Follow-up of chimerism status after allogeneic HLA-mismatched stem cell transplantation by detection of non-shared HLA alleles.
OBJECTIVE
Chimerism studies after allogeneic transplantation are usually performed using cytogenetic analysis, PCR-VNTR or PCR-STR. Here, we report an alternative method for following the chimerism status after an HLA-mismatched stem cell transplantation (SCT), detecting the presence of non-shared HLA alleles by reference-strand mediated conformation analysis (RSCA).


METHODS
We tested this new approach on allogeneic related haploidentical SCT, unrelated cord blood transplantation, and HLA-mismatched unrelated donor SCT. The quantification of the chimerism was performed by laser detection of fluorescent-labeled primers on an automated DNA sequencer.


RESULTS
In all cases this technique was able to detect mixed chimeras. The technique detected above 5% of residual cells when the analysis was based on HLA-class I and above 3% for HLA-class II. This sensitivity is similar to that of the PCR-VNTR analysis.


CONCLUSIONS
This method avoids the need to search for an informative locus (which is essential for PCR-VNTR or -STR). Moreover, we did not find the phenomenon of preferential amplification that is observed with most VNTR, thus avoiding the need for construction of standard curves to quantify mixed chimeras. We conclude that the detection of the non-shared HLA alleles by RSCA is a useful approach for chimerism follow-up after HLA-mismatched SCT.
Gallardo
D
D
Servicio de Hematología Clínica, Hospital Durán i Reynals, Avda. Gran Vía s/n, Km 2.7, 08907 L'Hospitalet, Barcelona, Spain. 27532dgg@comb.es
Rodríguez-Luaces
M
M
Aróstegui
J I
JI
Ferrá
C
C
Berlanga
J J
JJ
Querol
S
S
García-López
J
J
Grañena
A
A
D000483:Alleles; D004591:Electrophoresis, Polyacrylamide Gel; D005500:Follow-Up Studies; D005805:Genes, MHC Class I; D005802:Genes, MHC Class II; D006085:Graft Survival; D006680:HLA Antigens / Q000032:analysis*; D018380:Hematopoietic Stem Cell Transplantation / Q000706:statistics & numerical data*; D006648:Histocompatibility; D006801:Humans; D007834:Lasers; D009693:Nucleic Acid Hybridization / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA / Q000295:instrumentation; D013030:Spain; D014019:Tissue Donors; D014184:Transplantation, Homologous / Q000706:statistics & numerical data*; D016896:Treatment Outcome
D006680:HLA Antigens