BAG HISTO TYPE Rainbow QS6 User manual

EN Instructions for Use
HISTO TYPE Rainbow QS6
Test kit for the determination of HLA alleles on a molecular genetic basis
IVD 0123
REF 728221
Contents
1. INTENDED USE......................................................................................................................2
2. PRODUCT DESCRIPTION......................................................................................................2
3. TEST PRINCIPLE....................................................................................................................2
4. MATERIAL...............................................................................................................................3
4.1 Contents of the HISTO TYPE Rainbow QS6 kit.......................................................................3
4.2 Additionally required reagents and devices..............................................................................3
4.3 Validated real time-PCR Cycler................................................................................................3
5. STORAGE AND STABILITY....................................................................................................4
6. TEST PROCEDURE................................................................................................................4
6.1 Precautions and special remarks.............................................................................................4
6.2 DNA Isolation...........................................................................................................................4
6.3 Entering sample information into PlexTyper®Software............................................................5
6.4 Amplification ............................................................................................................................5
6.5 Export of results from QuantStudio™ 6 Flex System ...............................................................9
6.6 Evaluation and interpretation of the results............................................................................10
6.7 Import of the results file into the PlexTyper®software............................................................11
7. WARNINGS AND DISPOSAL INSTRUCTIONS ....................................................................12
8. KIT SPECIFICATIONS ..........................................................................................................13
8.1.1 Diagnostic sensitivity and specificity........................................................................13
8.1.2 Analytical sensitivity and measuring range ..............................................................13
8.1.3 Analytical specificity / cross reactive substances.....................................................14
9. LIMITS OF THE METHOD.....................................................................................................14
10. INTERNAL QUALITY CONTROL...........................................................................................14
11. TROUBLESHOOTING...........................................................................................................15
12. TRADE NAMES USED ..........................................................................................................15
13. EXPLANATION OF THE SYMBOLS USED ON THE LABELS..............................................16
14. LITERATURE ........................................................................................................................16
15. REVIEW HISTORY................................................................................................................16
Version: 02/2021 / Issue: 2021-06

Instruction for Use HISTO TYPE Rainbow QS6 Version: 02/2021
Page 2 of 16
1. INTENDED USE
The intended use of the HISTO TYPE Rainbow QS6 kit is the identification of HLA Class I and II
alleles using the QuantStudio™ 6 Flex System for PCR amplification. HISTO TYPE Rainbow QS6 is
an in vitro diagnostic test for tissue typing on a molecular genetic basis (see Product Description).
2. PRODUCT DESCRIPTION
HISTO TYPE Rainbow QS6 kits are used for the molecular genetic determination of HLA Class I
and II alleles at 11 loci: HLA-A, B, C, DRB1/3/4/5, DQA1, DQB1, DPA1 & DPB1. Kits are designed
to generally detect all alleles at the 11 loci; if any rare alleles are not detected the alleles are listed in
Kit Specific Information documents (KSI) which are available from the download section of the BAG
website. The primer and probe binding sites are listed there as well. The kit provides low to medium
resolution typing results of the common and well documented alleles using CWD list 2.1.0 which is
largely based on CWD 2.0.0 list1. The CWD list 2.1.0 used is available from the document download
section of the BAG website. Confirmed diagnostic results of HLA alleles are a prerequisite for a
successful organ transplantation.
3. TEST PRINCIPLE
The test is performed with genomic DNA as starting material. The DNA is amplified in a real-time
PCR with sequence-specific primers (SSP). The primers were specially developed for the selective
amplification of segments of specific HLA alleles or allele groups. The amplicons are detected using
sequence-specific fluorescence dye-labelled hydrolysis probes (TaqMan®-probes), which increases
the sensitivity and specificity of the test compared to the classical SSP.
If amplicons are present, the probes are hydrolysed by the Taq polymerase and a fluorescence
signal is generated to enable detection of the amplicon. Five different wavelength ranges of
fluorescence signals are measured by the optical detection unit of the real time PCR cycler. The
presence of a positive reaction is determined primarily by the Cq point, which is the point where
fluorescence signal increases beyond the baseline threshold. For amplification to be valid the
amplification must also achieve a certain threshold of fluorescence at the end of the PCR process.
This is to prevent false positive reactions.
Each PCR reaction also contains an internal amplification control (Human Growth Hormone gene
(HGH)) which is detected in a specific fluorescent channel.
To distinguish positive reactions from negative or irrelevant amplifications the ratio of the Cq of the
specific reaction compared to the Cq of the internal amplification is calculated. The thresholds for
these Cq ratios (CqR) vary from reaction to reaction and hence the PlexTyper®software is required
for the analysis of amplification data.

Instruction for Use HISTO TYPE Rainbow QS6 Version: 02/2021
Page 3 of 16
4. MATERIAL
4.1 Contents of the HISTO TYPE Rainbow QS6 kit
•10x 230 µl Plex Mix, ready to use, contains dNTPs, Taq Polymerase, reaction buffer.
•10x HISTO TYPE Rainbow QS6 plates for HLA-typing. The pre-pipetted and dried reaction
mixtures in each well contain HLA-specific primers and probes as well as HGH-specific
internal amplification control primers and probes (oligomixes).
•10x qPCR Seal
4.2 Additionally required reagents and devices
•Reagents for DNA isolation (validated extraction kits see 6.2)
•Real time-PCR Cycler (validated cycler see 4.3)
•Plate holder QS6 (REF 726321)
•Variable pipettes (0.5 –1000 µl) and pipette tips
•Application spatula for qPCR Seal
•Molecular grade DNAse free water
•Suitable plate centrifuge
•For QuantStudio™ 6 calibration please use BAG RT-PCR Universal Custom Dye Calibration
Kit REF 728260
4.3 Validated real time-PCR Cycler
Real time-PCR Cycler
QuantStudio™ 6 Flex System, Applied Biosystems / Thermo Fisher Scientific (QS6)
The following fluorophores are used in the HISTO TYPE Rainbow QS6 kits.
Fluorophore
Wavelength in nm
TAMRA
Excitation: 557
Emission: 583
FAM
Excitation: 495
Emission: 520
O560 (CAL Fluor®Orange 560)
Excitation: 538
Emission: 559
R610 (CAL Fluor®Red 610)
Excitation: 590
Emission: 610
Q670 (Quasar®670)
Excitation 647
Emission: 670

Instruction for Use HISTO TYPE Rainbow QS6 Version: 02/2021
Page 4 of 16
5. STORAGE AND STABILITY
The kits are shipped on blue ice. All reagents must be stored at ≤-20°C in temperature-controlled
devices. The expiry date is indicated on the label of each reagent. The expiry date indicated on the
outer label refers to the reagent with the shortest stability contained in the kit. The freeze-thaw cycle
testing has shown that up to 12 cycles for the Plex Mix has no detrimental effects on the quality of
the kit.
6. TEST PROCEDURE
6.1 Precautions and special remarks
Molecular genetic techniques are extremely sensitive methods and should only be performed by
qualified personnel with experience in molecular genetic techniques.
Special precautions must be met to avoid contamination and thus false reactions:
◆
Wear suitable gloves (preferably powder-free) during work.
◆
Use tips with filter insert or integrated stamp.
◆
Work in two different areas for pre-amplification (DNA-isolation, preparation of the reactions)
and post-amplification (detection); use two separate rooms if possible.
◆
Use devices and other materials only at the respective workplaces and do not exchange them.
6.2 DNA Isolation
The specimen material for the isolation of the genomic DNA must be sent in appropriate collection
systems. For genomic material from whole blood use only EDTA or citrate anticoagulants. The
presence of heparin may potentially inhibit the PCR reaction (2), therefore such collection systems
are not suitable and must not be used. It is recommended to use IvD certified kits for the DNA
isolation.
Validated DNA Extraction Kits:
•Qiagen QIAamp DNA Blood Kits (columns)
Both, the manual isolation and automated DNA isolation (QIAcube) are validated.
If the standard method established in the laboratory shall be applied for isolation of gDNA without
using one of the specified test kits, it must be validated by the user.
The HISTO TYPE Rainbow QS6 test requires 10 –20 ng DNA per well. The purity indices must be
in the following range:
•OD260 / OD280 = 1,5 and < 2,0
Higher values are an indicator for the presence of RNA, lower values
indicate protein contamination.
•OD260 / OD230 = > 1,8
Lower values indicate contamination with carbohydrates, salts or
organic solvents.

Instruction for Use HISTO TYPE Rainbow QS6 Version: 02/2021
Page 5 of 16
6.3 Entering sample information into PlexTyper®Software
It is mandatory to use PlexTyper®Software to analyse HISTO TYPE Rainbow data. It is advisable
to enter the sample information into PlexTyper®before setting up the PCR amplification to obtain
the unique RUN ID. All import files must be prefixed with a PT number.
When saving a test in PlexTyper®, the software assigns a Run ID to the test which is displayed in
red in the summary window shown below. This is a consecutive number prefixed with PT (PT1, PT2,
PT3….) which is linked to the sample identity and is used as a prefix on the PCR machine export file
to correctly link the PCR export to the sample identity.
This Run ID should be used when setting up the file identity in the PCR machine. If the PCR excel
export file is not pre-labelled with the RUN ID the file can be manually prefixed with the correct PT
Run ID before import into PlexTyper®.
6.4 Amplification
A pre-mix consisting of Plex Mix, water and DNA is made for the amplification that is subsequently
dispensed in the wells 1-95. In well 96 there is the negative control (no template control = NTC)
which should only contain water and Plex Mix. For other DNA concentrations the pre-mix has to be
modified accordingly (see below).
•The reaction volume for each PCR preparation is 10 µl
•For a single well the following reagents must be pipetted into a reaction tube:
2 µl Plex Mix
1 µl DNA specimen
7 µl Molecular grade water
A negative control (NTC) should be performed. Therefore prepare a PCR reaction with molecular
grade water instead of DNA.
2 µl Plex Mix
8 µl Molecular grade water