Establishment and optimization of a flow cytometric method for evaluation of viability of CD34+ cells after cryopreservation and comparison with trypan blue exclusion staining.
BACKGROUND
Trypan blue exclusion staining is probably the most frequently applied method (Method I) for assessment of viability in peripheral blood progenitor cell grafts after cryopreservation. Alternatively, a flow cytometry-based method (Method II) was established and optimized.


METHODS
In a first series of 22 autologous apheresis products, the influence of duration of antibody staining and red cell (RBC) lysis on viability was investigated. In a second series of 21 autologous and 1 allogeneic apheresis products, the effect of omitting the RBC lysis was evaluated. On the basis of the results of the first two series, 155 autologous and 57 allogeneic apheresis products were analyzed with Method I and the now optimized Method II.


RESULTS
Halving the incubation times did not influence the viability of CD45+ or CD34+ cells. Omission of RBC lysis resulted in a significantly (p = 0.022) increased median viability of CD45+ cells (75.8% vs. 71.0%) without any influence on CD34+ cells. In the third series, the median viability of CD34+ cells (96.9%) was significantly (p < 0.0001) higher compared with the viability of CD45+ cells (76.2%) and the viability determined by Method I (86.5%).


CONCLUSIONS
The viability of CD34+ cells was significantly higher compared with the viability of all white blood cells. The presented cytometry-based method is superior to the standard trypan blue method regarding the number of analyzable cells and documentation, regarding observer independence and standardization; it allows the analysis of the cells of interest for transplantation after minimal sample manipulation.
Humpe
Andreas
A
Second Department of Medicine of the University Clinic Schleswig-Holstein Campus Kiel, Dr Mildred Scheel Haus, Kiel, Germany. a.humpe@med2.uni-kiel.de
Beck
Christian
C
Schoch
Robert
R
Kneba
Michael
M
Horst
Heinz-August
HA
D018952:Antigens, CD34 / Q000378:metabolism*; D001781:Blood Component Removal; D002470:Cell Survival; D004396:Coloring Agents; D015925:Cryopreservation; D005069:Evaluation Studies as Topic; D005434:Flow Cytometry; D006461:Hemolysis; D006801:Humans; D017493:Leukocyte Common Antigens / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D014343:Trypan Blue / Q000378:metabolism*
D018952:Antigens, CD34; D004396:Coloring Agents; D017493:Leukocyte Common Antigens; D014343:Trypan Blue