Standardisation of multiplex fluorescent short tandem repeat analysis for chimerism testing.
To evaluate the origin of cells after allogeneic haematopoietic stem cell transplantation we optimised and evaluated two commercially available systems (AmpFlSTR Profiler Plus and GenePrint Powerplex-16) which are based on multiplex fluorescent short tandem repeat (STR) analysis. A standard procedure for interpretation of electropherographs was found essential to obtain reproducible results. On the basis of the relative length of donor and recipient alleles, TYPE-I (no shared alleles are used to calculate chimerism), TYPE-II (one shared and one unshared allele is used to calculate chimerism) or TYPE-III (not informative) allelic distribution types were distinguished. Also, stutter peaks were recognised as an important criterion to exclude a marker for analysis. Intralaboratory and multicentre evaluation of the AmpFlSTR Profiler Plus system showed that mixed blood samples could be determined with an absolute deviation of less than 2%. A sensitivity threshold was set at 5% for TYPE-I and 10% for TYPE-II markers since relative imprecision increases at low chimerism values. No significant difference of calculated chimerism values was observed between STR markers shared between both systems. By monitoring 26 allogeneic peripheral blood stem cell transplants, the applicability of the proposed method was demonstrated.
Nollet
F
F
Clinic and Laboratory of Haematology, AZ Sint-Jan AV, Ruddershove 10, B-8000 Bruges, Belgium.
Billiet
J
J
Selleslag
D
D
Criel
A
A
D000483:Alleles; D004247:DNA / Q000235:genetics; D017931:DNA Primers / Q000592:standards*; D004586:Electrophoresis / Q000592:standards; D005456:Fluorescent Dyes / Q000592:standards*; D005784:Gene Amplification; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D015588:Observer Variation; D016133:Polymerase Chain Reaction; D012015:Reference Standards; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods* / Q000592:standards*; D020080:Tandem Repeat Sequences / Q000235:genetics*; D018183:Transplantation Chimera / Q000235:genetics*
D017931:DNA Primers; D005456:Fluorescent Dyes; D004247:DNA