TGS TA Anti-Mitochondrial Antibodies (M2)
Contents
1. AUTOMATION The TGS TA Analyser instrument automatically performs all the operations envisaged by the dosage protocol addition of samples calibrators controls magnetic particles conjugates and chemiluminescence activation solutions to the reaction container magnetic separation and washing of particles measurement of the light emitted The system calculates the dosage results for the samples and controls by means of a stored calibration curve and prints a report that includes all the information related to the dosage and to the patient MATERIALS AND REAGENTS Materials and reagents supplied Magnetic particles coated with recombinant antigen containing immunodominant regions of PDC E2 BCOADC E2 and OGDC E2 mitochondrials MIT3 in Phosphate Buffer containing stabilising proteins surfactant Pro Clin 300 and sodium azide lt 0 1 as preservatives Monoclonal anti human IgG antibody marked with an acrinidium ester derivative conjugate in Phosphate Buffer containing stabilising proteins surfactant and sodium azide lt 0 1 as preservative Sample Diluent Solution Phosphate Buffer containing bovine serum albumin a surfactant an inert blue colouring agent Pro Clin 300 and Gentamicin SO as preservatives Human serum with low concentration of anti M2 IgG antibodies containing sodium azide lt 0 1 as preservative Human serum with high concentration of anti M2 IgG antibodies containing sod2. TGS TA AMA M2 REF YA500231 REF Y 850025 TGS TA Anti Mitochondrial A TECNO D tes Antibodies M2 R GENETIC INSTRUCTIONS FOR USE 8 C A CE EN English INTENDED USE The TGS TA Anti Mitochondrial Antibodies M2 AMA M2 test is a chemiluminescent immunoassay CLIA used for quantitative determination using the TGS TA Analyzer or IDS iSYS Multi Discipline Automated System of specific IgG class antibodies directed against Anti Mitochondrial Antibodies in samples of human serum or plasma EDTA Sodium Citrate This dosage is used as a diagnostic aid when assessing Primary Biliary Cirrhosis PBC This product must be used in strict compliance with the instructions given in this document by professional users CAUTION Medical decisions must not be based exclusively on the result of this test but must take into account all available clinical and laboratory data as a whole CLINICAL SIGNIFICANCE Primary biliary cirrhosis PBC is an autoimmune disease that causes a chronic inflammation of the intrahepatic bile ducts Progressive destruction of the liver cells leads to hepatic fibrosis and cirrhosis and over time may lead to hepatic insufficiency and to the need for a liver transplant The exact cause of PBC is unknown genetic factors linked to a dysfunction of the immune system are implicated as well as environmental factors such as interaction of the organism wit
3. 10 11 12 13 14 15 16 17 18 Hirschfield GM Heathcote EJ Antimitochondrial antibody negative primary biliary cirrhosis Clin Liv Dis 2008 12 323 31 Liu B Shi XH Zhang FC Zhang W Gao LX Antimitochondrial antibody negative primary biliary cirrhosis a subset of primary biliary cirrhosis 2008 28 233 9 Muratori P et al True antimitochondrial antibody negative primary biliary cirrhosis low sensitivity of the routine assays or both Clin Exp Immunol 154 2004 Berg PA Klein R Mitochondrial antigens and autoantibodies from anti M1 to anti M9 Klin Wochenschr 64 897 909 1986 Mackay IR Gershwin ME The autoantibodies of primary biliary cirrhosis clinico pathological correlations In van Venrooij WJ and Maini RN eds Manual of biological markers of disease Dordrecht Kluwer Academic Publishers 1996 C8 1 1 18 Bassendine MF Fussey SPM Mutimer DJ James PFW Yeaman SJ Identification and characterization of four M2 mitochondrial autoantigens in primary biliary cirrhosis Semin Liver Dis 1989 9 124 31 Miyakawa H et al Detection of antimitochondrial autoantibodies in immunofluorescence AMA negative patients with primary biliary cirrhosis using recombinant autoantigens Hepatology 248 2001 Moteki S et al Use of a designer triple expression hybrid clone for three different lipoyl domain for the detection of antimitochondrial autoantibodies Hepatology 103 1996 Mitchison HC Bassendine MF Hen
4. Based on the immunochemical structure 9 sub types of mitochondrial antigens have been classified named from M1 to M9 Only the anti M2 anti M4 anti M8 and anti M9 autoantibodies are specific to PBC and of these only the anti M2 antibodies specificity for pyruvate dehydrogenase are of diagnostic importance since they are present in high titres in almost all patients while the other three may be found in low titres and in any case are never found in isolation but are always associated with anti M2 antibodies In reality antigen M2 comprises 4 different autoantigens belonging to the structural complex of the pyruvate dehydrogenase enzyme to which 4 different types of non cross reagent antibodies correspond The antibodies most frequently detected are those acting against the E2 2 oxo acid dehydrogenase component those others are found less frequently and are normally associated with anti E2 antibodies The specific antigens have been identified as sub units of the pyruvate dehydrogenase complex PDC E2 of the 2 oxo acid dehydrogenase complex of the branched chain BCOADC E2 and of the 2 oxoglutarate dehydrogenase complex OGDC E2 The test to determine AMAs based on the use of the three combined antigens give higher performances than the IFA test making it possible to detect AMA antibodies in over two thirds of the serums of PBC patients who tested AMA negative with the IFA test Since the presence of AMA antibodies may pre
5. them in the analyser If there is no bar code on the sample or if it is not readable the sample identification data must be entered manually Select the required parameters for each sample Calibration The TGS TA Analyser instrument uses a memorised calibration curve master curve generated by the manufacturer for each batch of reagent cartridges The master curve parameters together with the calibrator concentration settings are stored in the DATA DISK and transferred to the instrument s data base IFU059TGS TA Version 01_EN 15 January 2015 Page 7 of 14 TGS TA AMA M2 REF YA500231 Calibrators A and B are used to recalibrate the master curve in both for the instrument used and for the reagents on board To perform recalibration analyse the two calibrators A and B in triplicate and the controls singly The concentration readings obtained with the controls make it possible to validate the new calibration Once recalibration of the master curve has been accepted and memorised all subsequent samples can be analysed without any further calibration except in the following cases when a reagent cartridge with a new batch number is loaded into the instrument when the control readings do not fall within the range of acceptability when the instrument maintenance procedure is performed when the validity of the recalibrated master curve has expired The validity of the recalibra
6. Solution Code No YC500009 Pack of 2 bottles containing 1 liter of ready to use solution TGS TA Cartridge Checking System Code No IS 601000 TGS TA Top Cap Set Code No YC500010 300 red top caps to close the calibrator containers after first use Manufactured by IDS France SAS 42 rue St phane Mazeau 21320 Pouilly en Auxois France and distributed by Technogenetics Srl Manufactured by IDS S A Rue E Solvay 101 B 4000 Li ge Belgium and distributed by Technogenetics Srl IFU059TGS TA Version 01_EN 15 January 2015 Page 4 of 14 TGS TA AMA M2 REF YA500231 Other Recommended Reagents TGS TA LIVER CONTROL SET Code No YA500233 Three 1 mL vials of negative human serum and three 1 mL vials of human serum positive for antic AMA M2 antibodies WARNINGS AND PRECAUTIONS The reagents supplied in the TGS TA Anti Mitochondrial Antibodies M2 kit are only for in vitro diagnostic use and not for in vivo use in humans or animals This product must be used in strict compliance with the instructions given in this document by professional users Technogenetics cannot be held responsible for any losses or damages caused by use not in conformity with the instructions supplied Safety precautions This product contains material of animal origin and therefore must be handled as if it contains infecting agents This product contains components of human origin All units of serum or plasma used to pr
7. document EP7 A2 has shown that the dosage performances are not influenced by the presence in the sample of the potentially interfering substances listed in the table below up to the tested concentration ieee internering Maximum tested concentration ubstances Free bilirubin 20 mg dL Conjugated bilirubin 20 mg dL Haemoglobin 1000 mg dL Tryglicerides 3000 mg dL Use of lipaemic haemolysed and turbid samples is not in any case recommended Analytical Specificity Cross reactions In order to assess potential cross reactions of the antigen used to sensitise the microparticles a study was conducted using 40 samples all with high levels of other antibodies and negative for anti AMA M2 IgG The samples used were subdivided as follows SS A 2 SS B 2 U1 snRNP 2 Jo 1 2 Scl 70 2 Cenp B 2 Sm 2 PR3 2 MPO 2 B2 GLI CL 2 Gliadin 2 t TG 2 CCP 2 GBM 2 dsDNA 2 TG 3 TPO 3 and Rheumatoid Factor RF 4 The study did not show any significant cross reaction to the antigen in the solid phase with the other antibodies Saturation effect at high doses Some immunological methods used to determine samples containing the analyte at extremely high concentrations may supply apparent levels of underestimated analyte Hook effect The method used in the TGS TA Anti Mitochondrial Antibodies M2 kit since it uses two incubations is not subject to this effect IFU059TGS TA Version 01
8. readings if analysed with the TGS TA Anti Mitochondrial Antibodies M2 kit EXPECTED VALUES The samples of 100 individuals selected randomly from the normal routine work of the laboratory were analysed to check the presence of anti AMA M2 IgG antibodies All samples analysed proved negative with an average reading of 0 7 AU mL and a standard deviation of 1 5 AU mL Using the results obtained the Limit of Blank LoB the highest reading that can be expected in a series of samples that do not contain the analyte was calculated The Limit of Blank determined as 95 percentile of the negative population proved equal to 3 8 AU mL with the Reagent batch no 2 CLINICAL SENSITIVITY AND SPECIFICITY A total of 664 samples were tested with the TGS TA Anti Mitochondrial Antibodies M2 kit 119 of the samples were from patients suffering from primary biliary cirrhosis PBC 66 from patients suffering from type 1 or type 2 autoimmune hepatitis AIH 1 or AIH 2 41 from patients with hepatitis C HCV 20 from patients with hepatitis B HBV 18 samples from patients with primary sclerosing cholangitis PSC while 400 samples were presumed normal originating from routine laboratory testing In the presumably negative population studied including 66 samples from type 1 or type 2 autoimmune hepatitis AIH 1 or AIH 2 41 with hepatitis C HCV 20 with hepatitis B HBV 18 with primary sclerosing cholangitis PSC and 400 normal s
9. SSAY PROCEDURE Scrupulously follow the instructions given in the operating manual of the instrument to obtain reliable analytical results Loading of reagents All the reagents supplied in the kit are all ready for use Before inserting the reagent cartridge in the system the magnetic particle container must be horizontally agitated by rotation in order to favour resuspension of the particles Avoid generating foam when performing this operation Place the reagent cartridge in the reagent area of the instrument using the guide provided and leave it to be agitated for at least 40 minutes before use IFU059TGS TA Version 01_EN 15 January 2015 Page 6 of 14 TGS TA AMA M2 REF YA500231 Positioning of the reagent cartridge simultaneously determines reading of the identity bar code If the cartridge label is damaged or if it is not readable the reagent cartridge identification data can be entered manually The instrument automatically maintains the magnetic particles constantly agitated If the reagent cartridge is removed from the instrument store it at 2 8 C in a vertical position in a dark place Loading of calibrators TGS TA calibrators are ready for use Leave the calibrators at room temperature for 10 minutes and then gently shake the contents either manually or using a vortex avoiding the formation of foam When using the calibrators for the first time remove the guarantee seal and the white sealing ca
10. TGS TA kit proved to be 420 AU mL BIBLIOGRAPHY 1 Lindor KD Gershwin ME Poupon R Kaplan K Bergasa NV Heathcote EJ Primary biliary cirrhosis Hepatology 2009 50 291 308 2 Crosignani A Battezzati PM Invernizzi P Selmi C Prina E Podda M Clinical features and management of primary biliary cirrhosis World J Gastroenterol 2008 Jun 7 14 21 3313 27 3 Giorgini A Selmi C Invernizzi P Podda M Zuin M Gershwin ME Primary biliary cirrhosis solving the enigma Ann N Y Acad Sci 2005 1051 185 93 4 Long SA Quan C van de Water J Nantz MH Kurth MJ Barsky D et al Immunoreactivity of organic mimeotopes of E2 components of pyruvate dehydrogenase connecting xenobiotics with primary biliary cirrhosis Clin Exp Immunol 2001 167 2956 63 5 Gershwin ME Mackay IR The causes of primary biliary cirrhosis convenient and inconvenient truths Hepatology 2008 47 737 45 Kaplan MM Gershwin ME Primary biliary cirrhosis N Engl J Med 2005 353 1261 73 Giorgini A Selmi C Invernizzi P Podda M Zuin M Gershwin ME Genetics and geoepidemiology of primary biliary cirrhosis following the footprints to disease etiology Semin Liver Dis 2005 25 265 80 8 James OF Bhopal R Howel D Gray J Burt AD Metcalf JV Primary biliary cirrhosis once rare now common in the United Kingdom Hepatology 1999 30 390 4 IFU059TGS TA Version 01_EN 15 January 2015 Page 13 of 14 TGS TA AMA M2 REF YA500231
11. _EN 15 January 2015 Page 12 of 14 TGS TA AMA M2 REF YA500231 A sample with an extremely high concentration above the measurement range of anti AMA M2 IgG confirmed the absence of the hook effect up to the concentration of 22000 AU mL Relative Sensitivity and Specificity The presence of anti AMA M2 IgG antibodies was determined using the TGS TA Anti Mitochondrial Antibodies M2 kit and an ELISA dosage method available on the market in 341 samples 119 of the samples were from patients suffering from primary biliary cirrhosis PBC 75 from patients suffering from type 1 or type 2 autoimmune hepatitis AIH 1 or AIH 2 41 from patients with hepatitis C HCV 20 from patients with hepatitis B HBV 18 samples from patients with primary sclerosing cholangitis PSC while 68 samples were presumed normal originating from routine laboratory testing 18 samples gave rise to discordant results between the TGS TA dosage and the ELISA dosage available on the market The relative concordance was therefore found to be 94 7 95 Confidence Interval 91 6 96 8 323 341 The relative sensitivity was therefore found to be 88 9 95 Confidence Interval 81 0 93 9 96 108 The relative specificity was found to be 97 4 95 Confidence Interval 94 2 98 9 227 233 Reference serum The quantity of AMA M2 IgG antibodies present in the Primary Billiary Cirrhosis HUMAN NIBSC code 67 183 sample measured with the
12. amples 15 samples tested positive and 504 negative Diagnostic specificity 97 2 530 545 Of the 15 samples that proved to be not negative 8 belonged to the group of patients diagnosed with AIH 1 or AlH 2 2 belonged to the group of patients diagnosed with HCV and 5 to the group of normal samples Of the 5 normal samples that tested positive 4 were also confirmed positive by a commercial ELISA method In the presumably positive population studied including 119 samples from patients diagnosed with PBC 87 proved to be positive and 32 samples were negative Diagnostic sensitivity 73 1 87 119 Of the 32 samples that tested negative 27 were also confirmed negative by a commercial ELISA method Based on the diagnostic specificity and sensitivity results the diagnostic agreement is 93 0 617 664 PERFORMANCES Caution the data presented do not represent the operating specifications of the kit but serve as experimental proof of how the kit works within these specifications in the manner envisaged by the manufacturer IFU059TGS TA Version 01_EN 15 January 2015 Page 10 of 14 TGS TA AMA M2 REF YA500231 Precision and Reproducibility The precision and the reproducibility of the TGS TA Anti Mitochondrial Antibodies M2 kit have been assessed using a protocol based on the guidelines given in Clinical and Laboratory Standards CLSI document EP5 A2 The precision was calculated by analysi
13. cede the onset of the symptomatic disease the ability to identify more accurately the presence of the PBC marker could contribute to early diagnosis and treatment and could slow down the progression of the disease PRINCIPLE OF THE METHOD The TGS TA Anti Mitochondrial Antibodies M2 kit for quantitative determination of specific anti mitochondrial antigen IgG employs an indirect two step immunological method based on the principle of chemiluminescence The specific antigen used to coat the magnetic particles solid phase and an anti human IgG antibody are marked with an acridinium ester derivative conjugate During initial incubation the specific antibodies present in the sample in the calibrators or in the controls bond with the solid phase During the second incubation the conjugate reacts with the anti M2 IgG antibodies sequestered by the solid phase After each incubation the material that has not bound with the solid phase is removed by aspiration and subsequent washing The quantity of marked conjugate that remains bound to the solid phase is assessed by activation of the chemiluminescence reaction and measurement of the luminous signal The generated signal expressed in relative light units RLU is indicative of the concentration of specific antibodies present in the sample in the calibrators and in the controls IFU059TGS TA Version 01_EN 15 January 2015 Page 2 of 14 TGS TA AMA M2 REF YA500231
14. drick A Bennett MK Bird G Watson AJ James OF Positive antimitochondrial antibody but normal alkaline phosphatase is this primary biliary cirrhosis Hepatology 1986 6 1279 84 Metcalf JV Mitchison HC Palmer JM Jones DE Bassendine MF James OF Natural history of early primary biliary cirrhosis Lancet 1996 348 1399 402 TECHNOGENETICS S r l Via Vanvitelli 4 20129 Milano Italy IFU059TGS TA Version 01_EN 15 January 2015 Page 14 of 14
15. h a number of infectious agents PBC is more frequent in women with a maximum incidence between ages 40 and 60 years with no racial difference but with a wide geographical variability ranging from 40 to 400 individuals in the general populations and higher frequency in Northern Europe and in the USA In half of the cases PBC is casually diagnosed when during other tests or screening anomalous levels of liver pathology markers are detected transaminases AST and ALT and above all cholestasis indexes y GT and Alkaline Phosphatasis Diagnosis is given by the presence of at least two of the following criteria positive result of a search for anti mitochondrial antibodies AMA at a sufficient concentration gt 1 40 persistence for over 6 months of alkaline phosphatasis higher than 1 5 times the upper limit of the reference range liver biopsy with a compatible histological context IFU059TGS TA Version 01_EN 15 January 2015 Page 1 of 14 TGS TA AMA M2 REF YA500231 Definite PBC can be diagnosed when all three criteria are met while the term probable PBC is used when only two criteria are met High AMA titres are the most sensitive and specific PBC immune serologic markers since they can be found in 90 95 of patients with a specificity close to 100 However it is worth underlining that the frequency of the antibodies detected depends on the group studied and the sensitivity may be lower
16. ion 01_EN 15 January 2015 Page 5 of 14 TGS TA AMA M2 REF YA500231 The components of the reagent cartridge must not be removed from the cartridge and reassembled Do not use the kit after its use by date PREPARATION OF THE REAGENTS The reagents supplied in the kit are all ready for use REAGENT STORAGE AND STABILITY Store the reagents supplied in the kit at 2 8 C in a vertical position in a dark place In these conditions unopened reagent cartridge and calibrators are stable up to the use by date After opening the reagent cartridge can be used for 60 days if kept in a refrigerator at 2 8 C or in the analyser After opening the calibrators can be used for 60 days if kept in a refrigerator at 2 8 C and if they have not been left in the analyser for more than 6 hours per session Do not freeze the reagents and calibrators SAMPLE PREPARATION AND STORAGE Dosage must be performed on samples of human serum and plasma EDTA Sodium Citrate Use of lipaemic haemolysed and turbid samples is not recommended If the dosage is performed after more than 8 hours separate the serum or the plasma from the clot from the red globules and from the separating tubes with gel Before being analysed samples may be kept in a refrigerator at 2 8 C for a maximum of 7 days If the dosage is to be performed after more than 7 days store the samples frozen lt 20 C Avoid repeated freezing and thawing A
17. ium azide lt 0 1 as preservative All reagents are ready for use Reagents 1 2 3 and 4 are assembled in a single kit forming the reagent cartridge IFU059TGS TA Version 01_EN 15 January 2015 Page 3 of 14 TGS TA AMA M2 REF YA500231 The Calibrator concentrations are expressed in AU mL arbitrary units and calibrated against an internal reference standard The concentration settings specific for each production batch are recorded on the DATA DISK included in the kit DATA DISK A Mini CD containing data regarding all the products in the TGS TA line Reagents Calibrators Control Serums updated to the last production batch with the exclusion of products that have expired at the date when the new DATA DISK was compiled Only the DATA DISK with the highest batch number needs to be kept to maintain the information required for correct operation of the system up to date Materials and reagents required but not supplied in the kit TGS TA Analyzer 120 Cod No TGS00001 TGS TA Cuvette Cube Code No IS CC1000 Pack of 960 cuvettes TGS TA System Liquid Code No IS CS1000 1 bottle containing 5 liters of ready to use solution TGS TA Wash Solution Code No IS CW1000 1 bottle containing 10 liters of ready to use solution TGS TA Trigger Set Code No IS CT1000 1 250 mL bottle of Trigger A pre trigger solution 1 250 mL bottle of Trigger B trigger solution TGS TA D SORB
18. ng the results of 20 replicates of four serums one negative and three positive with differing concentrations of anti AMA M2 IgG antibodies performed with two different batches of reagents in the same test session The concentration of the negative anti AMA M2 IgG N2 serum was always 0 0 AU mL with reagent batches nos 1 and 2 The table shows the results obtained with the 3 positive serums Reagents Average Concentration SD Sample BAEN NO j AU mL AU mL oe am aan a e JE re 1860 36 2 35 5 1 89 5 3 m ant a 100 0 o 2 96 oo 3 0 2 92 6 1 50 1 6 p 1 728 3 a FT 3 ay A EEEE EAEAN O AEE EEE 6 49 6 INAS 5j 55 3 i 8 The reproducibility was calculated by analysing the results of the determination of four serums one negative and three positive with differing concentrations of anti AMA M2 IgG performed singly in 30 different sessions with two different batches of reagents The concentration of the negative anti AMA M2 IgG N2 serum was always 0 0 AU mL The table shows the results obtained with the 3 positive serums Sample ney as A T CV P1 89 5 4 00 4 5 P2 97 2 4 27 4 4 P3 658 2 33 41 5 1 Analytical Sensitivity The analytical sensitivity of the TGS TA Anti Mitochondrial Antibodies M2 kit was assessed using a protocol based on the guidelines given in Clinical and Laboratory Standards CLSI document EP17 A In one case referred to as the Limit of De
19. oduce the reagents in this kit have been analysed with FDA approved methods and found not to be reactive due to presence of HBsAg anti HCV anti HIV1 and anti HIV2 However since no analysis method is able to guarantee the absence of pathogenic agents all material of human origin must be considered to be potentially infected and handled as such In the event of damaged packaging or accidental leakage decontaminate the area concerned with a diluted solution of sodium hypochlorite after putting on suitable personal protective equipment overall gloves goggles Dispose of the material use for the clean up and of the packaging involved in the leakage according to national regulations for disposal of potentially infected waste In the event of damaged packaging or accidental leakage do not use the reagents to perform the assay Some reagents contain sodium azide as a preservative Since sodium azide may react with lead copper and leaded brass forming explosive azides in piping it is recommended that reagents or waste are not poured down drains but are disposed of in compliance with the national regulations on disposal of potentially hazardous waste Operating precautions Reliable results can only be obtained by strictly complying with these instructions and scrupulously following what is written in the operating manual for the instrument The reagents supplied in the kit must be used only with the TGS TA Analyser system IFU059TGS TA Vers
20. or the system Interpretation of the results The dosage range measurable for TGS TA Anti Mitochondrial Antibodies M2 is 0 0 1000 AU mL Readings lower than 0 0 AU mL are extrapolated values the message OMR and or ORA appears and they are shown as equal to 0 0 AU mL Readings higher than 1000 AU mL are accompanied by the message OMR and or ORA and may be retested after suitable dilution The results of the samples may be interpreted in the following way AU mL Interpretation lt 10 The sample must be considered to be negative for anti AMA M2 IgG 2 10 The sample must be considered to be positive for antic AMA M2 IgG The above readings are to be considered only as suggested readings Each laboratory must establish its own reference ranges LIMITS OF THE ASSAY METHOD For diagnostic purposes the results obtained with the TGS TA Anti Mitochondrial Antibodies M2 kit and the TGS TA Analyser system must be used together with the other clinical and laboratory data available to the physician Bacterial contamination of the sample and heat inactivation may influence the result of the dosage IFU059TGS TA Version 01_EN 15 January 2015 Page 9 of 14 TGS TA AMA M2 REF YA500231 Heterophyllous antibodies present in human serum samples may react with immunoglobulin based reagents causing interference with in vitro immunological dosages Such samples may give rise to anomalous
21. p before placing them in the analyser If the calibrators have already been used the container will have a top cap red cap with no guarantee seal Remove the red closing cap before placing them in the analyser Place the calibrators in the samples area of the analyser see the analyser user manual on how to identify them in the analyser Bar code data must be entered manually if the label is damaged or if it is unreadable The readings for the IgG class anti AMA M2 antibody concentration in the calibrators are recorded in the DATA DISK and automatically transferred to the analyser At the end of the session the calibrator containers must be closed with the top caps red caps provided and stored at 2 8 C until they are used again The calibrators can be used for a maximum of four times Loading of controls Place the controls in the samples area of the analyser See the analyser user manual on how to identify them in the analyser If there is no bar code on the control or if it is not readable the control identification data must be entered manually If TGS TA Controls are used see the usage instructions provided The readings for the anti AMA M2 IgG antibody concentration in the TGS TA controls are recorded in the DATA DISK and automatically transferred to the analyser Select the required parameters for each control Loading of samples Place the samples in the samples area of the analyser see the analyser user manual on how to identify
22. tection LoD that is the smallest quantity of analyte that the method is able to measure the formula for calculating LoD LoB Cg SD in which LoB is the Limit of IFUO59TGS TA Version 01_EN 15 January 2015 Page 11 of 14 TGS TA AMA M2 REF YA500231 Blank SD is the estimated standard deviation of the distribution of the sample at low concentration and Cz is derived from 95 percentile of the standard Gaussian distribution was applied Three low concentration samples of analyte were used determined singly with one batch of reagents in 15 different tests The Limit of Detection of the TGS TA Anti Mitochondrial Antibodies M2 kit proved to be 5 7 AU mL In the other protocol calculation of the Minimum Detectable Concentration MDC is envisaged 20 replicates of the solution of the 0 AU mL Standard of the Master curve were used The average and the standard deviation DS were formulation on a batch of the kit The RLU value related to the average 2 6 DS was interpolated on the curve and the related concentration was obtained from this value The analytical sensitivity expressed as the Minimum Detectable Concentration is 0 3 AU mL The minimum detection values together with considerations of a clinical kind and the results of comparison with reference methods contributed to the definition of the cut off value Analytical Specificity Interferences A study based on the guidelines given in the CLSI
23. ted master curve for the TGS TA Anti Mitochondrial Antibodies M2 kit is 21 days Control of recalibration is performed automatically by the instrument Assay Press the start button 1 The system aspirates 100uL of Sample Diluent 40uL of Magnetic Particles 80uL of Sample Diluent and 10uL of the sample calibrators or control automatically pre diluted by 1 10 by the instrument The aspirated solutions and suspension are dispensed into the reaction cuvette The reaction cuvette is incubated in the rotor at 37 C for 10 minutes After this phase of incubation the magnetic particles are separated and washed 200 uL of conjugate are dispensed into the cuvette The reaction cuvette is incubated in the rotor at 37 C for 10 minutes Park wN After this last phase of incubation the magnetic particles are separated and washed and the cuvette is transferred to the reading chamber 7 The quantity of conjugate bound to the solid phase expressed in RLU is directly proportionate the concentration of anti AMA M2 IgG present in the sample 8 The responses obtained are interpolated on the calibration curve and transformed into concentrations QUALITY CONTROL To ensure the validity of the dosage control serums at differing levels of concentration at least one negative serum and one positive serum must be measured every day in which dosage is performed If your laboratory requires a more frequent use or a higher number of controls
24. to check the dosage results comply with the set quality control procedure If TGS TA control serums are used the expected average readings and the acceptability limits are those given on the DATA DISK included in the control pack too If different control serums are used before using them the readings expected with TGS TA reagents and system must be defined IFU059TGS TA Version 01_EN 15 January 2015 Page 8 of 14 TGS TA AMA M2 REF YA500231 If the control reading does not fall within the specified range of acceptability the related dosage results are not valid and the respective samples must be analysed again In this case before repeating the dosage a recalibration procedure must be performed CALCULATION AND INTERPRETATION OF THE RESULTS Calculation of the results The concentration of the anti AMA M2 antibodies present in the samples that are being tested is automatically calculated by the system The readings can be viewed on the display or printed The concentrations are expressed in AU mL Calculation of the analyte concentration in the sample takes place by reading the response obtained for each sample on a calibration curve processed by a logistic fitting system with four parameters 4PL Y weighted periodically corrected according to the responses obtained for dosage of the calibrators For detailed information on how the system calculates the results please see the operating manual f
Download Pdf Manuals
Related Search