A novel assessment of the quality of immunohistostaining overcomes the limitations of current methods.

Quality assurance has become an integral part of surgical pathology. Despite the development of interdisciplinary quality systems, however, the means for objective analysis in surgical pathology are limited. Immunohistostaining is a multi-factorial procedure that depends on the quality of reagents and antibodies employed in the process and on technical methodology. In the present study, we aim to establish a straightforward procedure for objective quality evaluation of the components involved in immunohistostaining. The quality of two of these components, the primary antibody and the automated staining device, was assessed by employing each component from two different sources, one serving as the test substance and the second as the reference. Assessment was performed by at least two pathologists in a blinded fashion using pre-established quality criteria and scores. The quality analysis of two automated devices revealed a significant difference between the reference and tested devices (3.5+/-1.7 and 4.2+/-1.5, respectively, P>0.05), while the analysis of two selected antibodies did not reveal any statistical difference. The described method provided objective quality assessment of selected components affecting immunohistostaining by elaborating numeric values that enabled statistical analysis. This approach is applicable to any given component in various surgical pathology procedures.

Collection and analysis of hematopoietic progenitor cells from cynomolgus macaques (Macaca fascicularis): assessment of cross-reacting monoclonal antibodies.

Previous studies have shown that hematopoietic progenitor cells can be isolated from human or nonhuman primate bone marrow (BM) cells. In the present study, we studied the cross-reactivity of 13 anti-human CD34, two anti-human c-Kit, and one anti-human CD133 monoclonal antibodies (mAbs) with cynomolgus macaque (Macaca fascicularis) BM cells, using flow cytometric analysis, cell enrichment, and clonogenic assay. Among the 13 anti-human CD34 mAbs assessed, six cross-reacted as previously reported by other groups. However, only three of these six mAbs (clones 561, 563, and 12.8) recognized cynomolgus CD34+ cells that formed progenitor colonies when grown in methylcellulose culture. Similarly, of the two anti-human c-Kit mAbs (clones NU-c-kit and 95C3) that were previously reported to cross-react with cynomolgus BM cells, only one (clone NU-c-kit) resulted in a similar outcome. The anti-human CD133 mAb (clone AC133) also cross-reacted with cynomolgus BM cells, although these cells did not give rise to colonies when grown in culture. These results suggest that antibodies that cross-react with nonhuman primate cells may not identify the hematopoietic cells of interest. In addition, while the CD34 mAb (clone 561) results in the selection of hematopoietic progenitor cells of all lineages when assessed in methylcellulose culture, the c-Kit(high) fraction (NU-c-kit) exclusively identifies erythroid-specific progenitor cells after growth in culture. It is important to consider these findings when selecting cross-reacting mAbs to identify cells of hematopoietic lineages in macaque species.

Flow cytometric assessment of CD15+CD117+ cells for the detection of minimal residual disease in adult acute myeloid leukaemia.

There is little information available regarding immunophenotypic monitoring of minimal residual disease (MRD) in acute myeloid leukaemia (AML). We investigated leukaemic cells co-expressing CD15 and CD117 (CD15+CD117+) in 72 adult AML cases at diagnosis. In 22 cases (31%) with various AML subtypes, more than 5% of leukaemic cells showed the CD15+CD117+ phenotype (range 5.22-55.48%). These 22 cases were younger and had a higher complete remission (CR) rate than the other AML cases, but the CD15+CD117+ cell percentage at diagnosis showed no correlation with the CR duration among the 72 cases. The CD15+CD117+ cell percentage showed a range of 0.00-0.08% in bone marrow cells from 10 haematologically normal subjects. We also investigated CD15+CD117+ cells in sequential bone marrow samples from 17 AML patients who achieved CR and who had had more than 5% CD15+CD117+ leukaemic cells at diagnosis. Because the CD15+CD117+ cell percentage varied among these AML cases, we calculated the percentage of MRD ¿MRD% = [CD15+CD117+ cells (%) in each sequential marrow sample] / [CD15+CD117+ cells (%) at diagnosis of the corresponding case] x 100¿. A high MRD% after 10 months of CR was significantly associated with a short CR duration (P = 0.0004), whereas continuation of a well-reduced MRD% was associated with a long CR duration. The leukaemic cells conserved the CD15+CD117+ phenotype in all of the eight cases who relapsed. Flow cytometric monitoring of CD15+CD117+ cells is simple and can be applied to a substantial fraction of AML cases. This monitoring may be useful for predicting relapse of adult AML.