guideline

Monitoring of chimerism following allogeneic haematopoietic

stem cell transplantation (HSCT): Technical recommendations
for the use of Short Tandem Repeat (STR) based techniques,
on behalf of the United Kingdom National External Quality
Assessment Service for Leucocyte Immunophenotyping
Chimerism Working Group

Jordan R. Clark,1 Stuart D. Scott,1 Andrea L. Jack,1 Helena Lee,2 Joanne Mason,3 Geoffrey I. Carter,4 Laurence Pearce,4
Tony Jackson,5 Hazel Clouston,6 Anne Sproul,7 Leigh Keen,8 Karen Molloy,9 Najeem’deen Folarin,10 Liam Whitby,1
John A. Snowden,11 John T. Reilly1 and David Barnett1
1
UK NEQAS for Leucocyte Immunophenotyping, Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield, 2Transplantation
Laboratory, Manchester Royal Infirmary, Central Manchester University Hospitals NHS Foundation Trust, Manchester, 3West Mid-
lands Regional Genetics Service, Birmingham’s Women’s NHS Foundation Trust, Birmingham, 4Molecular Diagnostics and Immuno-
phenotyping, Nottingham University Hospitals NHS Trust, Nottingham, 5Northern Genetics Service, Institute of Genetic Medicine,
The Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, 6Sheffield Diagnostic Genetics Service, Sheffield
Children’s NHS Foundation Trust, Sheffield, 7Department of Haematology, Western General Hospital, Edinburgh, 8Histocompatibility
and Immunogenetics Laboratory, NHS Blood and Transplant, Filton, UK, 9St James Hospital, Dublin, Ireland, 10Haematological
Medicine, Rayne Institute, King’s College Hospital NHS Foundation Trust, London, and 11Department of Haematology, Sheffield
Teaching Hospitals NHS Foundation Trust, Sheffield, UK

uses data obtained from the UK NEQAS LI Post-Stem Cell

Summary
Analysis of short tandem repeats (STR) is the predominant
method for post-transplant monitoring of donor engraftment.
It can enable early detection of disease relapse, level of
engraftment and provide useful information on the graft-
versus-host disease (GVHD)/graft-versus-tumour (GVT)
effect, facilitating therapeutic intervention. Harmonization
and standardization of techniques and result interpretation is
essential to reduce the impact of laboratory variability on both
clinical management and the results of multi-centre clinical tri-
als. However, the United Kingdom National External Quality
Assessment Service for Leucocyte Immunophenotyping (UK
NEQAS LI) has highlighted significant issues inherent in STR
testing that impact upon inter- and intra- laboratory variation.
We present here consensus best practice guidelines and recom-
mendations for STR chimerism testing, data interpretation
and reporting that have been drawn up and agreed by a con-
sortium of 11 UK and Eire clinical laboratories. This document
Transplant (SCT) Chimerism Monitoring Programme.
Keywords: chimerism, guidelines, short tandem repeats,
allogeneic haematopoietic stem cell transplantation.
Key recommendations
Pre analytical
1 EDTA is the preferred anticoagulant.
2 4–20 ml of peripheral blood or bone marrow should be
sufficient for chimerism analysis, unless cell fraction-
ation is being performed, in which case 20 ml should
be requested.
3 Samples should be stored and transported at ambient
temperature (15–25°C) if lineage-specific analysis is
being performed.
4 Lineage-specific cell separation should ideally be performed
within 24 h of venepuncture and certainly within 72 h.

Correspondence: Stuart Scott, UK NEQAS for Leucocyte

Immunophenotyping, Sheffield Teaching Hospitals NHS Foundation
Trust, c/o Pegasus House 4th Floor Suite, 463a Glossop Road,
Sheffield S10 2QD, UK.
E-mail: stuart.scott@ukneqasli.co.uk
Technical considerations
1 Purity assessment should be carried out on all lineage-
specific cell fractions separated.

ª 2014 John Wiley & Sons Ltd, British Journal of Haematology doi:10.1111/bjh.13073
Guideline
2 Where cell quantity is a limiting factor, lineage fraction
purity analysis may not be performed but this should
be stated on the final report.
3 To ensure optimization of thermocycling conditions and
exponential polymerase chain reaction (PCR) endpoint,
the quality (>1
8 260/280 nm ratio) and quantity (10–
25 ng) of the DNA extracted should be assessed and
recorded.
4 Suitable internal and external quality control proce-
dures must be in place.
Analytical considerations
1 Where possible, fully informative markers should be
used in the calculations of % chimerism.
2 Short tandem repeats (STR) markers with stutter peaks
coinciding with reciprocal donor or recipient peaks
should be excluded from the calculation, or normalized
by a validated algorithm.
3 It is recommended that STR markers suffering from
severe skewing caused by preferential amplification
should be avoided or normalized by a validated method.
4 The use of three or more markers, where possible, is
recommended as it allows the comparison of results or
reporting of a mean.
5 In the multiple donor setting, it is recommended that
% chimerism is calculated and reported for individual
cord donors.
6 A coefficient of variance (CV) ≤5% should be obtained
with a minimum of three STR markers used in the
overall % chimerism calculation.
7 If a CV of >5% is obtained, further investigation of
individual STR markers should be undertaken.
8 Where it is impossible to reduce the CV this should be
highlighted on the clinical report.
9 The limit of detection should be calculated and
reported to the end user (only applies to 100% donor
or 100% recipient results).
Post-analytical considerations
1 The final % donor or host results should be reported as
an integer.
2 Chimerism results should be reported cumulatively in
the form of a table or displayed longitudinally by graph
and include details of the sample type analysed.
3 The purity of the relevant fraction must be stated on
the final report to the end user.
4 The CV, number of markers used in the calculation and
the limit of detection should be included in the clinical
report.
5 It is recommended that chimerism results should be
reported within five working days of receipt of sample,
urgent samples within three working days.
2 ª 2014 John Wiley & Sons Ltd, British Journal of Haematology
Allogeneic Haematopoietic Stem Cell Transplantation
(HSCT) has become an important treatment for many malig-
nant and non-malignant disorders. Its use has increased with
the introduction of reduced intensity conditioning regimens,
improved human leucocyte antigen (HLA) matching and
donor availability (including cord blood) and better support-
ive care, all of which have improved the safety of the proce-
dure and extended its application to older age groups
(Passweg et al, 2012; Gratwohl et al, 2013). Despite such
progress, allogeneic HSCT remains a high-risk procedure,
with risks of infectious complications, graft-versus-host dis-
ease (GVHD) and graft failure. Disease relapse is also a
major cause of treatment failure, and an inverse relationship
with GVHD supports a significant role for a graft-versus-
tumour (GVT) effect in many diseases. In malignant diseases,
GVT may be encouraged by rapid reduction in immunosup-
pressive medications, donor lymphocyte infusions (DLI) or
other cellular therapy and by administration of immuno-
modulatory cytokines (Kolb, 2008). In non-malignant dis-
eases, graft failure may be prevented by optimizing
immunosuppressant medication (Lawler et al, 2009).
These pre-emptive therapeutic interventions are associated
with complex decision-making balancing the risks of relaps-
ing disease and/or graft failure with GVHD, which may be
potentially life-threatening. Such critical decisions are heavily
supported by laboratory testing of the relative mixture of
donor and recipient haematopoiesis, or ‘chimerism’, in sam-
ples taken from peripheral blood or bone marrow aspirates
(Bader et al, 2005a; Lion et al, 2012). Chimerism testing
should be supplemented with minimal residual disease
(MRD) assays, where available, although, in a number of dis-
eases (e.g. the myelodysplastic syndromes) the detection of
residual host haematopoiesis by chimerism testing may itself
be a surrogate marker of residual disease. However, persis-
tence or reappearance of recipient cells may also reflect sur-
vival of normal host haematopoietic cells, and therefore it is
important to monitor the dynamics of chimerism by serial
analysis at regular intervals. Furthermore, chimerism analysis
of whole blood or marrow has, at best, a sensitivity of
around 1%, and is therefore not a reliable method for the
detection of MRD, even when performed at short time inter-
vals. Sensitivity can be improved by analysing cell subsets,
and lineage-specific chimerism analysis, e.g. of the CD34+
fraction, is a proven means of detecting impending relapse in
acute myeloid leukaemia and acute lymphoblastic leukaemia
(Thiede et al, 2002). In addition, analysis of the CD3+ frac-
tion is commonly used to direct the use of DLI in an effort
to augment the GVT effect.
A number of methods are commonly used to evaluate chi-
merism status including: semi quantitative fluorescent poly-
merase chain reaction (QF-PCR) of short tandem repeats
(STRs or microsatellites), fluorescent in-situ hybridization
(FISH) of the X and Y chromosomes, quantitative real-time
PCR (qPCR) analysis of single nucleotide polymorphisms
(SNP) and insertion/deletions (Indels). Testing may be

performed on either peripheral blood, bone marrow aspirate or
on cell subsets separated by a variety of methods (i.e. lineage-
specific analysis). Irrespective of the techniques and samples
used for chimerism analysis, given the magnitude of clinical
risk associated with pre-emptive therapeutic interventions, it is
highly desirable that laboratory performance is monitored in
external quality assessment schemes, and, where available, gen-
eral and specific accreditation standards are satisfied.
Short Tandem Repeats (STR)
STRs are polymorphic 2–6 nucleotide tandem repeats distrib-
uted throughout the genome. The highly polymorphic nature
of STRs results in a high probability that a panel of STR
markers can allow for differentiation between individual ge-
nomes. Of the techniques mentioned above, STR is the most
prevalent method for monitoring chimerism because FISH is
limited to sex-mismatched transplants whilst SNP and Indels
analysis, despite their potential, have some limitations and
are yet to gain widespread adoption. Despite extensive use,
there are a large number of technical and quality issues asso-
ciated with the use of STRs in chimerism testing.
Quantitative STR analysis of chimerism is a procedure
requiring consideration of multiple factors and is therefore
inherently variable. Many commercially available kits in use
for chimerism analysis were originally designed for forensic
identification and have not been optimized for clinical evalu-
ation of donor chimerism. Additionally, there is a lack of
internationally recognized calibrants and there are no over-
arching best practice guidelines. This has been recognized by
the fact that many groups have attempted to standardize var-
ious technical aspects of the STR chimerism testing proce-
dure, including the development of a specific quantitative
chimerism assay (Nollet et al, 2001; Kristt et al, 2005, 2007;
Kristt & Klein, 2006; Lion et al, 2012).
The United Kingdom National External Quality Assess-
ment Service for Leucocyte Immunophenotyping (UK NE-
QAS LI) Post-SCT Chimerism programme has identified that
there is still a diverse array of methods and protocols in use,
with 70% of participants using one of nine different com-
mercial kits, whilst the remainder use in-house methods. The
programme has also observed variability in all steps of the
process, from template extraction through to electrophero-
gram interpretation and reporting.
The aim of this review guidance document is to provide a
critical and, where possible, evidence-based appraisal of chi-
merism analysis using STR techniques, so as to make recom-
mendations for current best laboratory practice and identify
areas for future research and development in this area. Ini-
tially, a meeting of UK participants in the UK NEQAS LI
Post-SCT Chimerism programme was convened in March
2012, with the following recommendations being produced
subsequently. This article primarily focuses on the technical
aspects of chimerism testing by STR and, although reference
is made to clinical background, it does not aim to
ª 2014 John Wiley & Sons Ltd, British Journal of Haematology
Guideline
comprehensively cover the clinical interpretation of chime-
rism tests and therapeutic decision-making, which will be
covered in a future guidelines document under the British
Committee for Standards in Haematology.
Pre analytical considerations
Mandatory information on request form
Prior to first chimerism analysis the following information
should be available, in line with local sample acceptance criteria:
Full patient demographics [e.g. full name, date of birth
(DOB), hospital reference number, National Health
Service (NHS) number, gender etc.]
Diagnosis
Donor type (i.e. related, unrelated)
Donor source(s) (e.g. peripheral blood stem cell harvest,
bone marrow harvest, or single/double/multiple cord
blood units or other relevant details)
Donor gender and key identifiers (e.g. donor registry
identification)
Transplant date
Transplant type [e.g. reduced intensity conditioning
(RIC), myeloablative].
It is encouraged that testing laboratories are included in
multidisciplinary team (MDT) meetings as these provide an
excellent forum from which to gain information.
Subsequent chimerism request forms should include the fol-
lowing information:
Full patient demographics (e.g. full name, DOB, hospital
reference number, NHS number, gender etc.)
Diagnosis
Sample type
Date and time bled (to aid single cell separation)
Lymphocyte count, latest full blood count
Urgency
Testing required (lineage-specific or whole blood)
Recent clinical activity and dates (DLI etc).
Frequency of testing
The frequency of testing should be determined by locally
agreed clinical standard operating procedures (SOPs). These
may vary depending on the disease and incorporate the abil-
ity to assess patients on an individual basis. National and/or
international guidelines should inform the local policy and
procedures as appropriate. Routine audit of clinical and lab-
oratory practice in relation to SOPs and clinical outcomes is
recommended.
Sample
Recipient pre-transplant material from which DNA can be
extracted must be available to enable genotyping of
3
Guideline
recipient alleles. Post-transplant buccal swabs are not ideal
for initial genotyping due to the issue of donor cell con-
tamination from GVHD lesions of the mouth. It is strongly
advised that donor material is also made available for geno-
typing purposes, although with sufficient experience, donor
genotypes can potentially be correctly inferred from post-
transplant samples.
Chimerism analysis can be performed on either periph-
eral blood (PB) or bone marrow aspirate (BMA). Although
for diseases primarily involving bone marrow, whilst it
maybe intuitive to consider BMA chimerism as being supe-
rior to PB in prediction of relapse, the evidence base is
limited and not considered sufficiently clear for any techni-
cal recommendation to be made at this time (Liesveld &
Rothberg 2008; Stumph et al, 2008). For serial monitoring,
the same sample material allows for consistency in trend
analysis.
Collection tube
EDTA is the standard anticoagulant for most molecular
workflows. EDTA chelates calcium and thus preserves cell
surface markers by preventing recirculation of surface recep-
tors and is therefore the preferred anticoagulant if lineage-
specific chimerism analysis has been requested.
Recommendation
EDTA is the preferred anticoagulant.
Sample volume
Accurate chimerism analysis depends on the extraction of
DNA from sufficient cells to prevent stochastic bias when
assessing low levels of chimerism. The volume of sample
extracted may need to be adjusted given the variability in cell
counts in the post-transplant patient. Additional volume may
be required in pancytopenic patients although a much smal-
ler volume will be adequate in the majority of occasions. It is
also advisable to perform a count on marrows prior to DNA
extraction.
As a general rule, 4–20 ml blood should be sufficient,
even when lineage-specific chimerism analysis has been
requested.
Storage and transport temperature
Whole blood for DNA extraction can be stored and trans-
ported at a wide temperature range, although temperatures
above 25°C are best avoided. It has been shown that a sam-
ple storage temperature range of 18–22°C is optimal for anti-
gen expression, whereas samples stored at 4°C show
decreased antigen expression (Ekong et al, 1993; Hanson
et al, 2013).
4 ª 2014 John Wiley & Sons Ltd, British Journal of Haematology
Recommendation
Samples should be stored and transported at ambient tem-
perature if lineage-specific analysis is being performed.
Time from venepuncture
Samples should be extracted as soon as possible after vene-
puncture because sample degradation may impact on quality
of results.
Lineage-specific cell separation should be performed as
soon as possible for maximum purity as cell surface markers
continually degrade. For EDTA samples, fractionation should
ideally be performed within 24 h, however, lineage-specific
separation can be performed up to 72 h without significant
loss of purity. Samples taken into anticoagulants other than
EDTA should be processed within 24 h. The cell fraction(s)
purity should be assessed and reported on all samples (Han-
son et al, 2013; European Federation for Immunogenetics
Standards version 6.1 2013, Section I)
Recommendation
Lineage-specific cell separation should ideally be per-
formed within 24 h of venepuncture.
Technical considerations
Lineage-specific cell separation
It has been proposed that lineage-specific chimerism analy-
sis can highlight relapse, or impending loss of engraftment,
earlier than whole blood analysis, thus enabling the
clinician to initiate salvage therapy (Bader et al, 2005b;
Mossallam et al, 2009; Rupa-Matysek et al, 2011; Breuer
et al, 2012). It is beyond the scope of these recommenda-
tions to suggest which lineages should be investigated in
which diseases; rather we will concentrate on the technical
aspects of cell separation that must be taken into consider-
ation.
There are a number of different methodologies to separate
cell lineages, the most common being magnetic-activated cell
separation (MACS), either manual or automated, and fluo-
rescence-activated cell separation (FACS). There are advanta-
ges and disadvantages to both:
MACS, a relatively cheap cell separation technique, relies
on positive/negative antigen expression and can be per-
formed in most diagnostic laboratories. Manual techniques
can be prone to purity variation; an issue overcome with
automated magnetic bead separation platforms. Conversely,
FACS enables complex lineage-specific cell separation based
on antigen density expression and also provides greater cell
lineage purity. However, it requires increased technical exper-
tise and access to expensive instrumentation.

Purity assessment
Willasch et al (2010) showed that CD3 purity post-separation
is variable: of 135 samples analysed, 62% had purity >90%,
27% had 70–90% purity with 11% having <70% purity. There
was even greater variation seen in other cell fractions. The rec-
ommended approach for purity assessment is flow cytometry
using fluorescently labelled monoclonal antibodies, as this is
the gold standard. The panel of antibodies used to assess purity
is dependent on the target cell lineage to be studied. For exam-
ple, if T lymphocyte lineage separation was undertaken using
anti-CD3, then that epitope of the CD3 antigen will no longer
be available to determine purity. Thus, an anti-CD3 antibody
binding at a different CD3 epitope must be used. Another
approach could be the use of a different T lymphocyte marker
(e.g. CD2) to the one selected for separation. Care should be
taken when designing a flow cytometry purity assay; many CD
antigens may not be specific to the cell subset being analysed
e.g. CD5 is found on both T lymphocytes and B lymphocyte
haematogones, and antibody blocking will cause erroneous
results. Alternatively, a secondary antibody can be employed
which would be applicable to any lineage tested (Hanson et al,
2013). A recent study has indicated that it is possible to deter-
mine purity of T lymphocytes fractions using PCR, with a
degree of agreement with flow cytometry, therefore making
this a viable option for laboratories without access to flow
cytometry (Hanson et al, 2013).
It is strongly recommended that purity assessment is
carried out on all lineage-specific cell separation fractions.
The purity of the relevant fraction should be stated on
the report to the end user.
Where fraction purity analysis has not been assessed e.g.
where cell quantity is a limiting factor, this must be stated
on the report.
DNA extraction
A variety of methods are available for the extraction of DNA.
The method utilized should yield DNA of sufficient quantity
and quality to facilitate the STR analysis method of choice.
However, when working with cell fractions that may contain
low cell numbers and in order to maximize DNA yield, spe-
cific protocols may need to be employed to obtain reliable
amplification of minor alleles, The EU-supported Eurochim-
erism Consortium has produced, for use in such circum-
stances, a standardized direct lysis DNA isolation method
(van der Burg et al, 2011). However, whilst the method cap-
tures an increased percentage of cellular DNA, it may also
produce DNA that is more contaminated with PCR inhibi-
tors and thus it is important that DNA purity is measured.
PCR input
Most commercial STR kits have been optimized for forensic
applications where only small quantities of DNA are available
ª 2014 John Wiley & Sons Ltd, British Journal of Haematology
Guideline
and 1–2 ng of DNA is advised. However, this amount of
DNA only corresponds to the nuclear material of 150–300
cells. In a clinical scenario where a minor DNA component
is being examined, there is a reasonable chance that the
DNA content of the cells in the minority is not input into
the PCR reaction given the stochastic bias particularly asso-
ciated with low template numbers. Below 10 ng, equating
to DNA from ~1500 cells, reproducibility has been shown
to be impaired (Lion et al, 2012). Studies have shown that
reproducibility is acceptable at 25 ng of DNA per reaction
(Nollet et al, 2001). Determining the optimum amount of
DNA template is crucial; therefore its measurement should
be performed as accurately as possible. In addition to the
measurement of the quantity, it is also useful to measure
quality.
To ensure adequate sensitivity and accurate quantifica-
tion (exponential PCR endpoint), the quality (>1
8 260/
280 nm ratio) and quantity (10–25 ng) of the DNA
template used in the assay should be assessed and this
recorded and reported internally.
Short tandem repeat PCR
For accurate quantification, the PCR reaction needs to stay
within the exponential (log2 linear) phase. An increase in
template must therefore be compensated by a decrease in
PCR cycle number, below that recommended for commercial
kits designed for forensic applications. Additionally, in order
to remain within the fluorescent detection thresholds of the
fragment analyser and to prevent fluorescence spectral over-
lap (often termed bleed through), it is important to limit
PCR cycle number and/or dilute PCR products prior to
analysis. For example with the Powerplex 16 assay (Pro-
mega, Madison, WI, USA), designed primarily for use in
forensic science, Nollet et al (2001) showed that reducing the
number of PCR cycles from 32 (as recommended by the
manufacturer) to 28 optimizes accurate quantification.
Different STR strategies for assessing chimerism exist, an
example of which is the use of multiplex to screen for the
best informative markers to facilitate optimal discrimination
between recipient and donor. Such an approach allows the
use of monoplex or duplex PCR in subsequent post-trans-
plant samples that may increase the sensitivity for detection
of minority populations. However, this pathway is more
expensive and laborious to perform. Alternatively, others use
their multiplex assay at all time points, which substantially
simplifies the procedure because all samples are run under
identical conditions. There is also the reassurance provided
by multiple markers giving similar results for calculated %
chimerism. However multiplex assays can be difficult to opti-
mize for uniform amplification across all markers, and may
be less sensitive.
The testing strategy, e.g. number of markers, in-house or
commercial kit, multiplex or monoplex, will fundamentally
affect both the sensitivity and reproducibility of the results.
5
Guideline

It is important for laboratories to make a choice based on
reproducibility, sensitivity, throughput and the cost implica-
tions of their testing strategy.
Capillary electrophoresis parameters
It is beyond the scope of this document to make recommen-
dations on capillary electrophoresis injection times and volt-
ages; however, it is extremely important that these settings
are validated when establishing the assay. Most, if not all,
laboratories base their quantification on relative peak heights
of host and donor alleles, however it is acceptable to use
peak area.
Other factors to be aware of when using this technique
include: over loading ‘saturation’, which makes quantifica-
tion of chimerism impossible [note: such results may still
allow for the reporting of 100% donor or recipient samples
(Fig. 1)], fluorescence spectral overlap ‘bleed through’ that
can artificially increase the peak height, or create artefactual
peak(s) potentially masquerading as a genuine low level
recipient or donor allele (Fig. 2). In both instances, a
reduction in injection time or dilution of the PCR product
may resolve the issue. To optimize the assay to achieve
the desired 1–3% sensitivity for detection of the minor
population, the minimum peak height that can be reliably
detected above background fluorescence must be assessed.
The detection limit (DL) can be calculated using the
following formula:
T
Homozygous recipient % DL ¼  100
A
T2
Homozygous recipient % DL ¼  100
A
In the above formula, T is the peak amplitude threshold
(e.g. the smallest relative fluorescent units detectable (RFU)
by the capillary electrophoresis in conjunction with the cho-
sen software) and A is the total measured DNA (either peak
height or area, dependent on which is routinely used)
(Kristt et al, 2007).
Therefore, in the simplest scenario where both recipient
and donor are homozygous for a marker and for the tech-
nique to achieve a 3% (%DL) detection threshold when
the minimum reliable height is 50 RFU (T), the major
population (usually donor) needs to give a peak height of
1500 RFU (A). If the recipient is heterozygous then the
donor peak must be 3000 RFU to confidently exclude 3%
recipient. When reporting a sample as 100% donor or
100% recipient, assay sensitivity should be stated on the
final report. In practice, as long as the minimum peak
height threshold is met, then the sensitivity value in the
standard report will suffice, however a caveat should be

D4S2366 D2S1338 D13S742 Penta D

105 175 245 315 385 455

4200
0
113·33 136·64 157·57 180·22 249·25 273·19 295·27 382·24 404·36 446·22
116·74 140·77 165·64 190·41 256·00 300·00 383·41 409·06

120·65 142·10 168·24 259·69 309·09 387·04 410·13

121·83 150·72 173·11 261·67 313·06 397·74

126·05 153·48 177·85 263·56 411·10

127·56 158·78 181·96 265·44 413·14

128·42 161·63 186·08 267·43 417·81

132·31 269·31
275·07

276·96

BMT 148184 02
D10S2325 D12S391 D18S51 D13S634 D18S535
105 175 245 315 385 455

4700
0

Fig 1. Markers D2S1338 (blue), D10S2325 (green), D12S391 (green) and D18S51 (green) are all saturated, therefore cannot be used in the calcu-
lation of mixed chimerism levels when mixed donor-recipient chimerism is present. Bleed-through into other dye channels is indicated by the
pink lines.

6 ª 2014 John Wiley & Sons Ltd, British Journal of Haematology

 Guideline

D4S2366 D2S1338 D13S742 Penta D

105 175 245 315 385 455

1700
0
116·63 140·77 162·62 186·07 235·39 259·79 312·43 385·45 400·53
120·54 153·48 177·94 255·95 280·83 387·15 410·35
121·63 157·57 181·95 261·68 389·43 411·10
124·45 161·64 190·40 263·56 397·64
125·53 168·29 265·55 413·14
128·46 172·63 267·33 417·92
132·39 269·43
133·36 273·20
275·19
277·07

BMT 148184 01
D10S2325 D12S391 D18S51 D13S634 D18S535
105 175 245 315 385 455

80

0
112·89 136·08 157·13 196·34 218·18 277·49 305·46 340·52 374·72 396·14 418·58 461·57 484·04
114·00 138·26 162·06 205·10 280·42 309·30 376·09 398·07 464·05 491·97
116·19 139·13 169·70 210·16 313·16 384.48 407·35 468·01
117·18 141·22 172·53 214·21 317·00 388·32 411·10 480·00
118·92 142·21 173·50 216·15 320·84 392·28
120·98 144·20 176·00 222·20 394·21
124·01 146·08 409·17
125·97 140·29
127·16 153·15
128·14 154·25
129.88
131.07
132.49

Fig 2. Bleed-through from blue into green, which could be misinterpreted as low level chimerism if the peaks happen to coincide with genuine
donor/recipient alleles. This can be avoided by analysing multiple markers, which will reduce the chance of inadvertently assigning bleed-through
as a genuine allele.

used if the desired sensitivity has not been reached on any
occasion.
Thus, the process needs to be optimized with reference to
the minimum and maximum peak heights that give sensitive,
accurate and reliable results, and these thresholds need to be
adhered to within local QC parameters.
It is recommended that optimum sensitivity is deter-
mined during validation, and minimum and maximum
peak height RFUs are agreed upon. Level of detection
should be stated in the final report when reporting 100%
donor or 100% recipient.
Quality control
Internal quality control. Internal quality control (IQC) is
critical in quantitative assays. When first developing tech-
niques for QF PCR STR chimerism analysis, it is important
to validate results against a second technique, such as XY
FISH, exchanging samples with an established laboratory or
using previously issued External Quality Assessment (EQA)
material. Additionally, further information of assay reproduc-
ibility can be obtained by testing previously analysed post-
SCT DNA at such a frequency that allows any technique drift
to be detected. As with all PCR reactions, non-template con-
trols (blanks) should be used to control for potential con-
tamination. All validation and IQC work should be recorded
and results retained in accordance with the Royal College of
Pathology guidelines (Wilkins, 2009).
External quality assessment. Participation in an accredited
EQA programme (where available) facilitates peer-to-peer
review. It is essential that laboratories compare their results
to those of other participants in ongoing EQA exercises; this
allows critical review of their results, and not just their per-
formance classification. In this way, trends can be identified
and rectified with minimal delay. International standards
state that participation in a suitable EQA/proficiency testing
is a mandatory part of a laboratory’s quality management

ª 2014 John Wiley & Sons Ltd, British Journal of Haematology 7

Guideline
system in order to attain accreditation to an International
Organization for Standardization standard.
It is recommended that suitable internal and external
quality control procedures be in place.
Analytical considerations
Choice of markers
Most laboratories will perform a multiplex STR PCR, at
least once, to choose the best markers with which to monitor
chimerism in post-transplant samples. Marker choice may
not be straightforward due to the factors listed below, and
may vary depending on the clinical status, including the
following:
1 markers may be affected by acquired or constitutional
changes, e.g., loss of chromosomes 5 and 7 in myelodys-
plastic syndrome and trisomy 21 in Down Syndrome,
2 particular markers will be more sensitive for detecting low
level recipient
3 some markers will be preferable in terms of accuracy in
the context of mixed donor-recipient chimerism.
STR exclusion criteria
There are a number of inherent problems in quantitative STR
analysis, all of which can affect the accuracy of analysis. The
following points highlight areas that must be taken into con-
sideration; a marker may be ideal with regard to one criteria
but its use precluded with regard to another.
Allelic constellations
A number of publications have described nomenclature for
post- HSCT STR allelic constellations (Nollet et al, 2001;
Thiede et al, 2001 and Watzinger et al, 2006). All of these
are acceptable but care must always be taken, irrespective of
nomenclature used, to ensure that the most appropriate
informative markers are used to calculate % chimerism. The
simplest approach is the one described by Thiede et al
(1999) that use the descriptors of: type 1 (fully informative),
type 2 (informative) and type 3. Using this nomenclature, a
fully informative, type 1 STR marker constellation (where
8 ª 2014 John Wiley & Sons Ltd, British Journal of Haematology
recipient and donor have no common alleles), is calculated
according to the following formula:
ðC + DÞ
% chimerismðdonor engraftmentÞ ¼  100
ðC + DÞ þ ðA + BÞ
where the recipient alleles are denoted A and B, and the
donor alleles C and D (Fig. 3i). Providing that all other
parameters have been accounted for (i.e. stutter, preferential
amplification, bleed through, etc.) this constellation is likely
to provide the most accurate and reliable result.
It is recommended, where possible, that fully informa-
tive markers should be used in the calculations of %
Chimerism.
Figure 3ii shows an example where the recipient and
donor are heterozygous and share one common allele. This
constellation is defined as informative. In such instances the
shared allele may be disregarded in the calculation:
C
% chimerism ðdonor engraftmentÞ ¼  100
A+C
When either the donor or recipient is heterozygous, and
one of the alleles is identical to the homozygous pattern of
the other individual:
C
% chimerism ðdonor engraftmentÞ =  100
AC
( 2 )+C
Whilst it is possible to calculate a % chimerism using the
above formula when a constellation profile is obtained as
shown in Figure 3iii, and because the calculation is based on
three alleles co-migrating at the same location it is more
prone to error, particularly saturation. Therefore it is recom-
mended that this marker constellation should not be used
in the calculation of % chimerism if possible.
Cord stem cell transplants
Umbilical cord blood (UCB) is increasingly used in both
paediatric and adult practice, especially where there is no
immediately sourced well-matched donor. To compensate for
the decreased number of stem cells in cord blood compared
to traditional donor sources, which can lead to an increased
risk of graft failure, double cord blood unit (dCBU)
Fig 3. Graphical representation of the allelic
constellations described in Thiede et al (1999).
A, B: recipient alleles; C, D: donor alleles; Post-
SCT: post stem cell transplantation.
 Guideline

transplantation may be used. Occasionally, more than two
cord blood units are used.
Monitoring % chimerism following cord blood procedures
presents unique challenges due to the use of multiple donors
and longer engraftment times. Marker choice is critical in
dCBU transplant, and laboratories performing chimerism
analysis may therefore need to consider increasing the STR
panel size to maximize the chance of finding at least three
fully or, more realistically, partially informative markers. It is
common for both cord donations to engraft initially, with
one then out-competing the other to provide long-term
donor haematopoiesis. However sometimes both cord units
persist in the longer term, resulting in a persistent state of
mixed donor chimerism. Thus three potential genotypes may
be present, confounding calculation of chimerism.
It is important to record % chimerism values for both
cord units when reporting results in patients who have
received dCBU. For example: Unfractionated (cord donor 1/
cord donor 2) PB=3/97%; PB CD3 = 4/96%; PB CD15 = 8/
92%.
Figure 4 shows the allelic constellation of cord 1, cord 2
and the recipient in a dCBU transplant at initial genotyping.
Figure 5 demonstrates a clinical sample at day 15-post
transplant. By application of the equations below, the %
chimerism of each cord can then be calculated.
Cord 1
% donor cord 1 ¼
Cord 1 þ Cord 2 þ Recipient 1 þ Recipient 2
100
Cord 2
% donor cord 2 ¼
Cord 1 þ Cord 2 þ Recipient 1 þ Recipient 2
100
It is recommended that % chimerism is calculated and
reported for individual cord donors. Further research is
warranted to ascertain the utility of chimerism assessments
in the dCBU transplant setting.

Cord 1
D10S2325 D12S391 D18S51
180 210 240 270
4900

4200

3500

2800

2100

1400

700

0
214·00 222·13

Cord 2
D10S2325 D12S391 D18S51
165 195 225 255 285
770

660

550

440

330

220

100

0
209·99 241·76

Recipient
D10S2325 D12S391 D18S51
165 195 225 255 285
4900

4200

3500

2800

2100
Fig 4. Double cord donor and recipient sam-
1400
ples needed to compile the genetic profile for a
patient undergoing double cordblood unit 700
transplantation. Displayed below each graph is 0
the allelic location of each peak. 218·04 233·76

ª 2014 John Wiley & Sons Ltd, British Journal of Haematology 9

Guideline
D10S2325 D12S391
165 195 R 225
1800
1500
C1
1200
900
600
300 C2
0 USA). The numbers under the peaks represent
174·34 193·98 206·06 214·00 222·05
177·59 210·03 217·90 225·73
Stutter interference
Stutter peaks are caused by slippage of the polymerase during
amplification of target STRs (Schraml & Lion, 2003). Stutter
most commonly manifests as the skipping or addition of
repeat units in the amplicon. Whilst it is technically possible
to compensate for stutter in chimerism calculations, when
stutter peaks are present and coincide with peaks of interest,
quantitation is impaired. However it may be necessary to
include such markers due to the limited availability of infor-
mative STR markers, particularly in the sibling transplant
setting. It is recommended that STR markers with coincid-
ing stutter peaks are either excluded or a stutter correction
calculation is applied (Van Deerling & Leonard 2000).
Preferential amplification
Shorter PCR products are preferentially amplified due to
their greater reaction efficiencies, resulting in allelic imbal-
ance and overestimation of the abundance of the shorter
amplicon. If alleles exhibit a large divergence in fragment
size, or preferential amplification is clearly severe, it may be
necessary to exclude the marker. A calculation for preferen-
tial amplification, specific to chimerism analysis and taking
into account the entire locus, has been devised and termed
Measurement Error (ME) (Kristt et al, 2007), and is available
within some automated software. A locally determined
threshold for ME can be set, beyond which markers can be
excluded. Alternatively, normalization is possible providing
sufficient validation has been performed; this may be by the
use of a correction factor or by the selection of informative
STR markers such that there is a zero net difference in the
size of amplicons, therefore theoretically mitigating the
effects of preferential amplification. It is recommended that
STR markers suffering from severe skewing caused by
preferential amplification should be avoided or normalized
by a validated method.
Statistical quality control analysis
Statistical analysis of STR markers is an extremely useful pro-
cess that helps ensure the reliability of the final reported
result. To increase reliability and to increase the statistical
10
D18S51
255 285
C1
R
Fig 5. Post-transplant sample taken at Day 15
shows 47% recipient, 39% cord 1 and 14%
C2 cord 2 when determined manually in GeneM-
apper (Life Technologies, Foster City, CA,
229·83 237·74 255·74 265·64 allele sizes in base pairs. Key; C1 = cord 1,
233·63 241·71 259·60 267·29 C2 = cord 2, R = Recipient.
power of analysis it is advisable to use several STR markers,
with a minimum of three STR markers being used in the
final % chimerism calculation unless not practically possible,
e.g., where three informative markers are not available, such
as in related transplantation.
Assessing the Coefficient of Variation (CV = Standard
Deviation/Mean 9 100) of the overall % engraftment is a
simple way of measuring the variation between STR markers.
Presuming that the STR exclusion criteria has been applied
as described above and the CV is ≤5%, it is implied that the
overall % engraftment results is reliable. Conversely, if the
CV is >5% this indicates that a high degree of variability
exists between individual STR markers thus making the
result unreliable. In exceptional circumstances, usually when
marker choice is limited or when a low % chimerism is cal-
culated, it may be necessary to accept a higher CV, but in
this scenario it may be worth checking individual results as
the high CV could be due to erroneous results, or phenom-
ena such as loss of heterozygosity in a leukaemic relapse
sample (Thiede et al, 2001). To overcome this, non-statistical
parameters such as locus error (LE) and DNA ME could be
used to exclude markers that are unreliable (Kristt et al,
2007). Alternatively, individual STR markers that are
outside  2 SD of the mean can be excluded to reduce the
CV to ≤5%. If this approach fails, reduction of the SD level
in 0
5 increments should be undertaken until the acceptable
CV level is reached.
It is recommended that a CV ≤5% is obtained with a
minimum of three STR markers used in the overall % chi-
merism calculation where possible. The CV and the num-
ber of markers should be stated on the final report. If a
CV of >5% is obtained, further investigation of individual
STR markers should be undertaken.
The above statistical analysis is time consuming and
costly, and therefore to assist in this process a number of
commercial computerized software packages are available as
well as those developed locally using basic spreadsheet
macros (Kristt et al, 2005, 2007). It is recommended that the
process be automated to reduce the risk of human/transcrip-
tion errors.
The use of three or more markers is recommended as it
allows the comparison of results or reporting of a mean.
ª 2014 John Wiley & Sons Ltd, British Journal of Haematology

Post-analytical considerations
It is recommended that results should be reported as a
final % donor chimerism (i.e. the mean value of all mark-
ers used) and as an integer, as the technique is not accu-
rate enough to report to decimal places. To assist the
identification of emergent trends, patient test results should
be reported cumulatively in the form of a table or, even
better, displayed longitudinally by graph. The European Leu-
kemiaNet MRD reporting software for RQ PCR is a good
example of how this can be done (Østergaard et al, 2011).
Recommendation
The sample type should be clearly recorded on each final
report and also on a longitudinal graph or table.
Discussion
Clinicians, on the basis of the results of chimerism analysis,
make major therapeutic decisions. These include dosing
and cessation of immunosuppression post-HSCT, transfu-
sion of donor lymphocytes or other cellular therapy, and
administration of immunomodulatory cytokines. Such deci-
sions potentially have both life-saving and/or life-threatening
consequences for the patient, as they may influence the
probability of relapse and/or GVHD. For the clinician, the
trigger for such interventions may depend on as little as a
few percentage points, or a progressive trend toward
increasing mixed chimerism in serial analyses, demonstrating
clear persistence of undesired recipient haematopoiesis.
Because of the potential rate of relapse or graft failure in
many disease states, there is often a need to intervene
promptly. Laboratories must be able to respond to the often
urgent clinical situations and prompt turnaround times of
no >5 working days is recommended, with urgent requests
being completed in no >3 working days. A delay of >5
working days, in reporting chimerism may compromise
clinical outcomes.
Any laboratory delivering results of chimerism testing
must be confident of the robustness of their assay in terms
of discriminating between full and mixed haematopoietic
chimerism at their defined sensitivity, and in what represents
a significant change or trend in whatever sample source or,
where performed, cell subsets are analysed.
Chimerism analysis is a complex procedure that involves
an accumulation of multiple technical steps, each of which
can introduce variability. This review, based on the proceed-
ings of a UK NEQAS LI chimerism forum held following the
introduction of an EQA/Proficiency Testing (EQA/PT) pro-
gramme for STR-based chimerism, has highlighted a number
of aspects of laboratory practice that need to be addressed to
ensure robustness of various methods in the clinical setting.
Notably, the appraisal was based on initial results of an
EQA/PT programme for analysis of whole PB, and results are
ª 2014 John Wiley & Sons Ltd, British Journal of Haematology
Guideline
not currently available for the EQA/PT of cell subsets sepa-
rated by various immunological methods that are delivered
routinely by the majority of UK laboratories (by our poll)
and many other laboratories worldwide. However, based on
UKNEQAS LI data, this additional step will inevitably intro-
duce a further area of variability in both intra- and inter-
laboratory variation.
There are therefore strong arguments that chimerism
analysis should only be performed by nationally standardized
and harmonized methods in laboratories accredited by a rele-
vant regulatory body (e.g. United Kingdom Accreditation
Service). Furthermore, to facilitate peer review and ensure
continuous quality monitoring of the assay, participation in
an accredited EQA programme (if available) is a requirement
of accreditation and therefore recommended. In conjunction
with a robust laboratory quality management system, it is
hoped that the use of these guidelines will enable laboratories
to optimize each of these processes, resulting in more reliable
and reproducible chimerism monitoring of allogeneic HSCT
patients.
Clinical HSCT services also need to have responsibility
and good communication with laboratories that provide chi-
merism testing. Increasing numbers of clinical services pro-
viding allogeneic HSCT have accreditation by JACIE [Joint
Accreditation Committee of the International Society for Cel-
lular therapy (ISCT) and European Society for Blood and
Marrow Transplantation (EBMT)], and there is now evidence
to support improved survival outcomes through the imple-
mentation of quality systems necessary for accreditation
(Gratwohl et al, 2011). Education and competency in the
clinical interpretation of chimerism testing is a mandatory
standard for relevant staff. Although the clinical evidence
base relating to chimerism testing is not complete, it contin-
ues to evolve in tandem with the broader field of cellular
immunotherapy. Regular interaction and dialogue between
clinicians and laboratory colleagues is recommended so that
all are updated with developments in the clinical application
of chimerism testing, as well as the robustness and limita-
tions of laboratory testing.
Disclaimer
The authors declare no conflict of interest.
Acknowledgements
We would like to acknowledge the input from all clinical lab-
oratories that attended the UKNEQAS LI chimerism forum
day.
Author contributions
JC and AJ organized the workshop, JC and SS wrote the
paper, HL, JM, GIC, LP, TJ, HC, AS, LK, KM, NF, LW, JAS,
JTR and BD contributed to and reviewed the manuscript.
11
Guideline
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