histo spot
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1. HISTO SPOT PCR Caps REF 726090 HISTO SPOT PCR Foils 726089 Thermal cycler Page 3 of 11 Instructions for use HISTO SPOT SSO kits Version 9 2013 Deionized water e Variable pipettes range 0 5 1000 ul and disposable tips 4 STORAGE 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 Individual strips of 8 wells may be opened the required number of wells for a test run can be snapped off and the unused wells returned to the opened foil bag and stored for future use with the kit Test wells stored in open foil pouches should be used within 30 days of being opened The other opened reagents should be used within 3 months The conjugate dilution must always be prepared afresh for each test run 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 false2. 49 0 6404 925 125 Fax 49 0 6404 925 421 service bag healthcare com Instructions for use HISTO SPOT SSO kits Version 9 2013 1 PRODUCT DESCRIPTION The HISTO SPOT SSO system is an in vitro diagnostic test for tissue typing of HLA alleles ona 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 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 t
3. are contained in the kit A single lot can contain many different batches 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 Reagents provided with the HISTO SPOT Reagent kit The reagents contained in one kit are sufficient for 96 tests Each reagent set contains BLOCKBUF Blocking Buffer ready to use contains 0 001 Proclin 150 HYBBUF Hybridisation Buffer ready to use contains 0 001 dye 0 1 sodium dodecyl sulphate 0 001 Proclin 150 STRGWASH Stringent Wash Buffer ready to use contains 0 001 dye 0 1 sodium dodecyl sulphate 0 001 Proclin 150 TBSWASH TBS Wash Buffer Tris Buffered Saline ready to use contains 20 mM Tris 0 003 dyes 0 001 Proclin 150 SUBS BCIP NBT Substrate ready to use 5 bromo 4 chloro 3 indolyl phosphate nitroblue tetrazolium chloride CONJ Conjugate Streptavidin Alkaline Phosphatase concentrate contains lt 0 1 sodium azide to be diluted 1 1666 in Blocking Buffer 3 3 Reagents and equipment required but not provided MR SPOT processor including HISTO MATCH software REF 726100 Pipette tips for the MR SPOT processor 1000 ul REF 726099 and 200 ul REF 726097 e DNA extraction reagents no salting out method Skirted PCR plates with lids or adhesive film
4. 726010 HISTO SPOT A 4D 96 tests 726020 HISTO SPOT B 4D 96 tests 726030 HISTO SPOT C 4D 96 tests DO BAG neam care Instructions for use HISTO SPOT SSO Kits Test kits for tissue typing of HLA alleles on a molecular genetic basis D CEs 726040 HISTO SPOT DRB1 4D 96 tests 726045 HISTO SPOT DRB3 4 5 24 tests 726050 HISTO SPOT DQB1 4D 96 tests 726060 HISTO SPOT DPB1 96 tests 726098 HISTO SPOT Reagent Kit Contents 1 PRODUC TIDES GRUP TION iaiccutsnnttscnsieiansssncns sonuielaseseensnentiebeeasueatenonaenerenanee 2 ESP IP INP Bi rescacecnatsacvetcesaceecsackttassactnccsiavutessdenaeasSaceansactaeccdactuceusieius 34 MATERIAL merian iain E E A E A cep ia wine EEE A 3 1 Reagents provided with the locus specific HISTO SPOT typing kits 3 2 Reagents provided with the HISTO SPOT Reagent kit o n 3 3 Reagents and equipment required but not provided sssseeeeeeeeeeeeees 4 STORAGE AND STABILITY sinnciivndsmnnbeuddnnnauedaunuemnsiouns 5 TEST a 2 O10 a 010 Seer eee ee aaaeei aii Ein 5 1 Safety conditions and special remarkS ccceeeeeeeeeeeeeeeeeeteeeeeeeeeeeeeeeeaaees 52 JOIN PRIS OAM earesned asassesvadeetcut onem ited araeted 5 39 PAMIPINIGATION sosa en ade etanetenie aaa adnate te E NEE A EEKE 5 4 Automated hybridisation assay on the MR SPOT DNOCESSOM sinistaisvcncdsneneavas 5 4 1 Reagent preparation cecee
5. B1 locus Mastermix for amplification of the HLA DRB3 4 5 loci Testwells with bound probes to type the HLA A locus Testwells with bound probes to type the HLA B locus Testwells with bound probes to type the HLA C locus Testwells with bound probes to type the HLA DRB1 locus Testwells with bound probes to type the HLA DQB1 locus Testwells with bound probes to type the HLA DPB1 locus Testwells with bound probes to type the HLA DRB3 4 5 loci Magnesium chloride solution Blocking buffer HYBBUF Hybridisation buffer Stringent wash solution Tris buffered saline Substrate ONJ O IO op Cl yO Co op Q gt n I Streptavidin Alkaline Phosphatase Conjugate Instructions for use in other languages see ttp www bag healthcare com ttp service bag healthcare com or phone 49 0 6404 925 125 Page 11 of 11
6. NA 15 30 ng ul Total volume for each amplification reaction is 20 ul Premix for multiple samples no of samples 2 x 10 pl Mastermix use 15ul premix no of samples 2 x 5 pl MgCl per sample 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 concentrations may cause amplification failures If a negative control should be done prepare one PCR reaction with distilled water instead of sample DNA Seal the amplification tubes with lids or adhesive film and shortly spin down the liquid Place 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 Annealing Extension 60 Sec 65 C Denaturation 10 Sec 96 C 20 Cycles Annealing 50 Sec 61 C Extension 30 Sec 72 C Hold 00 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 Page 5 of 11 Instructions for use HISTO SPOT SSO kits Version 9 2013 If other th
7. Regular wipe tests e g BAG Wipetest REF 7091 and 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 thermal cyclers heat blocks MR SPOT processor must be calibrated according to the manufacturers instructions Accuracy and temperature uniformity of thermal cyclers 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 data Failure in data transfer Manually transfer data using transfer USB drive No result Failure to grid image Perform manual gridding No Spots in well Failure to add mastermix to Repeat whole assay and PCR check PCR product on
8. ation of the result and function of the probes 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 alcohol Material used to clean spills including gloves should be inactivated before disposal e g in an autoclave Page 8 of 11 Instructions for use HISTO SPOT SSO kits Version 9 2013 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 Mastermix and Conjugate contain the preservative sodium azide The reagents co
9. cececccceeeeeeeeeseeneeeeeeeeeeeeeenenaaaeeeeeeeenena 5 4 2 Setup of the MR SPOT proceSso cccccsccscssescseeeceescseeeeseeecseeeess 5 4 3 Transfer of results to a PC for interpretation eeeeeeeeeeeeeeees 5 4 4 Interpretation Of results svicccericssesamrerereinsdenisusercistanorersixceneniteoieerense 6 WARNINGS AND PRECAUT IONS sessesseseeessesssserrnrrssserrrrrrnnresserrrrrrnnreset 7 SPECIFIC PERFORMANCE CHARACTERISTICS 2 eeeeeeeeeeeeeeeeeees Teal EAN OV esre essacteduscinseundedgoeacettgaeucee apaeacendodeuned Doe sas togeuees Igoe teasunseeedgoetees 7 2 PCR Amplification reaction siccs ccusesinesientsrceeseeacesunedoncemseeuaenisalenueseciaxedvelasenunes 7 3 Assay resolution ocx ccciac Sass ace cadccanivzascasuesaceeciccasaesesenacesieqmusesseaacaaieemacieesesentee 8 LIMITATIONS OF THE METHOD ceecceeeeeeee eter ee eteeennaeeeeeeeeeeeeeeeaaees 9 INTERNAL QUALITY CONTROL xiscscsciccecite distin deddinciiniceidindeeieedine 10 TROUBLESHOOTING x iresssikioini aa A EARE 11 TRADEMARKS USED IN THIS DOCUMENT PRODUCT n se 12 EXPLANATION OF SYMBOLS USED ON LABELING ssssseeeeessseeeeerenese BAG Health Care GmbH Auftragsannahme Ordering Amtsgerichtsstra e 1 5 Tel 49 0 6404 925 0 www bag healthcare com Tel 49 0 6404 925 450 35423 Lich Germany Fax 49 0 6404 925 250 info bag healthcare com Fax 49 0 6404 925 460 verkauf bag healthcare com Version 9 2013 Customer Service Tel
10. ermal 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 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 reagents and HISTO SPOT testwells 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 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 volum
11. es for the chosen number of strips Fill the required volumes of the reagents into the corresponding labelled reservoirs Place the test wells and the PCR plate into the appropriate blocks of the MR SPOT processor Note the correct arrangement of the PCR plate 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 The teststrips can be separated in single wells according to figure 1 if less than eight tests should be run If you are using separated wells be careful that they are sitting properly in the reaction plate holder and are not twisted against each other Page 6 of 11 Instructions for use HISTO SPOT SSO kits Version 9 2013 Separate testwells by snapping them off in a right angle ee Break off end pieces with the thumb Do not break the testwells upwards or downwards and do not twist the wells off Fig 1 Separating single testwells 5 4 2 Setup of the MR SPOT processor Switch on the MR SPOT processor The start up 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 5 4 4 Interpretation of results Open the HISTO MATCH software if th
12. gel Only control spots positive Failure to add DNA to PCR Repeat whole assay and or amplification failure check PCR product on gel False positive probes Too much DNA used or Check DNA concentarion conjugate concentration too Spin down conjugate before high not spun down use Exon dropout DNA concentration too high Check DNA concentration or DNA degraded run a gel with the DNA No result inconclusive result Mistake in conjugate dilution Repeat assay due to weak signals or poor amplification Check hybridisation Instrument malfunction temperature on instrument 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 Page 10 of 11 Instructions for use HISTO SPOT SSO kits 12 EXPLANATION OF SYMBOLS USED ON LABELING IVD For in vitro diagnostic use y Storage temperature LOTI Batch code 2 Use by REF Catalogue number BA Consult instructions for use Intended purpose HLA typing Mastermix A Mastermix for amplification of the HLA A locus Mastermix for amplification of the HLA B locus Mastermix for amplification of the HLA C locus Mastermix DRB1 Mastermix for amplification of the HLA DRB1 locus Mastermix for amplification of the HLA DQB1 locus Mastermix for amplification of the HLA DP
13. h 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 11 Instructions for use HISTO SPOT SSO kits Version 9 2013 3 MATERIAL 3 1 Reagents provided with the locus specific HISTO SPOT typing kits Testwells I X Testwells individually packed each strip containing 8 tests contains immobilized sequence specific oligonucleotide probes Mastermix X Mastermix ready to use contains biotinylated primers for the chosen locus dNTPs Taq polymerase reaction buffer 0 05 sodium azide MgCl2 Magnesium Chloride 6 mM ready to use contains 0 001 Proclin 300 X A B C DRB1 DRB3 4 5 DQB1 or DPB1 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 3 2 Kit e g HISTO SPOT A defines the locus tested Lot e g A084 A085 defines the layout and specificity of the probes that
14. he mastermix 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 plate containing biotin labelled amplicon is 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 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 eac
15. is 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 Page 7 of 11 Instructions for use HISTO SPOT SSO kits Version 9 2013 The colour of the circles around the probes indicate their function see IFU for the HISTO MATCH software for details 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 also functioning as a reference for the allele specific probes Positive allele specific probe o lo Jo lo Jo lo le o jo lo lelo le o lo Jo o e o olo lolo l oo o olol lololol e o lo lo lelolole bolbolololo 0 0 Jo Jo Jo Jo lclololelolole o l o lo jolole O Negative allele specific probe Figure 3 Schematic illustr
16. ntain 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 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 all HISTO SPOT SSO kits evaluation studies with pre typed DNA samples has been performed The results were compa
17. red to other typing methods e g SSP sequencing No discrepancies were observed between the typing methods For every lot the specificity of each probe was verified with DNA from reference samples 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 HISTO SPOT 4D typing kits usually give allele level results 4 digits if a common allele filter is used Criteria for the common allele filter are the following allele frequency 2 0 5 in at least one population with a sample size of at least 1000 samples on the website www allelefrequencies net Page 9 of 11 Instructions for use HISTO SPOT SSO kits Version 9 2013 8 LIMITATIONS OF THE METHOD Because of the high susceptibility of the PCR method to variations in DNA concentration and uality only DNA samples should be used that have a concentration between 15 and 30 ng ul Extreme care should be taken to prevent contamination of the kit reagents and other laboratory materials and equipment with amplicons or DNA
18. 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 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 11 Instructions for use HISTO SPOT SSO kits Version 9 2013 5 3 Amplification Use skirted PCR plates for the amplification because they have to be held down at the skirt by a clamp in the MR SPOT processor afterwards HISTO SPOT PCR Frameplates have been validated for this application plates from other suppliers have to be validated by the user For each sample to be amplified add the following reagents to each PCR tube 10 pl Mastermix 5 ul MgCl 5 wl Sample D
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