en plextyper software · plextyper software is designed for the interpretation and data management...

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PlexTyper Software

IVD

Instructions for Use Electronic instructions for use see www.bag-diagnostics.com

Version: 3.X-1/2020

CHANGE HISTORY Version Date Applies to Description

1 12.08.2020 PlexTyper 3.4.0.0 Initial release version for interpretation of

ERY Q and HISTO TYPE Rainbow kits

http://www.bag-diagnostics.com/

BAG Diagnostics GmbH PlexTyper Software Instructions for Use: 3.X-1/2020

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CONTENTS 1 Introduction ................................................................................................................................. 1

Limitations of the software ..................................................................................................... 1 2 Prerequisites ................................................................................................................................ 1

Hardware ................................................................................................................................. 1 Runtime environment ............................................................................................................. 1

3 Plextyper Software Structure ...................................................................................................... 1 Patients, samples and results .................................................................................................. 1 Kit files ..................................................................................................................................... 1

4 Installation and database management ...................................................................................... 2 PlexTyper installation .............................................................................................................. 2 Database .................................................................................................................................. 2

5 Starting the Plextyper Software .................................................................................................. 2 6 Initial Software Setup .................................................................................................................. 2

Activate license key ................................................................................................................. 2 Login ........................................................................................................................................ 3 Home screen............................................................................................................................ 3 Add users and assign roles ...................................................................................................... 4 Changing the user password ................................................................................................... 4 Import, view and update kits .................................................................................................. 5

7 TEST SETUP AND INTERPRETATION: HLA Typing ........................................................................ 7 Add patient and sample information, a test and prepare a run ............................................. 7 Transfer of data from the CFX cycler to PlexTyper ................................................................. 9 Interpretation HLA.................................................................................................................11

7.3.1 Calculation of positive / negative reactions and Quality Score by PlexTyper ...............11 7.3.2 Results histogram ..........................................................................................................12 7.3.3 Amplification Review: Show Cq ratio and fluorescence ................................................14 Interpretation tools ...............................................................................................................15

7.4.1 Change a reaction call ...................................................................................................16 7.4.2 Show all alleles positive with a reaction mix .................................................................16 7.4.3 Show results with mismatches .............................................................................................17 7.4.4 Search for allele reaction patterns .......................................................................................18

7.5 Results View ................................................................................................................................20 7.5.1 Assessing ambiguous results ................................................................................................21 7.5.2 Presentation of DP results ....................................................................................................22

7.6 Reports and exports ..............................................................................................................23 8 TEST SETUP AND INTERPRETATION BLOOD GROUP Typing ......................................................25

Create a sample/ test and prepare a run ..............................................................................25 Transfer of data from the CFX cycler to PlexTyper ...............................................................28 Interpretation Blood Groups .................................................................................................31

8.3.1. Calculation of positive / negative reactions and Quality Score by PlexTyper ...............31 8.3.2 Results histogram ..........................................................................................................32 8.3.3 Show Cq ratio and fluorescence ....................................................................................34 Interpretation tools ...............................................................................................................36

8.4.1 Change a reaction call ...................................................................................................36 8.4.2 Show all alleles positive with a reaction mix .................................................................36 8.4.3 Search for allele reaction patterns ................................................................................36 Results View ..........................................................................................................................37 Reports and exports ..............................................................................................................38

9 Literature ...................................................................................................................................40


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1 INTRODUCTION

This document provides instructions for the installation and use of the PlexTyper software. The

PlexTyper software is designed for the interpretation and data management of HLA and blood group

typing results obtained with the ERY Q and HISTO TYPE Rainbow real-time kits.

Limitations of the software PlexTyper software is designed to assist personnel experienced in HLA and blood group analysis by

suggesting typing results. However, any clinical or diagnostic results must be carefully reviewed by

a suitably qualified person to assure correctness.

2 PREREQUISITES

Hardware The application will run on most modern personal computers, however, it is recommended that the

computer has:

• minimum of 4 GB of RAM (8 GB recommended)

• A minimum video resolution of 1920 x 1080

• A dual core 2Ghz processor

• Enough available hard drive space for the database (each data file will need 280 KB space

and one HLA kit file needs around 30 MB).

Runtime environment The PlexTyper application is designed to run in the following environment:

• Operating system Windows 10 (64 bit only)

• .Net framework (usually already installed and updated with the operating system) as

execution environment – the latest version supporting the operating system will be chosen

as minimum requirement.

• The software requires access to an MS SQL database server, SQL Express 2017 edition.

3 PLEXTYPER SOFTWARE STRUCTURE

Patients, samples and results The PlexTyper application stores sample information and optionally patient information associated

with the sample. Results obtained from samples are linked and stored as well.

Kit files A range of kit files can be stored within the software; each lot can have different (updated) kit files. A kit file defines how each reaction mix reacts with the different alleles and the positive and negative thresholds for each reaction within a multiplex PCR. It also contains pre-calculated allele combinations for all possible allelic reaction patterns.


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4 INSTALLATION AND DATABASE MANAGEMENT

PlexTyper installation PlexTyper is downloadable from the BAG download server. To install the application, download and

run the setup.exe program to start the installer. There is no configuration required for the

installation; all prerequisites (.NET Framework and SQL Server) will be installed automatically.

Database PlexTyper uses SQL Server as a database server. SQL Server Express 2017 is provided with the

installation. With the 2017 Express version database size is limited to 10GB. The server is configured

to run in “User Instance” mode. The database can be located anywhere on your PC.

5 STARTING THE PLEXTYPER SOFTWARE

Installation of the application will place a shortcut on your computer’s desktop and a shortcut in the

Windows Start menu. Double click either of the shortcuts to start the application. On start up, the

application will attempt to connect to the local database. A splash screen will show the current

progress. The splash screen may open behind any current windows that are open.

6 INITIAL SOFTWARE SETUP

Activate license key When starting PlexTyper for the first time the license key must be activated. The license key is

usually provided on request by BAG Diagnostics per email.

Click on Activate your license and follow the step by step instructions. First you can choose between

online or offline (e.g. with QR code) activation. You will then be asked to enter your personal data

for registration. This will help BAG Diagnostics to inform you about updates or to provide other

important information. In a final step enter your license key and activate it.


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Login The application is provided with a default administrator user, you will

need to login with the username “Admin”, and the password “111111”.

Press Enter or click “OK” to continue and set your own user.

Home screen The home screen is the starting point in the

software. The main functions of the home screen

are:

• Start: Opens the menu

o Add Test: Upload the kit files and a create

new samples

o View Results: Show previous results

o View plates with no associated results:

Show tests which are already setup

o Add/Edit User: Add or edit users and

assign roles

o Edit Kit Releases: All imported kit files are

visible here and kits no longer used can

be archived.

• About: Application details

• Account: Change the password


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Add users and assign roles From the home screen choose Add/Edit User. The fields with * are mandatory to set up a new user.

1. Fill in the user details: User Name is the unique log in name for the user.

2. Roles: Select from the drop-down list.

• Admin: Can administer the software and users in addition to all other functions.

• Supervisor: Can perform most common tasks; adding samples, adding runs, analyse

results and authorize results.

• Technician: Can perform most common tasks; adding samples, adding runs and analyse

results.

3. Select the Save button

4. You can now log in using the new user

Changing the user password 1. Choose “Account”


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2. Enter your current and new password

3. Select the “Save” button to change the password

4. Choose “Start” to return to the home screen

Import, view and update kits Kits are distributed as .kit files. The file contains information relevant to a specific kit lot. It is

important to use the correct kit file for the physical kit that you are using. The Lot number of the

physical kit must match the lot number of the kit file. The kit file defines amplification thresholds

and specificities of the individual PCR.

You have to use the current KSI version of the kit. KSI is the Kit Specific Information - a change in

KSI means that there is a change in the reactivity in the file which may be due to new information

or corrections of errors. If there is a new KSI users will be informed via email and the new kit file will

be available from the download area on the BAG Diagnostics website.

To load a kit file first go to http://service.bag-diagnostics.com and navigate to the Kit Files HISTO

TYPE Rainbow or ERY Q section and download the relevant kit file for the kit you wish to use. Locate

the kit file either on the same computer you have installed PlexTyper on or use a suitable USB device.

Select the Add Test button.

http://service.bag-diagnostics.com/


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Choose the Load Kit button (see red arrow).

Navigate to and select the relevant kit file

The application will load the files and show you the contents.


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7 TEST SETUP AND INTERPRETATION: HLA TYPING

Add patient and sample information, a test and prepare a run To create a test you can either add a person’s identifiers or just add sample information without a

person’s identifiers.

To create a test for the HISTO TYPE Rainbow kit click on Add Test in the home screen. The setup

window opens:

It is possible to run a test with just a single

sample identifier, which must be entered

into ‘Sample ID 1’; to do this the ‘No

Person’ check box must stay checked. The

Sample ID 1 is mandatory and must be

unique. The second sample ID and the

other information is optional.

If you wish to add a person’s identifiers

then uncheck the ‘No Person’ box. This

will activate the Person identification box.


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Add Person identifiers into the form. The

Person ID 1 * is mandatory. The Person ID 1

must be unique and is typically the person’s

hospital number or patient number. It is also

compulsory to add a sample identifier in the

Sample Information form.

The other fields are optional.

To run a test a kit must be selected from the list on the right side must be selected. Then the Add

test button becomes active and can be pressed to add a test to the plate layout:

The Add Test and Remove Test buttons are used for kits or assays smaller than 48 reactions such as

the ERY Q kits, they are not active for 96 well kit options like HISTO TYPE Rainbow kit.

Test can now be removed again with the

Remove Test button or saved to proceed

with the run with the Save&Next button

1. Select Kit File

2. Press Add test button


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When saving the test, the PlexTyper software assigns a Run ID to the test which is displayed in red

in the summary window that is now opening. This is a consecutive number (PT1, PT2, PT3….) which

is used to identify the run on the cycler and link the results after the run to the defined test. After

saving the test the PCR is set up according to the instructions for use for the HISTO TYPE Rainbow

kits. The Run on the CFX real-time cycler must be named with the Run ID as a prefix to the Run File

Name (e.g. PT1_.xlsx). This is necessary to import the results file and re-connect them to

the test.

Transfer of data from the CFX cycler to PlexTyper

Export the data from the cycler: open the data with the CFX software and then export the Excel

2007 (.xlsx) file.


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Note: Only the “Quantification Amplification Results” is needed. It is

useful to delete the other files

View plates with no associated results: If PlexTyper is still open at the sample you wish to import

then you do not need to go through this step. If PlexTyper is closed, re-open the PlexTyper software

and import the raw data. From the home screen choose View plates with no associated results

under Plates. A list opens with all tests that are not linked with raw data yet. There is a global Search

field at the top of the table to search the whole table.

Double click on the test to be interpreted to open the

results summary window again then click on Import

File and select the file you have exported from the

CFX cycler.

Loading and processing the data might take a

minute, the bottom left corner of the screen shows

a progress bar. After that the results are shown.

Already imported results can be opened from the main menu by pressing View Results under Plates.

A list of results is presented, and you can choose one by double clicking. The Search function is

available here as well.

Search

Search


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Interpretation HLA

7.3.1 Calculation of positive / negative reactions and Quality Score by PlexTyper

For a reaction to be called positive in the PlexTyper software the results must be within three

separate threshold parameters. These thresholds are individually set for each reaction and channel

during QC of the assays and kits.

1) The data must be within the pre-set upper and lower Cq ratio thresholds. The Cq ratio is

derived from the target (allele) Cq divided by the Internal Amplification Control Cq.

2) The data must be above the pre-set ‘final fluorescence’ baseline threshold. This is the

fluorescence attained by a reaction in the final cycle of PCR.

3) The amplification plot (curve) must be statistically close to an ideal sigmoid shape for real

time PCR. This parameter ensures that any fluorescence artefacts are unlikely to be

misinterpreted as true amplification.

Quality Scores (QS) are attributed to all positive and negative

reactions. Positive reactions will have a score ranging from +10

to 0, all negative reactions will have a score ranging from 0 to -

10. The closer a QS value is to zero the closer it is to the

threshold limits. Scores of +10 are perfect positives, and scores

of -10 are perfect negative. A QS between 0 and -1 for a negative

reaction has more chance of being false negative than a reaction

greater than -1. If a positive reaction has a score of less than 3

then it is indicative of a weak reaction, or a possible false

positive. The QS can help the user focus on reactions that might

need manual override if the genotype obtained is questionable

or if no genotype at a particular locus is obtained.

The quality scores of reactions are a useful pointer to which data to review if a review of automatic

calls is required. Quality scores at or close to zero shown in results histogram are results that are

close to one or more thresholds.


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7.3.2 Results histogram

The results histogram shows all reactions from a test.

By default, they are sorted from positive to negative according to the results

quality score. Alternatively, they can be sorted according to Reaction Number.

The well numbers are in the order A1 = 1, A2 =2, through to H12 = 96.

The colour of the bar indicates the reaction channel where the reaction is detected. The green bar

above the histograms represents the internal amplification control. If it fails, the field turns white

with a “-“. The buttons in the right upper corner can be used to increase or decrease the

size of the histogram.

Internal amplification

control result

Reaction and well number

Reaction call: pos/neg

Quality score for specific reactions

Positive results with low QS Negative results with low QS


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Moving the mouse over the histogram elements opens a pop-up

window with additional information, e.g. the quality score:

Above the histogram there is a summary of the results for all loci on the one field level of resolution

and a list of the positive reactions. There is also a list of buttons to choose the loci displayed in the

histogram for review and editing:

At the top of the window, above the allele search function, there are functions to filter the alleles

and change the display mode for the alleles. By default, the CWD filter is chosen which includes all

the common (displayed in green) and well defined (displayed in blue) alleles. The CWD list is based

on the CWD 2.0.0 catalogue (Mack et al. 2013) but some entries have been corrected according to

current sequencing data (see complete list in Annex 1). The Common filter reduces the displayed

alleles to the common ones. Selecting All reveals all alleles including rare ones (displayed in grey).

The filters are also applied to the results display (see chapter 7.5) in the right window in the screen

and to the reports. Checking the Concatenate alleles button shortens the strings of alleles by

summarizing consecutive allele numbers with a hyphen (e.g. A*24:02:06-11).

By default, the results in the histogram are shown summarized on the one field level of resolution.

Pressing the expands the result to show the allele string combinations and the reaction patterns.

Moving the mouse over a string of alleles shows the complete list of alleles which might be quite

long if the All filter is selected.


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7.3.3 Amplification Review: Show Cq ratio and fluorescence

Double clicking on a bar in the histogram shows the fluorescence and the Cq ratio of the selected

reaction in comparison to previous tests. Note: opening a data review window prevents use of the

main program display. Close the window to reactivate user control of the main program.

This helps to estimate the quality of the result and this information can be used to decide if a

questionable reaction should be changed. The Cq ratio is shown on the x axis and the final

fluorescence on the y axis. Green dots indicate previous positive results with Cq quotients and

fluorescence values within the required specifications. Red dots show negative reactions from

previous tests. The blue triangle represents the currently selected reaction.


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The red, dashed lines are the thresholds used by PlexTyper to call the reaction positive or negative

based on the final fluorescence and Cq ratio. Positive results will be above the fluorescence

threshold and between the upper and lower CQ ratio thresholds. Click on the blue triangle on the

Data Review scatter plot to show the raw data curves for this reaction (target and corresponding

IAC channel signals).

Important note: visual interpretation of amplification curves for the purposes of converting a

reaction call is not recommended. Many reactions will produce what looks like satisfactory

amplification, but if it is outside of the thresholds it should be considered to be a false positive

reaction. Review of PlexTyper thresholds viewed on the data histogram are the recommended

method for considering whether to change a call for an individual reaction.

Interpretation tools There are some interpretation tools in the PlexTyper software that can be useful if the automatic

interpretation does not find a result, or finds a rare result where the user might want to consider if

there is a more common result.


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7.4.1 Change a reaction call

All user changes to results are recorded in the audit trail shown in the report. To review the effects

of changing a reaction call right mouse click on the relevant bar in the histogram to view the option

to change a call.

On right click on histogram two options are presented, choose Preview effect of change from + to

0 (or the other way around) to show what the result after the change would be. Then decide if you

want to Change the reaction or Cancel the change.

A changed reaction is indicated in red in the histogram as shown below. If you change a reaction

back again it stays highlighted even though the change is back to the original.

7.4.2 Show all alleles positive with a reaction mix

This function can be helpful if there is no plausible result or if a reaction does not make sense in a

reaction pattern and you would like to check which other alleles might be reacting with this mix.


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With a right mouse click on the bar in the histogram the option to Show Alleles with Positive Calls

is opened. As the list of alleles can become very long if all alleles are considered this function is best

used in CWD or common mode. If using for rare alleles in a large locus, it can sometimes take a few

minutes for the search results to be generated.

The list of alleles is shown in the results histogram together with the complete reaction pattern of

the alleles. The alleles are presented sorted by reaction pattern. This can be removed again with a

right mouse click on the histogram bar and choosing Clear alleles with Positive Calls.

7.4.3 Show results with mismatches

If the software does not find a result, or if you want to check other possible results, you can display

results with one to three mismatches by choosing the number of mismatches from the dropdown

list.

Then the software will search for all results that

are possible if one, two or three reactions would

be false positive or false negative.

Mismatches are indicated in red in the matching

pattern below the results histogram.

Only results with the selected number of mismatches are displayed, i.e. results with zero or one

mismatches are not visible if two mismatches are selected.


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7.4.4 Search for allele reaction patterns

Using the Search functions at the top of the results window allows access to three separate allele

search windows, however, if you do need to search for more in the same display merely type over

one of the existing searches. If the beginning of an allele name is typed into the search window the

autofill function provides a dropdown list of fitting alleles to choose from. The list depends on the

selected allele filter.

Use the Search and Clear buttons on the right side to apply the search or clear the search results.

The reaction patterns of the selected alleles are displayed above the results below the histogram

and can be compared to the detected reactions.

Search Clear


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7.5 Results View On the right side of the screen the results are shown in a table showing the one field resolution

results summary, the genotype as full list of possible alleles (reflecting the chosen allele filter), the

predicted phenotype (serological equivalent) and the approval status. From here the results can be

exported to a text file with the Export button and a pdf report can be generated with the Create

Report button.

Export Create Report


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There is a field with Kit Comments generated from the software regarding assay performance and

a field where the user can enter comments (User annotation of result) at the bottom of the window.

Additionally, there is a message if the no template control (NTC) has passed or failed.

A negative control (NTC) is used as contamination control. If DNA or contaminating amplicon is

inadvertently added to the NTC reaction a positive signal will occur. If the Cq is less than 36 it will

be detected as possible contamination by the PlexTyper software and a warning message is

generated. Amplification signals above Cq 36 in the NTC are regarded as PCR artefacts and are

disregarded.

7.5.1 Assessing ambiguous results

Results are considered ambiguous if there is more than one possible combination on the 1 field level of resolution (e.g. B*35 & B*51 or B*53 & B*78 as below). In case of an ambiguous result this is indicated in the results summary table.

Clicking on the Ambiguous button opens a window with the possible allele string combinations:

One of the options can be selected based on the frequency of the alleles or other additional

information. Without selecting one of the possible combinations the result will be reported as

ambiguous in the summary and with all possibilities as shown here at the end of the report. Pressing

the Change button transfers the selected result to the summary and the report.

Selected option


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The red button with the question mark indicates that the result shown is selected from a list of

ambiguous results. A right mouse click on this button presents the option to undo the selection or

view the ambiguity options again.

Ambiguities are determined based on the selected allele filter. Some results are unambiguous with

the CWD filter – like the one below – but turn ambiguous when all alleles are considered.

CWD Filter:

Two more allele combinations are possible for all alleles, but both have one allele string that only

contains rare alleles:

7.5.2 Presentation of DP results

The nomenclature of the DPB1 locus is different from the other HLA locus because most of the

alleles are not grouped into serologically defined 1 field resolution groups. Moreover, the HISTO

TYPE Rainbow kit uses primer and probe binding sites in exons 3, 4 and 5 where sequences for many

(rare) alleles are unknown. As a consequence, the PlexTyper software generates long results lists for

DPB1 – especially when All alleles is selected – with the algorithm used for the other loci.

Therefore, DPB1 alleles that have the same reaction pattern are summarised in amplification groups

in the kit files. For some DPB1 allele groups there is more than one reaction pattern resulting in


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several amplification groups named e.g. DPB1*01:02 G1 and DPB1*01:02 G2. If an allele has a

unique reaction pattern it is not named as a group.

Analysing DPB1 for ALL alleles can allow the user to see all of the common and rare alleles within a

DP group. e.g. in image below the rare alleles belonging to the DPB1*05:01 G1 are shown, whereas

DPB1*31:01:01:01 is the only DPB1 allele present.

Additionally, the amino acid motifs for the variable positions in pocket F (hypervariable region 6,

position 84-97) and pocket C (hypervariable region 3, position 55-57) are listed in the Serology

column for DPB1. These motifs are assumed to determine serological epitopes for the DP protein

possibly together with the variable position 31 in the DPA1 locus which is listed as well (Laux et al.

2003, Cano &Fernandez-Vina 2009, Hollenbach et al. 2012, El-Awar et al. 2017).

7.6 Reports and exports Clicking the Report button at the right upper corner of the

results window generates a report with the allele filter chosen

and displayed. It is possible to select if the positive reactions

and the assay history should be printed. Press Generate Report

and save the report in pdf format.


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The report contains all person and sample information, the overall summary of results and the

predicted phenotypes, the detailed results with allele strings (reflecting the chosen allele filter), the

positive reactions including information about failed NTCs and the audit trail.

It is possible to export the results to txt file by using the Export button. Choose a location for the file

and a file name, then press Export. The NHSBT format is not functional yet and should be unchecked.

The file contains sample number, the results and the kit used:

Export


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8 TEST SETUP AND INTERPRETATION BLOOD GROUP TYPING

Create a sample/ test and prepare a run To create a test, you can either add a person’s identifiers or just add sample information without a

person’s identifiers.

To create a test for the ERY Q kit click on Add Test in the home screen. The setup window opens:

It is possible to run a test with just a single

sample identifier, which must be entered

into ‘Sample ID 1’; to do this the ‘No

Person’ check box must stay checked. The

Sample ID 1 is mandatory and must be

unique. The second sample ID and the

other information is optional.

If you wish to add a person’s identifiers,

then uncheck the ‘No Person’ box. This

will activate the Person identification box.


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Add Person identifiers into the form. The

Person ID 1 * is mandatory. The Person ID 1

must be unique and is typically the person’s

hospital number or patient number. It is also

compulsory to add a sample identifier in the

Sample Information form.

The other fields are optional.

To run a test a kit file must be selected from the list on the right side. Then the Add test button

becomes active and can be pressed to add a test to the plate layout:

The Add Test and Remove Test buttons are used for kits or assays smaller than 48 reactions such as

the ERY Q kits, they are not active for 96 well kit options like HISTO TYPE Rainbow kit.

Test can now be removed again with the Remove Test button or saved to proceed with the run

with the Save&Next button . By default, the test will be placed in position 1.

3. Select Kit File

4. Press Add test button


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The test can be moved manually to each position on the plate by double clicking on the test and

pulling it to the wished position.

To set the test on the new position double click it again. All different ERY Q kits can be combined in

one run.

When saving the test, the PlexTyper software assigns a Run ID to the test which is displayed in red

in the summary window that is now opening. This is a consecutive number (PT1, PT2, PT3….) which

is used to identify the run on the cycler and link the results after the run to the defined test. After

saving the test the PCR is set up according to the instructions for use for the ERY Q kits. The Run on

the CFX real-time cycler must be named with the Run ID as a prefix to the Run File Name (e.g.

PT1_.xlsx). This is necessary to import the results file and re-connect them to the test.


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Transfer of data from the CFX cycler to PlexTyper Export the data from the cycler: open the data with the CFX software and then export the Excel

2007 (.xlsx) file.

Note: Only the “Quantification Amplification Results” is needed. It is

useful to delete the other files


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View plates with no associated results: If PlexTyper is still open at the run you wish to import then

you do not need to go through this step. If PlexTyper is closed, re-open the PlexTyper software and

import the raw data. From the home screen choose View plates with no associated results under

Plates. A list opens with all tests that are not linked with raw data yet. There is a global Search field

at the top of the table to search the whole table.

Double click on the test to be interpreted to open the results summary window again then click on

Import File and select the file you have exported from the CFX cycler.

Search


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After that the results appear.

Already imported results can be opened from the main menu by pressing View Results under Plates.

A list of results is presented and you can choose one by double clicking. The Search function is

available here as well.

Search

Use this bar to adjust

the window size


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Interpretation Blood Groups

8.3.1. Calculation of positive / negative reactions and Quality Score by PlexTyper

For a reaction to be called positive in PlexTyper software the results must be within three separate

threshold parameters. These parameters are individually set for each reaction and channel by BAG

during QC of the assays and kits.

1) The data must be within the pre-set upper and lower Cq ratio thresholds. The Cq ratio is

derived from the target (allele) Cq divided by the Internal Amplification Control Cq.

2) The data must be above the pre-set ‘final fluorescence’ baseline threshold. This is the

fluorescence attained by a reaction in the final cycle of PCR.

3) The amplification plot (curve) must be statistically close to an ideal sigmoid shape for real

time PCR. This parameter ensures that any fluorescence artefacts are unlikely to be

misinterpreted as true amplification.

Quality Scores (QS) are attributed to all positive and negative

reactions. Positive reactions will have a score ranging from +10

to 0, all negative reactions will have a score ranging from 0 to -

10. The closer a QS value is to zero the closer it is to the

threshold limits. Scores of +10 are perfect positives, and scores

of -10 are perfect negative. A QS between 0 and -1 for a negative

reaction has more chance of being false negative than a reaction

greater than -1. If a positive reaction has a score of less than 3

then it is indicative of a weak reaction, or a possible false

positive. The QS can help the user focus on reactions that might

need manual override if the genotype obtained is questionable

or if no genotype at a particular locus is obtained.

The quality scores of reactions are a useful pointer to which data to review if a review of automatic

calls is required. Quality scores at or close to zero shown in results histogram are results that are

close to one or more thresholds.


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8.3.2 Results histogram

The results histogram shows all reactions from a test. By default, they are sorted from positive to

negative according to the results quality. Alternatively, they can be sorted

according to Reaction Number.

The colour of the bar indicates the reaction channel where the reaction is detected. The green bar

above the histograms represents the internal amplification control. If it fails the field turns white

with a “-“. The buttons in the right upper corner can be used to increase or decrease the

size of the histogram.

Positive results with low QS

Negative results with low QS


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Moving the mouse over the histogram elements opens a pop up

window with additional information, e.g. the quality score:

For the detailed interpretation choose the single alleles button (see red arrow) to show the results

by selecting one or more of these buttons. To search for specific alleles, enter the allele in the Search

1 and press the button Search.

At the top of the window there is a filter function.

Quality score for specific reactions

Internal amplification control result

Reaction and well number

Reaction call: pos/neg


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By default, the filter is set on “Common”, only results with common alleles are shown. If there is

no result or an ambiguous result,

the filter must be set on “Rare” and the software will show you the new result.

8.3.3 Show Cq ratio and fluorescence

Double clicking on a bar in the histogram shows the fluorescence and the Cq ratio of the selected

reaction in comparison to previous tests. Note: opening a data review window prevents use of the

main program display. Close the window to reactivate user control of the main program.

This helps to estimate the quality of the result and this information can be used to decide if a

questionable reaction should be changed. The Cq ratio is shown on the x axis and the fluorescence

on the y axis. Green dots indicate previous positive results with Cq quotients and fluorescence

values within the required specifications. Red dots show negative reactions from previous tests. The

blue triangle represents the currently selected reaction.

The red, dashed lines are the thresholds used by PlexTyper to call the reaction positive or negative

using the final fluorescence and Cq ratio. Positive results will be above the fluorescence threshold

and between the upper and lower CQ ratio thresholds.


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Click on the blue triangle on the Data Review scatter plot to show the raw data curves for this

reaction (target and corresponding IAC channel signals).


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Interpretation tools There are some interpretation tools in the PlexTyper software that are mainly useful if the automatic

interpretation does not find a result or finds a rare result that is not very plausible.

8.4.1 Change a reaction call

This is a tool created only for the HLA analysis, see chapter 7.4.1. We do not recommend to use this

tool for blood group analysis, because we have only one probe for each allele and this would falsify

the results.

8.4.2 Show all alleles positive with a reaction mix

This is a tool created only for the HLA analysis, see chapter 7.4.2.

8.4.3 Search for allele reaction patterns

It is possible to show different allele reaction patterns using the Search function at the top of the

results window. If the beginning of an allele name is typed into the search window you will be

presented with a dropdown list of fitting alleles to choose from. The list depends on the selected

allele filter.


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Use the Search and Clear buttons on the right side to apply the search or clear the search results.

The reaction patterns of the selected alleles are displayed above the results below the histogram

and can be compared to the detected reactions.

Results View On the right side of the screen the results are shown in a table showing the results summary, the

genotype as full list of possible alleles, the predicted phenotype (serological equivalent) and the

approval status. From here the results can be exported to a text file with the Export button and an

XPS report can be generated with the Report button. There is a field with comments generated from

the software regarding assay performance (no user comments) at the bottom of the window.

Especially, if all alleles including the rare ones are considered, ambiguous results may occur. This is

indicated by an Ambiguous button.

Clicking on the Ambiguous button opens a window with the possible results. You can choose one of

the options by clicking on the black dot.

Export

Create Report


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Reports and exports Clicking the Report button at the right upper corner of the results window generates a report with

the allele filter chosen and displayed. Options exist to select printing the positive reactions and the

audit trail. Press Generate Report and save the report in pdf format.


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The report contains all person and sample information, the overall summary of results and the

predicted phenotypes, the detailed results with allele strings (reflecting the chosen allele filter), the

positive reactions including information about failed NTCs and the audit trail.


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It is possible to export the results to txt file by using the Export button. Choose a location for the file

and a file name, then press Export. The NHSBT format is not functional yet and should be unchecked.

9 LITERATURE

Cano P, Fernandez-Vina M, 2009, Two sequence dimorphisms of DPB1 define the immunodominant serologic epitopes of HLA-DP, Human Immunology (70), Issue 10: 836-843, doi.org/10.1016/j.humimm.2008.07.011

El-Awar N, Jucaud V, Nguyen A, 2017, HLA Epitopes: The targets of monoclonal and alloantibodies defined, Journal of Immunology Research, Article ID 3406230, 16 pages, doi.org/10.1155/2017/3406230

Hollenbach JA, Madbouly A, Gragert L, Vierra-Green C, Flesch S, Spellmann S, Begovich A, Norren H, Trachtenberg E, Williams T, Yu N, Shaw B, Fleischhauer K, Fernandez-Vina M, Maiers M, 2012, A combined DPA1~DPB1 amino acid epitope is the primary unit of selectin on the HLA-DP heterodimer, 2012, Immunogenetics (64): 559-569, doi 10.1007/s00254-012-0615-3

Laux G, Mansmann U; Deufel A, Opelz G, Mytilineos J, 2003, A new epitope-based matching approach for cadaver kidney retransplants, Transplantation (75): 1527-1532, DOI: 10.1097.TP.00000617.57702.8A

Mack SJ, Cano P, Hollenbach JA, He J, Hurley JK, Middleton D, Moraes ME, Pereira SE, Kempenich JH, Reed EF, Setterholm M, Smith AG, Tilanus MG, Torres M, Varney MD, Voorter CEM, Fischer GF, Fleischhauer K, Goodridge D, Klitz W, Little A-M, Maiers M, Marsh SGE, Müller CR, Noreen H, Rozemuller EH, Sanchez‐Mazas A, Senitzer D, Trachtenberg E, Fernandez‐Vina M, 2013, Common and well‐documented HLA alleles: 2012 update to the CWD catalogue, Tissue Antigens

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