Chimerism Status Analysis after Hematopoietic Stem CellTransplantation on Granulocytes and Mononucleated Cells of Peripheral Blood

Background Chimerism Status (CS) analysis is largely useful for evaluation of donor (D) cells engraftment after allogeneic haematopoietic stem cell transplantation (HSCT). The largest performed method is based on the evaluation of the DNA sequences “Short Tandem Repeats” (STR) and Variable Number Tandem Repeats” (VNTR). Their high polymorphism permits to differentiate donor (D) or recipient (R) origin of the analysed cells. PCR amplification and DNA sequencing permit to quantify D and R amount.

Patients and methods CS analysis after HSCT can be conducted on different cell populations in marrow or peripheral blood: whole blood cells, mononucleated cells, or single separated populations. A complete CS (CCS) usually correlates with a favourable outcome of HSCT, conversely a mixed CS may predicts a relapse of disease or a rejection of HSCT, particularly in the condition defined as increasing-mixed CS (IMCS), in which the amount of R origin cells increases along the time. In our study we employed a simple and easily reproducible method to analyse CS on peripheral blood cells at +30, +60, +90 and +120 days after HSCT. CS analysis was conducted separately on granulocytes and mononucleated cells, obtained after double gradient centrifugation. A semi-quantitative method, based on multiplex PCR amplification of 16 STR markers using a commercial kit (PowerPlex® 16 System–Promega), was applied to define the difference between D and R. We assumed the CS in granulocyte population as expression of myeloid engraftment. We also correlated the CS in mononucleated population, with the different leukocyte subpopulations (lymphocytes, monocytes) in peripheral blood count and with the reconstitution of lymphocyte subpopulations (B, T cells and NK cells) at the same time point of analysis, in order to compare the reciprocal course.

We evaluated 13 HSCT on 12 patients (pts) (1 pts was submitted to double HSCT for a rejection occurred after the first HSCT), 7 from sibling related donor (SRD) and 6 from matched unrelated donor (MUD). All the pts were affected by neoplastic disease in first (9) or next (3) remission: acute lymphoblastic (5) or myeloblastic leukaemia (4), mielodisplasia (1), chronic myeloid leukaemia (1), multiple myeloma (1). All the conditioning regimens were mieloablative, all the MUD-HSCT and one of the SRD-HSCT received Anti-Thymocyte-Globulin (ATG), 7/8 SRD-HSCT did not received ATG nor other T-depletion procedures.

Results: Evaluation on time + 30, +60, +90 and +120 days after HSCT was performed on 13 HSCT, except in two pts who stopped before (+30 and +60) due to their death for relapse or toxicity. A stable CCS in granulocytes was observed in 10/13 HSCT since the first control. 3/13 HSCT presented IMCS followed by relapse (2 pts) or rejection. In mononucleated cells population 10/13 HSCT presented CCS at the first control, 2/10 evolved to CCS in the successive times. The 3 pts who relapsed or rejected presented a IMCS in mononucleated cells too. Considering the different leucocyte populations we observed that the mononucleate cells amount was constituted for only 50% from lymphocytes, the remaining 50% was represented from monocytes. Therefore the analysis of lymphocyte subpopulations showed that T-lymphocytes were virtually absent (either T4 or T8) till to +120 in pts who received ATG, so in these ones CS analysis in mononucleated cells was performed only in monocytes, B lymphocytes and NK cells. Instead pts who did not received ATG presented complete and more rapid reconstitution of all the lymphocyte subpopulations (median at +60 day: T4 200, T8 120, B 60, NK160 × 10^9/l).

Conclusions: our method is simple and rapid in analysing CS. Granulocytes CS reflect the engraftment of myeloid lineage. Instead CS in mononucleated cells must be considered in the context of the type of transplantation (MUD or SRD, T-depletion or T repletion) and correlated with the single subpopulations of peripheral blood.