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Month, YYYY
DMSO Verification: A Case Study.
Steve Konings, MS. Lab Manager and Sarah Heidtke, QA/QC Specialist.
Froedtert Hospital/Medical College of Wisconsin
Blood and Marrow Transplantation Program
Milwaukee, WI
We recently compared the performance of dimethyl sulfoxide (DMSO), a component of our
cryopreservation medium, from our current vendor with three new vendors. This was prompted
by difficulty obtaining both the reagent and source documentation to qualify this reagent from
our current vendor. As we began to write the verification plan and obtain samples from vendors,
we also recognized the benefit of potentially qualifying additional vendors for this critical reagent
and obtaining better pricing.
The verification plan was written by the QA/QC Specialist and approved by the Laboratory
Director. The study used cells from HPC, Apheresis collections that exceeded the collection
goal and contained >0.4% CD34+ cells. All product samples were cryopreserved at 2e+8
cells/mL in a final volume of 10 mL per bag using our standard freezing medium of isotonic
electrolyte solution containing 4% human serum albumin and 10% DMSO. Initially DMSO from
our current vendor was compared to DMSO from two new vendors. All three bags were
cryopreserved in the same controlled rate freeze run as the product that was being
cryopreserved for clinical use. All freeze runs had acceptable freeze curves. Cryopreserved
products were transferred to vapor phase storage for at least 12 hours before thawing.
All three bags from a freeze run from the same product were thawed simultaneously (37 C
water bath) and washed (15 minutes at 500 x g) then stained for flow cytometry (No lyse, no
wash single platform method) at the same time and acquired sequentially using a BD Canto II
flow cytometer. Results from the first three products showed the percent CD34 recovered was
higher when using DMSO from either of the two new vendors than from our current vendor
(vendor C) (mean = 16.5% for vendor A and 4.5% for vendor B higher recovery) which was
significant only for vendor A (p=0.03 using a paired Ttest). However, while reviewing these
results, it was noted that the recovery of CD34 was also associated with the order in which
samples were tested after thawing; those tested first had the highest recovery and the sample
tested last had the lowest recovery. The other parameters that were assessed, including
recovery of viable TNC, recovery of viable mononuclear cells (MNC), and overall viability by 7AAD were not significantly different.
Based on results from the first three experiments, the decision was made to freeze cells from an
additional three products to assess if the difference in viable CD34 recovery was really
significant. DMSO from an additional vendor (vendor D) was obtained as well. The same
process was used to cryopreserve the cells. However, the bags were thawed, washed, stained
and acquired sequentially, rather than simultaneously such that the length of time from thaw to
flow acquisition was the same for all bags.
The results for the second three products were analyzed separately and together with the first
three experiments using an analysis of variance (ANOVA) to determine if differences in
outcomes were significant before comparing the new DMSO vendors with the current vendor.
No significant differences were seen for any of the outcomes as shown in Figure 1 below.
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Average 7-AAD Viability
100
80
% Viable
% Recovery Viable CD34
Average CD34 Recovery
60
40
20
0
80
60
40
20
0
A
P value
B
C
D
C
D
80
% Recovery TNC
100
% Recovery MNC
B
0.7730
Average TNC Recovery
Average MNC Recovery
80
60
40
20
0
A
P value
0.2866
A
P value
B
0.8963
C
D
60
40
20
0
A
P value
B
C
D
0.8543
Figure 1: Outcomes of freezing and thawing 6 products (vendors A-C), or 3 products (vendor D) using
DMSO from different sources. The data are shown as the mean and standard deviation of the
experiments. Differences in DMSO source were not significant using a one-way ANOVA.
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Factors considered in choosing a vendor for DMSO included CD34+ cell recovery, product
purity, availability of Certificate of Analysis, price, payment method, distribution source and
reliability of vendor. Since there was no significant difference in cell recovery or viability we
considered the other variables. Two vendors listed product purity at >99.0% and two vendors at
>99.9%. All vendors had a Certificate of Analysis for their product. Price quotes were obtained
from all vendors. One vendor accepted payment by purchase order (preferred by our
purchasing department) rather than check or credit card that the other vendors required. Two
vendors sell product directly while two vendors sell through a distributor. Reliability was judged
based on length of time in cell therapy industry and any previous experience we had with the
vendor. Based on these criteria, we determined that any of the four vendors could be an
acceptable source for DMSO. The vendors were ranked and one chosen, vendor D as the
primary source and a second vendor, vendor A, as the back up. We estimate an approximate
cost savings of 50% with our chosen primary source. Our first order has been received and
included all needed documentation and without any complications.
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