Enrichment of cell subpopulations applying automated MACS technique: purity, recovery and applicability for PCR-based chimerism analysis.
Enrichment of cell subpopulations is a prerequisite for lineage-specific chimerism analysis (LCA), a frequent approach in follow-up after allo-SCT. An efficient enrichment technique is Magnetic Cell Sorting (MACS) using the AutoMACS separator. However, evaluation of purity, recovery and applicability for PCR-based chimerism analysis of MACS-enriched subpopulations from post-transplant peripheral blood, providing reduced cell numbers and/or unbalanced proportions of subpopulations, is currently unavailable. We performed enrichment of CD3-, CD14-, CD15-, CD19- and CD56-positive subpopulations using 'Whole Blood MicroBeads' and AutoMACS separator in 137 prospectively collected peripheral blood samples from 15 paediatric patients after allo-CD3-/CD19-depleted SCT. Purity was assessed by immune phenotyping. Recovery and applicability for chimerism analysis was evaluated. Excellent purity >90% was achieved in CD14-, CD15-positive cells in 81%, 95% of the isolates and in 86% of CD3 and CD19 isolates, if ACC was >400 cells per mul. Median purity of CD56-positive isolates was 78.9%. Recovery >90% was between 93 (CD56) and 37% (CD15). Conventional and real-time PCR-based chimerism analysis was feasible in virtually all samples. Isolation of cell subpopulations by automated cell enrichment in post-transplant peripheral blood is feasible and fast providing excellent purity and recovery for routine lineage-specific chimerism analysis.
Willasch
A
A
Department of Pediatric Hematology, Oncology and Hemostaseology, Goethe University Frankfurt, Hospital for Children and Adolescents III, Frankfurt, Germany. andre.willasch@kgu.de
Eing
S
S
Weber
G
G
Kuçi
S
S
Schneider
G
G
Soerensen
J
J
Jarisch
A
A
Rettinger
E
E
Koehl
U
U
Klingebiel
T
T
Kreyenberg
H
H
Bader
P
P
D000293:Adolescent; D000328:Adult; D018941:Antigens, CD19 / Q000276:immunology; D017252:CD3 Complex / Q000276:immunology; D019002:CD56 Antigen / Q000276:immunology; D019070:Cell Lineage; D002469:Cell Separation / Q000379:methods*; D002648:Child; D002675:Child, Preschool; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods*; D006801:Humans; D016256:Lewis X Antigen / Q000276:immunology; D018950:Lipopolysaccharide Receptors / Q000276:immunology; D016131:Lymphocyte Subsets / Q000276:immunology*; D008280:Magnetics; D008297:Male; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011182:Postoperative Care; D020080:Tandem Repeat Sequences; D018183:Transplantation Chimera / Q000276:immunology*
D018941:Antigens, CD19; D017252:CD3 Complex; D019002:CD56 Antigen; D016256:Lewis X Antigen; D018950:Lipopolysaccharide Receptors