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1. C C DOT121v08 Instructions for Use for Biofortuna SSPGo HLA Typing Kits CE Revision 5 13 SSPGo Troubleshooting Guide Page 8 of 12 Probable Cause Incorrect concentration of DNA used No amplification in any reaction PCR inhibitors present in DNA sample Poor quality DNA sample used Reagents not fully re suspended Thermal cycler not set up correctly Electrophoresis problems Plates not sealed correctly Measure the DNA quantity by measuring at OD2g9 and ensure 50 100ng of DNA in total is added in a volume of 10ul per reaction Do not use heparinised blood Avoid DNA samples containing greater than 1 mg dL Hemoglobin Measure the DNA quality The OD2 0 280 ratio should be 1 66 1 94 by UV spectrophotometry Ensure that the DNA is fully re suspended in solution before use Ensure that the DNA sample was diluted in molecular grade water and does not contain more than 2 5mM Tris 0 25mM EDTA Ensure pellets are fully re hydrated on addition of DNA Ensure 10ul of DNA solution is used per reaction Ensure that the PCR program has been entered correctly according to the instructions for use Ensure that the thermal cycler s heated lid is engaged and sufficiently tightened Refer to the thermal cycler s instructions for use for further guidance Ensure there is power to the electrophoresis tank check the power pack and clean the electrodes Run the gel in 0 5X TBE buffer En2. Refer to your electrophoresis system manufacturer s instructions for use for specific equipment details Gels should be imaged using a UV gel documentation system with UV transilluminator Note Insufficient electrophoresis may lead to large amplicons above 600bp merging with control amplicon Please ensure the electrophoresis is sufficient to visualise such amplicons Electrophoresing too long may result in the loss of small amplicons into preceding wells on a gel 9 Interpretation SSPGo kits are designed so the results can be determined manually using interpretation tables available from www biofortuna com If you have trouble accessing the website please contact your local distributor The interpretation tables may include lot specific notes which may be relevant to interpretation Affix the gel photograph to the corresponding interpretation form by matching the kit and lot numbers Examine the gel image Each reaction should contain a positive control band Refer to the interpretation tables as this may be a different size in different SSPGo products Internal control bands might appear much weaker when allele specific bands are present If an allele specific band is present but a control band is not this should still be considered a positive result Ignore any bands less than 7Obp as these are unincorporated primers Determine the positive reactions Positive reactions are indicated by bands of the expected size as stated in the interpre
3. DOT121v08 Instructions for Use for Biofortuna SSPGo HLA Typing Kits CE Revision 5 Page 1 of 12 adi BIOFORTUNA SSPGo Instructions for Use for Biofortuna SSPGo HLA Typing Kits CE Revision 5 January 2014 C C DOT121v08 Instructions for Use for Biofortuna SSPGo HLA Typing Kits CE Revision 5 Page 2 of 12 1 Intended Use Biofortuna HLA SSPGo Kits are qualitative DNA based kits for determining HLA alleles in either low resolution kits or group specific amplification of alleles in intermediate medium level resolution kits The common definition of medium level resolution is where the majority of results are clearly defined at the two digit level e g DQB1 02 DQB1 05 High resolution is generally defined as the majority of alleles identified are defined at the four digit level such as DQB1 02 01 DQB1 05 01 This is an in vitro diagnostic product intended for use by trained personnel only 2 Introduction HLA molecules play a key role in immunity and recognition of self versus non self consequently HLA genotyping and HLA matching is mandatory prior to most forms of transplantation As HLA antigens restrict the specificity of T cell mediated immune responses HLA genotyping is a useful investigative tool in any immune disorder or any immune response to pathogens vaccines or medical treatment HLA genotyping can also be used to support disease diagnosis where certain HLA alleles have been shown to be significant
4. Date YYYY MM DD ma Rell lt 2 pe Storage Temperature Lot Number A Esa Distributed by Global Trade Item Number 16 Manufacturer Contact Details Biofortuna Ltd 1 Hawkshead Road Croft Business Park Bromborough CH62 3RJ UK T 44 0 151 334 0182 E info biofortuna com W www biofortuna com www biofortuna com 18 Translations Fran aise Traductions disponibles Deutsch bersetzungen verf gbar Espa ol Traducciones disponibles Italiano Traduzioni disponibili Cesk Preklady k dispozici Danske Tilg ngelige overs ttelser EAANVEs StaBEotEc WETADPAGELC Magyar Forditasok Norske Oversettelser tilgjengelig Polska Dostepne ttumaczenia Portugu s Tradu es dispon veis Poccuto lepeBonbi AocTynHbI Slovensk mu Preklady k dispoz cii T rk eviriler mevcut Svenska vers ttningar tillg ngliga www biofortuna com CE Page 12 of 12 BIOFORTUNA SIMPLY DIAGNOSTIC C
5. act with the PCR machines heated lid This may result in poor or failed PCR amplification PCR Parameters The following PCR parameters should be used Ensure ramp speeds of 1 C per second and enable the heated lid Please refer to the thermal cycler manufacturer s user manual for full instructions for use Thermal cyclers should be calibrated according to the American Society of Histocompatibility and Immunogenetics ASHI or European Federation of Immunogenetics EFI accreditation rules Denature 94 C 5 minutes Denature 96 C 15 seconds Anneal 66 C 50seconds 10 cycles Extend 72 C 30 seconds Denature 96 C 15 seconds Anneal 64 C 50 seconds 20 cycles Extend 72 C 30 seconds HOLD at 15 C for no more than 72 hours before running the gels If necessary the PCR plate can be stored at 2 8 C for up to 24 hours before running gels Always ensure that the plates are well sealed Gel Electrophoresis These instructions apply to horizontal agarose gel electrophoresis Prepare a 2 agarose gel in 0 5x TBE buffer When the gel is cooled to about 60 C add ethidium bromide to a final concentration of 0 5ug ml Cast gel and insert microtitre format combs e g 12x8 wells with 9mm spacing Once set remove the combs and cover gel in 0 5x TBE buffer Load the entire PCR product in sequence on to the 2 agarose gel noting the position of each reaction A 1000bp ladder is recommended to aid size determination Run gel for 20 minutes at 10V cm
6. compatibility and Immunogenetics or EFI European Federation of Immunogenetics accreditation rules Gel electrophoresis reagents agarose 0 5x TBE 1000bp DNA molecular weight marker 10mg ml Ethidium Bromide Gel electrophoresis equipment gel tanks power supply gel documentation system with UV transilluminator Software to assist in the manual analysis of SSPGo kit test results and the archival data storage can be downloaded from the Biofortuna website www biofortuna com Note any change in the specified conditions such as thermal cycler ramp rates may affect interpretation of the test results 6 Safety and Warnings For in vitro diagnostic use Tests should only be carried out by appropriately trained personnel All typing results should be verified by qualified personnel and if used for a clinical decision the results should be confirmed using another typing method Handle all reagents in accordance with Good Laboratory Practice Keep pre and post PCR areas separate Do not bring any post PCR materials back to the pre PCR area Biohazard Warning Treat all blood products as potentially infectious Biohazard Warning Ethidium Bromide is a potential carcinogen If used always wear gloves a laboratory coat and protective eye glasses Biohazard Warning Take care when using UV sources always wear gloves a laboratory coat and protective eye glasses Never view the UV light source directly Material Safety Data Sheets are ava
7. eaction in 10ul Thermal cycler problems Be sure to follow the manufacturer s guidance for the maintenance and calibration of your thermal cycler Check the PCR parameters are correct according to the instructions for use Gel errors Ensure that the same volume of reaction was added to each well between 5ul and 10ul Calibrate pipettors as described by the manufacturer s instructions Use fresh ethidium bromide solution Non specific DNA concentration problem Check the DNA concentration is neither too high nor low amplification Aim for between 50 100ng of DNA per reaction in 10ul Reactions loaded in the Check alignment of PCR and gel lanes incorrect order Prevent physical overflow from adjacent wells in electrophoresis by not overloading and making sure gel is set before removing combs New allele identified Previously un sequenced alleles may be present with a new amplification pattern If using old interpretation sheets then download a more current alignment update from www biofortuna com If this does not accommodate the new pattern you should check by using a different Biofortuna kit or attempt to identify the sequence by sequence based typing Amplification pattern is Incorrect interpretation of an Check the specific Interpretation Tables for correct band not interpretable artefact as a specific band size Check if all specific amplifications are correct in size or if an artefact carry over primer dimer ha
8. er Each plate or strip is provided sealed with a sheet or cap and individually packed within a foil pouch containing a desiccant bag e PCR sealing sheets or caps e 1x Instructions for use e Certificate of analysis e Interpretation tables and MSDS can be downloaded from the Biofortuna website www biofortuna com If you are unable to download from the website please contact your local distributor CleanAmp dNTPs are licensed from Trilink Biotechnologies Inc for use in Biofortuna SSPGo products C C DOT121v08 Instructions for Use for Biofortuna SSPGo HLA Typing Kits CE Revision 5 Page 3 of 12 5 Reagents and Equipment Not Supplied Appropriate calibrated pipettors and sterile tips e g P10 pipettor with 10ul filter tips DNA isolation kit equipment UV spectrophotometer Polypropylene tubes Sterile molecular grade water A thermal cycler with the following specifications should be used e 96 well thermal cycler with heated lid with a temperature of 104 C for oil free operation e Ramp rate of 1 0 C sec e Temperature range of 4 0 C to 99 9 C e Temperature accuracy of 0 25 C for the range of 35 C to 99 9 C e Temperature calibration traceable to a reference standard e Program the thermal cycler using the PCR Cycling Parameters in Section 8 below Note For specific thermal cycler information refer to the manufacturer s user manual Thermal cycler should be calibrated according to ASHI American Society of Histo
9. f the assay may be due to not tightening the lid sufficiently This can lead to evaporation and condensation of the PCR reaction half way up the PCR well and can lead to PCR failure Be sure to follow the manufacturer s guidance for the maintenance and calibration of your thermal cycler Check the PCR parameters are correct according to the instructions for use Ensure there is an adequate seal across all the wells Pay particular attention to the wells close to the edges of the PCR plate or strips Ensure the heated lid is enabled and sufficient compression is applied via the lid Ensure Biofortuna sealing sheets supplied are used For further supplies please contact your distributor No DNA present Ensure DNA is present in all wells Wrong volume Ensure 10ul of DNA solution is added to each reaction Too much DNA added Concentration of above 200ng or less than 20ng may cause PCR failure Contaminants in DNA may lead to sporadic or widespread failure to amplify DOT121v08 Instructions for Use for Biofortuna SSPGo HLA Typing Kits CE Revision 5 Page 10 of 12 Probable Cause Smeary gel image Check the concentration and purity of the DNA Adding too much DNA to the PCR reactions can result in smeary gel images Degraded or low purity can be the cause Obtain a fresh sample of DNA Weak amplification DNA concentration problem Check the DNA concentration is neither too high nor low Aim for 50 100ng of DNA per r
10. ilable from www biofortuna com 7 Storage and Stability Biofortuna SSPGo kits should be stored at 2 28 C Once PCR vessels are removed from the foil pouches the reagents should be re hydrated with DNA within 3 hours Refer to packaging for expiration date Do not use products after the printed date Do not use kits if the foil pouch is ripped or perforated or if there is no desiccant bag present C CE DOT121v08 Instructions for Use for Biofortuna SSPGo HLA Typing Kits CE Revision 5 Page 4 of 12 Using the sealing sheets or caps provided only ensure PCR vessels are sealed tightly after adding DNA as omitting this may lead to evaporation during PCR amplification Pay particular attention to edges and corners Note 1 If necessary the out of pouch non hydrated PCR plates and strips may be held for up to 3 hours prior to addition of DNA at a temperature of up to 21 C and humidity of no more than 60 Note 2 Once hydrated with DNA PCR strips and plates from freshly opened pouches can be stored for up to 24 hours at 2 8 C before the PCR step provided that the wells are well sealed to avoid evaporation 8 Directions for Use DNA Sample Requirements Each reaction in the test is optimised to utilise between 50 100ng of DNA but it is critical that each reaction should be re hydrated with exactly 10ul of liquid Therefore the test can only be performed with 10ul of DNA at 5 10ng ul Dilute the DNA to the required concentrati
11. ly associated with disease states Most HLA genes are highly polymorphic and generally DNA genotyping is required for accurate determination of HLA antigens PCR genotyping using Sequence Specific Primers SSP is a rapid method of HLA genotyping particularly suitable for situations where medium level resolution is required Biofortuna SSPGo kits all feature complete dried reactions including polymerase so that all the user has to do prior to PCR is add DNA Every effort is made to keep the kits updated with new IMGT HLA alignment releases Kit updates are available from www biofortuna com 3 Test Description PCR SSP is based on the principle that only primers with completely matched 3 terminals to a target sequence will amplify Mismatched primers do not yield positive amplification products An internal control primer pair which amplifies a conserved region of a housekeeping gene is included in every PCR reaction mix the internal control primer pair is an indicator of the integrity of the PCR reaction SSP genotyping generally uses multiple reactions that when analysed together indicate the genotype Visualisation of the amplified products can be achieved using agarose gel electrophoresis systems which separate the DNA fragments by size 4 Kit Contents e Polypropylene PCR plates or strips consisting of between 8 and 96 PCR wells kit dependant each well containing pre dispensed freeze dried primers polymerase dNTPs and buff
12. n Section 8 V Ensure the DNA contacts the dry reagents in each reaction prior to thermal cycling vi Seal the reactions with the sealing sheet or PCR tube caps provided Ensure the seal is as tight as possible to prevent evaporation Pay particular attention to edges and corners vii Place tray or strips directly into the thermal cycler Ensure the vessels are fully inserted into the block and the lid is fully compressed Failure to do so can lead to individual PCR failure due to PCR evaporation and condensation viii Run PCR program refer to PCR Parameters RE SUSPENSION NOTE Once PCR wells are removed from the foil pouches the reagents should be re hydrated with DNA promptly See Note 1 and Note 2 for additional information NO TEMPLATE CONTROL NOTE Some kits include a No Template Control NTC as the final reaction on the plate This reaction contains a purple dye to distinguish it from the rest of the reactions The NTC is designed to detect PCR contamination or genomic DNA contamination that may be present in the water used to re suspend your DNA If PCR contamination is present variable size amplicon s will be observed C C DOT121v08 Instructions for Use for Biofortuna SSPGo HLA Typing Kits CE Revision 5 Page 5 of 12 PCR PLATE STRIP HEIGHT PROFILE NOTE It is recommended that the height profile of plates and strips are equivalent when placed in the same PCR machine Different height profiles can cause poor cont
13. nge A total of 958 960 individual reactions distributed equally across three external sites were concordant to DNA samples resulting in an overall 99 7 concordance 0 995 LCL Lot to Lot reproducibility was performed on three lots of a representative SSPGo HLA Test kit by a single site operator using the DNA concentration range of 5 10 ng uL The lot to lot reproducibility for SSPGo HLA Type testing was 100 concordant to predicate device for 130 130 DNA samples tested across three 3 lots of the representative SSPGo HLA Type tests for a total of 390 tests C CE DOT121v08 Instructions for Use for Biofortuna SSPGo HLA Typing Kits CE Revision 5 Page 7 of 12 Concordant SSPGo HLA positive verification was obtained for Class and Class II alleles in comparative testing with LABType SSO tested clinical samples SSPGo HLA internal testing provided concordant results to reference DNA samples except for unavailable samples such as certain rare HLA DPB1 alleles and rare HLA B alleles and allele groups such as B 59 01 B 78 and B 83 01 The following rare allele groups were not confirmed B 59 B 78 B 83 DPB 21 01 DPB 26 01 02 DPB 27 01 DPB 28 01 DPB 29 01 DPB 31 01 DPB 34 01 DPB 35 01 01 DPB 39 01 DPB 45 1 DPB 46 01 DPB 51 01 DPB 55 01 DPB 59 01 DPB 63 01 DPB 81 01 DPB 85 01 amp DPB 105 01 12 References 1 Bunce M et al Tissue Antigens 1995 Nov 46 5 355 67 2 Saiki RK et al Nature 1986 Nov 13 19 324 6093 163 6
14. nsure correct performance Each reaction has been validated against a minimum of 47 well characterised cell line DNA samples Biofortuna recommend that any laboratory should internally validate any new typing products before use on clinical samples Only fully trained and qualified personnel should perform diagnostic typing and results should be cross checked by another trained member of staff 11 Clinical data An in vitro diagnostic device study was performed at five test centres comparing the performance of SSPGo HLA Typing Kits to the predicate device One Lambda Labtype SSO The locus typing completed for the SSPGo trial provided test results for HLA A HLA B HLA C HLA DQA1 HLA DQB1 HLA DRB1 3 4 5 HLA DPB1 DPA1 and HLA DQA1 05 DQB1 02 DQ8 The clinical performance of the SSPGo HLA Typing Kits provided an overall 98 3 100 concordance to the predicate device SSO method with not less than 95 confidence for genotyping of Class and II HLA alleles tested from DNA derived from whole blood at 5 10ng ul Excluding three 3 confirmed non concordant genotyping results 100 concordance was achieved for 1 222 samples for 3 lots of each test kit across clinical test sites in the US and UK Site to site reproducibility for SSPGo HLA Typing kits was performed by three sites using a representative panel of 8 SSPGo PCR reactions against supplied DNA samples formulated at 4ng ul and 11ng ul the extremes of the test kit concentration ra
15. on in sterile molecular grade water only Caution Ensure the final DNA sample does not contain more than 2 5mM Tris 0 25mM EDTA Only use DNA extracted from citrate and EDTA collected samples As heparin may inhibit PCR it is recommended that DNA should not be extracted from heparinised blood samples Haemoglobin has been shown to interfere with SSPGo HLA kits when present in DNA samples at greater than 1 mg dL DNA can be extracted using all of the traditional extraction methods Please ensure that the OD2 0 280 of the DNA sample falls between 1 66 and 1 94 as measured by UV spectrophotometry Pre PCR Directions i Remove an SSPGo plate or strip from a sealed pouch ii Note the product lot number of the assay iii Ensure all the freeze dried pellets PCR reagents are at the bottom of the plate tube wells prior to removing the seal or cap If not gently tap to get the pellet to the bottom of the tube Note that the first reaction of each test locus is always pale pink in colour to the rest of the kit Red when hydrated Some PCR plates contain a purple coloured integral no template control reaction in the last well of the plate iv Using sterile equipment pipette 10ul DNA solution into each reaction of the plate or strip See note in Section 8 on DNA Sample Requirements If the plate contains a purple coloured integral no template control then pipette 10ul of sample diluent without DNA to it See note on No Template Control i
16. s been misinterpreted as an amplification Reactions loaded in the Check alignment of PCR and gel lanes incorrect order Individual PCR failure Check all internal positive controls are present Re interpret without any missing reactions Small amplicons missing Electrophoresed too far small amplicons have run off the end of the gel or past the ethidium bromide front or are dispersed by entering preceding gel well Use DOT121v08 Instructions for Use for Biofortuna SSPGo HLA Typing Kits CE Revision 5 Page 11 of 12 Probable Cause electrophoresis conditions suitable for your gel system local distributor New allele identified in sample New alleles may occasionally be discovered that may give rise to an amplification pattern that does not correspond to an existing allele s Please contact your 14 Revision History Revision From version 4 to revision 5 Revision Date 21 January 2014 nce loa Section 5 Reagents and Equipment Not Supplied Kit lot number to be matched correctly with the lot number on the interpretation table Section 11 Clinical Data Addition of this complete section Section 14 Revision History Addition of this complete section DOT121v08 Instructions for Use for Biofortuna SSPGo HLA Typing Kits CE Revision 5 15 Guide to Symbols Used Number of Tests EC Representative Consult Instructions for Use Site of Manufacture In Vitro Diagnostic Expiry
17. sure 0 5ug ml of fresh ethidium bromide is used Check that there is sufficient UV illumination when imaging gels Refer to the gel tank and power pack manufacturer s instructions for further guidance Insufficiently sealed plates can lead to evaporation during PCR Biofortuna supplies recommended sealing sheets in the kit For further supplies please contact your distributor Ensure there is an adequate seal across all the wells Pay particular attention to the wells close to the edges of the PCR plate or strip DOT121v08 Instructions for Use for Biofortuna SSPGo HLA Typing Kits CE Revision 5 Page 9 of 12 Probable Cause Random drop outs of Gel errors control and or allele specific amplicons Thermal cycler problems Evaporation problems Sporadic failure due to DNA problems Ensure that all of the wells have been loaded onto the gel in the correct order and the same volume of PCR reaction was added to each one Calibrate pipettors as described by the manufacturer s instructions Check that the wells are properly formed in the gel Take care when removing combs as it is possible to tear the bottom of the wells Ensure that the agarose is fully dissolved before casting the gel Ensure that the gel is not run too long as smaller amplicons may run off the end Ensue the gel has ran long enough to allow bands to separate Use fresh ethidium bromide solution Failures particularly around the edge o
18. tation tables Be aware that there may be more than one product size in a given reaction these are multiplexed reactions and are noted on the interpretation tables C C DOT121v08 Instructions for Use for Biofortuna SSPGo HLA Typing Kits CE Revision 5 Page 6 of 12 Compare positive reactions with the interpretation tables A positive result in a reaction indicates the presence of at least one of the alleles listed against it on the interpretation table Any given allele may be amplified in several tubes if the allele is present there should be a positive reaction in all of the relevant reactions Control Band Direction of electrophoresis Allele Specific Band Figure 1 Examples of positive reactions indicated by the presence of allele specific bands and control bands reactions 1 3 negative reactions indicated by the presence of control bands but absence of allele specific bands reactions 4 7 and a failed reaction indicated by the absence of any bands reaction 8 Ensure the kit lot number is matched correctly with the lot number on the interpretation table Software to assist in the manual analysis of SSPGo kit test results and the archival data storage can be downloaded from the Biofortuna website www biofortuna com 10 Quality Assurance and Control Each SSPGo lot is checked for quality before any product leaves Biofortuna Samples of each kit lot are checked against a defined panel of human DNA samples to e
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