(For In Vitro Diagnostic Use) Particle Agglutination Test for Detection of Antibodies to Treponema pallidum using the Olympus PK7200/PK7300

Transcription

1 REF : EDMA CODE : (For In Vitro Diagnostic Use) Particle Agglutination Test for Detection of Antibodies to Treponema pallidum using the Olympus PK7200/PK7300

2 TABLE OF CONTENTS 1. INTENDED USE 3 2. SUMMARY OF TEST 3 3. PRINCIPLE OF PROCEDURE 4 4. REAGENTS 4 5. WARNINGS AND PRECAUTIONS 5 6. REAGENT PREPARATION 6 7. STORAGE 7 8. SPECIMEN COLLECTION AND PREPARATION 7 9. MATERIALS DIRECTIONS FOR USE QUALITY CONTROL INTERPRETATION INTERPRETATION OF RESULTS PERFORMANCE CHARACTERISTICS LIMITATIONS OF THE PROCEDURE REFERENCES 16 English-2

3 1. INTENDED USE SERODIA-TPPA Auto is intended for the qualitative screening of blood donors for the detection of IgM and IgG antibodies to Treponema pallidum (T. pallidum) in human serum or plasma using the OLYMPUS PK7200/PK7300, Automated Microplate Systems. The results obtained are provided to the user in a computer generated printout. All specimens which are repeatedly POSITIVE or indeterminate with SERODIA-TPPA Auto are considered POSITIVE for antibodies to T. pallidum by the criteria of the assay. For complete details on the setup and operation of the OLYMPUS PK7200/PK7300, refer to the appropriate Operator s Manual. 2. SUMMARY OF TEST The identification of T. pallidum antibodies aids in the diagnosis of syphilis caused by the microorganisms belonging to the genus Treponema and provides epidemiological information on syphilis. Serological tests for syphilis were first used in 1906 with the development of the nontreponemal complement fixation test by Wasserman. The nontreponemal tests measure both immunoglobulin G (IgG) and M (IgM) antilipid antibodies formed by the host in response both to lipoidal material released from damaged host cells early in infection and to lipid from the treponeme itself. Because of either the lipidic nature of the antigen or some unusual property of the antibodies, the antigen-antibody reaction remains suspended, and flocculation occurs, rather than agglutination or precipitation as in most other serological tests'. While useful in the diagnosis of suspected syphilis infection, nontreponemal tests are nonspecific. False positive tests may account for a significant proportion of POSITIVE results necessitating an additional test to identify the presence of specific antibodies to T. pallidum. Hemagglutination tests for T. pallidum gained wide acceptance since their emergence in the mid 1960 s 2,3,4 as a screening procedure or as a confirmatory procedure following a POSITIVE nontreponemal assay. Automation has enhanced the value of the test by significantly reducing the amount of time and labor needed to perform the assay 5,6. SERODIA-TPPA Auto has been developed to provide uniform reagents which are stable, easy to handle, and suitable for use on the OLYMPUS PK7200/PK7300, Automated Microplate Systems 7,8. The results obtained are provided to the user in a computer generated printout. All specimens which English-3

4 are repeatedly POSITIVE or indeterminate with SERODIA-TPPA Auto are considered POSITIVE for antibodies to T. pallidum by the criteria of the assay. For complete details on the setup and operation of the OLYMPUS PK7200/PK7300, refer to the appropriate Operator s Manual. 3. PRINCIPLE OF PROCEDURE The test is based on the principle of agglutination and pattern recognition. SERODIA-TPPA Auto utilizes gelatin particles sensitized with components of the pathogenic T. pallidum (Nichols Strain). The sensitized particles are added to the test mixture and reactants are allowed to settle in a patented, terraced microplate well. Agglutination occurs in the presence of T. pallidum antibodies in specimens. Visually, a POSITIVE test is a homogenous layer of particles. A NEGATIVE test would result in a compact dense button surrounded by a clear zone. The PK7200/PK7300 instrument will read the settling patterns of particles in each well based on the threshold settings chosen for each reagent. The PK7200/PK7300 determines the presence or absence of antibodies to T. pallidum using a CCD (charged coupled device) camera which captures the well image and allowing differentiation of agglutinated and unagglutinated patterns. 4. REAGENTS SERODIA-TPPA Auto is available in a kit sufficient to perform 1,900 tests: RECONSTITUTING SOLUTION (A) - 3 vials, 20 ml each. This reagent is used for reconstitution of the lyophilized SENSITIZED PARTICLES. The reagent contains a phosphate-buffered saline solution with 0.06% sodium azide. SAMPLE DILUENT (B) - 2 bottles, 300 ml each. The solution is used for the dilution of test specimens. The solution consists of a phosphate-buffered saline solution containing 0.10% sodium azide. SENSITIZED PARTICLES (C) - 5 vials of lyophilized, fixed gelatin particles sensitized with T. pallidum antigen. Each vial must be reconstituted with 10 ml of RECONSTITUTING SOLUTION. The reconstituted solution contains 0.08% (w/v) of sodium azide per vial as a preservative. Reconstituted particles are stable for 7 days and should be stored at 2-10 C for maximum stability. English-4

5 5. WARNINGS AND PRECAUTIONS SERODIA-TPPA Auto is for in vitro diagnostic use. 1) Avoid contamination of reagents or speciments with saliva, which can cause indistinguishable agglutination patterns. Do not mouth pipette any reagents. 2) The microplates must be clean before use. Improper washing of the microplates can adversely affect a test result by causing a false positive or false negative reaction. The suggested washing procedure can be found in the Standard Operating Procedure Manual for the PK7200/PK ) Avoid freezing of the reconstituted SENSITIZED PARTICLES. 4) Sodium azide is included as a preservative. Sodium azide has been reported to form explosive lead and copper azides in laboratory plumbing. To prevent azide buildup, flush with large volumes of water if solution containing azide are disposed of in the sink. 5) Visible signs of microbial growth or gross turbidity in the reagent may indicate degradation and warrant discontinuance of use. 6) Handle all specimens, control material and serum-based reagents as if potentially infectious. Refer to the US Center for Disease Control s guidelines on specimen handling 9. 7) Clean pipettes should be used to reconstitute all reagents. Clean glass or plastic containers should be used for reagent preparation. 8) Plasma is suitable for screening, however, field use has demonstrated a higher incidence of false positive results. Therefore, it is recommended to use serum specimen from the same draw for all repeat testing of initially POSITIVE or indeterminate results obtained from plasma sample. 9) The effects of specimen microbial contamination on this assay can not be predicted. 10) Carryover between specimens is a potential source of interference. 11) When specimen fails to be added to SERODIA-TPPA Auto, the potential exists for a false negative result to occur. 12) Inadequate adherence to the package insert can result in erroneous results. 13) The use of calibrated or verified equipment is required. 14) This kit is designed for use on OLYMPUS PK Instruments. If this kit is used by manual English-5

6 testing for the repeated test, please refer to the instructions for use of SERODIA-TPPA Auto UNSENSITIZED PARTICLES (REF : ) and SERODIA-TPPA Auto POSITIVE CONTROL (REF : ). 15) Quality assurance is given for each production lot. Do not use the reagents in combination with the kit of other production lots. 16) The containing sodium azide is classified per the applicable European Community (EC) Directives as Harmful (Xn). The following are the appropriate Risk and Safety classification. R-classification 22 Harmful to health after swallow S-classification 2 Keep away from children 13 Keep away from food, beverages and animal feed 36 Wear protective clothing when working with the material 46 After ingestion contact a doctor showing the bottle and label 52 Do not use in any living or social rooms 17) When disposing of reagents or vessels, separate them according to medical or industrial waste regulations. 6. REAGENT PREPARATION 1) Reconstitute, as needed, each vial of SENSITIZED PARTICLES with 10 ml of RECONSTITUTING SOLUTION. Replase the stopper and gently invert to assure thorough mixing. Allow to reconstitute at room temperature (15-30 C) for a minimum of 30 minutes or overnight at 2-10 C to ensure complete rehydration. 2) After the reconstitution period, gently swirl (DO NOT VORTEX) the rehydrated particles to assure thorough resuspension. Follow steps under heading, DIRECTIONS FOR USE, for use on the instruments. 3) SENSITIZED PARTICLES from the same lot may be pooled. The mixture is stable for 7 days from the earliest reconstitution date of the vials contained in the mixture. 4) SENSITIZED PARTICLES from one lot number should not be mixed with those of another lot number. 5) SAMPLE DILUENT from the same lot can be pooled for use on the instruments as long as good laboratory practices are followed. English-6

7 6) The date of reconstitution and the reconstituted expiry should be recorded on the reagent containers. Note: All reagents should be brought to room temperature (15-30 C) prior to use on the analyzer. 7. STORAGE 1) Store SERODIA-TPPA Auto at 2-10 C. DO NOT FREEZE. 2) SERODIA-TPPA Auto should not be used after the expiration date which is printed on the outside of the package. 3) Reconstituted SENSITIZED PARTICLES are stable for 7 days and should be stored at 2-10 C for maximum stability. DO NOT FREEZE. 4) Visible signs of microbial growth or gross turbidity in the reagents may indicate degradation and warrant discontinuance of use. 8. SPECIMEN COLLECTION AND PREPARATION SERODIA-TPPA Auto may be used with serum or plasma. Specimens for repeat testing must be obtained from the same draw. The sample should be free of particulate matter. If erythrocytes or other visible components are contained in the sample, remove by centrifugation to prevent interference with the test results. Samples should be centrifuged for a minimum of 10 minutes at a minimum of 1,000 x g within 10 hours of analysis on the PK analyzer. Specimens are acceptable for testing if they do not exceed any of the following values: Free hemoglobin 1,000 mg/dl Phospholipids 300 mg/dl Total bilirubin 40 mg/dl Samples which exceed these values may require alternate testing. Store sample specimens at 2-10 C. Specimen may be tested for up to five (5) days. Specimen may be stored at 20 C, if testing is to exceed five days. Avoid repeated freezing and thawing of specimens. Thorough mixing of frozen specimens is necessary after thawing and prior to testing. Allow the specimens to come to room temperature before testing (a minimum of minutes after thawing). English-7

8 Inactivation of serum specimen is not required but inactivated serum can also be used in the test. Serum may be heated for 30 minutes in a 56 C water bath without affecting the test outcome. Specimens to be heated should be at room temperature before placing in the water bath. Allow specimens to return to room temperature before testing (a minimum of minutes after heating). 9. MATERIALS MATERIALS PROVIDED IN SERODIA-TPPA Auto: RECONSTITUTING SOLUTION (A) SAMPLE DILUENT (B) SENSITIZED PARTICLES (C) MATERIALS REQUIRED BUT NOT PROVIDED: OLYMPUS terraced microplates A V-bottom reagent container and mixing comb for use on the PK7200 instrument Reagent container for the PK7300 instrument Pipetting devices for: 1.0 ml, 5.0 ml, and 10.0 ml OLYMPUS PK7200/PK7300 Automated Microplate System SERODIA-TPPA Auto CONTROLS 10. DIRECTIONS FOR USE The PK Instruments are programmable instruments whose operation is controlled by software defined by the user. Validated SERODIA-TPPA Auto parameters may be incorporated with routine operating files or defined in a separate file. If you are not familiar with this process, please consult the PK7200/PK7300 Operator s Manual. Example of working files for SERODIA-TPPA Auto are shown below for the PK7200/PK7300. Good laboratory practice dictates that each laboratory validates the operating parameters. English-8

9 PARAMETERS FUJIREBIO INC. has established recommended parameters for the instrument based upon application development testing with characterized samples. The final sample dilution must be in the range of 1/42. The following parameters will achieve this range. RECOMMENDED PARAMETERS PK7200 PK7300 Well Sample Diluted Sample Sample Diluent Sample Dilution Reagent Final Sample Dilution Temperature Setting Incubation Time 5 µm 16 µm 17 µl 15 µl 250 µl µl 1/ min. 5 µm Automatic 16 µm Setting 10 µl Automatic Setting µl 1/ min. RECOMMENDED THRESHOLDS PK7200 PK7300 Well (+) Limit P/C SPC LIA ( ) Limit Low High (+) Limit ( ) Limit LIA Selection BG/C 5 µm Low 16 µm Low 5 µm middle 16 µm middle : This kit is designed for use with 5 µm wells of OLYMPUS terraced microplates. In case 16 µm wells are used, the Thresholds are recommended. English-9

10 EXAMPLES OF SOFTWARE/HARDWARE SETTINGS FOR RECOMMENDED ASSAY CONDITIONS PK7200 PK7300 PARAMETERS Volume / Setting STROKE PIN PARAMETERS Volume / Setting Sample Volume 17 µl Sample Volume Automatic Setting Diluent Volume 250 µl G 0.25 Diluent Volume Automatic Setting Sample Dilution 16 Sample Dilution 12 Diluted Sample Volume 15 µl Diluted Sample Volume 10 µl Reagent Volume 25 µl Reagent Volume 25 µl Channel Name SYP Channel Name SYP Channel Designation (1-24) Channel Designation (1-24) Decision logic + / Decision logic + / Temperature Setting 30 Temperature Setting 30 Incubation Time 60 minutes Incubation Time 60 minutes THRESHOLDS THRESHOLDS 5 µm well 16 µm well 5 µm well 16 µm well SPC Low SPC Low High High P/C (+) Limit P/C (+) Limit ( ) Limit 0 0 ( ) Limit 0 0 LIA (+) Limit LIA (+) Limit ( ) Limit 0 0 ( ) Limit 0 0 LIA Selection 5 7 LIA Selection 5 5 BG/C Limit Low Low BG/C Limit middle middle In case the above THRESHOLDS are modifi ed, it is strongly recommended that the Thresholds must be validated by testing proper specimens. The Thresholds should be adapted to SERODIA-TPPA Auto with careful examination. Note: All reagents, diluents, and specimens should be at room temperature (15-30 C) prior to analysis. English-10

11 PK7200/PK7300 measurement procedure for SERODIA-TPPA Auto To use the reagents on the analyzers 1) Please confirm the setting of ion-exchanged water and the printer paper. 2) Input the assay parameters with reference to the RECOMMENDED ASSAY CONDITIONS. 3) Place the reconstituted SENSITIZED PARTICLES and SAMPLE DILUENT at the appointed place. 4) Replace the diluent lines of PK7200/PK7300 with SAMPLE DILUENT. 5) Be sure to place the OLYMPUS terraced microplates on the analyzer. 6) Set the samples on the rack. After that, place them on the rack feeder. 7) Proceed with sample analysis as described in the OLYMPUS PK7200/PK7300 Operator s Manual. 11. QUALITY CONTROL The SERODIA-TPPA Auto POSITIVE and NEGATIVE CONTROLS should be tested at the beginning and end of each batch of the samples tested, after the addition of reagents and after interruption or delays in processing. Refer to SERODIA-TPPA Auto CONTROLS package insert for compete details regarding this materials. Additional quality control testing may be performed by including other well-characterized specimens or reference sera. A single replicate of the POSITIVE CONTROL and a single replicate of a NEGATIVE control may be tested at the beginning and end of each batch of samples tested, after the addition of reagents, and after interruption or delays in processing. Additional quality control testing may be performed by the user by including other well-characterized specimens or reference sample. Perform the test as described under Section 10. DIRECTIONS FOR USE, using the POSITIVE and NEGATIVE controls as the specimens. The POSITIVE CONTROL should produce a POSITIVE (+) reaction and the NEGATIVE control should produre a negative ( ) reaction with the test. If the appropriate results are not obtained with the controls, all assay results within that batch are invalid and must be re-tested. Repeat testing making sure that the volume of control is sufficient English-11

12 for adequate instrument sampling. When control material repeatedly fails to perform as expected, contact your local distributor. 12. INTERPRETATION The PK7200/PK7300 instrument will read the settling patterns of SENSITIZED PARTICLES in each well based on the threshold settings chosen for each reagent. See the OLYMPUS PK7200/PK7300 Standard Operating Procedure Manual. Analyzer Reaction Interpretation sections, for complete details of the analyzer s interpretation of reactions. The threshold limits are programmed into the PK7200/PK7300 under the current operating conditions. As soon as possible after analyzer interpretation, results should be verified by visual judgment of the reaction pattern against the photometric data. All photometer results should be visually reviewed. Visually, a POSITIVE test is a homogeneous layer of particles. A NEGATIVE test would result in a compact dense button surrounded by a clear zone. Additional testing must be performed on any sample for which visual and analyzer interpretations do not agree. Refer to the Analyzer Reaction Interpretation section of the respective analyzer s SOP Manuals. The presence or absence of antibodies to T. pallidum is determined by the PK7200/PK7300 by a CCD camera, which analyses the well images and differentiates agglutinated and non-agglutinated patterns. The PK7200/PK7300 employs three assessment parameters for each microplate well containing SERODIA-TPPA Auto reagents and test specimens: * SPC Sharpness of the edge of the particle button * LIA Quantity of particles in the center of the well * P/C Ratio of the average light transmittance of the peripheral and central values The parameters, SPC, LIA and P/C, are compared to programmable thresholds to obtain an interpretation (+,,?) for each reaction. The most important parameter resulting from the image analysis system is SPC. If the SPC is determined positive, then either a positive or indeterminate LIA or P/C value will result in an overall positive results interpretation for the reaction. A positive SPC value together with a negative value for either the LIA or P/C will cause the channel result to be an indeterminate result. If the SPC is determined negative, then either a negative or indeterminate LIA or P/C value will result in an overall negative result interpretation for the reaction. A negative SPC value together with a English-12

13 positive value for either the LIA or P/C will cause the channel result to be an indeterminate result. Please refer to Table 1 for further clarification. Table 1: DECISION LOGIC FOR PK7200/PK7300 RESULTS INTERPRETATION Channel Results Interpretation SPC LIA P/C Positive + + or? + or? Negative or? or? Indeterminate ? +,, or? +,, or? English-13

14 When our recommended THRESHOLDS are adopted, overall PK7200 results interpretation will be as written Table 2. Table 2: DECISION LOGIC FOR PK7200/PK7300 RESULTS INTERPRETATION Channel Results Interpretation SPC Positive + Negative Indeterminate? 13. INTERPRETATION OF RESULTS (Table 3) A sample reported as NEGATIVE on initial screening is considered NEGATIVE for antibodies to T. pallidum and needs no further testing. An plasma sample which is POSITIVE or indeterminate (?) on initial screening with SERODIA-TPPA Auto, is considered initially POSITIVE by SERODIA-TPPA Auto, but prior to interpretation, the test may be repeated in duplicate using a serum specimen from the same draw. The duplicate tests must occur in the same run. If either duplicate is POSITIVE or indeterminate, the specimen is to be interpreted as repeatedly POSITIVE for antibodies to T. pallidum by the criteria of SERODIA-TPPA Auto. Initially POSITIVE specimens which are NEGATIVE in both of the duplicate re-tests are considered NEGATIVE by the criteria of SERODIA-TPPA Auto. Unless the repeated test is done on PK7200/PK7300, the specimens that are found Positive or Indeterminate with SERODIA-TPPA Auto should be retested by other test kits, such as SERODIA-TP PA, EIA and FTA-ABS. English-14

15 Table 3: INTERPRETATION OF RESULTS FOR SERODIA-TPPA Auto SERODIA-TPPA Auto Initial POSITIVE/Indeterminate (+/?) Repeat in duplicate with serum using SERODIA-TPPA Auto SERODIA-TPPA Auto POSITIVE / /+, +/+, /?, +/?,?/? SERODIA-TPPA Auto NEGATIVE SERODIA-TPPA Auto POSITIVE 14. PERFORMANCE CHARACTERISTICS A sample reported as POSITIVE on SERODIA-TPPA Auto is considered to be POSITIVE for IgG and/or IgM antibodies to T. pallidum by the criteria of SERODIA-TPPA Auto. POSITIVE results may indicate active, or past infections. The diagnosis of T. pallidum depends not only on the laboratory findings, but also on a carefully obtained history and a thorough physical examination. 1) Specificity When 14,106 random donor samples were tested, 43 samples showed positive results. As 24 of them were also positive by FTA-ABS, the specificity of SERODIA-TPPA Auto was 99.87% 10. 2) Sensitivity When 27 positive samples were tested, they showed positive results. The sensitivity of SERODIA-TPPA Auto was 100%. English-15

16 3) Reproducibility When three of in-house QC samples were tested 3 consecutive times respectively on PK7200/PK7300 according to the test procedure, all results are found to be within one doubling dilution. 4) Correlation result The results of the correlation test performed with 584 samples are shown in the table below. The correlation between SERODIA-TP PA and SERDIA-TPPA Auto was 99.80%. SERODIA- TPPA Auto (PK7200) SERODIA-TP PA Positive Indeterminate Negative Total Positive Indeterminate Negative Total LIMITATIONS OF THE PROCEDURE SERODIA-TPPA Auto has been shown to be safe and effective for the large scale detection of antibodies to T. pallidum in blood sample from blood donors when used in accordance with the instuctions provided with the kit. Serum must be used for any repeat testing on initially reactive or indeterminate results obtained from plasma samples. As with all serological tests for T. pallidum, interpretation of results obtained with SERODIA-TPPA Auto must take into consideration the donor s history and other clinical and/or laboratory findings. This product is for use only in screening blood donors and has not been evaluated as a serologic test for syphilis outside the blood bank setting. SERODIA-TPPA Auto may not be used to monitor the efficacy of the therapy or reinfection. 16. REFERENCES 1) Larsen SA, Hunter EF, Kraus SJ. A Manual of Tests for Syphilis. Washington DC: American Public Health Association, 1990 English-16

17 2) Rathlev T. Hemagglutination tests utilizing antigens from pathogenic and apathogenic Treponema pallidum. WHO Document WHO/VDT/RES 1965;L77:65 3) Tomizawa T and Kasamatsu S. Hemagglutination tests for diagnosis for syphilis. A preliminary report. Jap J Med Sci Bio 1966;19: ) Rathlev T. Hemagglutination tests utilizing antigens from pathogenic and apathogenic Treponema pallidum for the sero diagnosis of syphilis. Brit J Ven Dis 1967;43: ) Cox PM, Logan LC, Noris LC, Automated quantitative microhemagglutination assay for Treponema pallidum antibodies. Appl Microbio 1969;18: ) Cox PM, Logan LC, Noris LC, Automated quantitative microhemagglutination assay for Treponema pallidum antibodies. WHO Document WHO/VDT/RES 1969;69:174 7) Alpert N. Automated blood typing. Clin Inst Sys 1987;8:10 8) Gibbons D. Kano T, Edelmann M. A terraced microplate system for automated ABO and Rh Grouping. Am Clin Pdt Rev 1986;11 9) US Department of Health and Human Services. Biosafety in mirobiological and biomedical laboratories. HHS Publication(NH) , Washington U.S. Government Printing Office, May ) Sato S, Kishimoto S, Kuzuma K, Maeda R, Kato T, Ikeda H, Sekiguchi S Evaluation of Automated Syphilis screening for Blood Donors using Treponema Pallidum particle Agglutination Test (SERODIA-TPPA Auto). Japanese Journal of Transfusion Medicine, Vol 42. No.6 42(6) : , 1996 English-17

18 OLYMPUS PK7200 and OLYMPUS PK7300 are trademarks of Olympus Corporation, Tokyo, Japan. SERODIA is a trademark of FUJIREBIO INC., Tokyo, Japan Authorised Representative : Manufacturer : FUJIREBIO EUROPE B.V. Takkebijsters 69c 4817 BL Breda, The Netherlands TEL: FUJIREBIO INC. 62-5, Nihonbashi-Hamacho 2-chome, Chuo-ku,Tokyo, , Japan TEL: Revised in September, 2006 English-18