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 list
1
. 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.
