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

The colony-forming unit-granulocyte-macrophage (CFU-GM) assay, an esse ntial test in evaluation of the quality of autologous grafts of hemato poietic stem cells, has yet to be standardized. With this aim in view, we carried out a multicenter study of five commercially available cul ture kits for CFU-GM evaluation. Four kits were methylcellulose-based (H4431, H4434, H4435, StemBio(R)1d) and one was collagen-based (EasyCl one-Multi(R)), Using fresh and frozen samples of PBSC grafts, we compa red CFU-GM and burst-forming unit-erythrocytes (BFU-E) growth using th e 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 num bers were also higher with the EasyClone kit, but only when colonies w ere scored after May-Grunwald-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 number s correlated well with CD34+ cell numbers for all five kits for both f resh and frozen PBSC. In summary, our study shows that the EasyClone-M ulti and H4435 kits provide the best CFU-GM growth. The collagen-based EasyClone kit has the additional advantage of allowing gel staining a nd storage, which facilitates colony identification and, more importan tly, makes gel exchange possible for standardization of the CFU-GM ass ay.