IFU-HISTOSPOT-On-Call-Typing-V3-2014-EN
Contents
1. PCR Primers PCR Strips for locus specific amplification of HLA A B C 10 strips DRB1 DRB3 4 5 DQ DPB1 contains dried primers PCR Caps PCR caps 10 pcs PCR Buffer PCR buffer ready to use contains dNTPs Taq polymerase 1100 ul reaction buffer 0 05 sodium azide MgCl2 Magnesium chloride 6 mM ready to use contains 0 001 600 ul Proclin 300 With each kit there is a CD containing the batch file that has to be stored within the database of the HISTO MATCH interpretation software for details see Instructions for use for HISTO MATCH For each kit there are lots and batches e Kit e g HISTO SPOT A defines the locus tested e Lot e g A084 A085 defines the layout and specificity of the probes that are contained in the kit A single lot can contain many different batches e Batch e g A085 1 A085 2 A085 3 defines how a probe reacts in comparison to the control probes cut off values and defines the manufacture and expiry date of the strips 3 2 Reagents and equipment required but not provided MR SPOT processor including HISTO MATCH software REF 726100 HISTO SPOT Reagent Kit REF 726098 Pipette tips for the MR SPOT processor 1000 ul REF 726099 and 200 ul REF 726097 DNA extraction reagents no salting out method Thermal cycler Deionized water Variable pipettes range 0 5 1000 ul and disposable tips 4 STORAGE2. DRB1 DRB3 4 5 DQ DPB1 PCR Primers PCR strips with dried primers for amplification of the loci HLA A B C DRB1 DRB3 4 5 DQ DPB1 PCR Caps PCR caps PCR Buffer PCR buffer MgCl2 Magnesium chloride solution Instructions for use in other languages see http www bag healthcare com en Diagnostika Downloads or phone 49 0 6404 925 125 Page 10 of 10
3. also functioning as a reference for the allele specific probes Positive allele specific probe 4 O Negative allele specific probe 0 O 0 IO Jo o olol o o lo lolo 0 0 O 10 O 0 1 0 0 JO Jo Jo 9 0 0 Jo Jo Jo 0 0 0 jo o 0 0 10 0 0 0 eiclololelololele o le le lo lo jolole Figure 3 Schematic illustration of the result and function of the probes Page 7 of 10 Instructions for use HISTO SPOT On Call Typing Kit Version 3 2014 6 WARNINGS AND PRECAUTIONS HISTO SPOT is designed for in vitro diagnostic use and should be used by properly trained qualified staff All work should be performed using Good Laboratory Practices Biological material used for extraction of DNA e g blood or human tissue should be handled as potentially infectious When handling biological material appropriate safety precautions are recommended do not pipet by mouth wear disposable gloves while handling biological material and performing the test disinfect hands when finished the test Biological material should be inactivated before disposal e g in an autoclave Disposables should be autoclaved or incinerated after use Spillage of potentially infectious materials should be removed immediately with absorbent paper tissue and the contaminated areas swabbed with a suitable standard disinfectant or 70 a
4. com Fax 49 0 6404 925 460 Fax 49 0 6404 925 421 verkauf bag healthcare com service bag healthcare com Instructions for use HISTO SPOT On Call Typing Kit Version 3 2014 1 PRODUCT DESCRIPTION The HISTO SPOT SSO system is an in vitro diagnostic test for tissue typing of HLA alleles on a molecular genetic basis and provides low to medium resolution typing results It consists of the HISTO SPOT typing kits the HISTO SPOT reagent kit the MR SPOT processor and the HISTO MATCH interpretation software The HISTO SPOT typing kits contain all components required for the PCR reaction and testwells with immobilized sequence specific oligonucleotide probes for the detection of the PCR products The HISTO SPOT reagent kit contains the reagents for the hybridisation and detection and can be used in combination with all HISTO SPOT typing kits The MR SPOT processor is specifically designed to be used with the HISTO SPOT kits in order to process between 1 and 96 samples automating the process from hybridisation detection through to result interpretation The HISTO MATCH software is required to interpret the results The HISTO SPOT On Call Typing Kit is a combination of all tests necessary for an organ transplantation The kit is designed to make the workflow especially in the on call situation as easy as possible The amplification primers are pre dropped and dried in PCR strips and the SSO tests are combined in a holder
5. data transfer Failure in data transfer Manually transfer data using USB drive No result Failure to grid image Perform manual gridding Only control spots positive Failure to add DNA to PCR or amplification failure Repeat whole assay and check PCR product on gel False positive probes Too much DNA used or conjugate concentration too high not spun down Check DNA concentarion Spin down conjugate before use Exon dropout DNA concentration too high or DNA degraded check DNA concentration run a gel with the DNA No result inconclusive result Mistake in conjugate dilution Page 9 of 10 Repeat assay due to weak signals or poor amplification Check hybridisation Instrument malfunction temperature on instrument Instructions for use HISTO SPOT On Call Typing Kit Version 3 2014 11 TRADEMARKS USED IN THIS DOCUMENT PRODUCT Proclin is a trademark of Rohm and Haas company BCIP is a trademark of Sigma Aldrich Co Veriti is a trademark of Applied Biosystems 12 EXPLANATION OF SYMBOLS USED ON LABELING IVD For in vitro diagnostic use ri Storage temperature LOTI Batch code 2 Use by REF Catalogue number f Consult instructions for use HLA TYPING Intended use HLA typing Combistrip Testwells with bound probes for typing the loci HLA A B C
6. reagents when removing aliquots from reagent bottles The use of sterile disposable pipettes and pipette tips is recommended Do not use reagents with evidence of turbidity or microbial contamination Material Safety Data Sheets MSDS are available to download at www bag healthcare com 7 SPECIFIC PERFORMANCE CHARACTERISTICS 7 1 Evaluation For the HISTO SPOT SSO kits an evaluation study with at least 180 samples has been performed for each single locus The results were compared to other typing methods e g SSP ing No discrepancies were observed between the typing methods For every lot the specificity of each probe was verified with DNA from reference samples Page 8 of 10 Instructions for use HISTO SPOT On Call Typing Kit Version 3 2014 7 2 PCR Amplification reaction The alleles amplified with each HISTO SPOT SSO kit the HLA nomenclature release referred to and the exons that are amplified are given in the respective lot specific information This is found ona CD in each kit 7 3 Assay resolution The HISTO SPOT SSO typing system is designed to provide unambigious results at least at allele group level i e for two digits Different combinations of alleles that cross allele groups but have the same positive probe pattern are considered as ambigious 8 LIMITATIONS OF THE METHOD Because of the high susceptibility of the PCR method to variations in DNA concentration and quality only DNA samples should be
7. screen will appear Follow the process as indicated on the screen Details are described in the User Manual for the MR SPOT processor 5 4 3 Transfer of results to a PC for interpretation Transfer the data to the HISTO MATCH software via network or USB stick as described in the manual for the HISTO MATCH software Page 6 of 10 Instructions for use HISTO SPOT On Call Typing Kit Version 3 2014 5 4 4 Interpretation of results Open the HISTO MATCH software if this is not already installed it can be installed from the CD delivered with the MR SPOT processor and interpret the data as described in the manual for the HISTO MATCH software The images should look like the example shown in figure 2 and figure 3 gives a schematic illustration of the result and the functions of the different probes The colour of the circles around the probes indicate their function see IFU for the HISTO MATCH software for details Figure 2 Image of a result for HLA A Positional probes They are reacting with P the amplification primers in the mastermix and indicate that mastermix was added and that all reagents during the SSO assay were added correctly Furthermore they allow the software to locate the image The pattern is specific for the batch Amplification control for Exon 2 and Exon 3 in duplicate Those probes are universal for all alleles of the respective locus and show that the PCR was successful They are
8. use two separate rooms Amplicon should not be taken back into PCR set up area Use devices and other materials only at the respective places and do not exchange them 5 2 DNA isolation Prepare sample DNA by the laboratory standard method for DNA isolation for use in PCR preferably no salting out method The presence of heparin potentially inhibits PCR Therefore EDTA or Citrate Blood is recommended for typing The sample DNA should have a concentration of 15 30 ng ul Page 4 of 10 Instructions for use HISTO SPOT On Call Typing Kit Version 3 2014 5 3 Amplification For each combi test take one PCR strip PCR Primers with pre dropped amplification primers from the fridge Make a pre mix with the following components for each sample 80 ul PCR buffer 40 ul MgCl 40 ul Sample DNA 15 30 ng ul Pipet 20 ul of the pre mix into each well with the pre dropped primers and re suspend the primers with the pre mix Note It is important that the DNA concentration is in the range between 15 and 30 ng ul Higher concentrations may result in false positive probe reactions and lower concentraions may cause amplification failures OOOO Seal the PCR strips with the caps spin them down place them in the thermal cycler and amplify under the following conditions Programme Step Time Temperature No of Cycles First Denaturation 2 Min 96 C 1 Cycle Denaturation 15 Sec 96 C 10 Cycles Ann
9. used that have a concentration between 15 and 30 ng ul and a purity index extinction ratio OD2 0 OD290 between 1 5 and 2 0 Extreme care should be taken to prevent contamination of the kit reagents and other laboratory materials and equipment with amplicons or DNA Regular wipe tests e g BAG Wipetest REF 7091 and the negative controls with each assay are strongly recommended The hybridisation assay is a very temperature sensitive process Therefore the HISTO SPOT SSO kits should only be used in combination with the MR SPOT processor to ensure correct temperatures and incubation times All instruments e g pipettes thermocyclers heat blocks MR SPOT processor must be calibrated according to the manufacturers instructions Accuracy and temperature uniformity of thermocyclers may be tested with the BAG CyclerCheck REF 7104 9 INTERNAL QUALITY CONTROL Internal quality control of new lots of the HISTO SPOT SSO kits can be performed using a combination of DNA samples with known HLA type Internal positive controls are contained in each test well to ensure sucessful amplification and hybridization Negative controls to detect possible contaminations are recommended Use a PCR reaction without DNA in the subsequent hybridization assay as a negative control 10 TROUBLESHOOTING Symptom Possible problem s Potential Solution s Instrument Malfunction Numerous Refer to MR SPOT manual Error message at
10. AND STABILITY All reagents and components of the kits should be stored at 2 8 C The expiry date is indicated on the label of each reagent and is valid for the originally sealed reagents The expiry date indicated on the outer box label refers to the reagent with the shortest stability contained in the kit The opened reagents should be used within 3 months The conjugate dilution must always be prepared afresh for each test run Page 3 of 10 Instructions for use HISTO SPOT On Call Typing Kit Version 3 2014 5 TEST PROCEDURE 5 1 Safety conditions and special remarks Molecular genetic techniques are particularly sensitive methods and should be performed by well trained personnel experienced in molecular genetic techniques and histocompatibility testing The results from these tests must not be used as the sole determinant for making clinical decisions Transplantation guidelines as well as EFI standards should be followed in order to minimize the risk of false typings in the particular case of discrepancies in serological and molecular genetic methods Special safety conditions must be noted in order to avoid contamination and thus false reactions Wear gloves during work powder free if possible Use new tips with each pipeting step with integrated filter Use separate working areas for pre amplification DNA isolation and preparation of the reactions and post amplification hybridisation and detection Preferably
11. DO BAG neam care Instructions for use HISTO SPOT On Call Typing Kit REF 726070 Test kit for typing of HLA alleles on a molecular genetic basis 10 tests for HLA A B C DRB1 DRB3 4 5 DQ DPB1 IVD CEs Version 3 2014 Issued 2014 01 Contents 1 PRODUCT DESCRIPTION cutsiioniinia nina hahaha 2 Ze TEST PRINCIPLE wcnnccsncatncassusistasasainuauasaunsatacainussasaimiessuatenasaiainceieonsenssnuatnteusunsenauiiatncedeoasenaane 2 3 MATERIA e a med nats ee deinen nse am E A ston sented deelenen senso taieteasasenten ae 3 3 1 Reagents provided with the HISTO SPOT On Call Typing Kit ccccccccccsceseseeecseteeseeeees 3 3 2 Reagents and equipment required but not Provided sceesseeeesseseeeseeeeeseeeeeeseeeeseeeeeeees 3 4 STORAGE AND STABILITY acces cccadouetuvnnoeuteninieveseipee nid neers eroe einenixuainumenrensipoeriieredipeuacs 3 Be TESI PROGEDURE creceriracinr ar nA an R AAE A R RRR 4 5 1 Safety conditions and special rermarks cccccccceceeeeeeeeeeeneeeeeeeeeeeeeeesaaaaaeeeeeeeeeeessnaaaaeeeeees 4 52 EIA NS Oleg sainectecgsancuistnsanececesasae a ia sweake rane pie E E E inne A A E RE 4 53 AmplificatON eerie ree pretest ear EE E ae 5 5 4 Automated hybridisation assay on the MR SPOT PrOCESSO iersinii aaea 6 5 4 1 Reagent preparation acts tala dts cals neces enash es ete ececnand aa stcene eaciastce desea eainouesnenct 6 SAD Setup of the MR SPOT Processor ccciccccs
12. There is a special option for the procedure and the interpretation of this test combination in the HISTO MATCH interpretation software 2 TEST PRINCIPLE The test includes four basic steps DNA isolation PCR amplification hybridisation and detection data interpretation DNA isolation is performed on the clinical sample using the DNA isolation method established in the laboratory or using commercial kits Then the DNA is amplified in a locus specific PCR reaction using PCR strips PCR buffer and the MgCl solution provided with the kit The specificity of the amplification is governed by a set of biotinylated primers that have been designed to uniquely amplify the chosen HLA locus After the PCR amplification process the PCR strips containing biotin labelled amplicon are transferred to the MR SPOT processor MR SPOT adds hybridisation buffer to each well and then transfers each amplicon plus hybridisation buffer to a test well containing an array of immobilized sequence specific oligonucleotide SSO probes These probes are either single oligonucleotide probes or a combination of 2 or more individual probes immobilised in the same spot Mosaic Probes which have been designed to improve the identification of cis located polymorphisms The biotin labelled amplicon binds to those SSO probes that contain a complementary target sequence and can then be detected by a colourimetric reaction In order to prevent unspecific binding of the
13. amplicon on the surface of the test wells MR SPOT has blocked the wells with blocking buffer before transferring the amplicon After a stringent wash step to remove all unbound amplicon a streptavidin alkaline phoshatase conjugate is added to the wells and binds to the biotin labelled amplicon captured by the SSO probe After further wash steps BCIP NBT substrate is added which produces a blue purple colour when converted by the alkaline phosphatase The resulting coloured dots in the bottom of each test well are photographed by MR SPOT and the image is transferred into the HISTO MATCH software installed on the PC of the user The image analysis program of the HISTO MATCH software determines the intensity of each spot in the array and compares it to the intensity of the background From this data the positive and negative reactions are calculated The pattern matching program of the HISTO MATCH software determines the HLA type of the sample based on the specific hybridisation pattern Page 2 of 10 Instructions for use HISTO SPOT On Call Typing Kit Version 3 2014 3 MATERIAL 3 1 Reagents provided with the HISTO SPOT On Call Typing Kit The reagents contained in one kit are sufficient for 10 tests Each reagent set contains Combistrip Testwells for typing of the loci HLA A B C DRB1 DRB3 4 5 10 combitests DQ DPB1 negative control combined in a holder contains immobilized sequence specific oligonucleotide probes
14. ccscaiientecedeatcaceteasluaedetinccceucbvdessdasiciesieanbeastatucs 6 5 4 3 Transfer of results to a PC for interpretation sssseeesseesseeenrrrrerssrrrrnrrnnnrssrrrrrrrnn 6 SAA Interpretation of results eiccescreceslaccets cate kanepancednasd dathareeeicieid dedueatusiduarddediametidveleeden 7 6 WARNINGS AND PRECAUTIONS sincsciccsncnsecesacetacenstatietasnspieustatecetmsnsece R RE RR Ea 8 7 SPECIFIC PERFORMANCE CHARACTERISTICS cesseeseeesseessssseessssessseeessssssseeeeees 8 Fal Evaluate eean a a E A E tae piadseo Ea 8 7 2 PCR Amplification reaction c2 ctsseccdis ice ctacteynwwico nieces ass ode es ag Sis aid ages 9 7 3 ASSAY TOSOMIMON sirisser a EE acta E E EAEE AEE OE EA REEE EAN 9 8 LIMITATIONS OF THE METHOD ccccccccscc sete scccsssescessteccnesscsceescccdecnwnessecswtescecnwnesteessaccdecnse 9 9 INTERNAL QUALITY CONTROL cccscsivescanessecsanscnkssacessessanauessaeesveiteieseusseucsessteiereisteessacstavess 9 107 TROUBLESHOOTING sirrane aE N aE ATRAE E ATEAREN 9 11 TRADEMARKS USED IN THIS DOCUMENT PRODUCT ccccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 10 12 EXPLANATION OF SYMBOLS USED ON LABELING ccccccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 10 BAG Health Care GmbH Auftragsannahme Ordering Customer Service AmtsgerichtsstraBe 1 5 Tel 49 0 6404 925 0 www bag healthcare com Tel 49 0 6404 925 450 Tel 49 0 6404 925 125 35423 Lich Germany Fax 49 0 6404 925 250 info bag healthcare
15. ealing Extension 60 Sec 65 C Denaturation 10 Sec 96 C 20 Cycles Annealing 50 Sec 61 C Extension 30 Sec 72 C Hold o0 22 C The conditions are the same for all thermal cyclers however the overall time required for this step will vary according to the ramping speed of the specific thermal cycler The following thermal cycler models haven been validated with HISTO SPOT SSO Applied Biosystems PE 9600 PE 9700 use ramp rate of PE 9600 Veriti Biorad PTC 100 PTC 200 Mycycler Eppendorf Mastercycler EP Gradient S If other thermo cyclers are used the validation has to be done by the user It is generally recommended to use a ramp rate of 1 2 C sec Once the amplification step is complete the samples may be tested immediately or stored at 2 8 C for up to 5 days It is not necessary to make a gel to control the amplification It is also not always helpful because assay results may be good although there was only a very faint band visible on the gel Page 5 of 10 Instructions for use HISTO SPOT On Call Typing Kit Version 3 2014 If a gel should be done anyway you ahould not take more than 2 3 ul of the amplicon to do this The amplicon sizes for the different kits are given on the information CDs that can be found in every kit Hit Table in Excel format Second sheet Notes 5 4 Automated hybridisation assay on the MR SPOT processor 5 4 1 Reagent preparation Take HISTO SPOT reage
16. lcohol Material used to clean spills including gloves should be inactivated before disposal e g in an autoclave Blocking Buffer Hybridisation Buffer Stringent Wash Buffer and TBS Wash Buffer contain ProClin 150 and the Magnesium Chloride Solution contains ProClin 300 The reagents contain 0 001 preservative only nevertheless avoid contact with the skin and mucous membranes PCR buffer and Conjugate contain the preservative sodium azide The reagents contain lt 0 1 sodium azide which is not considered to be a harmful concentration Nevertheless avoid contact with the skin and mucous membranes Sodium azide may react with lead and copper plumbing to form explosive metal azides While disposing of sodium azide containing solutions down laboratory sinks flush the drains with a large volume of water to prevent azide build up All work with reagents should be handled with the appropriate precautions Wear eye protection laboratory coats and disposable gloves when handling the reagents Avoid contact of these materials with the skin eyes or mucous membranes If contact does occur immediately wash with large amounts of water Burns can occur if left untreated If spills of reagents occur dilute with water before wiping dry Do not expose Substrate to metals oxidising agents Disposal of all samples unused reagents and waste should be in accordance with country federal state and local regulations Avoid microbial contamination of
17. nts and HISTO SPOT testwells Combistrip out of the fridge and allow them to warm to room temperature Salt crystals may be observed in the hybridisation buffer and in the stringent wash solution If crystals are present warm reagents up to 30 C to dissolve Warm the whole content of the bottle not an aliquot The conjugate has to be diluted 1 1666 in blocking buffer The conjugate dilution must always be prepared afresh for each test run The conjugate has to be vortexed and spun down each time before before the dilution step The required volumes of the reagents will vary depending on the number of strips to be tested MR SPOT displays the required volumes for the chosen number of strips Fill the required volumes of the reagents into the corresponding labelled reservoirs Carefully open the PCR strips and place them into the sample block Make sure that the positioning and orientation of the PCR strip is correct see instructions on the touch screen Place the holder with the test wells Combistrip in the reaction plate Make sure that the positioning and orientation of the holder is correct see instructions on the touch screen Please make sure that there is no dirt or plastic particles in the reaction plate holder because this may disturb the heat transfer during hybridization 5 4 2 Setup of the MR SPOT processor Switch on the MR SPOT processor the internal PC and the touch screen The start up
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IFU HISTOSPOT On Call Typing V3 2014 EN