3. Standard procedures for the monitoring of cell chimerism at the Institute of Hematology and Blood Transfusion

#### 3.1 Informativity determination

The informativity determination always precedes the monitoring of cell chimerism. Recipient and donor DNAs are tested by a panel of highly polymorphic STRs and InDels by multiplex kits. VNTRs and SNPs are not used in our laboratory. Currently, the PowerPlex 16HS System (Promega, Madison, WI, USA) kit is used routinely for STR analysis; the kit contains 13 basic Combined DNA Index System (CODIS) core STR loci [24], sex-specific locus amelogenin, and 2 other pentanucleotide repeat polymorphisms Penta D and Penta E. For the genotyping of deletion–insertion polymorphisms (DIP), a Mentype DIPscreen (Biotype, Dresden, DE) kit is used that amplifies 33 DIP loci and amelogenin. A comparison of the donor's and the recipient's DNA profiles allows to select the specific informative markers suitable for the monitoring of cell chimerism during the posttransplantation course. We choose at least two informative polymorphisms located at different chromosomes specific for the recipient. It is necessary to take into account the potential cytogenetic changes that are associated with different types of cancer (such as genome instability, loss of heterozygosity, and chromosomal changes) [25, 26]. Only informative recipient alleles by at least n � 2 repeats outside stutter region (preferably by 2–4 longer) are used for calculation. An artifact of PCR, the so-called DNA stutter, is a result of strand slippage during DNA synthesis, showing up primarily one repeat before and, less frequently, one repeat after the true allele. The result of quantification is that such an allele would lead to an incorrect interpretation. It is always appropriate to quantify the minor genotype; therefore, in the case of graft rejection or graft failure (the donor's cells are present in <50%), we choose two informative polymorphisms specific for donor in the same way as the recipient.

### 3.2 Interpretation of chimerism status

Chimerism is a dynamic process, so the proportion of autologous cells after allo-HSCT can change during monitoring. It is therefore necessary to approach each patient individually and to select appropriate methods for quantification.

An overview and definition of the chimerism status are given in Table 2. Under optimal conditions, we can detect only the donor's genotype after allo-HSCT; thus, the recipient's hematopoiesis is completely replaced with the donor's graft. We interpret this as a complete chimerism (CC). In our laboratory the CC is detected by RQ-PCR, and, based on clinical validation, the significant detection limit was defined as ≤0.035% of the recipient's genotype (due to Monte Carlo effect as mentioned in point 2.2.3). The detection of recipient's genotype of less than 1% is interpreted as a microchimerism (range from ≥0.036 to <1%), and the presence of more than or equal 1% is interpreted as mixed chimerism (MC). The percentages of the individual alleles are then quantified by STR analysis. If we detect only the recipient's genotype or the donor's genotype less than 0.035%, transplant rejection and a complete recovery of the original hematopoiesis have occurred. Split chimerism can be seen in the analysis of cell fractions. This means that MC is detected in a certain leukocyte line, but in another cell line, it is CC. The analysis of cell subpopulations makes it possible to distinguish between residual malignant cells and nonmalignant hematopoiesis. More often, the fraction of monocytes, granulocytes, natural killer (NK) cells, T lymphocytes, and B lymphocytes is examined. The analysis of chimerism of T lymphocytes and NK cells can help, especially, as a guide to additional therapy in order to avoid graft rejection. The analysis of chimerism in cell fractions is particularly important for patients with a reduced intensity regimen before allo-HSCT or patients with autoimmune disease.

The interpretation of bone marrow samples is more difficult. Microchimerism is often detected as a result of the contamination of the primary sample by bone marrow stromal cells of the recipient. Therefore, microchimerism below 0.5% of the recipient's genotype is considered to be insignificant. The proportion of autologous hematopoiesis in the bone marrow can fluctuate over time, especially in the early period after allo-HSCT. In making a clinical decision, it is more important to watch the dynamics of chimerism and take into account the patient's diagnosis rather than the individual values of microchimerism or MC.


#### Table 2. Interpretation of chimerism status.
