Comparison of Simultaneous CD34+ and CD3+ Quantification with a Modified Stem Cell Enumeration Kit on Two Different Flow Cytometers.
BACKGROUND
Quantification of CD34+ cells in peripheral blood stem cell apheresis is normally performed by single platform flow cytometric measurements according to the ISHAGE protocol. Peripheral blood stem cell concentrates (PBSC) produced by apheresis normally contain many T cells. Those T cells can be used for production of donor lymphocyte infusion doses, if abundant amounts of CD34+ cells have been collected. Therefore, it is of interest to know both the CD3+ and the CD34+ cell count of allogeneic PBSC. This is the first study comparing the performance of a modified ISHAGE protocol allowing additional quantification of CD3+ cells on two different flow cytometers, the FACSCalibur and the FACSVerse, respectively.


METHODS
CD45+ and CD34+ cell concentrations were measured using a standard and a modified ISHAGE protocol including CD3+ cell quantification on both machines. All cell concentrations were measured using a Trucount bead based stem cell enumeration kit. The FACSVerse machine can additionally be equipped with a sample volume sensor allowing cell quantification without using beads. The samples analysed were taken from granulocyte-colony-stimulating factor mobilized peripheral blood stem cell apheresis procedures (pre- and post-apheresis, and apheresis concentrate).


RESULTS
There were no significant differences in cell concentrations measured by the standard and modified ISHAGE protocol, regardless of which machine had been used when using bead quantification. No significant differences between the results of the two flow cytometers using the modified ISHAGE protocol were observed. Pearson´s correlation was always > 0.96, and regression coefficients were higher than 0.93. The only significant differences were observed between bead quantification and volume sensor quantification on the FACSVerse machine.


CONCLUSIONS
The modified ISHAGE protocol can effectively be used on both flow cytometers tested, especially if bead quantification is used.
Julian
J
Strobel
Barbara
B
Hauck-Dlimi
Volker
V
Weisbach
Reinhold
R
Eckstein
Juergen
J
Zingsem
Erwin
E
Strasser
D018952:Antigens, CD34 / Q000097:blood*; D015415:Biomarkers / Q000097:blood; D001781:Blood Component Removal; D017252:CD3 Complex / Q000097:blood*; D002452:Cell Count; D002469:Cell Separation / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods; D006801:Humans; D000072916:Peripheral Blood Stem Cells / Q000378:metabolism*; D010641:Phenotype; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results
D018952:Antigens, CD34; D015415:Biomarkers; D017252:CD3 Complex; D011933:Reagent Kits, Diagnostic