Online supplementary information (SI)
Materials and methods
Isolation of DNA
A number of commercially available kits are known to provide adequate yield and quality of
DNA from peripheral blood specimens including, for example, the QIAmp DNA blood (Mini)
Kit (Qiagen, Hilden, Germany). The extraction of DNA from small cell numbers, such as
specific populations enriched by flow-sorting or magnetic bead-based separation, can be a
more challenging task in the context of chimerism testing, and different isolation protocols
were therefore tested. These included in-house methods based on rapid cell lysis with or
without Proteinase K and two commercially available kits including the QIAmp DNA blood
Mini Kit and QIAmp DNA Micro Kit (Qiagen, Hilden, Germany). The extraction protocols were
tested using 1000, 3000, 10000 and 30000 leukocytes as starting material. The quality and
quantity of DNA isolated by the indicated approaches was assessed by real-time PCR
analysis of albumin as control gene (19), and STR analysis using the PowerPlex 16® system
(Promega, Madison, USA) was performed to determine the suitability of the DNA as template
in the multiplex PCR setting. Owing to the importance of appropriate technique for DNA
isolation from limited cell numbers, details of the comparison between different
methodological approaches are described in a separate manuscript (17).
PCR amplification
Primers for the amplification of loci included in the EuroChimerism (EUC) panel were
adapted to permit annealing at 60°C, the temperature indicated by the manufacturer of the
commercial PP16® kit (PowerPlex 16®, Promega) in order to facilitate the use of similar
programs for all PCR amplifications within the study. The PCR programs used differed
slightly depending on the PCR instrument used, as recommended by the PP16® manual
(www.promega.com). Singleplex PCR reactions with EUC markers were performed in a total
volume of 25 µl containing 400 µM dNTPs, 2 mM MgCl2, 10 x PCR-buffer II (Applied
Biosystems (AB), Foster City, USA) 1 U AmpliTaq Gold® (AB) and 10 ng DNA template. The
primer concentrations varied according to the targeted STR locus and are indicated in Table
2. Multiplex reactions with EUC markers contained 800 µM dNTPs, 2 mM MgCl2, 10 x PCRbuffer II (AB) 5 U AmpliTaq Gold® (AB) and 10 ng DNA in a volume of 50 µl. Primer
concentrations for the established penta- and tetraplex reactions are indicated in Table 3.
The amplification programs of the singleplex and multiplex assays with EUC markers were
essentially identical to those recommended for the PP16® kit on different instruments, the
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only difference being the performance of two additional cycles (22 in total) in singleplex
assays during the second phase of amplification. PCR reactions using the PP16® kit were set
up and carried out according to the instructions provided.
Capillary electrophoresis
PCR amplicons generated by EUC- and PP16 primers were analyzed on different
instruments including the ABI 310, ABI 3100 and ABI 377 genetic analyzer systems. The
instruments were initialized according to the manufacturer´s instructions, and the PCR
products were initially loaded using standard injection parameters (5 kv, 15 sec). The time
and voltage of injection were appropriately adjusted in the presence of low peak heights (<
500 relative fluorescence units [RFU]) or off-scale signals, i.e. peak heights exceeding the
limit of linear quantification (> 6-7.000 RFU). Since these parameters strongly depend on the
apparatus used, the required modifications were determined individually for each type of
capillary electrophoresis device. To assess potential differences between measurements by
the instruments used, ten centrally prepared PCR products were distributed and analyzed on
each type of apparatus. The tests were performed in duplicate and the differences observed
were subjected to statistical analysis, as specified in the Results section. The peak height
permitting unequivocal distinction of specific signals from background noise was ≥50 RFU.
This level of fluorescence signals was therefore defined as the minimum requirement for the
assessment of chimerism. Consequently, signal heights of the dominant alleles had to be
≥6.000 RFU to permit the detection of minor alleles at the level of 0.8%. For the detection of
recipient cells at this level of sensitivity, lower donor signals (at the level of ≥3.000 RFU) may
be sufficient if the allelic constellation provided by the STR marker used is homozygous for
the recipient and heterozygous for the donor. Conversely, in the presence of certain allelic
constellations, the dominant alleles must be higher to ensure a detection limit of 0.8%: for
example, donor signals at the level of ≥12.000 RFU would be required if the recipient had a
heterozygous and the donor a homozygous allelic pattern. Since peak heights at this level
may be off-scale, and thus outside the area of correct computation by the capillary
electrophoresis instrument used, certain allelic constellations may provide a lower sensitivity
of detection.
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Definitions
Stutter peak formation
This phenomenon is caused by the so-called slippage of the polymerase during the
amplification process of microsatellite markers, leading to the formation of additional
products by skipping or adding one or more repeat units to the strands synthesized by PCR.
In fluorescence-based detection of PCR products, stutter peaks appear as additional PCR
products of lower height, mostly one repeat unit downstream and, less commonly, upstream
of the main signal (18). If, for example, the position of a recipient allele coincides with the
stutter region of a dominant donor allele, quantitative analysis of chimerism is severely
impaired. For chimerism analyses using the EUC marker panel, upstream stutter peak
formation was not found to have any impact on the quantitative assessment, and was
therefore eliminated as a criterion for the exclusion of individual markers.
Allelic constellation
The term describes the number of recipient and donor alleles (heterozygous/homozygous)
and the distance between individual alleles (indicated in nucleotides or tandem repeats) in a
given recipient/donor analysis by STR markers. The type of allelic constellation can be
conveniently described by the RSD code introduced by the EuroChimerism consortium (18).
The code is briefly summarized in Figure SI-1).
Informativeness
This term is defined as the probability of a microsatellite marker to yield an allelic pattern
eligible for chimerism analysis in any donor/recipient constellation. Moreover, the term
informativeness is also used for the actual availability of one or more eligible markers in a
specific donor and recipient constellation. The criteria of eligibility defined by the
EuroChimerism consortium have been published previously (18).
Required informativeness of multiplex microsatellite tests
For pre-transplant testing of the donor and recipient, multiplex microsatellite tests are the
preferred approach to identifying one or more markers compatible with the eligibility criteria
for quantitative monitoring of chimerism. According to the requirements established by the
EuroChimersim consortium, a multiplex test regarded as adequate for initial donor/recipient
testing should provide at least two eligible markers for further follow-up, even in the related
transplant setting. The availability of two or more suitable markers should permit the
comparison of independent measurements of chimerism or the calculation of the
mean of different measurements to ensure the highest possible accuracy of
quantitative chimerism analysis in situations of special interest.
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Allelic imbalance
This term describes the presence of unequal peak height (or peak area) of sister alleles
resulting from preferential PCR amplification of short alleles in comparison to alleles of
greater length (in bp). This phenomenon was assessed in the present study by evaluating
multiple data sets of heterozygous constellations and calculating the ratios between peak
heights of both alleles by regression analysis.
Detection limit (sensitivity)
The lowest (reproducibly) detectable dilution step of recipient DNA against the background of
donor DNA or vice versa.
Minimal threshold of peak heights (MTPH)
Minimal signal height of the dominant allele(s) permitting the detection of minor signals at the
desired limit of detection (0.8% when using the EUC panel). As specified above, the MTPH
value, which is indicated in RFU, depends on the constellation of donor and recipient alleles.
Reproducibility
Concordance between replicate tests.
Accuracy (Divergence; Residuum)
Discordance, indicated by the arithmetic difference, between the experimentally measured
and the expected (i.e. true) level of chimerism determined by STR-PCR analysis of well
defined dilutions of patient cells in donor cells (or patient DNA in donor DNA).
Dynamic range and linearity
In the present study, linearity of quantitative measurement of chimerism was assessed within
the range of 0.8% -100% recipient cells or DNA.
Statistical analysis
Impact of allelic imbalance on the analysis of chimerism
For the accuracy of measurements, the occurrence of allelic imbalance within IMCs was of
particular interest owing to the observation that longer PCR products of individual alleles tend
to yield disproportionally low peaks, thus distorting the measurements of allelic ratios. Allelic
imbalance was assessed by determining the total relative distance between the positions (P)
of donor and recipient alleles. For example, for IMCs of the type RRDD, the calculation was
AI = PD1+PD2-PR1-PR2, where PD1 is the position of the first donor allele, etc.
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Homozygous constellations were considered as two alleles at the same position. Hence, for
IMCs of the type RRD, the formula was AI = 2*PD-PR1-PR2; for IMCs of the type RD: AI =
2*PD-2*PR, for IMCs of the type RSD (where S stands for a shared allele between donor
and recipient; see Fig.1): AI = PD2 – PR1 etc.
Study design
The relevant parameters of the assay including detection limit, reproducibility, divergence
(accuracy), and linearity were determined by testing the dilution series of recipient in donor
DNA described above according to an incomplete balanced block design (20). First, 28 of
the available IMCs, including seven from each dilution series, were selected for the study. In
total, eight centers participated in this investigation, and at each center 14 different IMCs
were analyzed in duplicate assays. According to the experimental concept, each of the
selected IMCs was analyzed by 4 different centers. Two sets of 14 blocks of size four were
used according to the incomplete balanced block design (20). The assignment of the 28
IMCs to individual centers was balanced also with respect to the marker distribution and
allelic imbalance. The design was well balanced with respect to the marker distribution.
The particular selection of 28 IMCs ensured a widespread range of allelic imbalance (AI),
and their even distribution between different markers and dilution series. The AI values
ranged from -45 to 40 (indicating the maximum distance in base pairs between individual
donor and recipient alleles observed) and were not confounded by the selection of dilution
series or markers analyzed. Hence, the design was largely balanced for the factors center
and marker, dilution series and marker, and AI lacked any correlation with markers or dilution
series. The design was not balanced for the factors center and dilution series, but there was
sufficient variability to prevent these two factors from being confounded.
Statistical mehods
The final model of analysis specifically included the factors IMC, dilution step and allelic
imbalance. To determine the relatively complicated dependence of both mean and variance
of accuracy on the factor dilution step, a separate analysis for the dilution steps = 50%, 25%,
12.5% and the dilution steps ≤ 6% had to be performed. Based on this model, overall
confidence intervals accounting for the variation between different marker constellations are
provided.
The final model is of the form yij  b0  b1 xij   i   ij . Here index i refers to the IMC, index j
to the different observations of the i-th IMC, yij is the divergence and xij is the allelic
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imbalance. The random effect i and the error εij are independent such that  i ~ N (0,  int
ra )
2
and  ji ~ N (0, inter
) . Hence, the random effect indicates the variance between IMCs,
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whereas the error indicates the variance within IMCs. Based on this model, confidence
intervals of the divergence are provided for each individual IMC ( yˆ  inter Q(1 ) ) as well as
2
2
overall confidence intervals not including the information of IMC ( yˆ   int
er  intra Q(1 ) )
for α = 0.01 and α = 0.05, respectively.
Criteria of STR marker eligibility for quantitative chimerism testing (RSD code)
Judicious selection of appropriate microsatellite markers is an essential prerequisite for
reliable monitoring of post-transplant chimerism. The requirements of quantitative chimerism
analysis by STR-PCR show important differences to microsatellite testing for forensic
applications. The selection of marker panels for forensic analysis only requires the ability to
provide unequivocal genetic fingerprints in any individual. This type of analysis is qualitative
in nature, and the relative position of alleles is irrelevant as long as clear distinction between
individuals is provided. By contrast, chimerism analysis is based on quantitative assessment
of recipient- versus donor-derived microsatellite alleles, and the relative position of individual
alleles is important for precise calculation of chimerism. This is attributable to the fact that the
size (i.e. height) of alleles may be affected by the distance to neighbouring alleles which, in
turn, may render the quantitative analysis inaccurate (18). Dinucleotide repeat microsatellite
markers, for example, are highly polymorphic but are not suitable for chimerism testing due
to the occurrence of multiple stutter peaks interfering with quantitative measurement. Since
the distance between informative alleles is an essential criterion for accurate assessment of
chimerism levels, the frequency of individual alleles in the general population and the
distance between commonly occurring alleles play an important role for the adequacy of a
microsatellite locus for chimerism testing. Based on these considerations, it is obvious that
microsatellite marker panels designed for other applications, such as forensics, cannot be
readily used for chimerism testing.
The EuroChimerism microsatellite marker panel presented here has been selected based on
the requirements of quantitative chimerism analysis. In order to permit rapid assessment of
an allelic constellation, and to facilitate the evaluation of eligibility of any microsatellite marker
in a particular clinical specimen, the EuroChimersim consortium has introduced the RSD
code (18). This code describes the relative positions of recipient (R), donor (D) and shared
(S) alleles and reveals at a glance whether a microsatellite marker can be regarded as
adequate for the monitoring of chimerism in any given recipient/donor constellation. The RSD
code has been described earlier in detail (18), and examples of its application are displayed
in Fig. 1. In addition to providing a basis for the selection of appropriate markers for
chimerism analysis, the availability of a common designation of allelic constellations provided
by the RSD code facilitates the presentation and exchange of information between centers.
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Comparison of different fluorescence detection systems
The laboratories of the EuroChimerism consortium used a variety of instruments permitting
detection and quantitative analysis of PCR products labelled by a fluorescence marker.
Although preliminary experience indicated that the results of product quantification are
comparable regardless of whether polyacrylamide gel electrophoresis- or capillary
electrophoresis-based detection systems are used, the performance of the instruments used
in all participating laboratories was compared by testing centrally prepared fluorescencelabelled STR-PCR products containing equivalent amounts of recipient and donor-derived
DNA. The instruments tested included the ABI 310, ABI3100 capillary elecrophoresis and the
ABI 377 gel-based electrophoresis systems (all from Applied Biosystems). The accuracy and
reproducibility of PCR product analysis on different types of equipment were determined by
assessing the following variables: a) difference between duplicate runs on the same
instrument, b) effect of different injection parameters on the same instrument, c)
measurements by different types of instruments at the same center, and d) measurements
by the same type of instrument at different centers. Finally, a comparison across all centers
and instrument types was performed. For all parameters tested, the mean differences
observed were <2% in ≥90% of samples, indicating that comparable results can be obtained
regardless of the instrument used.
Allele frequencies of EuroChimerism markers
Detailed analyses and allele frequencies of the 13 selected EuroChimerism (EUC) STR
markers (Table 2) are presented in Tables SI-2a and 2b. Most of the markers display fournucleotide repeat motifs. Although some of them contain combinations of different
tetranucleotide repeat sequences (24), only the number of motif repeats was considered in
the designation of alleles. All fragment sizes of individual markers determined at the
participating centers were centrally converted into allele assignments based on the number
of repeats. Sequencing was performed in the presence of ambiguous findings. To validate
the allele assignment, a series of samples were exchanged between individual centers and
analyzed by sequencing. Interestingly, some alleles displaying identical fragment sizes
consisted of different repeat sequences (24). Alleles were defined by the number of motif
repeats in order to eliminate minor variations in the assessment of fragment length yielded by
various instruments used in the study (ABI377, ABI310, ABI3100, and ABI3700 Genetic
Analyzers, Applied Biosystems, Foster City, USA). However, the allele designations of STR
markers with complex repeat motifs (D17S1290, SE-33, D11S544 and MYCL1) reflect the
composition of multiple variable repeats of different length prevalent in these markers. The
predominant variable repeats are indicated in Table 5, and details of the complex repeats
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were described earlier (24). These microsatellite loci frequently display highly variable
alleles, and belong therefore to the most informative markers available.
Comparison of different fluorescence detection systems
The laboratories of the EuroChimerism consortium used a variety of instruments permitting
detection and quantitative analysis of PCR products labelled by a fluorescence marker.
Although preliminary experience indicated that the results of product quantification are
comparable regardless of whether polyacrylamide gel electrophoresis- or capillary
electrophoresis-based detection systems are used, the performance of the instruments used
in all participating laboratories was compared by testing centrally prepared fluorescencelabelled STR-PCR products containing equivalent amounts of recipient and donor-derived
DNA. The instruments tested included the ABI 310, ABI3100 capillary elecrophoresis and the
ABI 377 gel-based electrophoresis systems (all from Applied Biosystems). The accuracy and
reproducibility of PCR product analysis on different types of equipment were determined by
assessing the following variables: a) difference between duplicate runs on the same
instrument, b) effect of different injection parameters on the same instrument, c)
measurements by different types of instruments at the same center, and d) measurements
by the same type of instrument at different centers. Finally, a comparison across all centers
and instrument types was performed. For all parameters tested, the mean differences
observed were <2% in ≥90% of samples, indicating that comparable results can be obtained
regardless of the instrument used.
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Table SI-1. Aspects of chimerism analysis addressed in the EuroChimerism Concerted Action
■ Enrichment of cell subsets of interest
■ Methodologies of DNA extraction
■ Template amount required for adequate STR-PCR analysis
■ Establishment of eligibility criteria for STR markers in quantitative chimerism testing
■ Establishment of an optimized STR marker panel for quantitative chimerism analysis
■ Optimization of primer design, PCR reaction conditions and capillary electrophoresis
■ Signal evaluation (height vs. area) and calculation of recipient/donor chimerism
■ Establishment of multiplex STR-PCR reactions
■ Informativeness of the EuroChimerism panel with regard to provision of markers eligible for the monitoring of chimeris
■ Comparision of informativeness to a commercial STR kit for forensic analysis (PP16)
■ Detection limit of singleplex STR-PCR assays
■ Reproducibility and accuracy of quantitative analysis
■ Parameters affecting quantitative analysis
■ Comparison of different fluorescence detection systems
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Table SI-2 a . Tetranucleotide short tandem repeat (STR) allele frequencies.
Legend.
Allele numbers reflect the number of repeats of the STR allele. Allele 11.3 of D9S1118 and
18.3 of D12S391 are alleles with repeats including one trinucleotide sequence within the
tetranucleotide repeat sequences.
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Table SI-2b. .Short tandem repeat (STR) allele frequencies of complex repeat markers.
Legend.
The markers consist of multiple repeats of different length. Consequently, the allele number
does not directly reflect the number of repeats and is assigned based on the fragment length
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Figure SI-1. Schematic representation of the RSD code
Legend.
The peaks represent signals yielded by capillary electrophoresis of STR-PCR amplicons
provided by recipient, donor, and mixed chimeric specimens
R (capital letter) recipient allele outside the stutter peak areas of donor alleles
(distance >1 repeat unit*)
r (lower case letter) recipient allele within the stutter peak areas of donor alleles
(distance 1 repeat unit*)
D (capital letter) donor allele outside the stutter peak areas of recipient alleles
(distance >1 repeat unit*)
d (lower case letter) donor allele within the stutter peak areas of recipient alleles
(distance 1 repeat unit*)
S (capital letter) shared allele between donor and recipient
The sequence of the letters is arranged by allelic size, starting with the shortest allele.
The digits inserted between the letters indicate the number of nucleotides separating
neighbouring alleles
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