title: Use of collagen for standardization of PBSC graft quality evaluation: a multicenter comparative analysis of commercial collagen-based and methylcellulose-based colony-forming unit (CFU) assay kits.
abstract: The colony-forming unit-granulocyte-macrophage (CFU-GM) assay, an essential test in evaluation of the quality of autologous grafts of hematopoietic stem cells, has yet to be standardized. With this aim in view, we carried out a multicenter study of five commercially available culture kits for CFU-GM evaluation. Four kits were methylcellulose-based (H4431, H4434, H4435, StemBio1d) and one was collagen-based (EasyClone-Multi). Using fresh and frozen samples of PBSC grafts, we compared CFU-GM and burst-forming unit-erythrocytes (BFU-E) growth using the EasyClone kit to each of the methylcellulose kits. BFU-E and CFU-GM clonogenicity of both fresh and frozen PBSC was clearly inferior with the H4431 kit, which provides conditioned medium only. CFU-GM numbers obtained with fresh and frozen PBSC samples were significantly higher with the EasyClone kit than with the H4434 and StemBio kits. BFU-E numbers were also higher with the EasyClone kit, but only when colonies were scored after May-Grünwald-Giemsa (MGG) staining. Finally, although the H4435 kit provides higher doses of recombinant cytokines than the EasyClone kit, CFU-GM and BFU-E numbers obtained for fresh or frozen PBSC with both kits were similar. In addition, CFU-GM and BFU-E numbers correlated well with CD34+ cell numbers for all five kits for both fresh and frozen PBSC. In summary, our study shows that the EasyClone-Multi and H4435 kits provide the best CFU-GM growth. The collagen-based EasyClone kit has the additional advantage of allowing gel staining and storage, which facilitates colony identification and, more importantly, makes gel exchange possible for standardization of the CFU-GM assay.
authors.affiliation: Laboratoire d'Hématologie, CHU Grenoble, France.
authors.forename: P
authors.initials: P
authors.lastname: Mossuz
authors.forename: I
authors.initials: I
authors.lastname: Dobo
authors.forename: M C
authors.initials: MC
authors.lastname: Genevay
authors.forename: A
authors.initials: A
authors.lastname: Allegraud
authors.forename: M
authors.initials: M
authors.lastname: Dautel
authors.forename: A E
authors.initials: AE
authors.lastname: Niaussat
authors.forename: F
authors.initials: F
authors.lastname: Cousin
authors.forename: V
authors.initials: V
authors.lastname: Praloran
authors.forename: C
authors.initials: C
authors.lastname: Boccaccio
authors.forename: S
authors.initials: S
authors.lastname: Hermouet
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D002648:Child; D003094:Collagen; D003114:Colony-Forming Units Assay / Q000379:methods / Q000592:standards*; D005260:Female; D006085:Graft Survival; D019337:Hematologic Neoplasms / Q000628:therapy*; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D008297:Male; D008747:Methylcellulose; D008875:Middle Aged; D014182:Transplantation, Autologous
chemical_list: D008747:Methylcellulose; D003094:Collagen