3.3 Posttransplant monitoring

The monitoring of cell chimerism consists of analyzing 2–3 selected informative polymorphisms. The frequency of monitoring after graft transfer is performed under our standard days (D) +14, +21, and +28 after allo-HSCT in both adult and pediatric patients. Subsequently, in pediatric patients, the intervals of examination are every 2–3 weeks up to D + 180, once a month to the first year after allo-HSCT, later every 2–4 months up to 3 years after allo-HSCT, and every 6–12 months up to 5 years after allo-HSCT according to the dates of outpatient's controls. In adult patients, examinations from the second month after allo-HSCT are carried out in monthly intervals up to 2 years and, throughout the next period, at least every 6 months. The frequency of examinations depends on the patient's medical condition, diagnosis, the dynamics of their chimerism status, and especially the physician's decision. In cases of increasing microchimerism or MC detection after the previous period of CC, an intensive investigation scheme is recommended due to the risk of graft rejection or relapse of the primary disease.

In the first samples after allo-HSCT, the detection of MC can be expected. If the recipient's genotype fraction falls below 50%, we can interpret this as the so-called engraftment of the donor's cells. The MC gradually decreases until the patient reaches CC. The median achievement of CC is most often D + 21 or D + 28 after allo-HSCT and depends on the patient's diagnosis and many other factors such as the regimen of allo-HSCT or the quality of the graft.

The choice of method used depends on the patient's actual chimerism status. Due to the high sensitivity of the RQ-PCR method, in combination with STR analysis, it is advisable to use RQ-PCR for the monitoring of patients in cases where CC, microchimerism, or MC up to 10% has been detected. On the other hand, with a rising trend of MC, it is better to use only STR analysis for quantification. For more accurate determination, it is always necessary to analyze the informative polymorphisms that detect a minority genotype. This means that in cases where the MC increases over 50% of the recipient's genotype, it is preferable to select donorspecific polymorphisms for quantification.

#### 3.4 The importance of microchimerism

The introduction of the RQ-PCR method for the monitoring of cell chimerism as a part of routine examination has improved significantly the sensitivity of the assessment. Its high sensitivity of 0.035% allows for a much earlier detection of relapses than conventional methods (VNTR and STR). The importance of microchimerism detection was confirmed by us and many other studies [27–30], and we would like to present our retrospective data below.

## 3.4.1 Patients and methods

A group of 224 patients, from HLA-identical-related and HLA-identicalunrelated donors, who underwent allo-HSCT between 2011 and 2015 at the Institute of Hematology and Blood Transfusion, were enrolled in this study. Patients with early HSCT-associated mortality (less than 14 days), another allo-HSCT before the third year, with no RQ-PCR analyses or with a loss of follow-up were excluded. In total, 207 patients were eligible for analysis of cell chimerism dynamics. The test group was divided into 3 subgroups according to chimerism status 3 years after allo-HSCT: patients with CC (137), patients with microchimerism (38), and patients with MC (32). The patients' characteristics are listed in Table 3.


Abbreviations: AML, acute myeloid leukemia; CML, chronic myeloid leukemia; MDS, myelodysplastic syndrome; ALL and LBL, acute lymphoblastic leukemia and lymphoblastic lymphoma; MDS/MPS, myelodysplastic/ myeloproliferative neoplasms; CLL, SLL, PLL, chronic lymphoblastic leukemia, small lymphocytic lymphoma, prolymphocytic leukemia; PBPC, peripheral blood progenitor cells; BM, bone marrow.

#### Table 3.

Patient characteristics.

DNA from whole peripheral blood samples was isolated by means of a saltingout procedure [31] and diluted to a final concentration of 50 ng/μL. The combination of InDels by RQ-PCR, in conjunction with STR analysis by fragment analysis, was used to determine the chimerism status. Fragment analysis of the resulting PCR products was performed on an automated 3500 Series Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA), and data were processed by GeneMapper v5 software (Thermo Fisher Scientific). Quantification of MC was determined using the peak areas representing specific alleles. InDel analysis was performed by means of TaqMan technology. Rotor-Gene machine (Corbett Life Science, Sydney, New

#### Suitable Molecular Genetic Methods for the Monitoring of Cell Chimerism DOI: http://dx.doi.org/10.5772/intechopen.88436

South Wales) and Rotor-Gene 6 software were used for evaluation. The percentage of microchimerism was calculated by the ΔΔCT method. Data were normalized with the glyceraldehyde-3-phosphate dehydrogenase as a reference gene.

The impact of chimerism status on the 3-year overall survival of allo-HSCT patients and 3-year relapse rates was evaluated using GraphPad Prism 7 software (La Jolla, CA, USA). The logrank (Mantel-Cox) test was used for comparison of survival curves.
