Assessment of chimerism by next generation sequencing: A comparison to STR/qPCR methods.

Chimerism analysis is used to evaluate patients after allogeneic hematopoietic stem cell transplant (allo-HSCT) for engraftment and minimal measurable residual disease (MRD) monitoring. A combination of short-tandem repeat (STR) and quantitative polymerase chain reaction (qPCR) was required to achieve both sensitivity and accuracy in the patients with various chimerism statuses. In this study, an insertion/deletion-based multiplex chimerism assay by next generation sequencing (NGS) was evaluated using 5 simulated unrelated donor-recipient combinations from 10 volunteers. Median number of informative markers detected was 8 (range = 5 - 11). The limit of quantitation (LoQ) was determined to be 0.1 % recipient. Assay sample number/batch was 10-20 and total assay time was 19-31 h (manual labor = 2.1 h). Additionally, 50 peripheral blood samples from 5 allo-HSCT recipients (related: N = 4; unrelated: N = 1) were tested by NGS and STR/qPCR. Median number of informative markers detected was 7 (range = 4 - 12). Results from both assays demonstrated a strong correlation (Y = 0.9875X + 0.333; R2 = 0.9852), no significant assay bias (difference mean - 0.08), and 100 % concordant detection of percent recipient increase ≥ 0.1 % (indicator of increased relapse risk). NGS-based chimerism assay can support all allo-HSCT for engraftment and MRD monitoring and simplify clinical laboratory workflow compared to STR/qPCR.

Can novel methods replace the gold standard chimerism method after allogeneic hematopoietic stem cell transplantation?

After hematopoietic stem cell transplantation, chimerism assay is a useful approach to monitor the success of the transplant and to select the appropriate treatment strategy, such as donor leukocyte infusion or immunosuppressive drug dosage. Short tandem repeat PCR is the method that has been accepted as the gold standard for chimerism. However, it has not yet been sufficient to detect mixed chimerism in patients with minimal residual disease. Simultaneously, recent years have been marked by developing sensitive, high-throughput, and accurate molecular genetic assays. These novel methods have subsequently been adapted for the analysis of post-transplant chimerism. In this review, we discuss the technical features of both novel and conventional gold standard chimerism assays. We also discuss their advantages and disadvantages.

Chimerism Testing by Next Generation Sequencing for Detection of Engraftment and Early Disease Relapse in Allogeneic Hematopoietic Cell Transplantation and an Overview of NGS Chimerism Studies.

Chimerism monitoring after allogenic Hematopoietic Cell Transplantation (allo-HCT) is critical to determine how well donor cells have engrafted and to detect relapse for early therapeutic intervention. The aim of this study was to establish and detect mixed chimerism and minimal residual disease using Next Generation Sequencing (NGS) testing for the evaluation of engraftment and the detection of early relapse after allo-HCT. Our secondary aim was to compare the data with the existing laboratory method based on Short Tandem Repeat (STR) analysis. One hundred and seventy-four DNA specimens from 46 individuals were assessed using a commercially available kit for NGS, AlloSeq HCT NGS (CareDx), and the STR-PCR assay. The sensitivity, precision, and quantitative accuracy of the assay were determined using artificially created chimeric constructs. The accuracy and linearity of the assays were evaluated in 46 post-transplant HCT samples consisting of 28 levels of mixed chimerism, which ranged from 0.3-99.7%. There was a 100% correlation between NGS and STR-PCR chimerism methods. In addition, 100% accuracy was attained for the two external proficiency testing surveys (ASHI EMO). The limit of detection or sensitivity of the NGS assay in artificially made chimerism mixtures was 0.3%. We conducted a review of all NGS chimerism studies published online, including ours, and concluded that NGS-based chimerism analysis using the AlloSeq HCT assay is a sensitive and accurate method for donor-recipient chimerism quantification and minimal residual disease relapse detection in patients after allo-HCT compared to STR-PCR assay.

One fits all: a highly sensitive combined ddPCR/pyrosequencing system for the quantification of microchimerism after hematopoietic and solid organ transplantation.

OBJECTIVES: A combined digital droplet PCR (ddPCR)/pyrosequencing assay system was developed that demonstrated advantages applicable to multiple qualitative and quantitative molecular genetic diagnostic applications. Data for characterizing this combined approach for hematologic stem cell transplantation (HSCT) and allele quantification from graft-derived cell-free (cf) DNA in solid organ transplantation (SOT) is presented. METHODS: ddPCR and pyrosequencing assays targeting 32 SNPs/markers were established. ddPCR results from 72 gDNAs of 55 patients after allogeneic HSCT and 107 plasma-cfDNAs of 25 liver transplant recipients were compared with established methods/markers, i.e. short-tandem-repeat PCR and ALT, respectively. RESULTS: The ddPCR results were in good agreement with the established marker. The limit of detection was 0.02 % minor allele fraction. The relationship between ddPCR and STR-PCR was linear with R2=0.98 allowing to transfer previously established clinical STR-PCR cut-offs to ddPCR; 50-fold higher sensitivity and a variation coefficient of <2 % enable the use of low DNA concentrations (e.g. pre-sorted cells). ddPCR detected liver allograft injury at least as sensitive as ALT suggesting that ddPCR is a reliable method to monitor the transplant integrity, especially when other biomarkers are lacking (e.g. kidney). CONCLUSIONS: Combining pyrosequencing for genotyping and ddPCR for minor allele quantification enhances sensitivity and precision for the patient after HSCT and SOT. The assay is designed for maximum flexibility. It is expected to be suitable for other applications (sample tracking, prenatal diagnostics, etc.).

New methods for the quantification of mixed chimerism in transplantation.

Background: Quantification of chimerism showing the proportion of the donor in a recipient is essential for the follow-up of hematopoietic stem cell transplantation but can also be useful to document an immune tolerance situation after solid organ transplantation. Historically, chimerism has been quantified from genomic DNA, but with technological advances, chimerism from donor-derived cell-free DNA seems particularly relevant in solid organ transplantation. Methods: The reference method was until recently the short tandem repeat technique, but new innovative techniques as digital PCR (dPCR) and NGS, have revolutionized the quantification of chimerism, such as the so-called microchimerism analysis. After a short review of chimerism methods, a comparison of chimerism quantification data for two new digital PCR systems (QIAcuity™ dPCR (Qiagen®) and QuantStudio Absolute Q (ThermoFisher®) and two NGS-based chimerism quantification methods (AlloSeq HCT™ (CareDx®) and NGStrack™ (GenDX®)) was performed. Results: These new methods were correlated and concordant to routinely methods (r²=0.9978 and r²=0.9974 for dPCR methods, r²=0.9978 and r²=0.9988 for NGS methods), and had similar high performance (sensitivity, reproductibility, linearity). Conclusion: Finally, the choice of the innovative method of chimerism within the laboratory does not depend on the analytical performances because they are similar but mainly on the amount of activity and the access to instruments and computer services.

Evaluation of a quantitative PCR-based method for chimerism analysis of Japanese donor/recipient pairs.

Chimerism analysis is a surrogate indicator of graft rejection or relapse after allogeneic hematopoietic stem cell transplantation (HSCT). Although short tandem repeat PCR (STR-PCR) is the usual method, limited sensitivity and technical variability are matters of concern. Quantitative PCR-based methods to detect single nucleotide polymorphisms (SNP-qPCR) are more sensitive, but their informativity and quantitative accuracy are highly variable. For accurate and sensitive chimerism analysis, a set of KMR kits (GenDx, Utrecht, Netherlands), based on detection of insertions/deletions (indels) by qPCR, have been developed. Here, we investigated informativity and validated the accuracy of KMR kits in Japanese donor/recipient pairs and virtual samples of DNA mixtures representative of Japanese genetic diversity. We found that at least one recipient-specific marker among 39 KMR-kit markers was informative in all of 65 Japanese donor/recipient pairs. Moreover, the percentage of recipient chimerism estimated by KMRtrack correlated well with ratios of mixed DNA in virtual samples and with the percentage of chimerism in HSCT recipients estimated by STR-PCR/in-house SNP-qPCR. Moreover, KMRtrack showed better sensitivity with high specificity when compared to STR-PCR to detect recipient chimerism. Chimerism analysis with KMR kits can be a standardized, sensitive, and highly informative method to evaluate the graft status of HSCT recipients.

Molecular Monitoring of Allogeneic Stem Cell Transplantation in Fanconi Anemia.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT) is the treatment of choice in patients with Fanconi anemia (FA). The aim of our study is to evaluate the impact and benefits of allogenic matched donor HSCT in a case of a 12 year-old girl with FA, who displayed good clinical evolution following 2 months post-transplantation. METHODS: In the pre-transplant phase, reference blood samples from the donor and recipient were collected on EDTA. The DNA from blood samples was extracted using an automated Maxwell® 48 RSC instrument (Promega, USA) with the Maxwell® RSC Whole blood DNA kit (Promega, USA). For DNA quantification, the PowerQuant System kit (Promega, USA) was used with the ABI 7500 Real-time PCR system (Applied Biosystems, USA). The amplification of the short tandem repeat markers was performed using the 24plex Investigator QS kit (Qiagen, Germany) on a ProFlex PCR System. Furthermore, the PCR products were separated and detected on an ABI 3500 Genetic Analyzer (Applied Biosytems, USA). RESULTS: Thirty days post transplantation, a complete chimerism (CC) was achieved with a full replacement by do-nor derived hematopoietic cells. Sixty days post transplantation, the CC status was maintained with improvement of hematological findings. CONCLUSIONS: In FA, chimerism monitoring after HSCT provides useful information regarding engraftment or possibility of post-transplantation complications such as graft versus host disease.

[Strategy Optimization and Clinical Analyze of Multiple Nucleotide Polymorphism Analysis in the Chimerism Detection after Allogeneic Hematopoietic Stem Cell Transplantation].

AbstractObjective: To investigate the sample selection, result correction and clinical application value of multi nucleotide polymorphism chimerism detection method based on Next-generation sequencing. METHODS: The chimerism samples from November 2018 to June 2020 were collected, and Pearson correlation coefficient (r) was used to analyze the consistency of bone marrow and peripheral blood results detected by MNPseq; according to the different information integrity before transplantation, the calibration model was constructed to analyze the correction value of the micro chimerism results in each model; the clinical results were retrospectively analyzed to verify the reliability and practicability of chimerism results correction and the clinical value of MNPseq method. RESULTS: The results of bone marrow and peripheral blood chimerism detected by MNPseq method were consistent with each other and showed significant correlation (r=0.985, P<0.01). The three groups of calibration models were constructed according to different pre-transplant information. For the no donor and pre-transplant patients information group, the correction value was 1%; while for the group with pre-transplant patients and without donor information, 0.61% of the chimerism rate and 13 heterotopic points were used as the correction value; 0.26% of the chimerism rate and 21.57% of the heterotopic points were used as the correction value for the group with pre-transplantation patients and donor information. After correction, the number of the patients with incomplete chimerism decreased from 276 (74.19%) to 141 (37.91%) (P<0.01). Among 18 (18/141, 12.77%) patients with incomplete chimerism, the results of MNPseq in the patients were 25-39 days earlier than those in STR and flow MRD, and the result showed statistical significance. CONCLUSION: MNPseq method can be used to monitor chimerism with peripheral blood instead of bone marrow samples, and the results can be corrected to detect the changes of graft status in vivo in a more timely manner.

Strategy Optimization and Clinical Analyze of Multiple Nucleotide Polymorphism Analysis in the Chimerism Detection after Allogeneic Hematopoietic Stem Cell Transplantation / 中国实验血液学杂志

AbstractObjective: To investigate the sample selection, result correction and clinical application value of multi nucleotide polymorphism chimerism detection method based on Next-generation sequencing.@*METHODS@#The chimerism samples from November 2018 to June 2020 were collected, and Pearson correlation coefficient (r) was used to analyze the consistency of bone marrow and peripheral blood results detected by MNPseq; according to the different information integrity before transplantation, the calibration model was constructed to analyze the correction value of the micro chimerism results in each model; the clinical results were retrospectively analyzed to verify the reliability and practicability of chimerism results correction and the clinical value of MNPseq method.@*RESULTS@#The results of bone marrow and peripheral blood chimerism detected by MNPseq method were consistent with each other and showed significant correlation (r=0.985, P<0.01). The three groups of calibration models were constructed according to different pre-transplant information. For the no donor and pre-transplant patients information group, the correction value was 1%; while for the group with pre-transplant patients and without donor information, 0.61% of the chimerism rate and 13 heterotopic points were used as the correction value; 0.26% of the chimerism rate and 21.57% of the heterotopic points were used as the correction value for the group with pre-transplantation patients and donor information. After correction, the number of the patients with incomplete chimerism decreased from 276 (74.19%) to 141 (37.91%) (P<0.01). Among 18 (18/141, 12.77%) patients with incomplete chimerism, the results of MNPseq in the patients were 25-39 days earlier than those in STR and flow MRD, and the result showed statistical significance.@*CONCLUSION@#MNPseq method can be used to monitor chimerism with peripheral blood instead of bone marrow samples, and the results can be corrected to detect the changes of graft status in vivo in a more timely manner.

Fumarylacetoacetate hydrolase gene as a knockout target for hepatic chimerism and donor liver production.

A reliable source of human hepatocytes and transplantable livers is needed. Interspecies embryo complementation, which involves implanting donor human stem cells into early morula/blastocyst stage animal embryos, is an emerging solution to the shortage of transplantable livers. We review proposed mutations in the recipient embryo to disable hepatogenesis, and discuss the advantages of using fumarylacetoacetate hydrolase knockouts and other genetic modifications to disable hepatogenesis. Interspecies blastocyst complementation using porcine recipients for primate donors has been achieved, although percentages of chimerism remain persistently low. Recent investigation into the dynamic transcriptomes of pigs and primates have created new opportunities to intimately match the stage of developing animal embryos with one of the many varieties of human induced pluripotent stem cell. We discuss techniques for decreasing donor cell apoptosis, targeting donor tissue to endodermal structures to avoid neural or germline chimerism, and decreasing the immunogenicity of chimeric organs by generating donor endothelium.

A practical guide to chimerism analysis: Review of the literature and testing practices worldwide.

BACKGROUND AND PURPOSE: Currently there are no widely accepted guidelines for chimerism analysis testing in hematopoietic cell transplantation (HCT) patients. The objective of this review is to provide a practical guide to address key aspects of performing and utilizing chimerism testing results. In developing this guide, we conducted a survey of testing practices among laboratories that are accredited for performing engraftment monitoring/chimerism analysis by either the American Society for Histocompatibility & Immunogenetics (ASHI) and/or the European Federation of Immunogenetics (EFI). We interpreted the survey results in the light of pertinent literature as well as the experience in the laboratories of the authors. RECENT DEVELOPMENTS: In recent years there has been significant advances in high throughput molecular methods such as next generation sequencing (NGS) as well as growing access to these technologies in histocompatibility and immunogenetics laboratories. These methods have the potential to improve the performance of chimerism testing in terms of sensitivity, availability of informative genetic markers that distinguish donors from recipients as well as cost. SUMMARY: The results of the survey revealed a great deal of heterogeneity in chimerism testing practices among participating laboratories. The most consistent response indicated monitoring of engraftment within the first 30 days. These responses are reflective of published literature. Additional clinical indications included early detection of impending relapse as well as identification of cases of HLA-loss relapse.

A one-step assay for sorted CD3+ cell purity and chimerism after hematopoietic stem cell transplantation.

A hematopoietic chimerism assay is the laboratory test for monitoring engraftment and quantifying the proportions of donor and recipient cells after hematopoietic stem cell transplantation recipients. Flow cytometry is the reference method for determining the purity of CD3+ cells on the chimerism of selected CD3+ cells. In the present study, we developed a single-step procedure that combines the CD3+ purity assay (using the PCR-based Non-T Genomic Detection Kit from Accumol, Calgary, Canada) and the qPCR chimerism monitoring assay (the QTRACE qPCR assay from Jeta Molecular, Utrecht, the Netherlands). First, for the CD3+ purity assay, we used a PCR-friendly protocol by changing the composition of the ready-to-use reaction tubes (buffer and taq polymerase) and obtained a satisfactory calibration plot (R2 = 0.8924) with a DNA reference scale of 2 ng/µl. Next, 29 samples (before and after CD3 positive selection) were analyzed, the mean cell purity was, respectively, 19.6% ± 6.45 and 98.9% ± 1.07 in the flow cytometry assay; 26.8% ± 7.63 and 98.5% ± 1.79 in the PCR-based non-T genomic detection assay. Our results showed that the CD3+ purity assay using a qPCR kit is a robust alternative to the flow cytometry assay and is associated with time savings when combined with a qPCR chimerism assay.

A chimeric mouse model to study human iPSC-derived neurons: the case of a truncating SHANK3 mutation.

Using human induced pluripotent stem cells (iPSC), recent studies have shown that the events underlying autism spectrum disorders (ASD) can occur during neonatal development. We previously analyzed the iPSC-derived pyramidal cortical neurons of a subset of patients with ASD carrying de novo heterozygous mutations in postsynaptic SHANK3 protein, in culture. We reported altered spinogenesis of those neurons. The transplantation of human iPSC-derived neuronal precursors into mouse brain represents a novel option for in vivo analysis of mutations affecting the human brain. In this study, we transplanted the neuronal precursor cells (NPC) into the cortex of newborn mice to analyze their integration and maturation at early stages of development and studied axonal projections of transplanted human neurons into adult mouse brain. We then co-transplanted NPC from a control individual and from a patient carrying a de novo heterozygous SHANK3 mutation. We observed a reduction in cell soma size of selective neuronal categories and in axonal projections at 30 days post-transplantation. In contrast to previous in vitro studies, we did not observe any alteration in spinogenesis at this early age. The humanized chimeric mouse models offer the means to analyze ASD-associated mutations further and provide the opportunity to visualize phenotypes in vivo.

Unexpected Short-Tandem-Repeat Patterns in Posttransplant Chimerism Testing: Investigation of 3 Cases with Help from Forensic Science.

Chimerism testing by short tandem repeats (STRs) is used to monitor engraftment after allogeneic hematopoietic stem cell transplantation (HSCT). Generally, STR alleles are stable and transferred from parent to child or from donor to recipient. However, 3 cases did not follow this norm. Additional work-up with help from forensic literature solved these mysteries. In case 1, the patient received HSCT from his son. The son shared STR alleles in 22/23 loci except Penta E, which was explained by repeat expansion in the son. In case 2, the patient had been in remission for 14 years after HSCT for lymphoma and developed repeat expansion in CSF1PO in granulocytes. In case 3, a pre-HSCT patient demonstrated 3 alleles, with 2 peaks taller than the third, in the FGA locus (chromosome 4). A combination of a triallelic variant and leukemia-associated trisomy 4 explained the finding. STR number variants are rare and clinically inconsequential but can overlap malignancy-associated, clinically significant changes.

Detection and Monitoring of Lineage-Specific Chimerism by Digital Droplet PCR-Based Testing of Deletion/Insertion Polymorphisms.

Analysis of specific leukocyte subsets for post-transplantation monitoring of chimerism provides greater sensitivity and clinical informativeness on dynamic changes in donor- and recipient-derived cells. Limitations of the most commonly used approach to chimerism testing relying on PCR-based analysis of microsatellite markers prompted us to assess the applicability of digital droplet (dd) PCR amplification of deletion/insertion polymorphisms (DIPs) for lineage-specific chimerism testing in the related stem cell transplantation setting, where the identification of informative markers facilitating the discrimination between donor-derived and recipient-derived cells can be challenging. We analyzed 100 genetically related patient-donor pairs by ddPCR analysis using commercially available DIP kits including large sets of polymorphic markers. At least 1 informative marker was identified in all related pairs analyzed, and 2 or more discriminating markers were detected in the majority (82%) of instances. The achievable detection limit is dependent on the number of cells available for analysis and was as low as 0.1% in the presence of ≥20,000 leukocytes available for DNA extraction. Moreover, the reproducibility and accuracy of quantitative chimerism analysis compared favorably to highly optimized microsatellite assays. Thus, the use of ddPCR-based analysis of DIP markers is an attractive approach to lineage-specific monitoring of chimerism in any allogeneic transplantation setting.

Predicting Outcomes of Rat Vascularized Composite Allotransplants through Quantitative Measurement of Chimerism with PCR-Amplified Short Tandem Repeat.

Chimerism has been associated with the induction and maintenance of tolerance to vascularized composite allotransplants (VCA). Although most VCA studies have examined chimerism using flow cytometry, we proposed that precision in the measurement of chimerism may be better approximated when complimentary polymerase chain reaction (PCR) is applied to a specific short tandem repeat (STR). We identified a STR, D10Rat25, which exhibited a ~20 bp difference in length between two rat strains (BN and LEW) often utilized as the donor and recipient in many allotransplantation studies. D10Rat25 was PCR-amplified and quantified with capillary electrophoresis. With pure LEW and BN DNA, a standard curve was constructed to measure chimerism with good linearity. When applied to rat VCA, the relationship between systematic (in peripheral blood) or local (at specific organ/tissues) chimerism to allograft outcomes was noted. We found that peripheral chimerism was elevated by up to ~9% postoperative month 1 (POM 1) but then reduced regardless of the final VCA outcome. However, differences in VCA skin chimerism between early rejection and POM 1 (shown as ΔChimerismPOM1-ER) were notable with respect to VCA outcomes. ROC analysis identified the optimum cutoff value as 17.7%. In summary, we have developed a reliable method to quantify the percentage of BN cells/DNA in BN-LEW chimeras. The detection limit was characterized, and the acquired data were comparable with flow cytometry. This method can be applied to solid organ and composite tissue allotransplantation studies.

NF-κB Duplications in the Promoter-Variant HIV-1C LTR Impact Inflammation Without Altering Viral Replication in the Context of Simian Human Immunodeficiency Viruses and Opioid-Exposure.

Recent spread of the promoter variant (4-κB) Human immunodeficiency virus-1 clade C (HIV-1C) strain is attributed to duplication of the Nuclear Factor Kappa B (NF-κB) binding sites and potential increased heroin consumption in India. To study the underlying biology of 4-κB HIV-1C in rhesus macaques, we engineered a promoter-chimera variant (4NF-κB) Simian Human Immunodeficiency Virus (SHIV) by substituting the HIV-1C Long Terminal Repeat (LTR) region consisting of 4 NF-κB and 3 Sp-1 sites with the corresponding segment in the LTR of SHIV AD8EO. The wild-type (3NF-κB) promoter-chimera SHIV was generated by inactivating the 5' proximal NF-κB binding site in SHIV 4NF-κB. CD8-depleted rhesus macaque PBMCs (RM-PBMCs) were infected with the promoter-chimera and AD8EO SHIVs to determine the effects of opioid-exposure on inflammation, NF-κB activation, neurotoxicity in neuronal cells and viral replication. Morphine-exposure of RM-PBMCs infected with SHIVs 4NF-κB, 3NF-κB, and AD8EO altered cellular transcript levels of monocyte chemoattractant protein 1, interleukin 6, interleukin 1ß, and Tumor Necrosis Factor α. Of note, divergent alteration of the cytokine transcript levels was observed with these promoter-chimera wild-type and variant SHIVs. NF-κB activation was observed during infection of all three SHIVs with morphine-exposure. Finally, we observed that SHIV AD8EO infection and exposure to both morphine and naloxone had the greatest impact on the neurotoxicity. The promoter-chimera SHIV 4NF-κB and SHIV 3NF-κB did not have a similar effect on neurotoxicity as compared to SHIV AD8EO. All SHIVs replicated efficiently at comparable levels in RM-PBMCs and morphine-exposure did not alter viral replication kinetics. Future in vivo studies in rhesus macaques will provide greater understanding of 4-κB HIV-1C viral immunopathogenesis and onset of disease in the central nervous system during morphine-exposure.

Treatment of primary immunodeficiency with allogeneic transplant and gene therapy.

The treatment of primary immunodeficiency disorders with allogeneic hematopoietic cell transplantation (HCT) has a history dating back to 1968 with the first successful transplant for a patient with severe combined immunodeficiency (SCID). The omission of conditioning for patients with SCID owing to their inability to reject allogeneic grafts and the increasing use of reduced intensity conditioning regimens often result in a state of mixed or split donor-recipient chimerism. The use of gene therapy (GT) via retroviral or lentiviral transduction of autologous CD34+ hematopoietic stem and progenitor cells is expected to correct only a portion of the hematopoietic stem cell compartment. The consequences of partial correction after either form of cellular therapy differ according to how the genetic deficiency affects immune cell development and function. Moreover, the conditioning regimen or lack thereof impacts the cell lineages at risk of partial correction. Advances in our understanding of immune reconstitution after HCT and GT for SCID, Wiskott-Aldrich syndrome, and chronic granulomatous disease are discussed.

Quantitative polymerase chain reaction technology in chimerism monitoring after hematopoietic stem cell transplantation: One center experience.

Chimerism status evaluation is a routine test performed in post-hematopoietic stem cell transplantation (HSCT) period. The aim of the study was to evaluate a quantitative polymerase chain reaction (qPCR) method (GenDx, Utrecht, the Netherlands) applicability for this purpose. The study included 74 recipient/donor pairs tested for informative markers: median of four and six informative markers was found for patients (related and unrelated donor, respectively). Higher sensitivity of qPCR method was confirmed by analysis of recipient post-HSCT samples (N = 800) among which microchimerism (0.1%-1% recipient DNA) was detected in 21.8% of cases. The ability to detect less than 1% of minor population, as opposed to the short tandem repeat (STR) method for which 1% is the limit, translated into earlier identification of a disease relapse for four patients in our study sample.