Volume 8, Issue 8, August – 2023 International Journal of Innovative Science and Research Technology

ISSN No:-2456-2165

Assessment of STRs Loci on Investigator Id-Plex

Plus Kit for Forensics and Chimerism Monitoring in
Morocco: A Case Series
CHBEL Faiza (PhD) 1,2*, EL ARJI Marzouk2, HARIF Mhamed3, YAHIA Hakima2, RACHID Mohamed3, MADANI Abdellah3,
AZEDDOUG Houssein 1, MOSSAFA Houssein 4, QUESSAR Asmaa3
1
Higher Normal School, Hassan II University, Bio-Geosciences and Materials Engineering Laboratory, Casablanca, Morocco.
2
Laboratoire National de Police Scientifique, 4 Rue Bnou Bouraid, Maarif, Casablanca, Morocco.
3
Service d’oncohématologie Pédiatrique, Hôpital 20 Août, Casablanca, Morocco.
4
Laboratoire Internationale du groupe Oncorad- Hay Hassani, Casablanca, Morocco.

Corresponding Author: CHBEL Faiza (PhD)*

Abstract:- Allogeneic hematopoietic stem cell Keywords:- Chimerism; STR; Morocco; Allogenic
transplantation is a curative treatment for malignant Transplanted Patients; Investigator IDPlex Plus;
hematologic diseases, leukemias, congenital and acquired Informativity; Polymorphism, Forensic Parameters
non-malignant diseases of a patient (recipient) by
replacing recipients cells by new one from a healthy I. INTRODUCTION
donor. Monitoring of hematopoietic chimerism after
allogenic transplantation is a useful tool for determining Allogeneic hematopoietic stem cell transplantation
the engraftment of donor cells and predicting the risk of (Allo-HSCT) is curative treatment for malignant hematologic
relapse of the original disease. Nowadays, diverses diseases, leukemias, congenital or acquired non-malignant
techniques are used and differ from laboratory to diseases [1, 2]. It consists to the substitution of damaged
laboratory, which make data exchange and comparaison hematopoietic cells of a recipient (patient) by a new one from
between them difficult. Amplification of short tandem a healthy donor. The coexistence of cells with different
repeats (PCR-STR) constitutes the gold standard method genetic origins (donor and recipient) in a patient after
for chimerism quantification, although more sensitive receiving a hematopoietic stem cell transplant (HSCT) is
PCR techniques have recently developed. This study was called “chimerism”. It’s defined by the percentage of cells of
carried out on 30 allografted patients in whom the status donor origin in the recipient patient, either in the blood or in
of chimerism was analysed by the use of STRs included in the marrow [3-5]. Chimerism monitoring is actually a routine
Investigator Id-plex plus kit (Qiagen). This is conducted diagnostic tool at allogenic transplant centers and it is useful
by taking samples of whole blood from these patients at for evaluating the stability of lymphoid and myeloid donor
different times in post-allograft from which we extract engraftment to distinguish the success or failure of the
DNA and amplify STR markers and genotyped by transplant, to predict the possibility of a relapse, and to apply
capillary electrophoresis. The STR profiles have been the opportune therapy by physicians [6-7]. Complete
generated for each sample including donor and recipient chimerism (CC) is characterized by a total replacement of the
samples taken before the allogeneic transplant (J0). patient's hematopoiesis by that of the donor, and a mixed
Profiles comparison obtained from the post-allograft chimerism (MC) by the persistence of the two types of cells
samples with the genotypes of the donor and the pre- (patient and donor). An increase in the percentage of
allograft recipient, permits to determine the status of chimerism refers to an amplification in the percentage of
chimerism in these different patients. This work was cells obtained from the donor, with a MC approaching CC.
preceded by analyzing a set of 219 individual from Mixed chimerism can have two different meanings: either it
Morocco with the objective to establish their usefulness is normal mature cells of the patient, having survived
for human identification. Allelic frequencies for the 15 conditioning or having matured from residual hematopoietic
short tandem repeat (STRs) loci were calculated. Results stem cells from the recipient; or they are residual malignant
show that the 15 loci are highly polymorphic. The cells that have survived the conditioning and may cause a
combined power of exclusion for the fifteen loci is relapse. MC can evolve to the loss of the graft or a relapse of
0.99999968 and the combined discrimination indice was the hemopathy [1,8]. Very early MC would generally be
0.999999999999999985. The combined matching explained by the persistence of normal recipient cells, and
probability for these loci reaches 1.59 10-18 which make not by the presence of malignant cells. Early analyses of
these loci very useful for personal identification casework recipient chimerism patterns increase the importance of
purposes in Morocco. predicting graft rejection as well as persistent disease or
failure [9].

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Hematopoietic chimerism classification was based on Insensitive methods cannot differentiate a 90% donor
chimerism analyses using quantitative and qualitative tests chimerism from a 95% donor chimerism. The difficulty is to
based on exploring genetic polymorphism for the define a threshold that is significant from a medical point of
identification of the recipient’s and donor’s cells [5, 10, 11]. view, but also quantifiable by the technique used. In general,
In the past, several markers that have been successively used, a CC is considered if the percentage of cells derived from the
such as the blood groups of the ABO system, or HLA donor is greater than 95% donor.
antigens. These techniques were time-consuming, poorly
informative, or studying a single lineage. They were Chimerism analysis should be carried out in the routine
generally uninformative in the event of a blood transfusion with efficient techniques in terms of power of discrimination,
[12, 13]. Conventional cytogenetics has made it possible to cost and time, thus recommendations are focused in the use
develop a genetic approach, by detecting either the sex of the of STR markers as it’s the most used and standardized
patient and the donor, or specific abnormalities of the disease techniques [29- 31]. The analysis of chimerism by STR–PCR
(Philadelphia chromosome in CML). Because this technique usually is frequently carried out with commercial kits
is not very sensitive (5-10%), and not quantitative because of originally designed for forensic purposes, including a large
the culture; it has been replaced by the FISH (Fluorescent in number of STR, and cover diverse global human populations
situ hybridization) using fluorescent probes specific for [32-35]. Although the frequency of alleles differs from one
sequences carried by the X and Y chromosomes allowing population to another [36]. The inclusion of loci with
more precise quantification (sensitivity less than 1%)[14]. important differences in a power of discrimination (PD)
across different populations is inefficient as some loci often
The use of molecular biology techniques has a good become less informative for chimerism analysis in some
impact on the quality of chimerism analysis results. It started populations [37]. Thus, some studies try to elucidate which
by the study of RFLP (Restriction Fragment Length are the most informative markers [38-40]. Few studies have
Polymorphism) [15, 16]. This technique is semi-quantitative, been conducted to evaluate the informativeness for human
insensitive (5-10%) and consumes a lot of biological identification of certain STR loci included in commercial
material. The introduction of markers discovered in 1985 kits: 8 autosomal loci of Promega Kits [41], 15 autosomal
[17] commonly called “genetic fingerprints” based on PCR- STRs loci included in Identifiler Kit [42 , 15 Y STRs
VNTRs (Variable Number Tandem Repeats) (size between included in Y-filer [43].
10 to 50 bp) [11, 18] allowed the development of chimerism
monitoring [19-22]. This study aims to assess either the genetic diversity of
the 15 STR loci (CSF1PO, FGA, TH01, TPOX, vWA,
Later, VNTRs analyses have been replaced by studying Amelogenin D2S1338, D3S1358, D5S818, D7S820,
“Microsatellite” sequences: STR (Short Tandem Repeats) by D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11)
PCR which are smaller, comprising 2 to 7 bp repeat [23]. included in the Investigator IDplex-plus kit (Qiagen, Hidden,
They are highly variable from one individual to another and Germany) to explore the potential of applying for human
distributed throughout the human genome and are therefore identification in a population sample and to assess their
highly informative. They are tool used as genetic fingerprints usefulness for chimerism after allogenic transplantation in
in forensic medicine and for paternity testing as we can Moroccan patients to define the most useful in the
analyze many loci at once on small quantities of DNA, even perspective to identify a minimal STR panel allowing easy,
degraded by multiplex PCR. Nowadays, the analysis of short fast and cost-effective monitoring of chimerism in allogenic
tandem repeats (STRs) by polymerase chain reaction (PCR) bone marrow transplantation.
combined to capillary electrophoresis is the most commonly
used procedure for chimerism quantification which II. MATERIAL AND METHODS
sensitivity is about 3–5%. In an allograft context, the small
amounts of DNA required make it possible to obtain a result  Population Analysis
from the immediate post-allograft period, when the patient is
still in aplasia, with hypocellular marrow [24].  Samples and Methods
For the generation of population data, 219 saliva
The study of SNP (Single Nucleotide Polymorphism) by samples on FTA cards were obtained from genetically
quantitative real-time PCR TaqMan® (ThermoFisher, USA) unrelated individuals. All donors read and signed a written
and Droplet digital PCR (ddPCR) [25] as well as small consent statement form in accordance of the Hensilki
insertions and deletions polymorphisms, can be detected by declaration. Direct genotyping analysis workflow was
allele-specific quantitative PCR [26, 27]. Quantification by conducted without DNA extraction: a 0.2mm punches were
these methods is therefore very precise in low percentages, deposited on a 96 well containing 150 µl of water, incubated
currently usable routinely (10-5 to 10-3) since their mutation for 10 min, and the water was discarded but 5µl left in the
rate is smaller than the mutation rate of STRs and may well. 10µl of PCR master mix for amplification of 15 STR
provide additional information in some cases where STRs loci included in the Investigator Idplex plus kit (Qiagen,
assays present sensibility issues, with potential use in Hiden, Germany) was added (following manufacture
monitoring residual disease in a context of standard protocol). Amplification was conducted on Veriti
conditioning or attenuated conditioning, once CC is reached thermocycler (Thermo Fisher Scientific) using manufacture
[13, 28]. The threshold from which a chimerism is considered protocol with slight modifications (27 cycles and expansion
complete depends on the sensitivity of the technique. time 60° C for 45 min). The post-PCR mix was added to a

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ISSN No:-2456-2165
96-well genotyping plate. The post-PCR mix consisted of Hi- amplification was conducted using 3-5 µl of DNA and
di formamide (9.3 μL per well) and Internal Lane Standard amplification and genotyping were conducted as mentioned
BTO-500 (0.7μL per well). One microliter of each PCR below. Genotypes were analyzed using the GeneMapper®
product was added directly to the corresponding well, ID-X v1.4 software (Applied Biosystems). As defined by UK
denatured for 3 min at 95 °C and cooled at 4 °C. Fragment NEQAS technical recommendations for chimerism analysis
separation was performed by capillary electrophoresis on an using STR-based techniques, only fully informative markers
ABI3130XL Genetic Analyser (Thermo Fisher Scientific). were selected [11,40]. Loci considered for chimerism
Data collected were analyzed using the GeneMapper® ID-X calculation were those showing more than two alleles and
v1.4 software (Applied Biosystems) for genotyping step. showing recipient alleles outside of the stutter positions of
the donor’s peaks, usually referred to as ‘type 5’ [11]. A
 Statistical Analysis of Data: minimum of three informative STR markers were used to
GENEPOP software (Version 3.3) [44] was used to avoid misinterpretation in case of genetic alteration involving
calculate the allele frequencies and to perform the exact test one locus or the increase of microsatellites mutations rate in
of Hardy-Weinberg [45]. The observed heterozygosity (Ho), hematological malignancy relapse. Markers showing stutter
expected heterozygosity (He), the power of discrimination interference were excluded because interpretation is hence
(PD), the probability of match (PM), the Polymorphic difficult.
Information Content (PIC) and the Power of Exclusion (PE)
were calculated using CERVUS software [46]. Chimerism calculation was based on the fluorescence
intensity of each allele designed as peak height ratio “PHR”
 Chimerism Analysis: that depends on PCR amplification yield based on the DNA
Our series includes 30 patients suffering from malignant concentration. The use of too much DNA induces the
(acute leukemia: LLA MLA…) and non-malignant diseases apparition of saturation and stutters that can influence the
(medular aplasia, immune deficiency..), with bone marrow chimerism interpretation. Thus, DNA concentration used
transplantation subjected to chimerism analysis. This study around 5-10 ng (and less DNA quantity should not be used
was approved by the ethics committee of 20 Aout Hospital to avoid the problem of stochastic amplification tending to
according to the declaration of Helsinki protocol and provide less reliable STR- profiles (low copy number) [47].
recipients and donors gave written informed consent before Based on the analysis method parameters for
bone marrow transplantation. Blood samples from the GeneMapper®ID-X v1.4 software for forensic purposes, it’s
recipient and the donor were taken before transplantation, so obvious that for reference samples, it’s usual to increase the
the genotypes of the donor and the recipient are known. Pic Height Ratio (PHR) to avoid misinterpretation of stutters,
Furthermore, recipient blood samples at various time points on the contrary, casework samples have to be interpreted
post-transplant (J30, 60, 90…..etc),. A total of 98 post-HSCT with lower PHR to permit mixture interpretation. The same
peripheral blood samples (2-5 ml) were collected since reflection has to be followed in the case of chimerism
January 2019 (See Table 2 for details), 250-300 µl of blood interpretation. In case of mixed chimerism analysis, the pic
were used for DNA extraction using the EZ1 Investigator height ratio = 50 RFU was very useful for some loci to detect
DNA extraction kit following the manufacturer’s instructions chimerism than the use of 100-150 RFU which could be
(Qiagen, Hiden, Germany) on EZ1 automate. Microsatellite misinterpreted as complete chimerism status (Figure1).

Fig 1 Difference in Allele Size Calling Depending on Pic Height Ratio (PHR) Parameters

The percent donor chimerism for each locus was estimated as the sum of donor allele areas divided by the sum of the areas
of all alleles in a given locus (donor + receiver) [48]. For each sample, we estimated the percent donor chimerism (mean,) and the
number of STRs microsatellites showing chimerism.

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III. RESULTS to 0.972 (D18S51). The probability of exclusion varies from
0.470 (CSF1PO) to 0.765 (D21S11) and the combined power
 Population Analysis: of exclusion of the 15 loci is 0.99999968. The combined
A total of 156 alleles were detected with corresponding matching probability for the 15 loci reached 1.59 10-18, that
allele frequencies ranging from 0.0023 to 0.4521 (see make the Investigator IDplex-plus kit (Qiagen, Hidden,
Table1). The most polymorphic STR marker was D19S433 Germany) a highly polymorphic tool for the human identity
with 18 alleles and the less polymorphic markers are in the Moroccan population.
(D3S1358, TPOX, D5SS818, D16S539) with 7 alleles. The
Hardy-Weinberg equilibrium exact test using 2000 shuffling  Chimerism Analysis:
showed two departures from the equilibrium for vWA and Our cohort includes 30 patients, with a sex ratio M/F of
D21S11 (a 5% significance level is taken, table 1). After 1.14 and ages between 4 and 63 years old, presenting
employing a Bonferroni Correction for the number of loci different pathologies (see Table 2). The myeloid acute
analyzed, these observations are not likely to be significant. leukemia (AML) and medullar aplasia (30% each) are the
The observed heterozygosity varies from 0.612 for D21S11 two major cause of allogenic hematopoietic stem cell
to 0.8447 for D8S1179. The mean observed heterozygosity transplantation (ASCT) followed by acute lymphoid
across all loci reach 80%. As expected, the number of alleles leukemia (ALL) (20%) (Figure2). In fact, malignant diseases
is correlated with polymorphic information content PIC. The represent the major cause of ASCT than non-malignant
power of discrimination (PD) varies from 0.906 (D16S539) diseases (67% vs 33 %).

Fig 2 Distribution of Patients Based on the Type of Pathologies

Chimerism is a dynamic process with proportions variation of donor cells over time, the use of STRs gives reproducible
results and can assess samples at different time points to conduct longitudinal studies and produce an inclusive chimerism analysis
report (Table 2). As shown in Figure 3, the informativity of the STR markers varied widely, between 63.3% and 20%. The most
informative STR loci were D8S1179, D21S11, FGA, D18S51, D2S1338 and TPOX which individually allowed direct detection of
chimerism in more than 50% of the cases. In contrast, the least informative markers were D13S317 and CSF1PO. As expected,
informativity and heterozygosity tended to be correlated, although imperfectly (TPOX).

Fig 3 Loci Informativity for Chimerism Monitoring

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Chimerism status has been established for the patients and GVH disease in order to interfere with appropriate
included in this study as complete chimerism (CC) or mixed medical therapy. Chimerism, especially complete chimerism,
chimerism (MC) or absence of chimerism. We identified 37 has been associated with an increased risk of relapse in
samples showing mixed donor–recipient chimerism (MC) certain types of malignant diseases, such as leukemias and
while 47 samples showed complete chimerism (CC). For 14 lymphomas. From our cohort only 3 persons with an acute
samples a recipient profile has been detected as a leukemia disease die after relapse, all supporting a complete
consequence of the no engraftment success. Relationship chimerism status until D90 and D180 respectively. This is
between the nature of the disease and the success/or no of due probably to the expansion of recipient leukemic cells
engraftment was not established. surviving after conditioning therapy. The chimerism status
evaluation in these cases didn’t permit to detect of the high
In patients with non-malignant hemopathies, four risk of relapse after CC, and should be monitored otherwise
patients showed complete chimerism status (CC), however by conducting an STR-PCR chimerism analysis not on
mixed chimerism status (MC) was observed in five patients peripheral blood but from separated lineages blood cells (ex
and two patients showed transplant rejection. On the other T- and NK-cell) or bone marrow sample to permit the
hand, patients suffering from malignant hemopathies showed detection of any transformation earlier before the relapse
complete chimerism and mixed chimerism in nine and four expression [9] or like detecting residual leukemia in the form
patients respectively. Three patients showed transplant of MRD [2] , especially in those still alive with complete
rejection and three patients showed complete chimerism at remission after CC status. Only in this case, we can propose a
the beginning followed by mixed chimerism status very sensitive method like q-PCR to monitor chimerism as a
subsequent to D60 (1 patient) or D90 (2 patients). From our secondary tool besides STR-PCR markers.
cohort of 30 patients, 22 patients are still alive (until the
redaction of this article) and 8 died as mentioned in Table 3 However, it is important to note that the risk of relapse
from wish only 3 persons with an acute leukemia disease die in transplant recipients with mixed chimerism is generally
after relapse, all supporting a complete chimerism status until lower than the risk of relapse in non-transplanted individuals
D90 and D180 respectively. with the same malignant disease. The variation of the degree
of the MC may not be influenced by the disease but rather by
IV. DISCUSSION other factors including its stage at the time of ASCT, the
conditioning regimen, and the timing of the assay (time from
Chimerism monitoring is based on STR profil sampling until treatment should be considered as critical
comparaison after allogenic transplantation. The parameter). Additionally, the use of immunosuppressive
informativity of an STR in chimerism analysis could be therapy and other treatments can help to control the risk of
defined as its capability to distinguish between the donor and relapse in transplant recipients with chimerism.
the recipient cell components and implies that at least one
allele should differ between the recipient from those of the Moreover, the correlation between MC and relapse is
donor [49]. The distinction between donor and recipient still a controversial issue but seems to be more likely
alleles in the post-transplant genotype is easy when both the correlated in acute leukemia (2 patients with MC showed
pretransplant genotypes of the recipient and the donor are disease relapse) [53-54] It is important to note that
available to be compared with the post-transplant genotype. monitoring chimerism should be done in conjunction with
However, in routine medical practice, sometimes we lack one other forms of monitoring, such as clinical examination,
or two of those genotypes for comparison. In our case, the imaging studies, and laboratory tests, to identify potential
lack of donor/or recipient sample before transplantation signs of relapse as early as possible.
impacted the interpretation of one case only. Furthermore,
our STR evaluation criteria take into consideration either the V. CONCLUSION
potential influence of stutter peaks originated from
polymerase slippage during PCR that appear one repeat unit In conclusion, Investigator Id-plex plus kit offers either
smaller than the authentic allele and represent 4–11% of the a highly polymorphic tool for the human identity in the field
major component height, so they can be misinterpreted as a of forensics in the Moroccan population and a powerful and
true minor allele (39,50-51); and the peak height ratio relatively low-cost molecular method for helping in practice
imbalance. When the pre-transplanted and donor samples are medical patient care decisions for chimerism monitoring. The
available, the comparison of those genotypes with the post- use of the markers included in the Investigator IDplex plus
transplant genotype is easy even they share alleles. But in kit (Qiagen, Hidden, Germany) showed their usefulness and
case of lack of one genotype or the two genotypes, the we can choose those most polymorphic in our population to
interpretations became difficult. The use of polymorphic reduce the cost of analysis. Increasing our sampling will give
markers would help chimerism interpretation but will be more insight about the power of STR for allogenic
specific to population genetic polymorphism [52]. The FGA, transplantation monitoring.
D18S51, D21338 and D21S11 STR markers have been
reported before to be very useful for chimerism in Spanish  Conflicts of Interests: Authors declare no conflict of
population study [39]. interest.

The importance of chimerism study after ASCT is to

predict negative events like graft rejection, disease relapse,

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ISSN No:-2456-2165
ACKNOWLEDGEMENTS [11]. Thiede C, Bornhäuser M, Ehninger G. (2004)
Evaluation of STR informativity for testing:
Acknowledgements go to Pr Redouane Boulouiz for comparative analysis of 27 STR systems in 203
revision of the manuscript matched related donor recipient pairs.
Leukemia;18:248e54.
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Navarro, G., Buño, I., Castillejo, J.A., Rodríguez, A.I., stem cell transplantation using multiplex polymerase
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332.

Table 1 Observed Allele Frequencies and Statistical Parameters for Investigator Idplex plus Kit Loci in Moroccan Population:
TH0 D3S13 vW D21S TPO D7S8 D19S4 D5S8 D2S13 D16S13 CSF1P D13S3 D18S D8S11
Alleles FGA
1 58 A 11 X 20 33 18 38 38 O 17 51 79
- - - - - - - - - - - - -
0.00 0.002
5 68 3
0.17 - - - 0.01 - - - - - - - -
6 35 80 - -
- - - - 0.002 - - - - - - -
6.3 - 3 - -
0.23 - - - 0.01 0.004 - - 0.0046 - - - -
7 29 24 6 - -
0.00 - - - - - - - - - - -
7.3 23 - - -
0.18 - - - 0.45 0.107 0.050 - - - 0.0068
8 72 21 3 - 2 0.0160 0.0228 0.0959
0.22 - - - 0.18 0.114 0.032 - - - 0.0046
9 83 49 2 - 0 0.1187 0.0166 0.0365
0.12 - - - - 0.011 - - - -
9.3 10 4 - - - -
0.04 - - - 0.08 0.356 0.066 - - 0.006 0.0639
10 57 69 2 0.0023 2 0.0731 0.3356 0.0388 8
0.00 - - - 0.21 0.242 0.0160 0.258 - - 0.020 0.1142
11 23 92 0 0 0.2900 0.2783 0.2854 5
11.2 - - - - - - - - - - - - - - -
- - - - 0.02 0.143 0.1438 0.372 - 0.2991 - 0.139 0.1393
12 55 8 1 0.2988 0.3858 6
12.2 - - - - - - - - - - - - - - -
- 0.0046 0.00 - - 0.016 0.2489 0.198 - 0.1849 - 0.139 0.1941
13 23 0 6 0.0365 0.1096 6
13.2 - - - - - - - - - - - - - - -
- 0.0434 0.12 - - - 0.2511 0.022 0.0183 0.0068 - 0.142 0.2580
14 56 8 0.0023 0.0434 6
14.2 - - - - - - - - - - - - - - -

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- 0.3457 0.14 - - - 0.1096 - - - - - 0.122 0.1758
15 38 0.0046 0
15.2 - - - - - - - - - - - - - - -
- 0.2795 0.25 - - - 0.0388 - - - - - 0.146 0.0411
16 80 0.0434 1
16.2 - - - - - - - - - - - - - - -
- 0.1973 0.25 - - - 0.0137 - - - - - 0.154 0.0023
17 11 0.3174 0
17.2 - - - - - - - - - - - - - - -
- 0.1233 0.13 - - - 0.0023 - - - - 0.052 -
18 24 0.0799 - 5
- - - - - - - - - - - 0.00 - -
18.2 - 46
- 0.0052 0.06 - - - - - - - - 0.04 0.044 -
19 16 0.1005 57 4
19.2 - - - - - - - - - - - - - - -
- - 0.01 - - - 0.0023 - - - - 0.10 0.006 -
20 83 0.1575 50 8
- - 0.00 - - - 0.0023 - - - - 0.18 0.018 -
21 68 0.0594 95 3
- - - - - - - - - - - 0.00 - -
21.2 - 23
- - - - - - 0.0114 - - - - 0.18 0.004 -
22 0.0434 72 6
- - - - - - 0.0274 - - - - 0.20 0.002 -
23 0.0822 32 3
23.2 - - - - - - - - - - - - - - -
- - - - - - 0.0434 - 0.0776 - - - 0.13 - -
24 93
- - - - - - - - - - - - 0.00 - -
24.2 23
- - - - - - 0.0502 - 0.0274 - - - 0.06 - -
25 16
25.2 - - - - - - - - - - - - - - -
- - - - - - 0.0320 - 0.0091 - - - 0.04 - -
26 57
- - - 0.027 - - 0.0023 - - - - - 0.00 - -
27 4 68
- - - 0.095 - - 0.0023 - - - - - 0.00 - -
28 9 46
28.2 - - - - - - - - - - - - - - -
- - - 0.077 - - - - - - - - - - -
28.3 6
- - - 0.164 - - - - - - - - 0.00 - -
29 4 23
- - - 0.002 - - - - - - - - - - -
29.2 3
- - - 0.137 - - - - - - - - - - -
29.3 0
- - - 0.146 - - - - - - - - - - -
30 1
- - - 0.006 - - - - - - - - - - -
30.2 8
- - - 0.025 - - - - - - - - - - -
31 1
- - - 0.116 - - - - - - - - - - -
31.2 4
- - - 0.011 - - - - - - - - - - -
32 4

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ISSN No:-2456-2165
- - - 0.130 - - - - - - - - - - -
32.2 1
- - - 0.002 - - - - - - - - - - -
33 3
- - - 0.041 - - - - - - - - - - -
33.2 1
34 - - - - - - - - - - - - - - -
- - - 0.009 - - - - - - - - - - -
34.2 1
- - - 0.006 - - - - - - - - - - -
35 8
N° 7
9 7 9 16 7 10 18 7 12 8 8 14 14 10
allele
0.79 0.731 0.74 0.612 0.67 0.748 0.776 0.7763 0.7808 0.6986 0.7260 0.83 0.835 0.8447
HO 45 0 6 9 0.8265 3 56 6
0.81 0.750 0.81 0.886 0.70 0.770 0.8359 0.748 0.8390 0.7224 0.7454 0.85 0.851 0.8285
HE 36 4 7 7 8 0.7740 12 2
0.23 0.3665 0.00 0.000 0.15 0.282 0.9162 0.316 0.4910 0.1647 0.9035 0.3568 0.70 0.701 0.1664
P 82 20 9 21 9 0 15 5
0,93 0,93 0,87 0,95
PD 7 0,895 9 0,975 1 0,913 0,953 0,897 0,958 0,913 0,870 0,898 9 0,972 0,947
0,62 0,62 0,47 0,69
PE 3 0,518 9 0,765 5 0,562 0,677 0,526 0,691 0,560 0,470 0,530 8 0,749 0,655
0.78 0.78 0.66 0.83
PIC 4 0.706 6 0.872 3 0.736 0.815 0.708 0.822 0.737 0.669 0.707 1 0.863 0.804

 HO: observed heterozygosity; HE: expected heterosygosity; P: Hardy-Weinberg equilibrium exact test; PD: power of
discrimination; PE: power of exclusion; PIC: polymorphism information content

Table 2 Summary of the Patients Data and Chimerism Status Evaluated using Investigator IDplex Plus STR Markers
AG se se
transplant
N° E xe xe pathology J30 J60 J90 J180 J240 J365 J398 J600 775 J900 J1000 status
status
R R D
MC
MC MC, MC
P1 63 F F CML CC 73 x x x x x x Alive CR
77% 67% 89%
73%
P2 35 F M AML CC CC CC CC x x x x x x x Alive Relapse
transplant
P3 5 M M AML x x R R x x x x x x x Alive
rejection
MC
P4 56 F F AML CC CC 91,5 x x x x x x x x Dead CR
%
CR,
MC MC MC deceased
MC
P5 62 F M AML 90,5 67,4 72,2 x x x x x x x Alive after
50%
% % % COVID-
19
MC
MC
P6 44 M M AML 72,6 x x x x x x x x x Dead Relapse
92%
%
MC MC MC MC MC
P7 35 F F AML x x x x x x Dead CR
84% 88% 88% 92% 93%
P8 44 F M AML CC CC CC x x x x x x x x Alive Relapse
MC MC MC
MC
P9 25 M F AML 80,7 78,8 85,9 x x x x x x x Alive Relapse
92%
% % %
P1
45 M M AML CC CC CC x x x CC x x x x Alive CR
0

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P1 X
22 M M AML CC CC CC CC X X x x x x Alive CR
1
P1
47 F M LLA-B CC x x x x x x x x x x Alive RAS
2
P1
22 M M LLA-T CC CC CC x x x x x x x x Alive Relapse
3
P1
37 M M LLA-T R R R R X X x x x x x CR
4
P1
39 F M LLA-T x CC CC CC x x x x x x x Alive Relapse
5
P1
35 F M LLA-T x x x x x CC x x x x CC Dead CR
6
P1
18 M M LLA-T CC CC CC CC x x x CC x x x Dead CR
7
post
P1
34 M F SMD R R x x x x x x x x x Dead transplant
8
infection
P1 Fanconi MC
17 M M x x x x x x x x x x Alive CR
9 anemia 87%
CR, death
P2 MC due a
25 F M Hodgkinien CC CC x x x x x x x x Alive
0 95% transplant
lymphoma
toxicity
CR,
P2 immun autologos
4 M F x R R x x x x x R R x Alive
1 deficiency reconstitut
ion
MC MC
P2 Medullary MC
25 F F 81,5 92,2 x x x x x x x x Alive CR
2 aplasia 91%
% %
MC MC MC
P2 Medullary MC MC
19 M F 75,4 85,9 76,9 x x x x x x Alive CR
3 aplasia 93% 86%
% % %
P2 Medullary
20 F F CC CC CC CC x x x CC x CC x Alive CR
4 aplasia
P2 Medullary
21 F M x CC x CC x x x x x x x Alive CR
5 aplasia
P2 Medullary MC MC MC
28 M F x X X X x x x x Alive CR
6 aplasia 75% 61% 64%
Relapse
P2 Medullary
20 M F x R R x x x x x x x x Alive after 2
7 aplasia
ASCT
P2 Medullary
29 F M CC CC x x x x x x x x x Alive CR
8 aplasia
MC
P2 Medullary MC
34 M M X X 80,3 X X X x x x x Dead CR
9 aplasia 96%
%
P3 Medullary
17 M F CC CC CC CC x CC x x x x x Alive CR
0 aplasia
(R: recipient; CC: complete chimerism, MC: mixed chimerism; CR: complete remission)

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Table 3 Status of the patients at the end of this study and main causes of death.
Patients still alive: 22 patients Dead patients: 8
 Relapse (3)  Relapse (3)
 No engraftment (1)  Non engraftment (1)
 Complete remission (17) or Autologous recovery (1)  Post transplantation infection (1)
 COVID-19 (1)
 Digestive GVH disease (1)
 Pulmonary fibrosis (grapht toxicity) (1)

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