Instructions for use of 1 % Sheep-Erythrocytes-Suspension
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1 Instructions for use of 1 % Sheep-Erythrocytes-Suspension Ready to use for CFT Order No.: E-420, E-423, E-430, E-433, E-450, E-453 Intended Use The 1 % Sheep-Erythrocytes-Suspension is a ready-to-use reagent that is applied in diagnostics with the Complement Fixation Test (CFT) (DIN ). It contains buffers and erythrocytes in concentrations required by DIN for CFT. The 1 % Sheep-Erythrocytes-Suspension is designed to be used by professionals. Diagnostic Importance The Complement Fixation Test (CFT) is a classical serological test designed to measure serum levels of specific antibodies against antigens of e. g. bacteria, viruses, parasites and fungi. Antigen-antibody complexes activate and exhaust complement. These properties are utilized in the CFT. Complement is only activated in the presence of IgG and IgM antibodies. A high CFT titer can indicate the presence of IgM or a high level of IgG antibodies which in turn may be a hint for a recent infection. CFT is performed according to DIN Test Principle The CFT is an indirect test. First of all, the complement of the sample is inactivated by incubation at 56 C. In the test system, specific antigen is added to the test sera. If specific antibodies against the antigen are present, an immunocomplex is formed. The added complement is activated by this immunocomplex and exhausted. Due to the addition of sensitized erythrocytes (Hemolytic System), the erythrocytes are lysed if complement has not been exhausted (e. g. no immunocomplex was formed = negative reaction). If the complement was used up by antigen-antibody complexes, the erythrocytes remain intact and form a button at the bottom of the microtest plate (Hemolytic Inhibition = positive reaction) after centrifugation. The semi-quantitative analysis of the antibody concentration of a serum is possible by endpoint-titration of the serum. E-420, E-423, E-430, E-433, E-450, E-453/ Seite: 1 / 5
2 Safety measures The 1 % Sheep-Erythrocytes-Suspension is an in-vitro-diagnostic agent and designed to be used only by professionals who master the laboratory work techniques. The 1 % Sheep-Erythrocytes-Suspension is composed of blood components from sheep (erythrocytes) in a buffer solution. Generally, all products of animal origin are potentially infectious and must be handled and disposed of according to the safety instructions for biohazardous substances. Do not smoke, eat or drink in areas in which specimen or kit reagents are handled. Wear disposable gloves, laboratory coat and safety glasses while handling kit reagents or specimen. Wash hands thoroughly afterwards. Disposal For disposal please follow the relevant statutory requirements! Package Contents Name Volume Order No. 1 x 10 ml E x 10 ml E % Sheep-Erythrocytes-Suspension 1 x 100 ml E x 100 ml E x 50 ml E x 50 ml E-453 Additional material required but not provided for the CFT The additional test reagents must be obtained from an appropriate certified supplier (e. g. Virion\Serion Würzburg) and have to comply with DIN Amboceptor Antigen / Control Antigen for CFT Aqua dest. Complement Positive and negative control sera CFT Buffer Microtest plates in U-form Micropipettes with tips for 100 µl and 1000 µl Test tubes for inactivation Vortex mixer Plates Incubator 37 C (± 1 C) Water bath 56 C (± 1 C) Centrifuge with adapter for microtest plates (optional) E-420, E-423, E-430, E-433, E-450, E-453/ Seite: 2 / 5
3 Storage and Stability of Reagents Storage at 2 C - 8 C until expiry date (see label). Avoid prolonged exposure to light. Do not use the reagents after the expiry date. Stability after first Opening To avoid contamination for the removal of reagents from the bottle aseptic techniques should be applied. The stability after first opening is given for up to seven days if stored at 2 C 8 C. Transport Stability Transport stability is given for seven days at 37 C (result of an internal study). Sample Material Sera are used for the CFT. Contaminated samples and samples with particles are not suitable for the CFT. Samples can be stored for 5 days at 2 C 8 C. For long-time storage freeze at 20 C. Avoid repeated freeze-thaw cycles. Pretesting The pretesting procedure of CFT is performed in accordance with the general conditions of DIN or the instructions for use of a certified CFT supplier Preparation of the 1 % Sheep-Erythrocytes-Suspension Carefully shake the 1 % Sheep-Erythrocytes-Suspension. Hemolytic System is prepared with amboceptor in accordance with the general conditions of DIN or the instructions for use of a certified CFT supplier Test Procedure The test procedure of the complement fixation is performed in accordance with the general conditions of DIN or the instructions for use of a certified CFT supplier Prepare CFT as common, e. g. according to DIN Incubate CFT preparation overnight (16 20 hours) Prepare Hemolytic System with 1 % Sheep-Erythrocytes-Suspension and Amboceptor according to DIN Incubate for 15 minutes at 37 C. Add Hemolytic System Continue with test procedure and analysis of CFT according to DIN E-420, E-423, E-430, E-433, E-450, E-453/ Seite: 3 / 5
4 Test Analysis and Interpretation of Results The test analysis and interpretation of results is performed in accordance with the general conditions of DIN or the instructions for use of a certified CFT supplier Non-Specific Reactions In the CFT some sera exhibit a so-called Autogenous Inhibition. The term Autogenous Inhibition covers anti-complementary serum properties. In the test batch this phenomenon is displayed as a positive CFT reaction (Hemolytic Inhibition) that is also seen in the serum control (batch without addition of antigen). Autogenous Inhibition can be caused by circulating immunocomplexes, rheumatoid factors, drugs (Dextrane) or is observed with hemolytic or contaminated sera as well as after repeated freezing and thawing of the sera. By pre-treatment with undiluted complement these sera can be tested in the CFT. Follow the instructions for use of a certified CFT supplier Limitation of the Test The final clinical evaluation may not be based on the results of a single diagnostic method. The attending physician has to take into account anamnesis and further laboratory findings of the patient. Microbial contamination or repeated freeze-thaw cycles of the samples may lead to false test results and misinterpretation. Follow the instructions for use of a certified CFT supplier Troubleshooting The proper use of working techniques and strict compliance with the instructions for use are indispensible requirements to obtain reliable results. In particular, accurate sample preparation and precise pipetting of the reagents are prerequisites. 1. Hemolysis of supernatant Microbial contamination of the reagents Storage damage: Failure to comply with indicated temperatures 2. Brown or agglutinated erythrocytes Storage damage: Failure to comply with indicated temperatures Performance Characteristics of 1 % Sheep-Erythrocytes-Suspension The 1 % Sheep-Erythrocytes-Suspension represents only a part of the reagents for the CFT. In addition, reagents of other manufacturers are necessary for the CFT. The performance characteristics are measured by the titration of a standard complement and with a chosen standard CFT reagent set from the manufacturer Virion\Serion Würzburg. E-420, E-423, E-430, E-433, E-450, E-453/ Seite: 4 / 5
5 Detection Sensitivity and Effective Range The detection sensitivity and effective range was determined by the titration of 2 complement lots of different producers with 10 lots of 1 % Sheep-Erythroyctes-Suspension that were used for production of the Hemolytic System. Results Lot No. Hemolytic System Titer Complement :64 1: :64 1: :64 1: :64 1: :64 1: :64 1: :64 1: :64 1: :64 1: :64 1:64 Titer Complement 2 Specificity, Sensitivity, Interassay Variation The specificity, sensitivity and interassay variation were determined by titration of 5 control sera with standard reagents CFT Campylobacter of Virion\Serion Würzburg. In total, 4 lots of 1 % Sheep-Erythroyctes-Suspension were tested which were used to produce the Hemolytic System. Lot No. Hemolytic System KS-1 KS-2 KS-3 KS-4 KS-5 1:40 ± 1 titer 1:30 ± 1 titer 001 1:20/40 < 1:10 1:10/20 < 1:10 < 1: :40 < 1:10 1:20 < 1:10 < 1: :40 < 1:10 1:20 < 1:10 < 1: :40 < 1:10 1:20 < 1:10 < 1:10 Symbole I r L E T M in-vitro-diagnostic reference or order number lot number expiry date store between x and y degree celsius Manufacturer Literature 1) Deutsche Norm: Serodiagnostik von Infektionskrankheiten, Komplementbindungsreaktion (KBR), DIN 58969, Teil 10, Ausgabe November 2003; Beuth Verlag GmbH, Burggrafenstraße 6, Berlin 2) Lennette, E. H., Schmidt, N. J.: Diagnostic procedures for viral and rickettsial infections. 5th edition, pp , New York 1979 E-420, E-423, E-430, E-433, E-450, E-453/ Seite: 5 / 5
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