Quantitative Assessment of Hematopoietic Chimerism after Allogeneic Nonmyeloablative Stem Cell Transplantation.

Objective:

1. To establish a method for quantitative analysis of hematopoietic chimerism by polymerase chain reaction (PCR) based on short tandem repeat (STR) locies.

2. To investigate the correlation between the kinetics of chimerism and hematologic engraftment, graft rejection, disease relapse and graft versus host disease (GVHD) after allogeneic nonmyeloablative peripheral blood stem cell transplantation.

3. To guide implementation of therapy at an early stage and to improve patients life quality.

Method:

1. Cell dilution experiments were performed by mixing mononuclear cells (MNCs) obtained from peripheral blood samples of unrelated individuals to test the sensitivity, accuracy and linearity of the assay. Quantitative assessment of hematopoietic chimerism was performed by short tandem repeat-polymerase chain reaction(STR-PCR), polyacrylamide gels, silver staining and analyzed by Image Analysis System.

2. 28 patients received nonmyeloablative stem cell transplantation were evaluated. The conditioning regimen included fludarabine 30mg/(m²·d)×6d, busulphan 4mg/(kg·d)×2d, CTX 600mg·d⁻¹×2d, ±Ara-C.

Peripheral blood were collected before and after transplantation in different period and the chimerism were analysed by this method.

Results:

1. Sensitivity varied from 1.25% to 5% depending on the STR locies being tested. vWA and D16S539 appeared better sensitivity from 1.25% to 2.5% than other locies. The quantitative results showed a linear correlation between the percent chimerism calculated and the DC proportion mixed(R² =0.97~0.98, P<0.001). As an average of all informative markers, concordance between the calculated and mixed DC percent showed minimal deviation from 1(R²=0.996).

2. On day 30, one patient failed to engraft, 22 cases formed complete chimerism (CC) and 5 cases were of mixed chimerism (MC).

The time of molecular engraftment preceded the recovery time of neutropils and platelets. One patient with MC rejected grafts. The incidence of aGVHD in group CC was significantly higher than that in group MC(P<0.05). There was no significant difference in the incidence of cGVHD and disease relapse between group CC and group MC(P>0.05). One patient relapsed in CC status lacking a transitional MC interveral. Patients received an early implementation of therapy transferred from MC to CC or got a longer disease-free time.

Conclusions:

1. STR-PCR is a sensitive, precise and reliable method for quantitative analysis of hematopoietic chimerism. Selecting sensitive markers to analyze is an economic approach for monitoring of chimerism after transplantation.

2. Sequential and quantitative detection of chimerism may be of great value to predict engraftment, graft rejection, disease relapse and high risk of GVHD.

Furthermore it can provide a basis for early intervention of clinical treatment posttransplantation.