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DynaChip™ Software Manual

Software Version 1.1.x

Rev 002

DynaChip Software v1.1.0 Manual Page 2

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Contents: Page Section 1. Software Overview ....................................................................... 4

Loading the software ......................................................................................... 4 First-time installation.......................................................................................... 4 Upgrade installation - Database Upgrading........................................................... 4 Using the software............................................................................................. 5 Software Summary ............................................................................................ 7 Overview – Browse Menu ................................................................................... 9 Making changes: ............................................................................................... 9 Browse Patients:...............................................................................................10 Browse Requestors: ..........................................................................................10 Browse Samples: ..............................................................................................11 Browse Tests: ..................................................................................................12 Browse Runs: ...................................................................................................13 To Finalise a Run ..............................................................................................13 To Import data.................................................................................................13 To print a Report ..............................................................................................13 Editing a Run: ..................................................................................................14 Browse Frequencies – Population tables for vPRA ................................................15

Section 2. Workflow ....................................................................................16 Overview – Wizards Menu .................................................................................16 Test Wizard - overview......................................................................................16 Finalising a Run and generating a worksheet.......................................................18 Assay steps:.....................................................................................................22 Alarms during assay..........................................................................................22 End of Assay ....................................................................................................23 Run Imports.....................................................................................................23

Section 3. Analysing Data............................................................................24 Specificity Tab..................................................................................................24 aPRA ...............................................................................................................25 vPRA ...............................................................................................................25 Masked Antigens ..............................................................................................25 Substance Tabs ................................................................................................26 Searching for HLA antigens................................................................................27 Working with spots and cut offs .........................................................................27 Statistics Tabs ..................................................................................................29 Antigen Table...................................................................................................29 Excluding antigens............................................................................................29 Tail analysis Tab...............................................................................................30 Basis of Tail analysis: ........................................................................................30 To use Tail analysis: .........................................................................................30 Reports............................................................................................................31

Section 4. Administrative Features .............................................................33 Users...............................................................................................................33 Updates ...........................................................................................................33 Settings ...........................................................................................................33 System Registry................................................................................................33 User Registry Overrides.....................................................................................34 Security Settings...............................................................................................34 DynaChipTM Processor .......................................................................................34 Manual control..................................................................................................35


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Section 5. Tips and FAQs .............................................................................36 Tips:................................................................................................................36 Sorting.............................................................................................................36 Frequently Asked Questions...............................................................................36 Troubleshooting................................................................................................38


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Section 1. Software Overview Loading the software

The DynaChipTM software can be launched from the shortcut which should be installed on the desktop of the computer. Alternatively, the program is at C:/Program files/Dynal/DDS/DDS.exe The program will start up with a splash screen that displays which version number is being used:

First-time installation If this is the first time the software has been installed, then a new database should have been created. However, a newly created database does not contain any product information or antigen frequency data. To apply these, see ‘Updates’ in section 4 of this manual. The update files can be found on the software installation CD in the folder ‘Updates’. Product updates for newly released batches and products are supplied on CDs in the relevant kits. Upgrade installation - Database Upgrading Part of the new version of the software is that changes have been made to the database structure. If the software is being run connected to an old database, an error message will occur:

Selecting ‘Yes’ opens the Database Settings dialogue box:


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Ensure that the correct database is selected, and then select the ‘Upgrade’ option. This will apply the updates to the existing database – no changes will be made to existing data. Note that once a database has been upgraded, all copies of the software that connect to this database will also need to be upgraded – different versions cannot share the same database. Using the software

The screen will open showing the options to Login or Exit – no work can be performed without a user logged in. Using the settings option under the Administration menu, the database to connect to can be chosen, if not currently set correctly.

When the user clicks on Login, a dialogue box will open


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The correct user name should be chosen from the drop-down menu, or typed into the top box, and then the password entered. Users can be set up with different privilege levels – either ‘User’ or ‘Supervisor’ or ‘Administrator’. These levels affect the access of the user to advanced features within the software. NB – The first time the software is used, ‘Administrator’ will be the only available option, and a password will need to be set. Passwords are case sensitive. To create new Users, login as an Administrator-level user. Only these users can access the Browse Users screen, where users can be added, disabled or deleted.


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Software Summary

The DynaChipTM Software is based around an SQL Server database. The database stores results from individual Tests – each Test relates to one DynaChipTM, and these are the basic units of the database.

Each Test must be linked to a Sample. Several Tests can be linked to the same sample – e.g. if a repeat test is required, or a titration. Samples can also be linked to a Patient, although this is not strictly necessary. Several Samples can be linked to a Patient – i.e. samples taken over a period of time. Each Test must have a Product associated – at the time of writing, there is only one Product released, but it may be possible in the future to choose between, for example, Screening, Specificity or Single antigen Products. At the time of creation, each Test automatically includes the details of the User logged on. Each Test can also have a Requestor – e.g. name of the Doctor or Clinician or Department requesting the test be carried out. Tests must be placed in a Run, and the Run must be finalised to be performed on the DynaChipTM Processor. The Test location in the plate and the User who finalised the Run are also recorded. Once a Run has been performed, the raw data is added to the Test by importing. The User who imports the data is recorded. The data is then viewable through the software. Statistical tests are carried out to determine a list of possible specificities, and the final reviewed specificity is recorded. Specificities and data can be printed out or saved as PDF files for record-keeping.


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Workflow:

The software is also designed to enable use of macros, written in JScript or VBScript. The use of these macros would permit transfer of data between LIMS systems or Microsoft Excel and the DynaChipTM software. For more detail on parameters needed to write these macros, please contact your local sales representative or Technical Services ([email protected]).


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Overview – Browse Menu

All the layers of data stored in the database can be accessed from the Browse Menu.

The first five items in the ‘Browse’ menu open up a date-filtered view of the stored data, with the Filter Options tab at the top left of the view. To see an alternative subset of the data in the database, open the Filter Options tab by moving the mouse over it, and enter the details to filter with. To see all data in the database, use the ‘Clear’ button. Making changes: All Users can edit data. To ensure database integrity, Tests cannot be deleted once created, and for other types of records, only un-associated data can be deleted. The final item – ‘Browse Frequencies’ – opens a view of data which is only editable by Administrator-level users. In all views, changes made are not implemented until the record is saved – by clicking the icon of the disk with a blue arrow.

To discard changes, click instead the icon with a red cross.


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Browse Patients: Opens a list of Patients. Details of Patients can be seen, and the list can be filtered based on any of these details by using the ‘Filter Options’ tab in the upper left of the window. Any Patient record can be opened by selecting the Edit icon, or by double-clicking the record.

Opening a Patient record shows three tabs: Patient Details allows editing of details, and addition of any comments. Sensitising Events allows entering of patient genotype and any data about sensitising events. Patient Samples allows viewing and opening of any samples associated with that Patient.

Note: Not all samples will have a Patient associated, as there is no requirement. Browse Requestors: Opens a list of Requestors. Opening a Requestor record allows viewing of details associated with that Requestor.


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Browse Samples: Opens a list of Samples. Details of Samples can be seen, and the list can be filtered based on any of these details by using the ‘Filter Options’ tab in the upper left of the window. Any Sample record can be opened by selecting the Edit icon, or by double-clicking the record.

Opening a Sample record shows two tabs: Sample Details allows editing of details and addition of any comments. In addition, an existing Patient can be associated to the Sample. Test Details allows viewing and opening of any Tests associated with that Sample. In addition, a new Test can be requested on the Sample.


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Browse Tests: Opens a list of Tests. Details of Tests can be seen, and the list can be filtered based on any of these details by using the ‘Filter Options’ tab in the upper left of the window. Any Test record can be opened by selecting the Edit icon, or by double-clicking the record. This view has many columns (more than viewable at once on most screens), the order of which can be changed by dragging using the mouse.

Tests have a status flag: Unlisted – Test exists, and is currently editable, but is not currently attached to a Run. This can be done using an ‘Edit Run’ screen. Listed – Test is attached to a Run, but is still currently editable Finalised – Test is attached to a Run which has been finalised, and is therefore not editable. However, results have not yet been returned from the DynaChip Processor. Analysed – Test has been carried out, and results are available for review. To view results open the Test and choose the Results tab.


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Browse Runs: Opens a list of Runs. Opening a Run shows the Tests to be carried out/that have been carried out in that Run. This is the main screen used to access data and print reports. Depending on the stage in the workflow, each Run has a particular status, with coloured icons to indicate status:

To Finalise a Run for processing on the DynaChip Processor, select the Finalise icon: This exports a file to the DynaChip Processor, and can only be carried out on a computer connected (either directly or via the network) to a DynaChip Processor. When a Run is finalised, no editing can take place of any Tests or other details associated with the Run. At this point, a worksheet is generated to assist work in the lab. This can be printed or saved.

To Import data from a Run that has been carried out, select the Import icon: This will open a dialogue box of the location where data is stored by the DynaChip Processor. The most recently carried out Run will always be stored in the highest-numbered folder. Data is automatically associated to the correct Run. If an error occurs during import, there will be a dialogue box asking if you wish to accept the import. Always select ‘No’, and then contact Technical Services.

To print a Report, first select the Run, then select the icon . This icon will only be available when an Imported Run is selected. See the Report section of this manual for more information.


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Editing a Run: The name of a Run (which is still editable) can be changed by opening the Run and choosing the ‘Run Details’ tab. The Tests on the Run can be changed by opening the Run and choosing the ‘Run Tests’ tab.

In this Edit Run window, there is a graphical representation of the 96 wells available for carrying out Tests in a single Run. Wells with a Test are highlighted in green, while wells without a Test that will be wasted if no Test is added have a purple outline.

To remove a Test from the Run, first select the Test. Then select the Delete icon:

To add a currently Unlisted Test, select an empty position. Then select the Add icon: This will open a window with a filtered view of all currently Unlisted Tests. To create a new Unlisted Test, either use the Test Wizard, or use the New Test icon while viewing a Sample.

To add all currently Unlisted Tests to the Run, select the Bulk add tests icon: This will fill the whole plate if there are that many currently Unlisted Tests. If this requires more strips than currently available from the kit, use the tickboxes above the strips to remove strips until the number of strips to be used matches the number of strips available. Note – Never deselect strips from the middle of the Run, only from the end. The instrument will process all wells between the first and the last on the layout as shown on this screen.

To save changes made, the Save icon must be selected – if the window is closed without saving, changes will be lost.


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Browse Frequencies – Population tables for vPRA The other item in this menu is ‘Browse Frequencies’. The Frequencies screen shows the population data used to generate virtual PRA (vPRA) values based on the results of the test. There are 5 available populations, of which 4 are initially blank. Administrator-level Users can enter data into these populations, to represent the local distribution of HLA alleles. To do this, open the table up to the allele level, and select the allele to be edited. Once edited, data can be saved and reloaded. The screen also allows vPRA values to be generated for a user-defined antibody specificity, for each of the populations entered. The user can type, or paste, the antibody specificity into the box at the top of the page. The PRA will be calculated in the blue rows above the Class I section and the Class II section of the table.


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Section 2. Workflow Overview – Wizards Menu

The Wizards menu always contains the Test Wizard – a simple interface that allows creation of Tests and Runs, using either existing data in the database or creating new records of Patients, Samples and Requestors. The menu can also include any Macros that have been written into the software, but the default installation does not include any. Test Wizard - overview Launch the Test Wizard from the Wizards menu:

The Test Wizard will open up in a new floating window. Each line in the window represents a Test. All columns are resizable, and the order can be changed by dragging.

The Run tickbox in the first column controls whether the newly-generated Test is immediately added to a Run, or just created as an Unlisted Test (for use when adding Tests to an existing Run). The Test ID box will automatically generate a Test ID based on the date. This can be edited to a more meaningful ID if required, but note that Test IDs must be unique within the database. The boxes with a red exclamation mark are required fields – Product and Sample. Products can be chosen from a drop-down list, on which there will usually only be one choice. The default layout only requires that Product is set for the first Test, and will be automatically used for subsequent Tests.


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The boxes with a blue question mark are optional fields, where existing data can be used, or new records can be created. If using/creating a Sample associated with a Patient, select or create the Patient first. To find existing data, typing the first few characters will open a list of the matching records in the database for selection. In the example shown here, typing ‘p’ in the Patient ID box shows three records. Double-click to select the matching record.

If existing details are selected correctly, the icon will show a red tick mark:

If creating a new record, type in the new details, and the icon will show a yellow star: to indicate that new data has been entered. In this screen, the new records that can be created by typing in new details are: Sample, Patient and Requestor. If a Patient has been chosen, clicking in the Sample ID box will open a list of all samples (if any) associated with the Patient for selection.

Select the chosen Sample, or enter new details. If new Sample details are entered at this stage, they will be associated with the chosen Patient. The most basic (and fastest) use of the Test Wizard is simply to select Product, and then simply enter Sample IDs. The barcode reader can be used to scan samples if barcode labelling is used in the lab. Once a Test has been entered correctly, press ‘Add Test’ to create the next Test. Alternatively, press the down arrow on the keyboard. The disk icon is used to show whether a Test has been created yet – this only takes place when ‘Next’ is selected. To remove a line, select each field containing data and press the delete key on the keyboard.

The other icon used in this window is the re-use option and is available for several columns. If enabled, when a new line is created, the same data from the previous line is used. Once all data has been entered or selected, click ‘Next’. If any tests were selected for, using the tick-box in the first column, these will be added to a new Run (the Run is given an


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automatically generated sequential name, which can be altered). If no tests were selected, the next window will appear blank; new data and Tests entered will be saved into the database for later use in an ‘Edit Run’ screen, either with ‘Bulk Add Tests’, or ‘Add Test’. Finalising a Run and generating a worksheet The next stage for a Run is to finalise the list of Tests and make the data available to the DynaChipTM Processor for experimental work to be carried out. This action is carried out by

selecting the Finalise icon in the Browse Run screen. A worksheet is generated, which can be printed or saved and may be used to ensure the User has the correct samples/Tests in the correct order. Finalising a Run will only proceed on a computer linked to a DynaChipTM Processor, as the data needs to transfer to the Processor module. A remote link can be set up from the DynaChipTM Processor tab of the Settings screen (only accessible by Administrator level users). Once finalised, the coloured icon in the System ID column for the Run will be yellow. The Run cannot now be edited, but it is possible to open the Run to view the Tests listed. At this point in the workflow, the assay should be carried out on the DynaChip Processor, before importing the data back into the database.


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Performing a Run using the DynaChipTM Processor Once the Run details have been finalised, the Run assay can be carried out using the DynaChipTM Processor. First, ensure that the Processor is powered on, and that the lid is closed. To launch the processor, select ‘Launch Processor’ from the main menu of the software.

This opens the DynaChipTM Processor software module, and initialises the instrument.

[If the Processor is not powered on, a dialogue box will open and give the options ‘Abort, Retry or Ignore’. Selecting ‘Ignore’ allows the DynaChipTM Processor module to open, but only to access archived images by selecting the ‘Archive’ button].


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To carry out the Run, choose ‘New Test’. This opens a screen with a drop-down menu.

Select the Run you wish to perform. The name of the Run will start with the date it was Finalised, and then show the Run ID Click ‘Next’.


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The following screen asks for identification of the strips to be used in the Run.

Select either barcode scanner or manual keyboard entry option. For keyboard entry type in the 12 digits below the barcode on the strip pouch e.g. 123450130006 N.B. Each strip has a unique barcode. The software will not proceed until all barcode numbers have been entered based on Test information supplied when creating the Run. Click ‘Next’. The final screen before starting a Run displays several tick-boxes (scroll down to see all boxes).


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These must be ticked (checked) to confirm correct instrument set-up before starting a Run. The User must check they have completed each point before selecting the relevant tick-box. The assay will not proceed until all boxes have been ticked. Click ‘Start’ and the instrument will proceed to carry out the assay. Messages show the timing of steps carried out. The instrument will pause at certain steps to ensure all wells are treated equally. The circle icon will continue to move for the whole process; this indicates that the assay is proceeding.

Assay steps: Step 1 – Wash chips and add Sample Diluent for Pre-blocking : 40 mins Step 2 – Aspirate chips and add Serum samples (diluted in Sample Diluent) : 60 mins Step 3 – Wash chips and add Detection Antibody (diluted in Sample Diluent) : 40 mins Step 4 – Wash chips and add Substrate : 40 mins Step 5 – Aspirate chips : Up to 40 mins (depending on number of chips) Step 6 – Image chips ready for software analysis : 5 mins

Alarms during assay If the cover is opened during an assay, an alarm will sound, and a dialogue box will appear on screen:


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The User must close the cover within 300 seconds, or the Run will be aborted. This is to ensure that results are comparable. If the cover is opened, and subsequently closed, the assay will proceed, but the length of time the cover was open is recorded in the message screen:

If, for any reason the Run needs to be aborted, press the ‘Abort’ button. This should only be used under extreme circumstances. Restart the computer and the instrument following this action. Chips should only be used to restart the same Run if the instrument had not yet reached the sample addition step. End of Assay At the end of the assay, the camera and software will carry out image analysis of the chips; during this process the computer screen will be non-responsive. Once complete, the screen will indicate that the Run is finished, and allow the user to wash the tubing and dismantle the instrument. At this stage, it is possible to view an image from the Run by clicking on one of the thumbnails. The Run data must now be imported to the main DynaChipTM software database. Run Imports Result files are stored on the computer in numbered folders at the location C:/Program files/Icono/Data/Tests/ To import Run data, click the Import button on the Browse Run screen. In the dialogue box that

opens, choose the numbered folder of the Run to be imported. The last Run performed always has the highest numbered folder. The software automatically associates data with the correct Run, and ensures that Runs are not imported more than once.


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Section 3. Analysing Data To analyse Run data, open a completed Run from the Browse Run screen, or view a Sample in the Browse Sample screen and choose a Test. There are several tabs available. This section explains each in turn. Specificity Tab

The software uses a variety of statistical analyses to generate a list of likely anti-HLA antibody specificities. These calculated specificities may differ from those calculated subjectively by an experienced serologist (for further information, see the section on Statistics Tabs below). Three calculations are carried out: +) Using all spots above the basic cut-off as positive [default cut-off is 0.10] ++) Using only spots above the second cut-off as positive [default cut-off is 0.25] +++) Using only spots above the top level cut-off as positive [default cut-off is 0.50] The utilisation of several cut-off values helps to analyse more complex sera.

The User can choose the specificity that best describes the serum by selecting the button next to the specificity. This displays the specificity in the ‘Chosen specificity’ box (blue for Class I, green for Class II). This specificity will be displayed in the report when printed. Alternatively, if none of the calculated specificities matches what the User believes to be the true specificity, the ‘Chosen specificity’ box can be edited. If this happens, none of the buttons next to the specificities will be highlighted.


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Also shown under the Specificity tab are: aPRA (assay Panel Reactive Antigen) – The aPRA returns the percentage of spots deemed to be positive (> lowest cut-off) , regardless of the specificity. vPRA (virtual Panel Reactive Antigen) – the % of a given population that would be expected to react with the determined specificity. This is based on the population frequency data accessible from the Browse menu. Note - It is important to note that the vPRA is based on specificity, so if no specificity is assigned the vPRA will be negative, regardless if there are positive spots or not. Masked Antigens – any antigens which are only present on spots which also contain antigens included in the calculated specificity.


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Substance Tabs

The tabs marked ‘Class I Substance’ and ‘Class II Substance’ allows access to all the data generated in a Test.

At the top of the data table the control spots RS1, PC2 and PC3 are always displayed. Positive results with these spots prove the correct addition of reagents to the Chip. These spots are:

Biotin marker spots - RS1. If positive, these spots prove the correct addition of the detection reagents – Detection Antibody & Substrate. If negative, results from the chip in question are invalid. These spots are also used for image orientation during image analysis.

Anti-Human IgG spot - PC2. If positive, these spots prove addition of serum sample to

the chip, as all serum samples contain Human IgG, irrespective of the presence of anti-HLA antibodies.

Human IgG spots - PC3. If positive, these spots prove the correct addition of the

detection reagents – Detection Antibody & Substrate. The table shows a result for each of the antigens tested. An antigen is positive if it is above the lower cut-off (default value 0.1 for +). There are two higher cut off values (default values 0.25 for ++ and 0.5 for +++) to allow calculations using only the strongest reactions. This helps


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with interpretation of complex specificities. An antigen is negative (-) if the value is below the lower cut off. It can be void if the software detects problems (e.g. dust) and inactivates both duplicate spots. Both negative and void spots are not included in statistical calculations – void spots affect the total number of spots in the calculation. All values have the local background for the spot subtracted – this removes the non-specific binding to the chip surface. Searching for HLA antigens Both the Class I and Class II Substance tabs show a box labelled ‘Search’. This can be used to highlight antigens or alleles within the data and bring them to the top of the view in the table. The HLA specificity can be entered in as much or as little detail as required – typing ‘B17’ will highlight all HLA-B57 and HLA-B58 antigens, but typing B5703 will only highlight HLA-B*5703 alleles. As many antigens or alleles can be entered as required, simply separate by a comma. Data can also be pasted into this box (e.g. patient HLA genotype) or copied from this box to the ‘Chosen Specificity’ box in the Specificity tab. An alternative way of highlighting a particular antigen or allele is to right-click on it within the table. A menu will appear, allowing the user to select or de-select that antigen or allele.

When particular antigens or alleles are highlighted, they will be sorted and viewed at the top of the table. This allows the user to examine all the values for a particular antigen or allele, without having to manually search through all the antigens or alleles in the table. Working with spots and cut offs The histogram shows data as an average of any duplicates of spots on the Chip. Any variation between the duplicates is shown as an error bar. To look at the data for individual spots, double-click the entry, and it will expand to show the spots. The likely cause of a discrepancy will be dirt or dust on the Chip. It is likely that the higher value will be the error, as there are few cases where a spot will record a false negative value. If a spot is giving an erroneous value, there is the option to declare it inactive by clicking in the Result column. The analysis software also tries to detect spots with dirt or dust interference, so some spots may already have been deactivated. In the example shown here, the weaker duplicate spot has been inactivated by selecting the spot 1872 and selecting ‘inactivate’ from the drop-down menu. The override column shows that one spot has been overridden and that this has changed the call for the whole substance (from + to ++).


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To alter cut off values for a given antigen, drag the lines on the histogram. If the alteration changes the result (e.g. changing an antigen from ++ to - ) then the ‘Override’ column will show ‘Yes’ in red. This change to the result is also noted on the report. Values cannot be changed – only the result. In the example shown here, the cut-off for spot 9 has been adjusted below 0.083 by dragging the cut-off line. The override column shows that this has changed the call for the substance (from – to +).


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Statistics Tabs

Antigen Table The anti-HLA antibody specificity analysis in this version of the DynaChipTM Software is based entirely on statistical calculation. With a Run open, the statistical data can be viewed by clicking on either of the tabs ‘Class I Statistics’ or ‘Class II Statistics’. Two calculations are performed – 1. Chi-squared with Yates’ correction and 2. R-values for goodness of fit for a putative specificity A cut-off is applied to the table, and any antigens with statistical values above the cut-off are used in the specificity table (viewed using the ‘Specificity’ tab). To explain the table: : Each antigen is considered as a broad antigen (e.g. A9), a split antigen, (e.g A24) and an allelic antigen, (e.g. A2403). A chi square is carried out for each broad, split and allelic antigen. In each case, four values are determined: TP – True Positives – the no. of positive spots which contain the antigen in question. FP – False Positives – the no. of positive spots which do not contain this antigen. FN – False Negatives – the no. of negative spots which contain the antigen. TN – True Negatives – the no. of negative spots which do not contain the antigen. All calculations are independent of the other antigens. To give an example: There are 49 Class I spots on a Chip. If there are 10 spots on the Chip containing A0201, and they are all positive with a particular serum, while all other spots are negative, the values are TP=10, FP=0, FN=0, TN=39. This gives the highest possible statistical value (R=1.000). If, however, with a different serum, only 2 of the A0201 spots are positive, but 8 other different spots are also positive , the values are TP=2, FP=8, FN=8, TN=31. This gives a much lower statistical value, indicating a lower likelihood of the reactivity being due to the A0201 antigen. [Further explanation of statistical analysis for HLA, and references for further reading, can be found in Part 5 of the book Histocompatibility Testing (Bidwell & Navarrete, Imperial College Press, 2000, ISBN: 1860941567)] The options on this screen allow changes to cut-off values for R-Value and Chi-squared:

Combination of R-squared and Chi-squared used, The sort order Level (allelic, split, broad or all) the specificity generated will be.

The final option, signal strength, allows the statistics to be viewed based on all positive spots, or only those over the two cut-off values set on the Substance screen. Excluding antigens Antigens can be excluded from the table by typing them into the Excluded Antigens box, or by right-clicking alleles from the Substance Tabs and selecting ‘Exclude’. Excluding an antigen means that it does not get added to the specificity list and also provides a visual aid to interpretation – it does not have an effect on the statistical figures. The only effect on the statistics sheet is to cross out the antigen within the table.


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Tail analysis Tab

Basis of Tail analysis: Tail analysis is the iterative process of looking for anti-HLA antibody specificities in the data that may be partially obscured by other, more statistically significant specificities. It can help interpretation of more complex data, providing assistance for the user. The calculation involves selecting the specificity with the highest statistical value, and excluding it from a recalculation of the statistical analysis. This can then be repeated until the statistical values are too low to be significant, or until all the positive values have been assigned to a specificity. To use Tail analysis: With selected Run open, choose the sample of interest from the grid. Next, choose the tab ‘Tail Analysis’ from the far right of the result view bar. This opens the tail analysis view. The screen is spilt into two equal parts – Class I and Class II. Results are automatically generated based on the default parameters. Parameters that can be adjusted include: Antigen Analysis level – in common with other parts of the software, analysis can be carried out at different levels of allelic definition: ‘Allelic’ carries out the calculations with each individual allele (eg. B*5703 will be separate

from B*5701). ‘Split’ carries out the calculation with each two digit allele (eg. B5703 will be in the same

calculation as B5701 – they both come under B57). ‘Broad’ carries out the calculation with each serological group of alleles (eg B57 will now be

grouped with B58 – they both come under B17)

Analysis order – the antigen to be removed from the analysis on each iteration is the one that has the highest values of R and Chi squared, the analysis can be prioritised on either R value or Chi squared. Analysis strength – in some samples with a higher background, a more powerful analysis of specificities is possible using statistics based only on the spots with the strongest reactivities. Number of iterations – the calculations can be continued until all potential specificities have been determined, or stopped after a given number of iterations.


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Reports

Reports are generated by selecting ‘Print Report’ from the Browse Run screen. The dialogue box that opens allows the selection of options before output occurs.

The first screen gives the possibility to choose the Tests to include in the report, while the second screen gives the choice of tables to include in the report.

The final screen, Output Options, gives the choice of how to output the report. It can be sent straight to the printer, saved as a PDF for future use, or viewed as a preview. From the preview screen it is also possible to save the file as a .ndr file, but not a .PDF. To save as a .PDF, close the preview window using the exit door, and generate the report again. [.NDR files are usable through the Tools menu on the main screen of the software, where there are options to convert .ndr files to PDF files, or to print them.]


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The report shows details at the top. There are different coloured boxes containing the Test data, the Sample data and the Patient data (if available). Next are the aPRA values, then the specificity table (including masked antigens and vPRA). The third column of the specificity table uses a single letter to denote the basis of the specificity – A = All positives M = Medium (++ and +++) positives S = Strong (+++ only) U = User defined. If selected, Tail Analysis results are displayed next.

The rest of the report shows the tables of data for the Test. Antigens are shown at the split level, and there is a column showing if a result has been overridden by the User. If selected in the Print Report dialogue box, there are separate tables for Class I and Class II, as well as separate tables for Class I Statistics and Class II statistics. If used, the statistical tables are reported in two columns, to save space on the page. A quirk in the reporting system means that these statistical tables must be read in this order: 1 2 3 4 5 6 7 8 Etc.


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Section 4. Administrative Features Users

Administrator level users have control of all other users e.g. only Administrators can create and edit other users. Users can be marked as deleted, but this is permanent. A temporary removal of a User from being able to use the software can be affected by marking them as disabled. Administrators can also alter the number of attempts to logon a user is allowed before their account is locked, with the exception of the initial “Administrator” account. Should this occur, Administrators are the only users who can re-enable the user and if necessary reset their password. Updates

Under the Administration menu there are two update options available: ‘Load Product Updates’ allows the addition of product updates e.g. DynaChipTM with

increased numbers of antigens spotted. Also required when first setting up a new database.

‘Load Antigen Updates’ allows the addition of updates that add new information to the allele frequency table. These updates will be made available through our website (www.invitrogen.com)

Settings

The Administrator is the only user that can change all settings. Settings allow various changes to the workings of the software, and caution should be exercised in making alterations. If in doubt, don’t make the change, and contact your local sales representaive or HLA Technical Services ([email protected]) to ask for help. It is possible to make changes that will render your copy of the software inoperable without expert help. Within Settings there are 5 tabs: System Registry This tab controls the database being accessed by the software. The first option – Server Connection Timeout – allows an increase to the period of time the software will wait for a response from the database during startup, before aborting. Use this option if the database connection is very slow. The second option allows the user to determine the database accessed by all users on the computer where the alteration is made. There will be very few circumstances where this is needed – for example if IT staff decide to change the location of the server – and should not be changed without consulting with qualified staff.

mailto:[email protected]


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Note: In an NT environment only users logged on to the PC as an Administrator can make these changes. User Registry Overrides This tab allows the same controls as the previous tab, but at a Computer User level rather than applying the settings to all Users on the computer. Settings on this tab override settings on the previous tab, for the User concerned. Note: This applies to the computer user account not the software users. Security Settings Allow NT logon synchronisation – if enabled, the software attempts to use the logon ID and password of the person using the PC as the logon ID and password for the DynaChipTM software. You can only do this if the username and passwords are both the same, if only the usernames are the same then you will need to enter the password and you will be prompted to synchronise the password, no check is made to see if the passwords are the same. Prompt to logoff instead of exiting – if enabled, when the user clicks exit while logged on, the software will display a prompt to suggest logging off instead of exiting the software altogether. DynaChipTM Processor This tab allows control over the display of the buttons for using the DynaChipTM Processor. ‘Launch Processor’ and ‘Finalise Run’ will not work on a computer not attached to a DynaChipTM Processor. Use this tab to disable these buttons on computers not attached to an instrument. It also contains an option for defining the location of the files the DynaChipTM Processor uses. This option can be used for a computer in a location remote from the DynaChipTM Processor – define the folder on the computer attached to the DynaChipTM Processor across the network, to allow workers in the lab to carry out the Run. The final option should not be required by users, and should not be altered, unless specifically instructed to do so during a support call.


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Manual control Import and export of files to the DynaChipTM processor can also be carried out manually. This tab allows the definition of which User Levels can see these buttons. There is also an option to define the default location to find import files. Again, this can be used for a computer based in a remote location from the lab – set the path across the network.


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Section 5. Tips and FAQs Tips:

When the edit boxes do not automatically appear, press F2 to bring up the edit box. If new items do not automatically appear, e.g. after running a macro or creating a new Run, press F5 to refresh view. If while editing a field you realise you have made a mistake before the change is accepted press Esc to undo changes to the edit field. This is useful when the system refuses to accept the change. Sorting All tables with non-editable data displayed can be sorted by any of the headings in order to find the most relevant data. Just click the heading to carry out the sort. Click the same heading again to carry out the sort in the opposite direction. The order of the columns is also editable – move the column to its new location by clicking on the column heading and dragging. Frequently Asked Questions

Q: Previous Runs are no longer visible in the ‘Browse Run’ screen / a newly created Run is not visible in the ‘Browse Run’ screen. Why is this? A: The ‘Browse Run’ screen has a filter that allows powerful searches for Runs. The filter action is only carried out

i) When the window is first opened (the default filter is for Runs created during the last 3 months)

ii) When the ‘Filter’ button is selected (from the filter tab, accessible at the upper left of the window)

iii) When the user presses the F5 key to refresh the view Q: How can a Run be renamed? It isn’t possible in the Browse Run screen. A: To rename a Run, open the Run and select ‘Run details’ near the top of the screen. Here, the Run ID is an editable field (for Runs that have not yet been finalised). The Run ID is also editable on the final screen of the Test Wizard. Q: Where do the factory installed frequencies come from? A: They are compiled from a combination of sources including internal data and data from www.allelefrequencies.net. They are intended to be representative of a Caucasian population – users can add more appropriate data, if needed, to the 4 available user-defined frequency tables. Q: Why are there three different sets of data on the specificity tab? A: The three sets of data reflect the results of the statistical calculations carried out using three different cut-off values. Each of these calculated specificities may differ from those calculated subjectively by an experienced serologist, but they should indicate some of the most likely specificities, and help guide the user through interpretation of the data. Q: Which of the three different sets of data should be used? A: All data should be reviewed by an experienced serologist. If the calculated specificity matches the specificity derived by the experienced serologist, selection of the radio button next

http://www.allelefrequencies.net


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to the calculated specificity places the data in the ‘Chosen Specificity’ box. If the calculated specificity differs from the derived, the ‘Chosen Specificity’ box should be edited. Q: What other help does the DynaChipTM software offer the user? A: The Tail analysis tab can help with deriving specificities. On the Substance screens, typing in antigens to the search box highlights all instances of those antigens within the panel Q: What is the typical workflow with this software? A: Create Run using ‘Test Wizard’ (under ‘Wizards’ on main menu)

Finalise Run (button on ‘Browse Run’ screen) Launch Processor (on main menu) & carry out assay on DynaChipTM Processor Import data (button on ‘Browse Run’ screen) Analyse data (open Run from ‘Browse Run’ screen, and review data) Print / save Report (button on ‘Browse Run’ screen)

Q: After viewing a preview of a Report, it was saved as a .ndr file. Why is this, and how can it be printed? A: A report can be saved as a PDF file from the main ‘Print Report’ dialogue box, but not from the preview screen. There are tools to use an .ndr file in the ‘Tools’ subsection of the main menu.


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Troubleshooting OBSERVATION POTENTIAL CAUSES and PREVENTATIVE ACTIONS All spots are positive / highly positive

Wash buffer bottle empty – ensure bottle is sufficiently filled before assay start

Serum antibody titre too high – retest serum at higher dilution.

In some cases, serum may contain antibody against spotting buffer or other component

Other instrument error - please contact Technical Services ([email protected])

Almost all spots are positive / highly positive

Detection Antibody too concentrated – check that 1:100 dilution was used

High Serum antibody titre – retesting serum at higher dilution may help to determine specificities.

All spots are negative If biotin marker spots (RS1) are negative, reagents have not been added correctly. Test is invalid. Check correct volumes of Detection Antibody and Substrate were added to bottles in DynaChip™ Processor.

All spots are negative except control spots

If biotin marker spots (RS1) are positive, check spot PC2. If these spots are negative then serum sample was not added. Check that serum sample plate was in correct position and orientation.

If either PC2 is positive, then serum sample was added. The serum has no anti-HLA antibodies. This is a result within the normal range

All controls and marker spots are weak (reactivity values <0.2).

Detection Antibody too dilute – check that 1:100 dilution was used

Detection Antibody is temperature sensitive. Ensure that this reagent is kept on ice when not stored at 2 – 80C.

Spots give reactivity values below zero (e.g. -0.1)

Check correct concentration of wash buffer was used – below zero values are likely if water is used instead of Wash buffer. This is the most likely cause if the effect applies to the majority of Chips in a Run.

Some serum samples may react non-specifically with the chip surface, causing ‘below zero’ values. The effect can be reduced by titration of the serum sample.

A spot / several spots were shown as inactive or void in software

Dust on chip – keep instrument cover closed as much as possible. Software algorithm reduces false positives by inactivating spots where anomalies are detected.

Majority of spots were shown as inactive or void in software

Misalignment of grid during image analysis. Test is invalid. Please contact Technical Services ([email protected])


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Index Alarms ........................................... 22 aPRA.............................................. 25 Assay steps .................................... 22 barcode.......................................... 21 Browse Frequencies ........................ 15 Browse Menu....................................9 Browse Patients .............................. 10 Browse Requestors.......................... 10 Browse Run .................................... 36 Browse Runs................................... 13 Browse Samples.............................. 11 control spots................................... 26 cut off ............................................ 28 cut-off............................................ 24 DynaChipTM Processor...................... 19 Excluding antigens .......................... 29 filter............................................... 36 Finalising a Run .............................. 18 Macros .............................................8 Masked Antigens............................. 25

refresh ...........................................36 rename...........................................36 Reports...........................................31 Run Imports....................................23 Search............................................27 Searching .......................................27 Settings ..........................................33 Software Overview ........................... 4 Sorting ...........................................36 specificity........................................24 Specificity .......................................24 Statistics.........................................29 Substance.......................................26 Tail analysis ....................................30 Test Wizard.....................................16 Troubleshooting...........................38 Updates..........................................33 Users..............................................33 vPRA ..............................................25 Workflow: ..................................... 8

Documents

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