User Manual xHLA
Contents
1. A 0104N 73 10 13 10 117 33 48 5 30 0103 ka A 0104N A av Exon h i G G 1 lt gt Alignment Nuc Number A70104N A iT amp C C jG T C jA T G amp C j amp CE je jc C jG jA JA C JC je jt je jc iT Figure 15 Alignment details usign A 2633 and A 01CD NMDP code The allele code list returned to A 0CD is A 0103 and A 0104N The alignment in the IMGT HLA website can also be visualized on line easily just clicking the button SJ This operation executes an integrated browser as show in Figure 16 Notice that xHLA uses old nomenclature to generate maps but since nomenclature has changed in april during web sites queries they are translated to new nomenclature The xHLA system still uses the old nomenclature because HLA MAtchmaker program was not updated until now amp i RaR Eg pf eveesii iii ee Go Reset eae E Databases Tools EBI Groups Training Industry About Us Help Site index BY amp z 5 IMGTHLA Sequence Alignments Release 3 0 0 01 April 2010 Sequence Alignment Results SUBMIT ANOTHER ALIGNMENT JOB For most browsers hold Ctrl or Apple and press or to change the size of the text Further instructions ere available here AA Codon 20 15 10 1 A 01 01 01 01 ATG GCC GTC ATG GCG CCC CGA ACC CTC CTC CTG CTA CTC TCG GGG GCC CTG GCC CTG acc CAG ACC TGG GCG G GC A 33 01 01 NU er A 68 012. If an eplet is in the NEG set it means that pre antibodies against it were not found The blue eplets algorithm paints in blue color all eplets found both in NEG and POS sets If all eplets of a HLA molecule in POS set becomes blue it means that no antibody against this molecule has been found and it is an acceptable mismatch Figure 18 1 After executing this algorithm new two sets are created The acceptable and unacceptable mismatches sets The first one contains all molecules for whose there are no pre formed antibody and the second one contains all HLA molecules for whose there are pre formed antibodies against then Shortly the acceptable mismatches are the NEG set molecules of the POS set whose all eplets are in NEG set too The unacceptable mismatches are the POS set blue molecules of the POS set After executing this algorithm the cutoff can be or not be upgraded See in Figure 18 that if the eplets 44KM and 152RW were in NEG set also the cutoff would be upgraded to 2762 However because they are not in NEG set the cutoff remains 1500 44KM and 152RW are positives Eplets A 0301 333 B 1503 328 B 5001 3259 A 6602 OF 62QE 144KR 151AHE 161D 275EL POS set Tc cutoff sia B 4901 1444 A 1102 B 1513 Cw 0303 Ccw 1402 NEG set A 1101 9H 32L 41T 44RK 113YN 116L 145QRT 150TAH 151AHE 163EW 1 9H 32L 90D g 9411 113YD 21H 46RGE 69QRQT 77TVS 80VRN All 113YD 173K 184H 219
3. different level of details The data to generate a histocompatibility map is retrieved from several sources including a Laboratory private data Luminex donor and recipient s clinical and genetic data and spreadsheets b Public database web site data IMGT HLA database and NMDP site c HLA Matchmaker program One compatibility map is generated for each donor recipient pair and the recipient can be a highly sensitized one To illustrate its use the HLA typing in Figure 12 will be considered to generate a Class map The PRA results and any other data needed to simulate this example are available in www ufpi br LIB web site xHLA programa download Recipient A 3301 A 2402 A 6801 A 3301 B 1516 B 3501 B 7801 B 7801 Figure 12 HLAtypings used to generate the Histocompatibility map of Erro Fonte de refer ncia nao encontrada The Histocompatibility map The input data to generate a compatibility map are The recipient and donor s HLA typings the PRA results and cutoff Changing any of these variables the resulting map also will be different The map showed in Figure 13 is a complete one because the four inputted variables were provided If only HLA typings are provided the Eplets map is not generated because PRA results are needed to generate it On the other side if the donor s HLA typing is not provided the Alignment map is not generated because both HLA typings are needed to generate it The hi
4. from cutoff In this case there is a high positivity associated with this eplet The 7OKHA is similar to 62GE however it was found in the donor HLA typing On the other side the 44RE has a high negativity associated with this eplet because it was found in HLA molecules with normal values to far from cutoff Eplet Eplet Cay NEG Unique eplet at donor B NEG Be4901 1448 BeS701 4589 A 2402 4246 Bx0801 729 El POS Bx5703 10458 POS Be1401 409 Be2705 9636 Bx5801 3808 A 0201 3890 Be1402 152 Be2708 4962 5 POS Ax0203 4145 Be1510 696 B 4001 8698 0201 3890 A 0206 4670 POS Be4002 13009 A 0203 4145 A 2301 3023 Be0702 11083 Be4101 7231 A 0206 4670 A 2402 4246 Be1503 3282 B 4402 12633 B 5701 4589 A 2403 3911 Be2705 9636 Be4403 11354 Bx5703 10458 Be2708 4962 Be4501 8146 Bx5801 3808 Bx3801 9543 Be4701 8758 Be3901 7272 BxS001 3258 Be4201 7710 Be4006 13296 Be4801 12373 Gray Be5501 10289 Be4901 1448 Bx5601 10121 B 5901 8054 B 6701 7545 B 7301 11828 B 8101 5393 B 8201 10038 Figure 19 Three diferent eplets and their reports The report gives a more accurate vision about the selected eplet Histocompatiblity maps and searches in databases The xHLA program can use the sets acceptable and unacceptable eplets to perform searchers in databases in order to find the best worst matching to a donor or recipient This facility can increase
5. list of donors and recipients tab to see all at once In the case of the example JONH something or something JOHN will be retrieved 2 Type the HLA typing information in the HLA typing area Figure 11 Warning Some allele codes are not recognized by HLA Matchmaker program and in these cases the most frequent in population are used instead During allele codes typing the System presents them in green color if the typed allele is valid Invalid alleles are shown in black color Figure 9 ieee ed red sa Sq ODI Chooseacode O x Figure 8 Typing na allele code The list of codes is retrieved from IMGT HLA database 3 Define the Cutoff If PRA results are available to the selected patient they are presented in the PRA results tab Figure 9 in a HLA Fusion like report PRA results amp Potential donors pS Histocompatibility Map 371772010 5 1500 Ail PRA results fo this patient A Lot Cutoff gt al class 1 Lote 5 Cutoff 1500 Raw Sample NC Ns Raw NSNC Raw Normal Dif 14210 16 13491 16 13365 16 Min 76 Mean 4843 Max 14124 NEG Control 15 63 POS Control 6109 24 54 13054 16 57 17AR4 16 75 91 59 34 4n Summary HLA Fusion like report Figure 9 PRA results of a patient reported to User 5 5 5 5 S 14124 13389 13296 13009 12634 735 93 287 37A 4 If a potential do
6. the reliability and the chances of highly sensitizes patients in the waitlist to find an organ Exporting the eplets map The eplets map can be exported to MS Excel easily in order to facilitate the medical reports In this case choose one of the eplets sets Table 1 and click the button cs to export it XHLA and the LabSystem Gen Architeture The xHLA tool uses the architecture provided by LabSystem Gen to perform its tasks The LabSystem Gen provides tools and a framework that allow researchers to build a Laboratory Information Manager System LIMS without programming efforts The architecture of the System as it is shown in Figure 20 provides three layers Application layer Data access layer and Data storage layer On the top of the architecture is the Application layer xHLA is in this layer The first step towards the creation of a LIMS is to design the Data and Application models using the Designer tool The Data Model includes a domain specific data model describing the entities tables and relationships associations between tables that the researcher wants to manage The application model in turn describes properties that allow the researcher to customize the look and feel of the LIMS graphical user interface GUI As show in Figure 20 on top a Data and Application models can be created based on an experimental plan using the modeling tools provided by Designer In laboratories where data are stored in spreadsheets o
7. 01 G AA Codon 5 10 15 20 25 A 01 01 01 01 TCC CAC TCC ATG AGG TAT TTC TTC ACA TCC GTG TCC CGG CCC GGC CGC GGG GAG CCC CGC TTC ATC GCC GTG GGC A 33 01 01 wor AC won A 68 01 01 a Sa a San ae a eet SS a ae a Se es See ae a Sa a ae St a ee eet Se AA Codon 30 35 40 45 50 A 01 01 01 01 TAC GTG GAC GAC ACG CAG TIC GTG CGG TIC GAC AGC GAC GCC GCG AGC CAG AAG ATG GAG CCG CGG GCG CCG TGG A 33 01 01 ee eet anena es erent Seared Save SS ae i et en eee A 68 01 01 SS SS SS SS SS SS SS SS ee AA Coden ss 60 6s 70 75 A 01 01 01 01 ATA GAG CAG GAG GGG CCG GAG TAT TGG GAC CAG GAG ACA CGG AAT ATG AAG GCC CAC TCA CAG ACT GAC CGA GCG v Histocompatibility Map A 2 Figure 16 IMGT HLA website integrated to xHLA graphical interface The best Alignment map shows the columns exon 2 and exon 3 with zero mismatches because clinically has been reported that mismatches in these two exons increase the chances of graft failures During searches in waitlists or databases the xHLA System tries to find a donor recipient pair with no mismatches in these two exons Eplets map To generate an Eplet map the minimum inputted data are Recipient s HLA typing PRA results Luminex csv files and the eplets for each HLA molecule In molecular terms a particular HLA molecule may prese
8. 14 Description name HIGHLY SENSITIZED RECIPIENT Y EAE at UIs A B Cw DRB1 DRB345 DOA DOB DPA DPB 3301 1516 6801 7801 Options Tree PRA results p Potential donors pS Histocompatibility Map Date mm dd yy Classe Lot Cutoff 1 1500 class 1 Lote 5 BeadID Raw Sample NC INS Raw INSNC Raw Normal Dif P ae cma 16 75 5 14124 0 Mean 4843 88 13491 16 31 5 13389 735 Max 14124 99 13365 16 59 5 13296 93 NEG Control 15 63 Dala area POS Control 6109 24 54 13054 16 34 5 13009 287 57 12684 16 40 5 12633 376 82 12691 16 115 5 12565 68 62 12447 16 64 5 12373 192 77 11916 16 77 5 11828 545 36 11888 16 65 5 11812 16 87 11825 16 94 5 11721 91 95 11639 16 100 5 11528 193 58 11394 16 30 5 11354 174 34 11154 16 60 5 11083 271 73 10548 16 79 5 10458 625 70 10402 16 103 5 10289 169 71 10157 16 26 5 10121 168 68 10150 16 38 5 10101 20 80 10099 16 50 5 10038 63 47 9675 16 29 5 9636 402 The screen is divided in four main areas i Tool box ii Options Tree iii Data area and iv Main menu area The Tool box is composed of three buttons Close application Login Logoff and Turn on Turn off web link Figure 4 The green red ball indicates that a link to web is not available or the link is slow at the moment To activate the web link you just click once in this icon and it will become green The Login logoff button opens the login dialog box to user lock the station or to log as another user Q Close applicatio
9. 17 Sometimes the cutoff cannot be easily determinate and the default initial value becomes 500 e class 1 Lote 5 Cutoff 1500 Min 76 Mean 4843 Max 14124 NEG Control POS Control 15 63 6109 24 BeadID Raw Sample NC NS Raw Nswe Raw 40 32 2852 2466 848 807 789 16 16 Figure 17 The chosen cutoff value was 1500 because the higher Dif value encountered was 854 beadID 17 and 63 All values above Cutoff is added to the POS set and all values below cutoff including itself is added to the NEG set An additional set name GRAY ZONE is also created This set is the PRA results whose normal values are too close to the chosen cutoff value up or down xHLA uses these two sets to create the following five Eplets sets Table 1 Eplets sets provided by Eplet Map 1 Acceptable mismatches No pre formed antibody against these HLA molecules 2 Unacceptable mismatches There are pre formed antibodies against these HLA molecules 3 Gray Zone mismatches These molecules are too close to cutoff 4 No mismatches These molecules have the same eplets if compared to recipient s HLA typing molecules 5 Anti donor mismatches If a donor was provided the differences between donor and recipient are presented here 6 All eplets Union of the POS and NEG sets The blue eplets algorithm Acceptable and unacceptable mismatches
10. User Manual XHLA Beta version 2010 June Summary 10104 12 Uk see eet ene ne nr rr en Er ener err cere enn re eer cee enone renee 2 MATEO ION isiin ai Ee a Ea a EE a Ea KE a a a 3 D mo limitatlons sisena ine etree eer ica EEEa E e aE S a Eeee aE ai 3 Installing XALA earner ne ner n EEEE ne AEE eer 4 Admin user and password default oiejeertceccestceseecciesteeetieetieetie eed edited 4 Downloading NMDP allele code list eccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeneeeeeees 4 Downloading IMGT HLA data ccccccccceeeeeeeeeeeeneeeeeeeeeeeeeeenaeeeeeeeeeeeseeseneneeeeees 5 SILA M din SOR aes occas owe nieces tous ia a Ea Aa EE EA DEEA ANER EAI E 5 Options i ps us eeneioe Eni oiis ebenei ENN Eu iein 7 General Workflow Processing PRA ReSullts ceceeeeeeeeeeeeeeeneeeeeeeeeeeees 8 General Workflow Generating Histocompatibility Maps ceeeeeeeeee 10 Adding deleting and navigating Local Database cccccccscseeeeeeeeeeeeeeeeees 13 Compatibility Histocompatibility Map sssce2c20 2 25 gseceactvenceastceaeeenieecoenns ceteuavieeneene 14 aligo Droo a E tener rere tre era ene renee ere Rete rre ere net eter rree rer renter 14 The Histocompatibility map 0 0 0 0 eeeeeeeeee teeter eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeetes 15 Alignment Map seen eee ee 16 Eplets Mapsi aiea cle ease ele aa eta eee aa ewe bose etna cleans ele bene 18 The blue eplets algorithm Acceptable an
11. W 267PE 49S 46RGE 65QKR 69QRQT 77TVS 80VRN 113YD 184H 219W 267PE 62QE 90D 144KR 151AHA 152HA 163RW 275EL 41T 144KR 44RK 113YN 151AHA __152HA 116L 163RW Gray zone cutoff 100 i eplet appears at least one time in NEG set E eplet NEVER apperars in NEG set Figure 18 The dance of eplets algorithm 65QKR was found both in NEG and POS sets Another important analysis is to see if the eplet in both POS set and NEG set appears only in gray zone The gray zone is determinate by user and is a values around cutoff value By default xHLA determinates the gray zone as Cutoff value 100 In the example gray zone ranges from 1400 to 1600 normal value Eplets found as neg and pos in this zone can be a false positive ou false negative So it is better not to find the eplet only in gray zone Farther from the Cutoff is found na eplet more positive or negative is the eplet Eplet report In order to facilitate compatibility analyses once the User clicks an eplet a report to it is generated and showed as seen in Figure 19 The eplet 32L was found in NEG set POS set and Gray zone set Although it is an acceptable mismatch once it is both in NEG and POS sets it can be a false negative because this eplet is only in the negative part of the Gray zone The eplet 62GE is only in the POS set and additionally it was found in three molecules as the possible unique positive eplet including in the molecule B 5801 very far
12. aboratory Unique Number 6214 IDescription name F ee 7 HIGHLY SENSITIZED RECIPIENT Y Recipient C Donor DRB345 HLA typing PRA results amp Potential donors IS Histocompatibility Map Date mm dd yy Classe Lot Cutoff gt 3 71772010 1 5 1500 i hili S class 1 Lote 5 Histocompatibility a EE T ee Pan Sample NC NSNC Raw Cutoff 1500 is lt study s area Min 76 Mean 4843 88 13491 16 5 Max 14124 99 13365 16 5 NEG Control 15 63 POS Control 6109 24 54 13054 16 5 57 12684 16 5 5 5 82 12691 16 62 12447 16 Figure 11 Local database form 2 Type the information of the donor recipient The HLA typing is stored in fields A B Cw DRB1 and son on Haplotype 1 is the upper line and the haplotype 2 is the lower line The Description name field is required 3 Click the icon to save the information Deleting Locate the record see step 1 in General Workflow Generating Histocompatibility Maps section Click the delete button Navigating To navigate use buttons prior and next t Canceling To cancel an input click cancel button x Compatibility Histocompatibility Map Introduction xHLA generates a histocompatibility map to assist researchers and clinicians in their pre and post transplantations decisions The map is divided in two major parts and each part can be visualized in
13. ace G U I The desktop based GUI is a generic program that dynamically build forms for users manage data based on the look and feel specified in the Application model The web based GUI is a set of web pages created dynamically by the Web Server Application and as desktop based G U I forms they are based on Application model and are used for users manage data xHLA as shown in Figure 20 uses the eDA framework functionalities to manage data in its local relational database and to access data existing in web resources Report BUGS Please any bug report to claudio demes ufpi edu br Manual revision Unfortunately this manual was not revised until 05 05 2010 Any comments about errors and grammar please help us to improve it reporting to us
14. ait ii Deleting a program Figure 6 top Click Del button to delete the selected program 2 Select the appropriate program m SINGLE_ABCLS1 AO04LOT O05 xs C HLAMatchmakerProgram SINGLE_ABCLS1A04LOT OOLxIs E HLAMatchmakerProgram SINGLE_BDRDQLOT 004 xls C HLAMatchmakerProgram SINGLE_ABCLS1 A04LOT O02 xIs HLAMatchmaker Program C NHLAMatchmakerProgram SINGLE_BDRDQLOT 007 xls C HLAMatchmakerPraogram SINGLE_ABCLS1A04LOT O005 x s 3 Select the desired CSV file generate by LUMINEX equipment with PRA results Process button Available PRA Files H Donors recipients to be processed Cus Name A A 7 LIB PRA single lot05 170310104303 csv Char between name and Lab Unique _ decimal symbol Processed LIB PRA single lot5 270110_ID4167 csv Name Lab unique number Date mm dd yy Values Trimmed Mean Filename a LIB PRA single Il ot 270110_1D4168 csv LIB PRA Single Il pi6 260110 1D4168 csv 6411 3 17 2010 5 29 6889 28 28 36 24 71 22 33 25 97 31 68 42 31 LIB PRA single lot05 170310 LIB PRA Single lot5 pl6 260110_ID4165 csv NONAME xxxx 6207 3 17 2010 16 34 3652 64 35 59 33 73 25 11 35 5 28 54 110 74 LIB PRA single lot05 170310_ LIB_PRA single lot05 170310_ID4303 csv RAR 37172m R 2R ARAR 54 33 R1 2R 11 15 17 78 1837 3471 321R PRA sinale IntN 170310 Figure 7 Processing PRA results If the CSV file is a valid one th
15. d unacceptable mismatches 20 ey Ce Mls 6 6 Ieee ener eter eee en ee ee ee ee 21 Histocompatiblity maps and searches in databases ceeeeeeeeeeeeeeeeeeees 22 Exporting the eplets MAD iorsicisesscsesoeseeetenuseesteadesontendetieseneaveusunisoeesvavesaesecssaueeens 22 xHLA and the LabSystem Gen Architeture cceecceeeeeeeeeeeeeeeeeeeeenneeeeeeeeeeees 22 Report BUGS srne E E eee este wie RER E EEEE A 25 Manual revision cccccccecceeececceceeeececcececuececaueceecaeecuecseaueceeseeseecueeaeeseeseenees 25 Introdution XHLA is a graphical tool based on LabSystem Gen architecture which was developed to assist researchers in pre and post transplant studies in Histocompatibility laboratories It provides a fast way to researchers manage data of donors and recipients and to study the compatibility between them The tool has the following features 1 2 Graphical and ease to use interface Data of donors and recipients are stored in a centralized and secure relational database Access Compatibility studies use both local and public domain data provided by Web resources The tool generates a compatibility map in order to assist clinicians in their transplant decisions Donors and recipient s data stored in other systems or in spreadsheets existing in the laboratory can be used in the compatibility studies easily Since uses LabSystem Gen architecture new web resources and algorith
16. directory if wished HLA Matchmaker Directory It is where HLA Matchmaker programs must be stored Figure 5 bottom Before copy a version of the HLA Matchmaker program to this directory be sure that the PANEL sheet was properly filled up and that information about beads lot were correctly inputted xHLA only accepts Omni Lambda Once a copy of the HLA Matchmaker program is coppied to this directory they can be added to xHLA System i Addind a new HLA Matchmaker program Click Add button to load a new HLA Matchmaker program and follow the sequence showed in Figure 6 During the addind process xHLA retrieves information existing in HLA Matchmaker spreadsheet to determinate the lot and class I or Il of the program However it is better to set them manually or correct this information if retrieved erroneously after step 3 of Figure 6 A new program has to be loaded every time version or lot information PANEL sheet change HLAMatchmaker Program GH SINGLE_ABCLS1A04LOT 001 B SINGLE _ABCLS1A04LOT 002 HLAMatchmaker program SINGLE _DROQLOT 004 Program HLAMatchmaker a E SINGLE _DRDQLOT 005 E SINGLE_DRDQLOT 006 B SINGLE_DRDQLOT 007 Lota 0 Class fi SINGLE_ABCLS1A04LOT 003 HLAMatchmaker Excel File Figure 6 Adding an HLA Matchmaker program Follow steps 1 to 4 After clicking button OK 4 several minutes can be needed Please w
17. e data is showed in the matrix showed in Figure 7 Click the Process button on detail to processe file Once a CSV file is processed the PRA results loaded from CSV files are visualized in the form Local Database Histocompatibility map node A record in the local database is added for each recipient processed for the first time WARNING xHLA uses internally a unique number named Laboratory Unique Number or LUN Figure 3 Data Area This number is used to identify a donor recipient internally by the System and it is a sequential unique number that is created for each patient social security number can be used as LUN for example The files containing PRA results must be produced considering this important pre requisite The sample description must contain the patient name and this number one number per patient The description must set as shown below Patient name specialCaracter aboratoryUniqueNumber Where specialCaracter can be _ Avoid using or Usage example JohnSmith_9431 In LIB laboratory it is used the underscore _ as the special character If another character is going to be used by Lab please inform the System in the text edit box named Char between name and Lab Unique Figure 7 top 4 Search the recipient in the Local Database form to check PRA results after processing operation To access this form click the Histocompatibility map node Options Tree and locate a recipient donor as ex
18. le codes of the donor recipient pair are compared to each other and if they don t match their nucleotide sequences are retrieved from IMGT HLA database and aligned b The alignment mismatches are counted and summarized in the Alignment map Figure 13 2 c Each line of the Alignment map is summary of the alignment performed The details of this alignment are showed in detail by the Detailed alignment map Figure 14 This detailed map contains three matrixes of data Alignment matrix 1 Mismatches matrix 2 and Number of mismatches by exon matrix 3 Number of mismatches by exon Mismatches 16 217 e 19 20 Exon 1 Exon 2 Exon 3 Exon 4 Ezon 5 Exon 6 Exon 7 Exon 8 ant Nuc Number 15 A 330103 61 6 10 276 72 0 0 0 425 Ax680107 6 lc e e e e A 330103 x x e x x 1 Exon 1 1 T 1 1 xta 3 2 lt gt Alignment 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 Nuc Number 65 66 67 es es 70 7n 72 73 74 A 680107 CCT G GGCGG amp e jt jc C je jA jC T C IC JA T G JA G G t A T IF A 330103 2 2 2 2 j2 jz B j2 j2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Exon 1 lt Recipient Donor Recipient HIGHLY SENSITIZED RECIPIENT Y A 6801 gt x A 3301 5 A 2 2 a aaa Onor ar Figure 14 Details
19. ms can be added in order to improve tool functionalities Automation and integration of the HLA Matchmaker and HLA Fusion programs We are improving xHLA tool in order to generate more valuable reports to works HLA matchmaker algorithm These changes are being done every day and new versions will be deployed as soon as possible Demo limitations The demo version has the following limitations 1 Advanced searchers to local database are not available These searchers allow researchers to find the best scored potential donors to a specific recipient or the best recipients to a specific donor These searchers are performed both in local database and external databases 2 Data stored in other databases or spreadsheets external data can not be integrated to be used in data analyses Installing xHLA The installation procedure is straightforward 1 Download file Application_xHLA zip 2 File must be placed in directory C Otherwise a configuration procedure is needed 3 Unzip file 4 Locate file C Application_xHLA xHLAb exe 5 Double click xHLA exe file Admin user and password default Default user Admin Default password Admin Downloading NMDP allele code list The NMDP allele list code must be downloaded every day In order to download the allele code list please do the following procedure Go to menu File and choose the option Download NMDP Data A confirmation dialog box is shown Figure 1 Choose Ye
20. n Login logoff Figure 4 Tool Box area Ble roro Turn on Turn off web link The Main Menu Area has the same options showed in Options Tree and three additional ones 1 Exit Download IMGT HLA Data and Download NMDP Data File Menu These databases are used in generation of the compatibility map You must download NMDP allele code list every day to have a more accurate analyses The Options Tree allows user to access the main functions of the application easily Click over each node existing in the options tree and the Data area right side shows the appropriate information Options tree The options tree is a dynamic tree that provides direct access to the System functionalities There are three main nodes in the tree Histocompatibility map PRA processing and Password Figure 3 Selecting the Histocompatibility map node the Data Area shows the form Local Database data donors and recipients repository and the compatibilities maps generated detailed later The PRA processing node shows in Data Area the form Process files Using this form PRA results generated by laboratory s equipments are loaded and processed The Password node shows the form Password that provides functionalities to change user s password and to create new users Figure 5 shows the PRA processing form and form Local Database is show in Figure 3 Hisrocompatibili
21. nor is available but not include yet add him her into Local Database see Adding deleting and navigating and Local Database section If he she is already in database goto step 5 If no donor is available go to step 6 5 If a potential exists and he she was stored previously in in the Histocompatibilty study s area Figure 10 Click add button 1 and locate the donor in the dialog window Search Type the name of the desired donor in the Search for edit box and press Enter The donors retrieved are listed in the matrix 3 Choose one and click OK button 4 to add him her Click Cancel button if you decided to abort database select the Potential operation donors tab Figure 10 Steps to add a potential donor to a recipient B 6 Click the Histocompatibility map tab to generate the histocompatibility map Adding deleting and navigating Local Database Patient and donors data are stored in a Local Database Local repository Thos section explains how to maintain data stored in it Adding 1 Click the Histocompatibility map node Option trees The local database form will be available as shown in Figure 11 Click the icon H in the tool bar This will add a new blank record elbe gt 26 TE as amp Recipient and Donor s data i List of donors and recipients Donors and gt recipients data Database ID 57 User 1 L
22. nt non linear sequences of amino acids eplets potentially shared by other HLA molecules HLA molecules that share the same eplets for which the patient has preformed antibodies and can be potentially recognized contraindicate transplantation On the other hand eplets that are not recognized by preformed antibodies in the renal transplant recipient in theory offer no danger to the transplant As a result any HLA molecule consisting of only eplets not recognized by the antibodies of the patient are acceptable for transplantation Such molecules are now known as HLA acceptable mismatches The elucidation of involved eplets can be achieved by running the HLA Matchmaker HMM algorithm and the xHLA generates the Eplets map based on outputs generated by this program The eplets map generation in based on Cutoff value These values will determinate which HLA molecules are positively recognized and negatively recognized by antibodies So the cutoff generates two main sets of data in the PRA results POS and NEG The determination of the cutoff is crucial in results and the xHLA produces a report basead on PRA results to assist the researcher in this determination Figure 11 Histocompatibility Studys are The report shows the PRA results ordered from highest value to lowest value The idea is to try to determinate in which moment the Normal value decreases rapidly mostly cases In the example the cutoff was determinate as 1500 Figure
23. of the alignment between A 6801 and A 3301 The Alignment matrix shows the alignment of the nucleotide sequences retrieved from IMGT HLA This matrix has four or more lines Nucleotide position recipient s allele code one or more donor s allele codes and the Exon number If one position is pink colored it means that there is a mismatch in that position If an asterisk is showed in one position it means that the nucleotide is unknown in that place and they both are counted as mismatches The Mismatches matrix shows the mismatching nucleotides found during alignment process and the Number of mismatches by exon NME matrix summarizes the Mismatches matrix exon by exon The Aligment Map showed in Figure 13 is the union of all Number of mismatches by exon matrixes generated to the donor recipient pair The generation of the Alignment map using NMDP codes as input is also possible however the xHLA System needs to retrieve allele list codes from the NMDP web site and combine them The result is an Alignment map showing all alignments possible Figure 15 is an example The donor s A 01CD creates two lines in the Alignment matrix because the allele list codes returned to this NMDP code are A 0103 and A 0104 So the A 2633 recipient is aligned to each allele of the list Number of mismatches by exon Mismatches Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6 Exon 7 Exon 8 mm Nuc Number 1 2 2 A 0103 73 10 12 10 117 33 48 5 30 2633
24. plained in General Workflow Generating Histocompatibility Maps section General Workflow Generating Histocompatibility Maps A Histocompatibility map can be generated if the donor and recipient s HLA typings are available However maps generated to highly sensitized patients require PRA results To load PRA results related to a patient see General Workflow Processing PRA Results section The steps to generate a histocompatibility map are described below 1 Search the recipient a Click the Histocompatibility map node Options Tree b The form Local Database Figure 11 is shown There are two C main areas Donors and recipients area top and Histocompatibilty study s area bottom Click a field of the form Donors and recipient s area This field will be considered by the System as parameter in searchers For instance click the Description name field if you desire to search for a record using its description information as search parameter d Click the Locate text box on top and it will become Search for text box e Type information in the Search for text box that will be used as search parameter Example type JOHN if Description was selected as parameter f Click the button Locate In the example all records containing the word JOHN are returned by the System g If one or more records match they will be presented to User If more than one is retrieved click the
25. r legacy systems an automatic approach using reverse engineering is also possible In this process they are used as input to generate the Data model However the Application model is still defined using the Designer modeling tools Designer 4 Web based application 1 data model model fo TAVS a I G U experimental and public data experimental and model data relational schema Figure 20 LabSystem Gen s general architecture In the middle layer is the eDA framework The eDA framework provides transparent access to data stored in databases and performs three major tasks to Application layer 1 It retrieves data stored in public HLA databases on web These data can be used in complex analysis performed by bioinformatics tools developed in LIB 2 It provides functions to store and to retrieve experimental data stored in local relational database and 3 It interprets a designer s data model and generates a relational schema in the local database transformation This generated relational schema stores a copy of the Designers model for future reference and the experimental data Public HLA databases and the local database are in the Data layer The local database is a backend MySQL relational database provided by System that can be accessed by applications through the eDAframework Experimental data can be collected and managed using the Graphical User Interf
26. s to confirm operation and the updated data will be downloaded and unzipped Please restart application after this operation Depending on your web link speed this operation can take several minutes Confirm Wd Figure 1 Operation confirmation before downloading NMDP Data Downloading IMGT HLA data The IMGT HLA database can be downloaded every time a new version of the database is deployed This operation can take several minutes depending on your web link speed Please restart application after this operation Go to menu File and choose the option Download IMGT HLA Data A confirmation dialog box is shown Figure 1 Choose Yes to download only missing files If you choose No the entire database is downloaded and updated locallly Confirm Wd Figure 2 Choose NO to download entire database Choose Yes to download missing files xHLA Main Screen HLA Se File Password PRA processing Histocompatibilty map Main menu ole e oof box Control tool box B Histocompatibility map y e e j Local database FP PRA processing Sh gt 8 amp Locate J Password gt gt amp Recipient and Donor s data b List of donors and recipients a Figure 3 xHLA main screen Database ID 57 User 1 Laboratory Unique Number 62
27. stocompatibility map is the eplet and alignment maps together and information existing in one complements the data existing in the other pS Histocompatibility Map Eplets ma p 5 Cutoff is 1500 Gray zone is cutoff 7 100 9411 Anti donor 3 NEG Normal il L T aaah i f f2 Pos B5301 10101 3411 B 3501 7807 B 5901 8054 44RE 116L Be3501 7807 jarr IMGT HLA website B 5701 4589 Ax3201 7279 oF 62QE 69RAHT 76ESI Bx5703 10458 B 5801 3808 Be4101 7231 9H 32L 41T 44RK 177DT 180E CW 1502 9611 i E nas Alignment map m Recipient Donor Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6 Exon 7 Exon 8 A 3301 A 2402 0 17 14 0 0 A 6801 A 3301 61 6 0 Ax2402 Be1516 Be3501 73 22 8 Ax3301 Be3501 Be7801 DRB1 1503 DRB1 1406 14 Histocompatibility Map A 1 A 2 B 1 Figure 13 A complete histocompatibility map with two parts Eplets map 1 and Alignment map 2 Eplets maps are generated based on HLA Matchmaker program and eplets data and they are important to highly sensitized patients The Alignment map is generated using data provided by IMGT HLA database and its advantage lays on the fact that PRA results are not needed to generate it Alignment map The alignment map is generated using the donor and recipient s HLA typings as follows a For each locus the alle
28. t05 170310_ 6381 3 17 2010 05 10448 28 24 95 25 73 13 08 25 84 20 35 164 2 LIB PRA single lot05 170310_ 6295 3 17 2010 6 43 9427 03 82 52 22 89 14 61 24 4 27 57 61 34 3 LIB PRA single lot05 170310_ 3 6212 3 17 2010 6 81 2708 37 56 62 38 45 19 63 42 56 41 19 30 12 LIB PRA single lot05 170310 lt gt lt gt Input directory C Input Singles Change Output directory D Output Change HLAMatchmaker root directory C HLAMatchmakerProgram Change Figure 5 PRA processing and Password forms General Workflow Processing PRA Results Read this section carefully before using the xHLA program 1 First time using xHLA select the Input Output and HLA Matchmaker directories PRA processing node a Input Directory CSV files containing PRA results generated by laboratories equipments Luminex must be stored in this directory whose default value is c Application_xHLA Input If you wish click Change button to set a new directory path Figure 5 The PRA files stored in this directory are shown in Available CSV files area Figure 5 on left WARNING Do not allow or copy any other file format to the Input Directory because xHLA can fall into an endless loop buggy b Output Directory It is used by xHLA as a place to temporary C storage while processing data The default value is c Application_xHLA Output Figure 5 bottom Click button Change to set a new
29. ty map 14 Matchmaker Program SNE Ree enn ane Ned Oe F Class Lote 5 Add Del J Password 7 Available PRA Files Hw 7 Donors recipients to be processed peaa i A CLASS 1 and LOT 5 1B PRA single lot05 170310_1D4303 csv Char between name and Lab Unique _ decimal symbol v Processed Yes L ani LIB PRA single lots 270110_ID4167 csv Lab unique number Date mm dd yy Values Trimmed Mean Filename a LIB PRA single Il lot 270110_1D4168 csv 1 LIB PRA Single Ii pl6 260110 ID4166 cov 6411 3 17 2010 5 29 6889 28 28 36 24 71 22 33 25 97 31 68 42 31 LIB PRA single lot05 170310 LIB PRA Single lot5 pl6 260110_1D4165 csv 6207 3 17 2010 16 34 3652 64 35 59 33 73 2511 35 5 28 54 110 74 LIB PRA single lot05 170310_ LIB_PRA single lot05 170310_ID4303 csv Selected CSV file data 1683 54 33 61 26 01 15 17 25 18 32 34 71 33 LIB PRA single lot05 170310_ 3 11161 65 16063 4 203 32 124 53 160 47 15 LIB PRA single lot05 170310_ Available CSV files 97 65 108 29 33 34 28 63 28 57 35 36 68 73 LIB PRA single lot05 170310_ PRA results 6220 3 17 2010 6 31 8254 11 97 64 32 06 21 04 31 56 3851 64 764 LIB PRA single lot05 170310_ 6324 3 17 2010 6 68 4451 93 90 65 38 52 20 49 41 06 57 8 138 31 LIB PRA single lot05 170310_ 6327 3 17 2010 6 92 7315 36 538 85 35 28 36 89 29 35 5289 93 50 LIB PRA single lot05 170310_ 6320 3 17 2010 6 68 8663 89 76 72 35 96 25 21 43 89 40 13 103 2E LIB PRA single lo
Download Pdf Manuals
Related Search