Mossuz P, Dobo I, Genevay M C, Allegraud A, Dautel M, Niaussat A E, Cousin F, Praloran V, Boccaccio C, Hermouet S
Laboratoire d'Hématologie, CHU Grenoble, France.
J Hematother. 1998 Aug;7(4):351-9. doi: 10.1089/scd.1.1998.7.351.
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.
集落形成单位-粒细胞-巨噬细胞（CFU-GM）测定是评估自体造血干细胞移植质量的一项重要检测，但尚未标准化。鉴于此目标，我们对五种市售的用于CFU-GM评估的培养试剂盒进行了多中心研究。四种试剂盒基于甲基纤维素（H4431、H4434、H4435、StemBio1d），一种基于胶原蛋白（EasyClone-Multi）。我们使用外周血干细胞移植的新鲜和冷冻样本，将使用EasyClone试剂盒的CFU-GM和爆式红系集落形成单位（BFU-E）生长情况与每种甲基纤维素试剂盒进行比较。仅提供条件培养基的H4431试剂盒，新鲜和冷冻外周血干细胞的BFU-E和CFU-GM克隆形成能力明显较差。使用EasyClone试剂盒从新鲜和冷冻外周血干细胞样本中获得的CFU-GM数量显著高于H4434和StemBio试剂盒。EasyClone试剂盒的BFU-E数量也更高，但仅在May-Grünwald-Giemsa（MGG）染色后对集落进行计数时如此。最后，尽管H4435试剂盒提供的重组细胞因子剂量高于EasyClone试剂盒，但两种试剂盒从新鲜或冷冻外周血干细胞中获得的CFU-GM和BFU-E数量相似。此外，对于新鲜和冷冻外周血干细胞的所有五种试剂盒，CFU-GM和BFU-E数量与CD34+细胞数量均具有良好的相关性。总之，我们的研究表明，EasyClone-Multi和H4435试剂盒提供了最佳的CFU-GM生长。基于胶原蛋白的EasyClone试剂盒具有允许凝胶染色和储存的额外优势，这便于集落识别，更重要的是，使凝胶交换成为可能，从而实现CFU-GM测定的标准化。
