Abstract
Recently, high-dose chemo- and radiotherapy for newly diagnosed young patients with multiple myeloma has been established in member centers of the Nordic Myeloma Study Group (NMSG). The treatment includes supportive use of autologous hematopoietic blood progenitors harvested by leukapheresis. Safe and adequate hematopoietic support with minimal toxicity requires optimization of the number and quality of harvested progenitors. We have therefore established a workshop consisting of the 11 laboratories within the NMSG with the aim of developing recommendations for enumeration of CD34-positive cells.
In this first workshop report, we discuss technical variables affecting the enumeration of progenitors harvested by leukapheresis and make specific recommendations. The products are analyzed within 4 h following erythrocyte lysis using the Ortho lysing solution for 8–10 minutes. A sample containing 0.5−1.0 × 106 nucleated cells is incubated with the test or control antibody (anti-CD34 = HPCA-2PE and Ms IgG1PE from Becton Dickinson) at dilutions of 1:10 to 1:40 in a final volume of 1 ml. The samples are incubated 15 minutes at ambient temperature, washed two to three times, and fixed with 1% paraformaldehyde (150 μl/pellet) for a minimum of 10 minutes. Flow cytometric analysis is performed on 50,000 cellular events with debris eliminated. The estimation of CD34-positive cells can be performed on an FL2-side-scatter plot after marking of a positive population containing >50 events. Using quadrant statistics, this population can be identified in the upper left quadrant, among cells with the side-scatter profile of small lymphocytic cells. The negative control must be judged negative by the analyzer and the small number of events subtracted from the positive population.
Subsequent calculation of the total number of CD34-positive cells per kg patient weight is based on the CD34 estimate and the total number of nucleated cell harvested. Allowing for variations in estimation of progenitors, a safe progenitor number appears to be >2.0 × 106 CD34-positive cells per kg, which can be harvested in two to three leukapheresis procedures.
Future workshops will focus on multiparametric evaluation of CD34-positive subsets with an attempt (i) to define megakaryoblast subsets, (2) to optimize quantitative polymerase chain reaction for evaluation of tumor cell contamination, and, finally, (3) to establish recommendations for the leukapheresis procedure per se.
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