- 1 - Chapter: DNA Based Testing Section: Application - ASHI-U
Contents
1. Table 2 Thermal cycler conditions for amplification Hybridization Be sure hybridization buffer components of the probe mix are solubilized and that the beads are thoroughly suspended Turn on the Luminex Instrument and XY platform to allow for 30 minute laser warmup Fe Qe IN pi Warm probe mix in a 55 60 C heat block for at least 5 10 minutes Sonicate briefly 15 sec then vortex probe mix for about 15 seconds Aliquot 5 uL of locus specific PCR product into a thermal cycler 96 well plate Aliquot 15 uL of probe mix into each well When aliquoting probe mix to more that 10 wells gently vortex probe mix after each set of 10 Seal plate with Microseal film Hybridize samples under the following incubation conditions 97 C for 5 minutes 47 C for 30 minutes 56 C for 10 minutes 56 C HOLD While samples are hybridizing prepare a 1 200 dilution solution SA PE mixture Combine 170 uL Dilution Solution DS and 0 85 uL 1mg mL SA PE per sample Keep in the dark at 2 room temperature until ready to use It is recommended to prepare enough Dilution Solution Mixture sufficient for n 1 samples to account for pipetting loss See Table 3 Dilution Solution may be warmed at 45 C for 5 minutes but must be at room temperature before making the mixture Vortex just before adding to the mixture to ensure all components are in solution Discard any unused portion NOTE Do not cancel hybridization program be2. Range 988 4000 C Bd 13 Range 1000 4000 DR Bd 34 Bd 35 Range 6 23 Bd 35 Range 6 28 Bd 35 Range 9 19 Bd 35 Range 8 18 Range 1000 5182 DQ Bd 34 Range 1000 5069 Bd 35 Range 8 18 31 One Lambda LABType SSO Plate Layout SE n PCR Set Up Denaturation Hybridization Labeling Pos QC ID per 1 sample per allele per 1 sample 13 8 ul D Mix Denaturation buffer 2 5 ul SAPE stock 0 5 ul Batch File Name Aul Primer Neutralization buffer 5 ul SAPE buffer 49 5 Hybridization buffer 34 ul Bead Mixture 4 ul Tech Name ash buffer 480 ul One Lambda rSSO PCR Preparation A Locus samples 2 x 13 8 ul D Mix DR locus samples 2 x 13 8 ul D Mix samples 2 x 4 ul Primer samples 2 x 4 ul Primer samples 2 x 0 2 ul Taq samples 2 x 0 2 ul Taq 2 ul DNA added separately per well tested 2 ul DNA added separately per well tested B Locus samples 2 x 13 8 ul D Mix DQ Locus samples 2 x 13 8 ul D Mix samples 2 x 4 ul Primer samples 2 x 4 ul Primer samples 2 x 0 2 ul Taq samples 2 x 0 2 ul Taq 2 ul DNA added separately per well tested 2 ul DNA added separately per well tested low Locus samples 2 x 13 8 ul D Mix samples 2 x 4 ul Primer samples 2 x 0 2 ul Tag l 2 ul DNA added separately per well tested Hybridization samples 2 x 34 ul Hybridization buffer
3. D 5 2 6 2 4 Determining when antigen or allele redefinition and retyping are required D 5 2 6 2 5 Assignment of haplotypes if reported D 5 2 6 2 5 1 If haplotypes are assigned based upon population frequencies this must be clearly indicated on the report and relevant references or sources must be stated D 5 2 6 2 5 2 Reports must include an explanation of recombination when this occurs D 5 2 6 3 Ensure that typing for class or class II antigens or alleles employs a sufficient number of antisera monoclonal antibodies and or DNA markers to clearly define all the antigens alleles for which the laboratory tests D 5 2 6 4 Use HLA typing terminology that conforms to the latest report of the World Health Organization W H O Nomenclature Committee for factors of the HLA System Potential new antigens and or alleles not yet approved by this committee must have a designation that cannot be confused with W H O terminology 38 LITERATURE CITED RECOMMENDED READING References One Lambda LABType SSO Typing inserts Gen Probe Lifecodes Quick Type SSO Typing inserts One Lambda LABType Visual software manual 100 User s Manual Luminex ASHI Standards R Phycoerythrin Conjugated Streptavidin Product Insert ASHI Procedure Manual 4th Edition 2000 HLA Beyond Tears Introduction to Human Histocompatibility 2nd Edition Glenn E Rodey 2000 EN ch GY 9 OT PS Ser Je e Recommended Reading HLA Beyond Tears Introduction t
4. samples 2 x 34 ul Hybridization buffer samples 2 x 4 ul Beads samples 2 x 4 ul Beads samples 2 x 34 ul Hybridization buffer DQ locus samples 2 x 34 ul Hybridization buffer samples 2 x 4 ul Beads samples 2 x 4 ul Beads samples 2 x 34 ul Hybridization buffer samples 2 x 4 ul Beads Labeling total number of samples Batch file name samples 10 x 0 5 ul SAPE stock samples 10 x 49 5 ul SAPE buffer Technologist Date FREQUENTLY ASKED QUESTIONS Question What do you do if the control results do not fall within the listed range Answer The acceptable control ranges were created by our own laboratory after analyzing the data from the control beads of approximately 20 different samples We continuously monitor these values to come up with the range Every laboratory will need to have their acceptable control ranges and corrective actions Question What could cause low fluorescence Answer Low fluorescence could be caused by leaving too much liquid in the sample well prior to addition of the SAPE thereby diluting the fluorescent tag Be sure to perform a visual check of the wells prior to adding SAPE If it appears there is too much re wash the plate and flick for the One Lambda assay off the wash buffer a little bit harder Exposure to light could also cause the intensity of the fluorescence to decrease Question Can the correct results be obtained
5. Click on Acquisition Details or Diagnostics to visually see the data acquisition Allow 2 to 3 samples to acquire before leaving the instrument Check to make sure the majority of the beads fall within the circles probes Once satisfied you can let the instrument run until acquisition is complete NOTE Acquisition time per sample will vary between 10 to 90 seconds depending on the bead count Data Acquisition Instrument Shut Down 1 2 5 Click on Wash 2 times placing Deionized water in the reservoir takes about 30 seconds Click on Sanitize once placing 20 bleach in the reservoir Click on Wash 3 times placing Deionized water in the reservoir wash rinses off the probe Click on Soak leaving Deionized water in the reservoir soak empties sheath fluid inside the probe and replaces it with deionized water Check waste reservoir and empty as necessary NOTE Refer to the Luminex User s Guide for recommended weekly and monthly maintenance schedule Data Analysis A Launch HLA Fusion software m S 22 wW Click on LABType icon looks like double helix in top tool bar Click on folder icon on top left corner of screen Browse to Luminex Raw Data gt Output Find sessions to be analyzed Highlight all low definition sessions to be analyzed Click Open Sessions will populate on left side of screen under CSV file name It may be necessary to click Include Imported CSVs in upper left corner to make ses
6. 100 identifies the fluorescent intensity of PE phycoerythin on each microsphere HLA assignment is identified using One Lambda software Specimen Genomic DNA extracted from ACD solution A or B EDTA or ficoll separated blood buccal mucosal scrapings paraffin embedded tissue cultured cells ortissue biopsy and stored at 2 8 C in the pre PCR room or 70 C DNA extracted from heparin preserved blood cannot be used in this assay Optimal DNA concentration 20 ng ul Controls Positive Control previously run DNA UCLA DNA exchange proficiency samples or CAP samples Negative Control No DNA purified DNA RNA free water Materials RSSOT1A A locus typing kit RSSO1B B locus typing kit RSSO1C C locus typing kit RSSO2B1 DRB1 locus typing kit RSSO2QB1 DQB1 locus typing kit The above typing kits are dissembled and stored at the following conditions Unopened bead vials Store at 70 C until expiration date Opened bead vials Store at 2 8 C until expiration date Denaturation buffer Store at room temperature 20 24 C until expiration date Neutralization buffer Store at room temperature 20 24 C until expiration date Hybridization buffer Store at room temperature 20 24 C until expiration date Wash buffer Store at room temperature 20 24 C until expiration date Water ultra pure DNAse and RNAse free Sigma W4502 Store at room temp 20 24 C until exp date
7. A comparison of One Lambda vs Lifecodes kits Amount of Processing DNA Req d PCR Time Time from for PCR Hyb to SAPE One Lambda 20 ng ul 2 hours 1 5 hours Lifecodes 10 200 ng ul 1 5 hours 1 hour SAPE R phycoerythrin conjugated streptavidin NOTE Lifecodes is a division of Gen Probe PROTOCOLS Procedure l Analysis of HLA Class and Class Il Loci by Reverse PCR SSOP Using the One Lambda SSO System Purpose To describe the process of identifying the HLA alleles carried by a patient or donor Since the proteins coded by the HLA genes are expressed on the cell surface and are involved in transplant rejection and graft versus host disease the information regarding HLA types is used to help match recipient and donor for solid organ and bone marrow or stem cell transplantation It is also used to identify the presence or absence of specific HLA antigens or alleles that have been found to be associated with an increased risk for certain diseases or for vaccine development or other immunological studies Extracted DNA is amplified using biotinylated group specific primers The PCR product is biotinylated which allows it to be detected using R Phycoerythrin conjugated PE streptavidin SAPE The PCR product is denatured and rehybridized to probe conjugated to fluorescent coded microspheres The beads carrying the PCR products are detected with SAPE using the LABScan 100 A flow analyzer the LABScan
8. R Phycoerythrin conjugated strepavidin buffer SAPE Cat LT SAPE PROTECT FROM LIGHT Store at 2 8 C until expiration date Deionized water Store opened bottles for 2 weeks at room temperature 20 24 C 70 Ethanol Store at room temperature 20 24 C until expiration date 20 Bleach Store at room temperature 20 24 C until expiration date Sheath Fluid Luminex Corporation Store at room temperature 20 24 C until expiration date Luminex Calibration and Control Beads PROTECT FROM LIGHT Store at 2 8 C until expiration date Equipment LABScan Flow Analyzer Centrifuge 13 000 x g Vortex mixer with adjustable speed Thermocycler 15 50 ml disposable tubes thin walled PCR tubes microamp base and tray holder Tray seal one Lambda SSPSEA300 PCR pad 1 5 ml microfuge tubes Bead resistant tips Pipets MLA adjustable pipets 96 well 0 2ml tube plate Multi channel pipet Whatman V bottom plates 96 well cold tray or wet ice IMPORTANT Bead Handling and Storage LABType beads must be evenly distributed before dispensing Always mix beads vigorously by pipetting up and down several times or by vortexing for 30 seconds Protect beads from light during usage and storage Store beads at 20 C ina tightly covered container until ready to use NOTE Once beads are thawed store at 2 8 C and use within 3 months Do not refreeze Procedure Amplification Set Up done in pre PCR room 1 Setup a tray of
9. at room temperature for approximately 15 minutes before placing sample tray in it Using a multichannel pipet add 5 ul of Neutralization buffer Vortex Note the color change from pink to pale yellow If color has not changed to pale yellow add 1 ul of Neutralization buffer Vortex Place PCR tray containing neutralized product on the pink 96 well cold tray Another option place 96 well sample tray on ice Detection continued Hybridization 9 Turn the thermocycler on to the 60 C hold program before proceeding 10 Combine appropriate volumes of Beads and Hybridization Buffer to prepare Bead Hybridization mixtures see SSOP worksheet or the table below Bead Mixture and Hybridization Buffer Volumes of Tests Hybridization Buffer Beads ul samples loci ul 680 Note 1 Make sure the beads are well mixed prior to use Note 2 Make sure to add 2 extra rxns for each calculation to allow for sample loss during pipetting 11 Add 38 ul of Bead Hybridization mixture to appropriate wells 9 12 13 14 15 16 17 18 19 Cover tray with tray seal and vortex thoroughly at low speed Remove from tray holder and place PCR tray into the pre warmed thermocycler 60 C hold program Place PCR pad on top of the tray Close and tighten lid Incubate for 15 minutes After incubation place tray in a tray holder remove seal and quickly add 100 ul of Wash Buffer to each well Cover tray with
10. reproducibility of results Corrective Action Deviation from an acceptable range or specifications in any of the quality control standards for rSSO Luminex assays must be resolved immediately Resolution may include but is not limited to discarding reagents mechanical repairs performed by authorized service representatives repeating assays or performing confirmatory testing of the assay using a previously tested sample Any corrective action must be documented NOTE The following forms are examples of what are used at Henry Ford Hospital Other laboratory forms will vary ES Ne E SSO Reagent Quality Control Form Reagent Tested New Lot Exp Date Current Lot KE New Lo pmen o New Lo pmen o Nowtot orNewshipment New Lo iment o ew Lo pmen o A Locus Kit B Locus Kit C Locus Kit DR Locus Kit DQ Locus Kit These PE Conjugated are all part Streptavidin SAPE of the kit or New Shipment N or New Shipment GER E New Lot_or New Shipment New Lot_or New Shipment N or New Shipment ew Lol New Lot_or New Shipment New Lot or New Shipment Primer mix denaturization buffer Neutralization buffer New Lot_or New Shipment New Lot_or New Shipment N Hybridization buffer Wash buffer or New Shipment ew LO New Lot or New Shipment SAPE buffer Samples Tested 1 Name Lab 2 Name Lab Results 1 A
11. 60 C 00 15 a ae 72 C 00 20 Step 4 Hold 72 C 10 00 Upon completion of thermocycling amplified DNA is now ready to be tested 1 y e oo Step 3 Cyle 30 ae i 1 Amplified DNA can be stored at 2 8 C 20 C or 70 C if not proceeding immediately to next step It is recommended but not required that amplification is confirmed by running 2 5 ul of the PCR product on an agarose gel Detection Denaturation Neutralization NOTE Make sure a thermocycler is available for use before starting this phase of assay 1 2 Remove tray containing amplicons from refrigerator or freezer Let the tray sit at room temperature 20 24 C for 15 minutes before using Spin tray for approximately 15 seconds before removing caps Place a clean 96 well AB Gene tray in a tray holder Transfer 5 ul of each amplified DNA sample into the corresponding well of the 96 well plate Make sure locations and sample numbers correspond to your original PCR tray If not please make sure you note this on your Luminex layout sheet Original tray may be resealed and stored at 20 C indefinitely Using a multichannel pipet add 2 5 ul of Denaturation Buffer to the bottom of each well containing amplified DNA Cover with tray seal vortex plate and incubate at room temperature for 10 minutes mixture will change to a shade of pink when properly mixed NOTE Immediately remove the pink 96 well cold tray from the 20 C freezer it must stay
12. Locus B Locus C Locus DR Locus DQ Locus Expected Results 2 A Locus B Locus C Locus DR Locus DQ Locus Exp Date Acceptability Criteria The result obtained with testing the new lot or shipment should be identical to the expected result Acceptable Unacceptable Technologist Date Supervisor Date Director Date HLA DNA Typing Coversheet for rev PCR SSOP One Lambda LABType Date Batch File Name Luminex Used Hybridization temperature C Washing temperature C PCR Amplification Reagents Reagent Lot HLA A Kit Bead HLA B Kit Bead HLA DRB1 Kit Bead HLA DQB1 Kit Bead HLA C Kit Bead B Primer DR Primer DQ Primer C Primer el gt ay x 3 2 4 AQ Expected Results Cw Comments Date Initials DNA Extraction PCR Amplification Hybridization and Detection Reactions Computer Analysis Verify Transcription Hil PCR Hybridization Detection Reagents Reagent Lot Number Exp Date Denaturation Soln Neutralization Soln Hybridization Soln Wash Buffer SAPE Stock Soln SAPE Buffer II Ranges adjusted per manufacturer s insert as well as monitoring of data acquired by testing in house RAW DATA Entered onto Spreadsheet Y or N Pos QC Exon 3 Bd 32 Range 956 4000 Bd 32 Range 800 4000 Bd 32 Range 1000 4000 Pos QC Exon 2 Pos QC Neg Bead A Bd 13 Range 990 4000 B Bd 13
13. be verified and documented at least every six months D 4 1 8 Control Procedures D 4 1 8 1 For each test system the laboratory must have control procedures that monitor the accuracy and precision of the complete analytical process D 4 1 8 2 The laboratory must establish the number type and frequency of testing control materials using if applicable the performance specifications verified or established by the laboratory D 4 1 8 3 The control procedures must D 4 1 8 3 1 Detect immediate errors that occur due to test system failure adverse environmental conditions and operator performance D 4 1 8 3 2 Monitor over time the accuracy and precision of test performance that may be influenced by changes in test system performance environmental conditions and variance in operator performance D 4 1 8 4 The laboratory must D 4 1 8 4 1 For each test system perform control procedures using the number and frequency specified by the manufacturer or established by the laboratory when they meet or exceed the requirements in this section D 4 1 8 4 2 Perform the following at least once each day that specimens are assayed or examined D 4 1 8 4 2 1 For each quantitative procedure include two control materials of different concentrations D 4 1 8 4 2 2 For each qualitative procedure include a negative and positive control material D 4 1 8 4 2 3 If reaction inhibition is a significant source of false negative results include a control mate
14. each laboratory will have their own software unique naming of their patient list etc See Appendix for an example Open Luminex sotftware From the Home Menu select New Multi Batch icon Enter your file name in the Multi batch name dialog box i Ex 052505TG date of PCR amplification initials of the tech running the Luminex Click on New Batch 10 11 12 13 14 15 16 17 Highlight the template you wish to run this is specific for the catalog number of the reagent kit Click on Select Enter the necessary information under the Batch Info dialog box Under name type in the date of the PCR amplification plus the Loci plus the Lot Ex 052505A L 5 Enter your tech initials and any description you wish Click on Load Patient List Select the load list you want Verify that the samples are loaded in the correct location as shown on your Luminex Plate Layout run sheet If not drag the highlighted square box around 1A to the desired location Click on Finish Continue doing steps 4 through 12 until all your samples are loaded This needs to be repeated for each locus Once done click on Finish again to save the Multi Batch file Vortex the samples prior to loading on the Luminex Click on Eject retract to open the plate holder load your plate and click on retract When ready click on Start plate You will see the pressure gauge going up pressure needle must be between the two green arrows
15. if the incorrect catalog information was used during the data acquisition on the Luminex machine Answer Yes The batch needs to be replayed using the replay batch button The data will need to be renamed and resaved with a different name The individual files will now be named DAT PA2 etc You will need to match up the patient names with the tray layout to properly match up the results with the correct patient in the analysis program Question What do you do if the analysis program gives you no exact match Answer Look at the possible answers to see if there are any suggestions for beads to change If so manually adjust the bead to get an exact match If there are no suggestions look at the summary of all the beads and see if any of them are close to the cutoff value for becoming positive Adjust the bead or beads and see what you get There is not a set number of acceptable adjustments That decision is left up to your lab supervisor or director ASHI STANDARDS D 4 1 3 6 Components of reagent kits of different lot numbers must not be interchanged unless otherwise specified by the manufacturer D 4 1 3 7 If commercial kits are used the manufacturer s instructions must be followed unless the laboratory has performed and documented validation testing to support a deviation in technique or analysis D 4 1 7 4 For thermal cycling instruments the appropriate target temperatures must be achieved Accuracy of these temperatures must
16. less than 10ul of buffer is left in the wells If more than 10ul remains repeat wash centrifuge flick steps Add 70 ul of Wash Buffer to each well Gently mix by pipetting up and down several times with multi channel pipettor 26 Transfer samples to a white Whatman V bottom reading plate Cover tray with tray seal Keep tray in the dark and at 2 8 C until ready to read NOTE Make sure your reading tray is properly oriented before you transfer the samples 27 For best results read samples immediately Be sure to thoroughly mix the samples immediately before reading NOTE If not acquiring samples immediately cover tray with tray seal and keep at 2 8 C until ready to read Sample Reading or Detection Luminex reads samples from 1A thru 1H then 2A thru 2H etc Zomm Dom gt 8 SF SP SF SP SP SP SP SP For use with Luminex IS v2 3 Data acquisition Luminex Machine Daily Start Up and Calibration The LABScan is an advanced flow analyzer that requires daily maintenance and calibration Refer to the LABScan user manual Luminex User s Guide for all necessary maintenance operation Daily maintenance includes routine start up and shutdown procedures The instrument must be calibrated as part of the start up routine In addition calibrate the instrument whenever the d Cal Temp temperature shown on the system monitor is more than or 2 C Note Allow the Calibration and Control bottles to come t
17. strip tube for each locus under the hood Layout of tray should include one positive control and one negative control for each locus tested 2 Fill out a Luminex tray layout sheet see attachment page 26 in the same order the samples will be placed in the PCR tray Label tubes to identify the locus for each group of samples in the appropriate space on the worksheet Negative control should be set up last 10 11 12 13 14 Thaw DNA amplification primers and D Mix Keep all reagents cool until ready to use Vortex D Mix and primers for 15 seconds Spin briefly Using the table below as a guide mix indicated volume of D Mix and primers When doing calculations for your mixture allow for 2 extra reactions to account for reagent loss during pipetting Vortex for 15 seconds Amplification Mixture Rxns D Mix ul Amplification Taq 13 8 138 276 8 690 Add Taq polymerase immediately before use to your D Mix primer tubes Pipette 2 ul of DNA into the bottom of your PCR tubes Add 18 ul of D Mix primer taq solution into each well containing DNA Cap tubes tightly Close lid and tighten NOTE Spin tray in centrifuge for 15 seconds to force all liquid to the bottom of the tubes before placing in the thermocycler Run LABType SSO PCR program as shown below SSO PCR Program Temperature and Incubation Time Step 1 Ho 96 C 03 00 1 Step 2 Cycle OC 00 20 TI 72 C 00 20 i O 20 e
18. that are specific for each of the individual HLA loci The PCR product is biotinylated which allows it to be detected using a fluorescent tag The amplified product is denatured using a pH change to make a single strand of DNA This is then hybridized to a series of probes specific for nucleotide sequences that are used to define the HLA alleles The probes are bound to a series of fluorescently coded beads Luminex technology Stringent washes are performed to wash off any PCR product that does not exactly match the sequence detected by the probe The bound PCR product is labeled with streptavidin conjugated with a PE fluorescent tag SAPE and detected using the LABScan system Using a flow cytometry analyzer the intensity of PE fluorescence on each bead will be measured Software is used to assign positive or en Bei negative reactions based on the strength of the fluorescence signal and identify HLA alleles that are present Lifecodes method for DNA amplification also uses labeled locus specific primers to produce biotinylated amplified DNA followed by hybridization to a mix of sequence specific probes bound to fluorescent beads However the Lifecodes method differs from the One Lambda method in the following ways Lifecodes employs equimolar amounts of both forward and reverse primer to generate a double stranded DNA product There is no separate denaturation step Hybridization takes place in a thermocycler for 45 minutes then
19. tray seal Note Leave thermocycler on until the labeling step is complete Centrifuge tray for 5 minutes at 1300 x g After centrifugation remove the PCR tray from its base and quickly hold the tray upside down and gently flick off the wash buffer while holding the tray in a horizontal position Repeat wash steps 2 more times for a total of 3 washes NOTE After the final wash be sure that less than 10ul of buffer is left in the wells If more than 10ul remains repeat wash centrifuge flick steps Prepare 1X SAPE solution during the third centrifugation See SSOP worksheet or the table below as a guide SAPE and SAPE Buffer Volumes of Test SAPE stock soln ul SAPE Buffer ul EE 495 0 990 0 1237 5 2475 0 Note Make sure to add 10 extra reactions to allow for sample loss during pipetting Detection continued Labeling 20 21 22 23 24 25 After the last centrifugation and flick place tray in tray holder Add 50 ul of 1X SAPE solution to each well Place tray seal on tray and vortex thoroughly at low speed Place tray in the pre heated thermocycler 60 C Place PCR pad on top of tray Close and tighten lid Incubate for 5 minutes After incubation remove tray seal and quickly add 100 ul of Wash Buffer to each well Cover with tray seal and centrifuge tray for 5 minutes at 1300 x g Remove PCR tray from centrifuge and quickly flick off wash buffer NOTE After the final wash be sure that
20. A C Typing kit LM DRB Lifecodes HLA DRB Typing kit LM DQB Lifecodes HLA DQB Typing kit Refer to product insert for storage conditions Reagents not provided in above kits e R Phycoerythrin Conjugated Streptavidin SA PE 1mg mL catalog 628511 Protect from light e Luminex Sheath Fluid catalog 628005 Store at room temperature 20 24 C until expiration date e Recombinant Taq Polymerase store at 20 C until expiration date e Nuclease free water store at room temperature 20 24 C until expiration date e Luminex calibration beads Cal 1 Cal 2 Con 1 and Con 2 Store at 2 8 C until expiration date Protect from light e 70 Isopropanol or 20 bleach store at room temperature 20 24 C until expiration date Equipment e Vortex mixer e PCR tubes and caps e Thermal cycler e Microseal Film e 96 will PCR reaction plates e Bath Sonicator e Microcentrifuge e Barrier filter pipette tips e Pipettors e Heat Block Important Reagent Handling Probe mixes and SAPE are light sensitive Keep away from light and do not freeze Warm the beads at 55 60 C for at least 5 10 minutes to thoroughly solubilize components in probe mixture Sonicate briefly 15 sec then vortex probe mix for about 15 seconds to thoroughly suspend the beads All temperatures must be precisely maintained Fluctuations as little as 0 5 C can affect results At the hybridization stage samples should not remain in the d
21. Chapter DNA Based Testing Section Application Modules Module Reverse SSO by Luminex Author s Sharon Skorupski CHT Henry Ford Hospital Detroit MI Tamara German Henry Ford Hospital Detroit MI Susan Saidman ssaidman bidmc harvard edu Toni Lyrenmann tlyrenm1 fairview org Date Prepared 08 20 2006 OBJECTIVES Understand the basic technique used to perform rSSO testing using the One Lambda and the Gen Probe Lifecodes software Understand the similarities and differences between the One Lambda and Gen Probe Lifecodes SSO Typing kit Understand how to interpret test results and provide the final answer the patient s HLA typing result Understand and identify the ASHI standards that pertain to the compliance of the assay all pertinent equipment and the quality control measures required for this test system E A INTRODUCTION The sequence specific oligonucleotide SSO assay for HLA typing utilizes a single PCR reaction per each HLA locus This is followed by hybridization to a series of labeled sequence specific probes that are used to determine which specific allele or group of alleles is present In the forward SSO technique amplified DNA is attached to a membrane and each membrane is hybridized with a different probe Since multiple probes are required for even low resolution typing numerous membranes are needed In the standard dot blot format multiple amplified DNAs are applied to each large membrane All the D
22. NAs are then simultaneously hybridized with each probe Therefore this dot blot format is much better suited to high volume batch testing The rSSO aka Reverse Sequence Specific Oligonucleotide probe technique is a variation of the SSO methodology In this case the probes are attached to a bead or membrane and labeled amplified DNA is added for hybridization rSSO is much more suited to lower volume individual patient testing The two most common methods of rSSO testing involve either a color change on a nitrocellulose membrane or a fluorescent tag that is read by a flow cytometer Both methods allow for small batches of patient samples The nitrocellulose membrane method involves a PCR reaction with biotinylated primers followed by hybridization and detection The nitrocellulose strip has multiple probes attached to it The biotinylated PCR product is mixed in a well with the strip If the amplified product will recognize specific binding sites on the probes and will attach to the strip at those specific probes After addition of various reagents and incubations the final product is detected by a color change at the location of the probes where the DNA is bound The probes are identified by comparing them to a control strip and then analyzed using a computer program The second rSSO method with fluorescent probes is utilized by both One Lambda and Gen Probe Lifecodes In the One Lambda method extracted DNA is amplified using primers
23. SA PE is added and the sample is read in the LABScan system There are no wash steps with LifeCodes method HLA assignment is done using LifeCodes software The rSSO methodology is commonly used due to its relatively quick turn around time good reproducibility and ability to give a clear cut answer The extensive number of antigenic probes represented on the assay beads results in fewer ambiguities therefore requiring almost no need for further testing to clarify the final answer Currently there are assay bead kits available which can result in allele level typings This module will describe in detail the fluorescent label methods for both One Lambda and Gen Probe Lifecodes SSO kits METHODOLOGIES Commercial kits containing all required reagents are available Such kits have undergone extensive quality control measures to determine the appropriate working conditions of the assay The manufacturers provide training for using their product and continuous technical support Validation of the assay is required by the laboratory prior to clinical use regardless of the quality control provided by the manufacturer There are currently two commercial suppliers of this methodology One Lambda and Gen Probe Due to the easy availability of commercial kits the significant amount of quality control required and the constant discovery of new HLA alleles performing this test as a home brew method is not an easy task and not recommended
24. als must be established over time by the laboratory through concurrent testing of control materials having previously determined statistical parameters D 4 1 8 4 12 Results of control materials must meet the laboratory s and as applicable the manufacturer s test system criteria for acceptability before reporting test results 35 D 4 1 8 4 13 The laboratory must document all control procedures performed D 4 1 8 4 14 If control materials are not available the laboratory must have an alternative mechanism to detect immediate errors and monitor test system performance over time The performance of alternative control procedures must be documented D 4 1 8 4 15 Laboratories must adhere to their policy for quality control of each lot and shipment of reagents Reference material must be used for quality control whenever possible D 4 1 8 5 Laboratories performing nucleic acid testing must have written criteria or protocols for preventing DNA contamination using physical and or biochemical barriers for assays involving amplification of templates D 5 Application and Test Systems D 5 1 General Standards D 5 1 1 Test systems D 5 1 1 1 Test Systems selected by the laboratory must be performed D 5 1 1 1 1 following the manufacturer s instructions or as modified and validated by the laboratory and or D 5 1 1 1 2 as developed and validated by the laboratory and D 5 1 1 1 3 in a manner that provides test results that are within the laboratory
25. ct typing and the sample test should be repeated e There is a separate threshold table for each locus e These threshold tables are Lot specific be certain that the Lot on the threshold tables matches the Lot of the typing kit e If anormalized value for a particular probe falls above the maximum threshold for a negative assignment and below the minimum value for a positive assignment the sample should be considered as indeterminate for this probe The sample should be typed first assuming the value to be negative and then again assuming the value to be positive e See EXPECTED VALUES section for further information on threshold values 24 LIMITATIONS OF THE PROCEDURE The PCR conditions and assay conditions described require precisely controlled conditions Deviations from these parameters may lead to product failure All instruments must be calibrated according to the manufacture s recommendations and operated within manufacture s prescribed parameters Beads must be pre warmed and well suspended prior to use This ensures that the hybridization buffer components are in solution The incubations performed at 47 C and 56 C require strict temperature control 0 5 C A thermal cycler should be employed Temperature should be verified within wells of the 96 well thermal cycler plate The temperature within wells and among wells should not vary more than 0 5 C Time of the incubation performed at 56 C is cri
26. der to preserve the integrity of the assayed samples the following guidelines will be adhered to by the technologist performing the assay No aliquot of a specimen shall be returned to the original tube To prevent cross contamination of specimens only one specimen shall be opened and handled at a time This is especially critical when the opened tube is the original specimen All tubes must be prelabeled prior to aliquoting of specimens Clean disposable pipets or pipet tips must be used for each individual specimen This is to prevent commingling and or contamination of specimens during pipetting When aliquoting specimens from their original container to another such as transferring of peripheral blood samples to freezing vials only one sample is to be opened and handled at a time All labels must be rechecked prior to transfer In performance of the assay plan layout sheet carefully and adhere to the layout This is necessary for sample tracking during the testing procedure Keeping samples in alphabetical order helps with tracking If specimen mix up is suspected during the course of testing repeat the assay on a fresh aliquot from the original specimen tube Request a fresh specimen by contacting the physician or nursing staff if needed Report all questionable specimens to the supervisor or laboratory director The technologist should verify that control beads have proper fluorescent intensity for each individual sample
27. e Intensity FI must be greater than or equal to 500 2 Negative Control bead Fluorescence Intensity Fl must be less than or equal to 80 NOTE If any or these benchmarks are out of range please consult the supervisor or lab director for further instructions Data Calculations 1 The Fluorescence Intensity generated by the Luminex Data Collector Software or equivalent contains the FI for each bead or probe bound to the bead per sample The percent positive value is calculated as a Percent Positive Value 100 x FI Probe n FI Probe Negative Control Fl Probe Positive Control Fl Probe Negative Control i A reaction with the percent positive value higher than the pre set cut off value for that probe will be considered positive ii A reaction with the percent positive value lower than the cut off value will be considered negative Troubleshooting NOTE In the event that the test system is inoperable due to assay problems notify supervisor or director immediately One Lambda customer support is available for troubleshooting The following are examples of some problems that may be encountered 1 Positive DNA control results give inappropriate results a Ifthe wrong answer is obtained for the positive control it must be investigated before the run is accepted 2 Reactivity seen in the negative control well a The entire run will need to be repeated to ensure there was not any DNA contamination 3 No Amplification o
28. e free of contamination D 5 2 2 6 Ensure that reagents used for primary amplification are not exposed to post amplification work areas D 5 2 2 7 Ensure that reagents used for secondary amplification are stored in a contamination free area D 5 2 2 8 Define criteria and perform quality control testing to confirm specificity for each lot and shipment of primers and probes D 5 2 2 9 For each new lot of kits D 5 2 2 9 1 Perform parallel testing using the number of samples determined by the Director or designee for the size of the kit and frequency of use D 5 2 2 9 2 When possible include testing of alleles known to have demonstrated weak false negative amplification with previous lots of the same kit D 5 2 2 9 3 When possible include testing of new primer probe sets that have changed from the previous lot D 5 2 2 10 Test each new shipment of kits to demonstrate that the integrity of the kits has not been compromised during shipment This can be accomplished by D 5 2 2 10 1 Testing with reference DNA samples and assessing the results or D 5 2 2 10 2 Testing with non critical clinical samples and assessing the quality of the reactions and 36 the ability to give a clear interpretation of the results or D 5 2 2 10 3 Testing the new lot or shipment in parallel with the old lot D 5 2 2 11 Ensure that each lot and shipment of primers or probes is monitored to confirm stability and performance of the primers or probes D 5 2 2 12 En
29. f patient sample a All patient samples that do not amplify are re extracted and repeated If the same results are obtained after the second amplification consult with the supervisor or director Verify the DNA concentration and purity using the spectrophotometer 4 Ambiguous Alleles a Ambiguous allele combinations should be resolved using PCR SSP If these groups cannot be identified by PCR SSP then PCR SBT sequence based typing must be performed Family studies may be helpful in some cases b If an unusual or infrequently seen combination of alleles is presented as a possible answer it is acceptable to report out the common typing with a footnote explaining that the lab is unable to rule out the rare allele combination and that further testing is available upon request 5 Homozygosity of patient alleles a Samples that exhibit homozygosity may be reported if the integrity of the probe signals are valid no weak probe signals All homozygous test results must include a statement indicating that a second unidentified allele may be present and family studies are recommended b Note If two alleles belonging to the SAME GROUP are identified the sample is NOT considered to be homozygous For example DRB1 03XX where 03XX may be DRB1 0301 0302 or 0301 0305 This sample is homozygous at the group specific level but clearly heterozygous at the allele level 6 Positive control Fluorescent intensity Fl low a Ifthe FI of the positive c
30. fore removing the tray from the thermal cycler of Dilution SAPE Samples Solution DS 1 170uL 0 85uL 5 850uL 4 25uL 10 1700uL 8 5uL 20 3400uL 17uL 50 8500uL 42 5uL Table 3 8 At the 56 C hold while the tray is on the thermal cycler add 170 uL of the prepared dilution solution SAPE mixture It is critical to dilute all samples within 5 minutes 9 Remove the sample tray from the thermal cycler and place in the Luminex Instrument For best results assay the samples immediately using the Luminex Instrument Samples can be read up to 30 minutes after being diluted If not read immediately protect samples from light DATA ACQUISITION USING QUICK TYPE SOFTWARE Luminex Instrument Luminex Machine Daily Start Up and Calibration e See Procedure I Data Acquisition Luminex Machine Daily Start Up and Calibration on page 10 of this module 22 CREATING A BATCH Note The following protocol is from the Ohio State University Hospitals and has been edited for use in this module e Open Quicktype for Quick Type software e Click Create Automated Batch e Click Get Samples e Scroll to the patient list created by your lab this will vary as every laboratory will have a different method of creating their patient lists e Name your Session this batch name will be unique to your laboratory e Choose the Lot for your kit from the pull down menu e Click Set Name pull down tab and click
31. g on skin or in eyes or on clothing Keep tightly sealed Wash hands thoroughly after handling Flush spill area with large amounts of water 3 Denaturization Buffer and Neutralization buffer are corrosive and may cause burns In case of contact immediately flush eyes or skin with a copious amount of water for at least 15 minutes while also removing contaminated clothing see MSDS 4 Proper PPE must be worn according to the procedure being performed A labcoat and disposable gloves must be worn when performing this procedure 5 Pre PCR room Precautions All pre amplification steps must be performed in a Pre PCR room All persons performing this procedure must wear clean gloves disposable lab coats and shoe covers The work area must be cleaned with 10 bleach and 70 ethanol before and after use Place a protective plastic backed lab mat or sterile field on the bench before assembling the reagents and supplies needed for the PCR Keep reagents and supplies confined to the lab mat Discard the mat after each PCR set up 6 Aerosol resistant tips must be used throughout the setup procedure Procedure ll Analysis of HLA Class and Class Il Loci by Reverse PCR SSOP Using the Lifecodes SSO Kit Purpose To describe the process of identifying the HLA type of a patient or donor This information is used to help match recipient and donor for solid organ and bone marrow or stem cell transplantation It is also used to identify the presence or abse
32. iluted state at 55 C for more than 5 minutes It is recommended to assay the amplified samples as soon as possible Samples can be stored up to 3 days at 2 8 C prior to use For longer storage store at 20 C up to one week The amplified product can only be frozen and thawed once Repeated freezing and thawing will result in degradation of amplified samples Procedure DNA amplification PCR Allow the master mix to warm to room temperature 18 30 C 2 Gently vortex for approximately 10 seconds Spin briefly 5 10 seconds in microcentrifuge to bring contents to the bottom of the tube 3 Using Table 1 below prepare components for amplification for n 1 reactions Amount per PCR Component sample reaction Lifecodes Master Mix 15 uL Genomic DNA 10 200ng uL Total of 200ng Taq polymerase 0 5 uL 2 5U To 50uL final Nuclease free water volume ees E NOs Table 1 Reaction components for amplification Pipette the appropriate amounts of DNA into the PCR tubes Aliquot the amplification mix into the PCR tubes containing the genomic DNA Cap tubes tightly to prevent evaporation during PCR Place samples in the thermal cycler and run program See Table 2 95 C for 5 min Number of cycles 1 95 C for 30 sec 60 C for 45 sec 72 C for 45 sec Number of Cycles 8 95 C for 30 sec 63 C for 45 sec 72 C for 45 sec Number of cycles 32 72 C for 15 min
33. ions NOTE Contamination of the Pre Amplification Area Must Be Avoided Precautions That Must Be Taken Upon Entering and While Working in the Pre amplification Area a Wash hands in sink before or as you enter the room b Clean shoe covers must be worn at all times Do not reuse these covers Be careful when putting these covers on as shoes floor have a high potential for tracking amplicons Do not step on the floor of the pre amplification room with an uncovered shoe c Change gloves often d Do not open the door or handle items in the refrigerator freezer without wearing clean gloves e Do not wear a lab coat into this room that has been worn in an area of PCR product processing f Put on a clean disposable lab coat when processing samples A fresh coat must be put on prior to working under the hood Coats are not to be worn for more than one day g Paperwork cannot be brought into this room The only exception is freshly Xeroxed worksheets which have been cautiously handled h Sticky mats must be monitored daily for dirty build up in order to maintain effectiveness ao27 The workbench must be thoroughly cleaned with fresh 10 bleach and 70 ethanol after use Caution must be maintained when bringing consumable items into the area Do not put items onto the workbench if the outer packaging which may have been exposed to contaminated areas has not been carefully removed Sample Integrity In or
34. le on thermal cycler to verify parameters are within acceptable ranges Low Median Fluorescent Intensity Value MFI Warm dilution solution at 45 C for 5 minutes before use and vortex Store at room temperature Replace R Phycoerythrin Conjugated Streptavidin Multiple SSO failures or sample fails to yield a valid HLA typing result Allele specific amplification Amplification conditions not within specific parameters Run thermal profile on thermal cycler to verify parameters are within acceptable ranges DNA sample contaminated DNA partially degraded Re isolate DNA from blood sample Evaporation during hybridization step If not using an entire plate leave one row empty on each side of the plate to allow tight sealing QUALITY CONTROL ASSURANCE Purpose Dee To describe the standards involved in maintaining appropriate quality control measures to ensure the validity and accuracy of the rSSO Luminex methodology The quality control of these assays is dependent on both mechanical performance and biological reagents Some of the quality control standards are accounted for in daily laboratory QC Some standards are performed at longer intervals i e pipette calibration and others are needed every time the assay is run Materials Same as above Equipment Same as above Procedure The following procedure is used at Henry Ford Hospital Other laboratories will have variat
35. less than or equal to 50 result must be flagged for supervisor and or director review prior to release of results Low bead count is usually the result of flicking too hard while washing the plate If result is consistent with common haplotype inheritance patterns and or family typings result may be released If not consistent or unsure of result repeat detection step only If count still low and result consistent with first typing report result If result is not consistent repeat test from primary amplification 9 Unexpected Family Haplotypes a If unexpected family haplotype results are obtained notify supervisor or director immediately and i Review all result data for analysis or transcription errors ii Contact transplant coordinator to confirm family relationships iii Repeat DNA extraction and PCR from original vial or obtain a new sample iv Consult director for further action if problem not resolved Limitations of Assay The LABType SSO system combines an HLA locus specific DNA amplification process and DNA DNA hybridization process The procedure as well as the equipment calibration described in this procedure must be strictly followed DNA amplification is a dynamic process that requires highly controlled conditions to obtain PCR products that are specific to a target segment of HLA gene s The procedure including proper DNA concentration proper PCR conditions proper temperature and storage of reagents mu
36. mposition of the probe and that achieve the defined specificity D 5 2 3 5 Establish criteria to determine positive or negative hybridization results for each probe using nucleotide sequences reference DNA and or manufacturers QC data D 5 2 3 6 Ensure that each probe used gives an adequate signal and allows detection of alleles in a heterozygous individual D 5 2 3 7 Document the specificity and sensitivity of the labeling and detection methods e g demonstrate correct signal strength for a control sequence in the laboratory before results are reported D 5 2 3 8 If there is reuse of nucleic acids probes or targets bound to solid supports have a validated procedure for re hybridization assays and include controls to ensure that the sensitivity and specificity of the assay are unaltered D 5 2 6 Laboratories performing HLA typing must D 5 2 6 1 Ensure that the level of resolution of HLA typing is appropriate for the clinical application and is based on established criteria D 5 2 6 2 Have written criteria or protocols for D 5 2 6 2 1 Preparation of cells or cellular component isolations for example solubilized antigens and nucleic acids as applicable to the HLA typing technique s performed D 5 2 6 2 2 Selection quality control and usage of all typing reagents and components D 5 2 6 2 3 The assignment of HLA antigens and alleles and for distinguishing common null alleles as appropriate for the clinical use of the test results
37. n the approximate number of alleles and the distinguishing characteristics e g sizes sequences of the alleles that are amplified D 5 2 2 24 Have acceptable limits of signal intensity for positive and negative results If these are not achieved acceptance of the results must be justified and documented D 5 2 2 25 Adhere to the established criteria for accepting or rejecting an amplification assay or document the justification for acceptance of an assay when acceptance criteria are not met D 5 2 2 26 Have two independent reviews and interpretations of the data D 5 2 2 27 When applicable interpret data using the IMGT HLA or other appropriate nucleotide sequence database The database that is used must be updated at least every six months D 5 2 2 28 When applicable document in laboratory records which version of the IMGT HLA or other appropriate nucleotide sequence database was used for allele interpretation D 5 2 3 Laboratories performing SSOP methods must D 5 2 3 1 Define the specificity and critical polymorphic sequence of each primer and probe D 5 2 3 2 Label probes by a method appropriate for the testing procedure e y E D 5 2 3 3 Ensure that hybridization conditions for maintaining sensitivity and specificity have been established D 5 2 3 4 Ensure that pre hybridization hybridization and detection are carried out under empirically determined conditions of concentration and stringency that are determined by the length or co
38. nce of specific HLA antigens or alleles that have been found to be associated with an increased risk for certain diseases During amplification once the limiting primer is exhausted the remaining primer uses the double stranded product as a template for generation of single stranded DNA This method generates both double stranded and single stranded products that upon denaturation will both participate in the hybridization reaction The PCR product is biotinylated which allows it to be detected using R Phycoerythrin conjugated PE streptavidin SAPE A flow analyzer the LABScan 100 identifies the fluorescent intensity of PE phycoerythin on each microsphere HLA assignment is done using LifeCodes software Specimen DNA extracted from whole blood cord blood stain cards and buccal swabs using any preferred method DNA extracted from blood preserved in heparin cannot be used in this assay The presence of alcohol detergents or salts may adversely affect DNA amplification Final DNA concentration should be 10 200 ng uL Absorbance measurements of the DNA sample at 260 and 280nm should give a ratio of 1 65 to 2 0 DNA can be used immediately after isolation or stored at 20 C for up to 1 year Controls DNA positive control can be any previously run DNA sample Negative control No DNA purified DNA RNA free water Reagents LM A Lifecodes HLA A Typing kit LM B Lifecodes HLA B Typing kit LM C Lifecodes HL
39. nt choose Print Report select Print Preview then Bead Ranking and Print This will open a printing option To print the information on the screen click Print Report To save the report click on the Save icon The report will be saved as a PDF on a location assigned by the user MANUAL DATA IMPORT FROM LUMINEX IS SOFTWARE To import the data manually go to Setup option on home page of Quicktype for Quick Type and select Manual Data Import A screen will appear select the appropriate lot number of the kit and click on the binocular to search for desired batch that has been saved in a known file Then double click on the output csv file of that batch and click Next That will transfer the analysed data to the Reports and Re Analysis icon Go to Report and Re Analysis and select the appropriate session To print choose Print Report select Print Preview then Bead Ranking and Print This will open a printing option For screen analysis just click Print Report Caution e To obtain reliable results there must be sufficient data gathered by the Luminex Instrument e Collect at least 60 events for each SSO e The LIFECODES Probe Mix es contain one or more SSO designated CON exon 2 or exon 3 200 exon 2 or 300 exon 3 that hybridize to all alleles These act as internal controls to verify that the PCR reactions and hybridizations worked e If the minimum value is not obtained for these SSO s the sample may not produce the corre
40. o Human Histocompatibility 2nd Edition Glenn E Rodey 2000 39
41. o room temperature prior to use 1 Turn on the Luminex Turn on the computer Verify that the sheath delivery system is on 2 Double click on the Luminex icon on the desktop 3 When the Luminex software loads laser Warm Up usually starts automatically If not click Warm Up This process takes about 30 minutes 1800 seconds 4 During or after Warm Up is complete click on Prime Click on OK Instrument will prime for 30 seconds 5 Repeat this step 10 11 12 13 14 Click on Alcohol Flush Click on eject Fill reservoir with 70 alcohol Click on OK This takes about 5 minutes removes air bubbles in the lines Click on Wash Click on eject Fill the reservoir with Sheath Fluid Click OK Cick on Wash and OK to repeat Perform instrument calibration using Luminex Calibrator and Control reagents reagents must be at room temperature prior to use Use a 96 well Whatman V bottom reading plate white Calibration reagents are CAL1 and CAL2 Control reagents are CON1 and CON2 Vortex vigorously prior to use Add 3 4 drops of each of the reagents to the calibration plate Make sure the sample location you are dispensing the reagents into matches the sample location on the drop down menu Click on New CAL Target from the maintenance menu to enter or confirm the lot number of the calibration reagents If necessary update new information found in the kit When finished click on CAL1 on the menu to proceed with the fi
42. on Lot again e Designate your starting well using the pull down menu under Start Position e Sign your initials in the Setup box e Submit to Luminex by clicking on Submit Batch to Luminex RUNNING AN ASSAY ON THE LUMINEX e Open Luminex software Click on Open Batch it is a blue box with a yellow arrow on top This will open a box with your batch listed as well as any other unread batches Select your batch e Click Eject Place your tray in the Luminex machine and click Start e After acquiring the data Quick Type will pull the raw data amp automatically analyze it INSTRUMENT SHUT DOWN See Procedure Data Acquisition Instrtument Shutdown on page 12 of this module NOTE Please refer to the Luminex User s Guide for recommended weekly and monthly maintenance schedule RESULTS GENERATING A REPORT e Click on the Reports amp Reanalysis box A calendar box will open Select the date you ran your batch amp hit O K this will list the batch on the left hand side of the rectangular white box e Double click on the session A list of all patients in the batch will appear e By individually clicking a sample name the analysis for that sample will appear as a graph n or AE Double clicking any box on the graph will enlarge that screen and allow you to work on it Click Graph Edit On to adjust the curve if you want to set your MFI cutoff at a certain number Click Graph Edit Off amp Save when finished adjusting To pri
43. ontrol bead is less than 800 results must be flagged for supervisor and or director review prior to release of results This could be due to incomplete binding of fluorescent tag during hybridization and detection If the majority of the samples appear to have signal above 800 it is most likely a problem with the sample itself and not the entire run If other loci for the sample worked fine it is not likely to be a problem with the extracted DNA Repeat the test for that sample only b Ifthe entire run has low intensity it is most likely a problem with the detection step Repeat the detection step for all samples SAPE will lose its intensity the longer it is sitting around in its prepared formula Try to use the SAPE up in a timely manner 7 Negative control Fluorescent Intensity Fl high a Ifthe FI of the negative control bead is greater than or equal to 80 result must be flagged for supervisor and or director review prior to release of results b This could be due to inadequate washing Splashing or contamination could cause a false positive in the negative well The contamination may have occurred during the preparation of the PCR product or during hybridization or detection step If the Fl is above 80 on the negative control sample for the run repeat the run If the FI is above 80 for an individual sample determine if the test needs to be repeated on a case by case basis 8 Bead Counts a b CG d If bead count is
44. re amplification work areas with wipe tests D 5 2 2 21 1 Monitor potential contamination using a method that is at least as sensitive as routine test methods and that uses appropriate testing primers At least one negative no nucleic acid and one positive control must be included in each amplification assay D 5 2 2 21 2 It is recommended that each wipe test amplification be run without added DNA and with added DNA as a control for wipe test inhibition D 5 2 2 21 3 If contamination and or inhibition is detected clean the area to eliminate the contamination or source of inhibition and document re testing as well as the measures taken to prevent future contamination D 5 2 2 21 4 Document acceptable electrophoretic conditions used for each gel electrophoresis D 5 2 2 22 If the size of a nucleic acid is a critical factor in the analysis of the data D 5 2 2 22 1 In each gel include size markers that produce discrete electrophoretic bands spanning and flanking the entire range of expected fragment sizes D 5 2 2 22 2 The amount of DNA loaded in each lane must be within a range that ensures equivalent migration of DNA in all samples including size markers D 5 2 2 23 Define and document the specificity and sequence of primer targets The genetic designation e g locus of the target amplified by each set of primers must be defined and documented For each locus analyzed the laboratory must have documentation that includes the chromosome locatio
45. rial capable of detecting the inhibition D 4 1 8 4 3 For each electrophoretic procedure include concurrent with patient specimens at least one control material containing the substances being identified or measured e g molecular weight markers D 4 1 8 4 4 Perform control material testing before resuming patient testing when a complete change of reagents is introduced major preventive maintenance is performed or any critical part that may influence test performance is replaced D 4 1 8 4 5 Over time rotate control material testing among all operators who perform the test D 4 1 8 4 6 Test control materials in the same manner as patient specimens D 4 1 8 4 7 When using calibration material as a control material use calibration material from a different lot number than that used to establish a cut off value or to calibrate the test system D 4 1 8 4 8 Establish or verify the criteria for acceptability of all control materials D 4 1 8 4 9 When control materials providing quantitative results are used statistical parameters for example mean and standard deviation for each batch and lot number of control materials must be defined and available D 4 1 8 4 10 The laboratory may use the stated value of a commercially assayed control material provided the stated value is for the methodology and instrumentation employed by the laboratory and is verified by the laboratory D 4 1 8 4 11 Statistical parameters for locally obtained control materi
46. rst calibration Perform a Wash step after calibration Repeat step 10 for CAL2 CON1 and CON2 Perform a Wash step after each calibration Verify that all calibration has passed Click on Diagnostics menu Green indicates pass and red indicates failure Contact supervisor or director if calibration fails also fill out corrective action form Print the calibration and control reports Go to File and click on Print Report Click on Calibration Trend Report Select Cal 1 Print Do the same for Cal 2 and print i Click on System Control Trend Select Con1 and print Do the same for Con2 and print Data Acquisition Collection of sample data In order to obtain valid data two parameters count and fluorescence intensity must be monitored for each data acquisition Count represents the total number of beads that has been analyzed and should be above 50 A significant reduction in the count suggests bead loss during sample acquisition or assay and can void test results Fluorescence intensity Fl represents a PE signal detected within the counted beads FI varies based on the reaction outcome The FI for the positive control probe is expected to be 800 to 4000 depending on the bead pool and lot Significant reduction in the FI for the positive control probe suggests inadequate sample quantity and or quality poor assay efficiency or instrument failure and can void test results 1 Create your patient load list This will vary as
47. s stated performance specifications for each test system D 5 1 2 Evaluation of Test Systems D 5 1 2 1 The laboratory must have a system to identify assess and document patient test results that appear inconsistent with the following relevant criteria when available D 5 1 2 1 Patient age D 5 1 2 2 Sex D 5 1 2 3 Diagnosis or pertinent clinical data D 5 1 2 4 Distribution of patient test results D 5 1 2 5 Relationship with other test results D 5 2 2 Laboratories performing Amplification based nucleic acid testing must D 5 2 2 1 Use a method to prepare DNA that provides sufficient quality e g purity concentration and quantity to ensure reliable test results Written protocols must specify the minimal acceptable sample in terms of volume or numbers of nucleated cells If tests are performed without prior purification of nucleic acids the method must be documented and validated in the laboratory D 5 2 2 2 Ensure that samples are stored under conditions that preserve the integrity of the nucleic acids that will be tested D 5 2 2 3 Ensure that template quantity and quality are sufficient to provide interpretable data for a locus or loci or allele s D 5 2 2 4 Ensure that the amount of amplification template in each amplification reaction is in an acceptable range D 5 2 2 5 Ensure that aliquots of all batches of reagents solutions containing one or multiple components utilized in the amplification assay are demonstrated to b
48. sion populate Highlight first session Check that catalog is correct If not select correct catalog from pull down list Click Import After session imports it will populate in Navigator on right side of screen In Navigator click once on session to be analyzed Wait for Retrieving Session Information window to close Analyzing Sample at bottom of screen indicates progress of session analysis Wait until this is complete Click on Exon 2 and Exon 3 if applicable Lowest positive control values will populate at the top Note low positive controls if lt 500 Fl sample needs to be repeated or amplification failures samples will need to be repeated Click on Min Bead Count Note samples that have low bead count Every laboratory will need to set their criteria if the sample requires repeating If pre analysis is to be done click the exclude box next to all failed amps including negative control Click on Position samples should now populate in order Check list of samples against plate map Initial at bottom of locus to indicate this double check has been done Reviewing technologist will repeat this check and initial as well Double click on first sample in session Check in Utilities to verify serological equivalent function is activated 1 Click on Utilities on top tool bar 2 Select Molecular Product Configuration gt Molecular Analysis Configuration 3 Computer Assigned Serology at bottom of screen sho
49. st be followed Reagents for the pre PCR process must be physically separated from reagents used for the detection and post PCR product procedures Frequent wipe tests to check for contamination are required The results of this test are dependent upon proper storage conditions of all reagents All reagents are temperature sensitive SAPE and the bead mixture is also light sensitive Multiple freeze thaw cycles is not recommended for any of the reagents except for the D mix When compared to SSP SSO has more ambiguities because the probes used in SSO can interrogate sample DNA at only one region per test whereas SSP can interrogate sample DNA at two regions per test However LABType kits include specialty probes that target at least two non contiguous regions at the same time which has reduced the ambiguities previously found in SSO methods A list of resolution limitations is provided with each kit All equipment and instruments must be calibrated according to the manufacturers instructions Interferences Isolated DNA samples should not be resuspended in solutions containing chelating agents such as EDTA which are above 0 5mM in concentration Precautions As with any exposure to human blood products wear proper personal pretection at all times 2 Ethidium bromide which is used in agarose gel electrophoresis is a known carcinogen Handle with appropriate caution It can be absorbed through the skin Avoid splashin
50. sure that oligonucleotide probes and primers are stored under conditions that maintain specificity and sensitivity D 5 2 2 13 Verify that the conditions for primer extension e g polymerase type polymerase concentration primer concentration concentration of nucleotide triphosphates are appropriate for the template e g length of sequence GC content D 5 2 2 14 Ensure that for each set of primers conditions that influence the specificity or quantity of amplified product have been demonstrated to be satisfactory for the range of samples routinely tested D 5 2 2 15 Set the number of cycles at a level sufficient to detect the target nucleic acid but insufficient to detect small amounts of contaminating template D 5 2 2 16 Monitor the quantity of specific amplification products e g gel electrophoresis hybridization D 5 2 2 17 Recognize and document ambiguous combination s of alleles for each template primer or template probe combination and have procedures available to resolve these as appropriate for the clinical use of the test results D 5 2 2 18 Define and document the genetic designation e g locus of the target amplified by each set of primers or hybridized with probes D 5 2 2 19 Define the specificity and sequence of each primer and or probe D 5 2 2 20 Routinely monitor for contamination of pre amplification areas by the most common amplification products that are produced in the laboratory D 5 2 2 21 Routinely monitor p
51. tested The technologist should verify that the control beads on each individual patient sample give the expected results The technologist should verify that all bead counts are acceptable 28 5 The technologist should verify that the calibration and control beads give acceptable results prior to acquisition of data on the Luminex machine 6 All reagents should be examined for expiration dates Do not use reagents which have exceeded the manufacturer s expiration date All reagents should be visually examined for turbidity or contamination Discard any reagents found to be turbid or contaminated 7 Validation of all new shipments of reagents regardless of whether or not they are new lot numbers must be performed This can be done either by testing samples in parallel with the current lot or by testing previously tested samples on the new lot alone Ensure that the proper result is obtained Acceptable results include no amplification seen for the negative control and the expected answer same answer that was obtained with the original testing for the positive samples It is preferable to validate the complete kit as it is received in order to test out every reagent 8 Computer software programs used for analysis must be validated for accuracy A manual check of results against the computer graded analysis should be performed to document accuracy A parallel analysis of previously analyzed data is also recommended to display
52. tical and should not exceed a total of 15 minutes This includes the 10 minute incubation plus no more than 5 minutes to dilute all the samples with Dilution Solution SA PE mixture Once diluted the samples are stable at room temperature 18 30 C for up to 2 hours protect from light Since a full 96 well plate can take up to 1 5 hours to run through the Luminex Instrument the analysis should be started no more than 30 minutes after dilution to ensure that the last sample is analyzed within the 2 hour limit Do not mix components from different kits and lots Due to the complex nature of HLA typing qualified personnel should review data interpretation and typing assignments TROUBLESHOOTING PROBLEM POSSIBLE CAUSE SOLUTION e 95 gt os Low Bead count Probe Mix not well suspended Prewarm sonicate and vortex probe mix and repeat assay Instrument not Functioning properly Out of calibration Calibrate instrument Sample flow path blocked Remove and sonicate needle Perform backflush CON Threshold Failure Sample failed to amplify or amplified poorly Low DNA Concentration Check DNA concentration and purity Salts in Master Mix are out of solution Heat Master Mix at 37 C for 5 minutes vortex gently and spin down briefly Poor Taq Polymerase Use only Recombinant Taq Amplification conditions not within specific parameters Run thermal profi
53. uld be checked If it isn t click on it and click OK to notice that pops up SAVE If analysis looks acceptable click on Assign or Alt A to move to next sample If analysis indicates a false reaction for a specific bead click on the bead pull down list and select the bead in question If it is plausible that the cutoff should be changed make the adjustment Note change made in comments under lower left quadrant If adjustment results in clearly assigned alleles click on Assigned Sero at bottom of screen to get the serological equivalents to populate Continue to click Assign or or Alt A until all samples for that locus have been analyzed Repeat analysis steps l U for remaining loci Sample Reports A In FUSION click on Reports on top tool bar B In Session window type in Session Name no loci Make sure asterisk remains in place e g SAMPLERUN Click FIND C All loci tested for that sample set will populate on top right of screen D Check small square under Include for all loci E On bottom of screen click on Sample and Locus This will sort samples in correct order F Click on Reporter at top of screen G Click on Generic Typing and Molecular Custom H Check box next to 1 Sample Per Report l Click on View Report J Reports will populate on right side of screen This will take awhile K Click on Printer icon Click OK Assay Validation Benchmark 1 Positive Control bead Fluorescenc
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