A comparison of fluorescent in situ hybridization and multiplex short tandem repeat polymerase chain reaction for quantifying chimerism after stem cell transplantation.
OBJECTIVE
Despite the great utility of chimerism analysis after allogeneic stem cell transplantation, a gold standard method for its quantification has not yet been defined. The objective of the present investigation was to compare the sensitivity (detection limit) and the quantification accuracy of fluorescent in situ hybridization with specific probes for the sex chromosomes (XY-FISH) and multiplex short tandem repeat polymerase chain reaction (STR-PCR) revealed by capillary electrophoresis for the quantification of chimerism after stem cell transplantation.


METHODS
A first experiment was performed on two sets of artificial cell mixtures from two sex-mismatched healthy donors mixed in different proportions (% male: 100, 75, 50, 25, 10, 5, 3, 1, 0.1, 0). In a second experiment, 58 samples obtained from 10 selected patients with different clinical courses and chimerism evolution after sex-mismatched stem cell transplantation, which had been studied by XY-FISH, were retrospectively analyzed by STR-PCR. In a third experiment, 60 unselected prospective samples belonging to 15 patients (5 of whom had also been included in the retrospective study) were analyzed by both XY-FISH and STR-PCR.


RESULTS
Both techniques showed high quantification accuracy and were highly reproducible. The sensitivity of both approaches reached 1% under standard conditions. Moreover, the use of long injection times for the capillary electrophoresis (30 and 50s vs. the standard 10s) resulted in an increase of sensitivity of the STR-PCR assay up to 0.1%, which has interesting clinical implications.


CONCLUSIONS
Considering the high sensitivity and quantification accuracy of multiplex STR-PCR and the fact that this assay is sex-independent and can be applied to virtually all patients, STR-PCR could be considered as the method of choice for chimerism quantification after stem cell transplantation when high sensitivity is not a requirement.
Buño
Ismael
I
BMT Unit, Dep. of Oncology, Hosp. G. U. Gregorio Marañón, Madrid, Spain. ibuno.hgugm@salud.madrid.org
Nava
Paola
P
Simón
Ainhoa
A
González-Rivera
Milagros
M
Jiménez
José Luis
JL
Balsalobre
Pascual
P
Serrano
David
D
Carrión
Rafael
R
Gómez-Pineda
Alfonso
A
Díez-Martín
José Luis
JL
D000293:Adolescent; D000328:Adult; D002675:Child, Preschool; D046528:Chimerism; D005260:Female; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods / Q000592:standards*; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012680:Sensitivity and Specificity; D033581:Stem Cell Transplantation / Q000592:standards*; D020080:Tandem Repeat Sequences / Q000235:genetics*; D014019:Tissue Donors; D018183:Transplantation Chimera / Q000235:genetics