This document describes the validation of a new STR-based assay using the Eurochimerism (EUC) primer set to detect chimerism in bone marrow transplant patients. The validation showed that the assay can reliably detect chimerism levels down to 5-10% by analyzing control samples containing varying percentages of donor DNA. Results from patient samples tested with both the new EUC assay and the previous assay were over 90% concordant, demonstrating that the new assay meets the diagnostic accuracy standards required for clinical use.

1. The STR based assay using the EUC primer set is a reliable and robust assay for detecting chimerism. The validation criteria have been met andThe STR based assay using the EUC primer set is a reliable and robust assay for detecting chimerism. The validation criteria have been met and
validation of test performance has shown that it can accurately detect chimerism, meeting the required diagnostic standards which has also beenvalidation of test performance has shown that it can accurately detect chimerism, meeting the required diagnostic standards which has also been
confirmed by analysis of External quality assessment scheme samples.confirmed by analysis of External quality assessment scheme samples.
Conclusion
Development phase-Level of detection of the assay
Validation of a new STR based assay to detect chimerism in bone marrow
transplant patients
Ryan Pepper (1)
, Jayne L Duncan (2)
, Nicola Williams (2)
, Thomas Kerr (2)
(1)
. University of the West of Scotland (2)
Laboratory Medicine, Southern General Hospital, Glasgow.
Development phase
Asses diagnostic and technical use of the process.
Ensure measurements obtained are relevant to the diagnostic question
Ensure the analyte can be unambiguously identified
New test
Test performance
Accuracy of the test must meet the required diagnostic standards
Figure 2:
Before implementing a diagnostic test it must undergo validation, a formal
requirement for accreditation of laboratories according to ISO standard 15189.
The key components of validation are detailed in the ACCE framework and
include: Analytical validity, clinical validity, clinical utility and ethical and social
considerations. In the laboratory Analytical validity is assessed according to
figure 2.
Validation Development phase
In the developmental phase PCR amplification of control DNA with the
EUC primer sequences produced allele sizes within the expected size
ranges. PCR conditions were optimised and DNA quantification was
standardised to ensure amplification remained in exponential phase to
allow quantitative determination of alleles present.
Figure 3:
Amplification of STR marker D12S1064 in two control
samples
To assess the level of detection STR status of 5 control samples was
assessed. Chimeric mixes of 90% donor, 50% donor, 20% donor, 10% donor
and 5% donor were made for each primer set. Analysis showed that we can
reliably detect chimerism present at 5-10%
Test Performance-Sample comparison
To complete the validation 22 Samples were run in parallel using
both protocols. Results were concordant in over 90% of cases for
all lineages analysed. Differences are likely to be caused by
possible saturation on the Beckman CEQ 8000 and DNA quality.
In biology a chimera is as an organism with two distinct cell lineages. Post
transplant analysis of patient/donor cell chimerism is used to aid
assessment of successful engraftment, detect graft versus host disease (an
inflammatory disease, unique to allogeneic transplantation, where immune
cells from the donor attack host cells) and in malignant haematological
disorders regular monitoring can alert to disease relapse.
The aim of this project is to validate a new STR based assay using primers
designed by the Eurochimerism (EUC) consortium. This assay analyses 11
STR loci in one PCR multiplex and will replace the assay currently used in
the laboratory which analyses 8 STR loci in separate PCR reactions. We will
also utilise the 3130xl Genetic Analyser, which offers increased performance
over the previously used Beckman CEQ 8000 sequencer, allowing scientists
to save time, reduce costs and increase productivity.
In the laboratory chimerism is assessed by analysing the relative mixture of
donor and recipient DNA extracted from peripheral blood and bone marrow
aspirates (Figure 1).
Introduction Chimerism Analysis
Quantitative Fluorescent (QF) PCR analysis of short tandem repeat
(STR) sequences is used to distinguish donor (red) from recipient
(green) DNA and a percentage donor chimerism (blue) is calculated
using peak height and peak area.
Figure 1
Figure 4
50% donor chimerism
5% donor chimerism
STR marker D12S1064
Donor 135bp & 147bp
Recipient 143bp &141bp
Comparative Results For Chimerism
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Sample
%donorchimerism
Beckman CEQ
ABI 3130xl
Figure 5: