Comparative evaluation of commonly used clones and fluorochrome conjugates of monoclonal antibodies for CD34 antigen detection

CD34(+) cell enumeration is currently the most appropriate technique for hematopoietic graft quality control. At the same time, numerous CD34 mAb have become commercially available. This study was designed to compare the commonly used clones 8G12 and QBEND-10 with the new clones 581 and BIRMA-K3. All available fluorochrome conjugates were tested: FITC, PE, and PE-Cy5 or PerCP for QBEND, BIRMA, 581, and 8G12 and FITC and PE for 581. Bone marrow from healthy donors (n = 5) and leukapheresis samples (n = 16) were stained, according to each manufacturer's protocol and analyzed using the FACScan(R). The following parameters were evaluated: % CD34(+) cells detected and percentage of deviation from the median within each sample; mean channel fluorescence intensity of the CD34(+) cells; resolution index (median channel fluorescence intensity of CD34(+) cells/monocytes), % overlapping of CD34(+) cell and monocyte fluorescence; proportion of CD34(+) events after blocking with the same unlabeled clone; values of compensation requirements. Tables with results for each evaluated parameter separately were created, and rank points were applied. These scores represented the quality performance of the studied clones and fluorochrome conjugates and may be summarized as follows: 581 and 8G12 produced the best results, followed by BIRMA-K3 and QBEND10. The fluorochrome sequence was PE, PE-Cy5, PerCP, and FITC. However, all PE conjugates of the studied clones provided highly comparable results and conditions for CD34(+) cell enumeration. When antigen coexpression must be studied and another dye than PE must be applied for CD34(+) cell discrimination, the PE-Cy5 conjugates should be preferred.