**4.3 Methods**

Whole peripheral blood samples were collected for DNA extraction from both the donor and recipient before transplantation in order to determine an informative marker for two molecular methods mentioned previously. The blood samples of leukaemia patients (AML, ALL and CML) after allo-hSCT (N = 65 pairs) were collected at regular time periods (before allo-hSCT and 30 days, 100 days, 6-month, 1 year, 2 years and 3 years after allo-hSCT) at the Department of Haematology and Transfusion, Comenius University Medical School Bratislava. Isolation of the DNA and chimerism testing was provided in two diagnostic laboratories: Laboratory Diagnostics Medirex, Bratislava and Bone Marrow Transplantation Unit, Bratislava, Slovak republic, EU. RQ-PCR was performed by the real-time PCR system using SYBR green and 12 pairs of specific primers for two allelic variants of DNA polymorphism and GAPDH as endogenous gene control. The cT (threshold cycle) values of informative patient's DNA markers before allo-hSCT were used as calibrator for the relative quantification. The PCR reaction mixtures and DNA were placed on 96-well reaction plates in duplicates separately, not as multiplex amplification set. Relative quantification was evaluated by gene expression software study [40]. The PCR-STR analysis was performed using commercially available STR multiplex amplification kits with fluorescently labelled PCR primers. The quantification of donor and recipient DNA marker's signal was provided by comparing the fluorescence intensity given by the peak area of analysed fragments [20].

#### **4.4 Results**

We screened 65 related and unrelated donor/recipient pairs by both molecular methods, and we found at least one informative marker for each laboratory approach mentioned above. The parallel quantifications of DNA (two different informative DNA marker sets used) were provided by both methods, and the estimated chimerism was compared at the same time period after the allogeneic haematopoietic stem cell transplantation. We found that our results were identical only in 2% and the discrepancy was noticed also in 2% between the two methods

*Monitoring of Chimerism in Rare Haematological Malignant Diseases after Allogeneic… DOI: http://dx.doi.org/10.5772/intechopen.89845*

used. In the case of 1–50% mixed chimerism (MC), similar results were obtained. However, complete chimerism (CC) estimated by the fragment analysis was evaluated as mixed chimerism (MC) by the real-time PCR in 94% patients, mainly in the first half of a year of the post-transplantation monitoring. The example of the parallel monitoring of one patient is shown in **Figure 8**. From this presented results of patient chimerism monitoring, we can see that 30 days after allo-hSCT mixed chimerism was detected by both methods used (FA-MC and RQ PCR-MC). Also 1, 2 and 3 years after allo-hSCT, both methods showed identical complete chimerism results (FA-CC and RQ PCR-CC). But the discrepancy was revealed 100 days after allo-hSCT where complete chimerism was detected by the method FA (FA-CC), but simultaneously mixed chimerism was detected by RQ PCR method (RQ PCR-MC) in the same patient sample.

#### **4.5 Conclusion**

The parallel chimerism monitoring of post allo-hSCT leukaemia patients was provided for 3 years. Discrepancy between complete chimerism (CC) detected by the fragment analysis (FA) of PCR-STR DNA markers and mixed chimerism (MC) detected by the real-time PCR of SNP and NP DNA markers was due to the different sensitivity of two methods used. It is also important to note that in different molecular diagnostic approaches also, two different DNA marker sets were used. RQ PCR had the higher sensitivity (<1%) for the detection of the autologous DNA markers than FA (1–5%), so it is better for earlier revealing of eventual relapse. On the other hand, the quantification of donor's DNA markers is more precise estimated by the FA. Both methods compared above are suitable for chimerism assessment after

#### **Figure 8.**

*Parallel monitoring of chimerism of one leukaemia patient after haematopoietic allogeneic stem cell transplantation (allo-hSCT) by two DNA molecular methods in different time periods. FA, analysis of PCR-STR DNA markers by fragment analysis following capillary electrophoresis on Genetic analyser; RQ-PCR, analysis of SNP and NPs DNA markers by relative quantification of SYBR green-based Real-time PCR; CC, complete chimerism (only donor's DNA markers detected in patient); MC, mixed chimerism (recipient's -autologous and donor's DNA markers detected in patient). 100 days after allo-SCT complete chimerism was detected by the method FA (FA-CC), but simultaneously mixed chimerism was detected by RQ PCR method (RQ PCR-MC) due to its different sensitivity.*

the allogeneic haematopoietic stem cell transplantation, although nowadays many new kits with different DNA marker sets (STR, SNP, NPs and DIPs) for chimerism monitoring are available.
