains a reagent cartridge for 50 determinations (REF ). Note: Cartridge design is protected under patent (US D565,741 / EC Design ) Reagent cartridge composition: Cartridge has 4 different vials with the following contents: A. 1 cylindrical vial of microparticle suspension coated with Toxoplasma antigen in a phosphate buffer. Contains < 0.1% Sodium azide. B. 1 vial of assay buffer. Contains < 0.1% Sodium azide. C. 1 opaque vial of tracer consisting of mouse monoclonal anti-human IgM labeled with isoluminol. Contains < 0.1% Sodium azide. D. 1 empty vial. Preparation See the figure below. Cartridge: Microparticles settle during shipment and storage and require mixing to resuspend. The first time that the cartridge is used, gently invert the cartridge 30 times avoiding foam formation. Bubbles may interfere with the instrument liquid sensors. Check for the complete resuspension of the microparticles. If the microparticles are not totally resuspended continue to invert the cartridge until the microparticles have been completely resuspended. If the microparticles do not resuspend or it is detected that the reagent seal is broken, DO NOT USE THE CARTRIDGE. Once the microparticles have been resuspended, place the cartridge on a solid surface and gently remove the Red Secure Shipping Tab from the cartridge. With the cartridge still on a solid surface, press the two tabs placed on the sides of the piercing cap (grey part) and apply pressure to the top portion of the cartridge until it snaps into a lock position. Once in the locked position the tabs should not be visible. Do not invert the open cartridge. Let the cartridge stand for a period of 5 minutes before loading it onto the instrument. Once the cartridge is placed on the instrument, additional periodic mixing is automatically performed on board.

3 Precautions The BIO-FLASH reagents are intended for IN VITRO diagnostic use. For professional use only. Sodium azide may react with lead or copper pipes and plumbing creating highly explosive metal azides. Flush drains with water thoroughly after disposing of the remains of reagents. WARNING: POTENTIALLY BIOHAZARDOUS MATERIAL. Dispose of all used materials in a suitable biohazardous waste container. Please do not reuse nor reintroduce any reagent in the cartridges or vials. Hazard class Not classified. Hazard statements None. Precautionary statements P280: Wear protective gloves/protective clothing/eye protection/face protection. P : IF IN EYES rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. Sample collection and storage Use fresh serum (including serum separator tube) or plasma (EDTA, Li-Heparin, Li-Heparin separator tube, Na-Heparin and Na-Citrate). Other anticoagulants should be evaluated before use. Liquid anticoagulants as Na-Citrate have a dilution effect resulting in lower concentrations for individual patient specimens. Samples should not be heat inactivated. Samples showing visible particles should be clarified by centrifugation. Refer to the CLSI H18-A3 and H21-A5 guidelines for further information on handling, transport, processing and storage of samples. Serum Serum samples can be stored at 2-8 C for 8 days. For longer periods, sera should be frozen at -20 C or colder. Samples can be freeze/thawed up to 3 times. Mix thoroughly after thawing. Inspect all samples for bubbles and remove all of them prior to analysis. Plasma Plasma samples can be stored at 2-8 C for 8 days. For longer periods, plasma samples should be frozen at -20 C or colder. Thaw plasma at 37 C. Thaw samples only once. Mix thoroughly after thawing. Inspect all samples for bubbles and remove all of them prior to analysis. Sample volume The sample volume required to perform a single BIO-FLASH Toxo IgM test varies depending on the type of sample container used. A test requires at least 15 µl plus dead volume, which is 200 µl for the recommended sample cup (REF ). Additional materials The following materials are not supplied with the reagent cartridge and must be purchased separately. REF REF BIO-FLASH Toxo IgM Calibrators BIO-FLASH Toxo IgM Controls Read carefully their corresponding inserts for more information. Do not use other calibrators. The information required by the BIO-FLASH instrument to calibrate the BIO-FLASH Toxo IgM assay is included in the barcoded vials. Using controls from other manufacturers may lead to unexpected results. Ensure that the BIO-FLASH instrument has enough quantity of the following consumables on board before running samples, calibrators or controls: REF BIO-FLASH Cuvettes Note: Cuvette design is protected under patent (US D560,816 / EC Design ) REF BIO-FLASH Triggers REF REF BIO-FLASH System Rinse BIO-FLASH Sample Diluent

4 Instrument / test procedure Refer to the BIO-FLASH Operator s Manual for the complete assay procedure instructions. Calibration Each cartridge barcode contains the Master Calibration Curve (MCC) valid for the reagent lot; however a Working Calibration Curve (WCC) is required for all tests. The WCC is lot specific and valid throughout the shelf life of the lot. A new WCC is indicated when controls report outside the acceptable range or when adjustments are made to the instrument. Read carefully BIO-FLASH Operator s Manual for configuring a WCC. Quality control Two levels of control are recommended for a complete quality control program. BIO-FLASH Toxo IgM Controls Negative and Positive are designed for this program. It is recommended that each laboratory establishes its own mean and standard deviation and a quality control program to monitor laboratory testing. Controls should be analyzed at least once every 24 hours each day of use. If controls are out of range, recalibration may be indicated. Refer to the instrument s Operator s Manual for additional information. Refer to Westgard et al. for identification and resolution of out-of-control situations. 10 Storage and stability Unopened reagents are stable until the expiration date shown on the cartridge label when stored at 2-8 C in the upright position. Once open the cartridge stability on board the BIO-FLASH instrument or stored at 2-8 C is 8 weeks. Interpretation of results The amount of analyte in every sample is determined from the emitted light (RLU) by interpolation in the stored Working Calibration Curve. BIO-FLASH Toxo IgM results are reported in S/CO (Signal/Cut-Off). This is a qualitative test; the numeric value of the result is only indicative of the amount of antibody present. The determination of anti-toxo IgM, using the BIO-FLASH Toxo IgM assay, helps to establish the diagnosis of recent infection. samples with a result < 0.80 S/CO are considered non reactive (negative). samples with a result 0.80 and < 1.00 S/CO are considered indeterminate (grey zone). Specimens interpreted as indeterminate should be retested. If the retest result is indeterminate, it should be reported as such and an additional test should be done using an alternative method or a second specimen should be obtained and tested. samples with a result 1.00 S/CO are considered reactive (positive). As in all sensitive immunoassays, there is the possibility that non-repeatable positive results occur. A negative result does not exclude the possibility of toxoplasma infection. A possible seroconversion must be confirmed by the quantitative determination of specific IgG e.g. with BIO-FLASH Toxo IgG REF by testing paired samples taken at 2-3 weeks interval. Limitations The result of a single sample is not sufficient for the diagnosis of toxoplasma primary infection and therefore the BIO-FLASH Toxo IgM results should be used in conjunction with other data; e.g., symptoms, clinical history, results of other tests and other appropriate information. Heterophile antibodies in human serum can react with reagent immunoglobulins, interfering with in vitro immunoassays. 11 The data obtained may indicate that presence of heterophile antibodies in the sample may lead to false positive results. Expected results Approximately one-third of the world s population possesses antibodies to toxoplasma, although there is a considerable variation in antibody prevalence between, and even within, countries. In England and Wales the prevalence increases with age by 0.5% - 1.0% per year to reach about 30% by the age of 40. However, in France 80%-90% of the population is antibody-positive by 40 years of age. 12 In a multicentre study performed on more than 16,000 serum samples from pregnant women in Barcelona (Spain), 28.6% of the women had toxoplasma antibodies. IgM specific antibodies were detected in 2.2% of women with anti-toxoplasma. 13 In a study performed in Norway during the period from 1992 to 1994, involving 35,940 pregnant women, 10.9% had toxoplasma antibodies before the onset of pregnancy. The existence of primary infection during pregnancy was found in 0.17% of the women who had not been infected previously. 14

5 Performance characteristics NOTE: The following data are representative; results in individual experiments may vary from these data. Method comparison BIO-FLASH Toxo IgM was evaluated in comparative studies with other commercial assays. Evaluations An internal evaluation and an external evaluation at Servei de Microbiologia in Hospital de la Santa Creu i Sant Pau in Barcelona (Spain) were performed. In the internal study 154 samples from different sources, including positive and negative sera for toxoplasma IgM antibodies, were tested with BIO-FLASH Toxo IgM in comparison with a commercially available EIA Toxo IgM method. In the external study 426 samples from different sources, including positive and negative sera for toxoplasma IgM antibodies, were tested with BIO-FLASH Toxo IgM in comparison with a commercially available MEIA Toxo IgM method. Results are shown globally. Indeterminate results were not used in calculations. Reference method BIO-FLASH IND NEG POS Total Toxo IgM IND NEG POS Total The following results were obtained for initial relative sensitivity, specificity and overall agreement: Relative Sensitivity Relative Specificity Overall Agreement N Value 95% CI Value 95% CI Value 95% CI % 81.1% to 94.0% 92.6% 89.8% to 94.9% 91.9% 89.3% to 94.0% The discrepant samples were tested with a commercially available ELFA Toxo IgM method. Three of them were resolved in favor of BIO-FLASH Toxo IgM. After this additional testing, the following results were obtained for resolved sensitivity, specificity and overall agreement: Resolved Sensitivity Resolved Specificity Resolved Overall Agreement N Value 95% CI Value 95% CI Value 95% CI % 82.2% to 94.7% 92.9% 90.1% to 95.1% 92.3% 89.7% to 94.3% A total of 92 samples of the external evaluation were from pregnant women. The figures of sensitivity and specificity remained similar to those of general population. CDC Panel The CDC Toxoplasma 1998 Human Serum Panel consisting of 32 positive serum samples and 65 negative serum samples for anti-toxoplasma IgM was assayed with the BIO-FLASH Toxo IgM assay and reported 96.9% Sensitivity and 93.8% Specificity. Precision Within run and total (run to run and day to day) precision were assessed (following CLSI EP05-A Guidelines) over multiple runs. Results are summarized in the following table: Mean (S/CO) Within run Total Negative Control SD SD Positive Control % CV 7.8% CV Around Cut-Off Level % CV 6.6% CV Interferences As assessed by the CLSI EP7-A Guidelines, BIO-FLASH Toxo IgM results are not affected by the following potentially interfering substances:

6 Potentially Interfering Substance Concentration % Interference Hemoglobin 500 mg/dl 10 Free bilirubin 18 mg/dl 10 Complexed bilirubin 18 mg/dl 10 Triglycerides 1300 mg/dl 10 Rheumatoid factor 800 IU/mL 10 Cross-reactivity 202 specimens with potential cross-reactivity with the BIO-FLASH Toxo IgM assay were tested against commercially available Toxo IgM assays showing the following agreement: Potential Cross-Reaction Agreement anti-rubella IgM 5/5 anti-cmv IgM (Cytomegalovirus) 5/5 anti-hiv (Human Immunodeficiency Virus) 9/9 HAMA (Human Anti-mouse Antibodies) 3/3 SLE (Systemic Lupus Erythematosus) 8/10 ANA (Anti-Nuclear Antibodies) 11/11 anti-ebv (Epstein-Barr Virus) 4/4 anti-pv B19 (Parvovirus B19) 10/10 anti-hsv 1 (Herpes Simplex Virus type 1) 4/5 anti-vzv (Varicella Zoster Virus) 4/4 Paraprotein IgG and IgM 6/6 Elevated IgG and IgM 6/6 Heterophile antibodies 2/4 RF (Rheumatoid Factor) 10/10 Pregnant women (including multiparous) 96/110