Karla Murcia , Melanie Dimapasoc , Bonnie Yip , Dale Hirschkorn

Melanie
1
Dimapasoc ,
Dale
1
Hirschkorn ,
and Mars
1
Stone
Systems Research Institute; San Francisco, CA United States
The quality of peripheral blood mononuclear cells (PBMC) isolated from whole blood has a significant impact
on subsequent analysis. Maximizing the recovery, viability and functionality of isolated PBMCs are essential
for reliable assessment of a variety of downstream applications, particularly cell-mediated immune responses.
The standard method for purification of PBMCs is the use of density gradient centrifugation. This method
requires precise layering of whole blood over density medium, careful pipetting of enriched cells after
centrifugation, and meticulous care throughout the labor intensive isolation procedure to avoid mixing of
layers before and after centrifugation steps.
SepMate® tubes are a new commercially available product that greatly reduces the time for optimal PBMC
isolation from whole blood, but requires validation to ensure that they perform equivalently to traditional
Ficoll methods. Here we compare the two methods for PBMC yield, viability, subset frequency and
functionality.
Objective
Introduction
1Blood
Bonnie
1
Yip ,
1.  To compare subset frequencies, immunophenotype and Tcell function between standard Ficoll density gradient
PBMC cell separation and a novel method using
STEMCELL Technologies SepMate tubes.
2.  To compare both methods of PBMC isolations from whole
blood processed immediately after phlebotomy, 24 hours
after phlebotomy as well as pre- and post cryopreservation.
3.  To determine if there were differences in inter-technician
variability between the Ficoll and SepMate methods of
PBMC isolations.
Methods
Karla
1
Murcia ,
PBMC Viability
Memory Subset Frequencies
PBMC Recovery
Leukocyte Subset Frequencies
Fresh
A.
B. A.
Fresh
B. B.
A.
A. 2.45%
11.07%
0 Hour
14.21%
B.
25.63%
B.
3.17%
9.69% 16.00%
9.70% 18.81%
11.29%
8.83%
Ficoll
24 Hour
Ficoll
SepMate
SepMate
9.88%
Monocytes
T-cells
61.07%
71.14%
B-cells
54.79%
72.27%
Figure 3. Mean viability of paired PBMCs isolated immediate after
phlebotomy and after 24 hours using traditional Ficoll and SepMate
methods (A). Mean viability post cryopreservation of paired PBMCs
isolated immediate after phlebotomy and after 24 hours (B).
Cryopreserved
D.
C.
PBMC Viability
Inter-Technician Comparison
PBMC Yield
A.
% of Parent Population (CD45+)
Figure 4. Mean recovery of paired PBMCs isolated immediate after
phlebotomy and after 24 hours using traditional Ficoll and SepMate
methods (A). Mean recovery post cryopreservation of paired PBMCs
isolated immediate after phlebotomy and after 24 hours (B).
B. A.
B.
Granulocytes
% of Parent Population (CD45+)
Cryopreserved
C.
2.54% 9.38%
14.04%
0 Hour
2.70%
11.62% 12.12%
Ficoll
D. 6.02%
9.62%
24 Hour
5.04%
12.21%
13.53%
13.70%
SepMate
Ficoll
SepMate
Monocytes
69.22%
73.56%
T-cells
70.67%
74.03%
Figure 1. Paired comparison of CD4+ Memory T-cell subset frequencies in both freshly and
cryopreserved PBMCs isolated by Ficoll and SepMate methods measured by flow
cytometry. Overall, there was no significant difference in subset frequencies in PBMCs
isolated using both methods in fresh or cryopreserved cells (p>0.05 for nearly all
comparisons).
ELISPOT
A. B.
% of Parent Population (CD45+)
Figure 6. Bland-Altman Plot of paired Ficoll (A) and SepMate (B)
PBMC isolation viability agreement. The mean viability of both
technicians was plotted against the viability difference. Both methods
showed insignificant variability between technicians (p>0.05).
Figure 8. Bland-Altman Plot of paired Ficoll (A) and SepMate (B)
PBMC yield agreement. The mean PBMC yield of both technicians was
plotted against the viability difference. The SepMate method showed
insignificant variability between technicians (p>0.05).
% of Parent Population (CD45+)
Granulocytes
Figure 5. Comparison of Leukocyte subset frequencies of PBMCs isolated by
Ficoll and SepMate methods measured by flow cytometry. There were no
significant difference between subset frequencies when comparing both
methods (p>0.05).
Results
Figure 2. Comparison of IFN-γ SFC in cryopreserved PBMCs isolated by Ficoll and
SepMate methods. T-cell function was measured by IFN-γ response to FEC antigen pool
using ELISPOT. No differences were observed in T-cell responses of PBMCs isolated
immediately and 24hours after draw (p>0.05).
B-cells
•  Viability in both standard Ficoll vs. SepMate method were similar (p>0.05).
•  Yield of PBMCs using both methods was comparable (p>0.05).
•  Recovery of PBMCs post-cryopreservation from samples processed immediately after phlebotomy and after 24 hours was
equivalent, with slightly higher yields in freshly processed blood using SepMate tubes.
•  Memory CD4+ subset frequencies were the same using both methods (nearly all parameters measured p> 0.05).
•  Leukocyte subset frequencies were comparable using both methods in all parameters measured (p>0.05 for all comparisons).
•  Over all there was no difference in the inter-technician variability of yield or viability.
•  No differences were observed in T-cell responses of PBMCs isolated in both methods (p>0.05).
Conclusions
•  Isolation methods using SepMate tubes performed comparably to
traditional Ficoll methods in all parameters measured.
•  Use of SepMate tubes reduce the time and care needed for optimal
PBMC recovery.
Acknowledgments
The authors gratefully acknowledge the assistance of Roberta Bruhn and John Heitman for guidance in
statistical analysis and the performance of the ELISPOT assay.
Comparison of Subsets, Immunophenotype and Function Between PBMC Isolation Methods