Transfusion Clinique et Biologique xxx (xxxx) xxx–xxx
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Letter to the Editor
Uncontrolled freezing of peripheral blood hematopoi-
etic cell at −80 ◦ C: Our experience!
Sir,
Cell therapies based on hematopoietic cells have become the
standard of care for a large number of clinical indications. These
indications include lymphoma (Hodgkin’s and non-Hodgkin’s)
other hematological malignancies, myelodysplastic syndromes,
etc. [1]. Hematopoietic cells can be stored at 2–6 ◦ C for 72 h
but for long-term storage, they need to be cryopreserved. Over
the years, peripheral blood hematopoietic cells (PBHCs) have
been cryopreserved in liquid nitrogen at −196 ◦ C, in vapour
phase nitrogen at −152 ◦ C or −135 ◦ C, and in mechanical freezers
at −80 ◦ C [2].
Here, we report an analysis of initial 5 cryopreserved PBHCs in
mechanical freezers at our centre in terms of CD34+ cells, viability
and cell engraftments in those patients.
All the five cases were planned for autologous PBHCs collection
and the details of their diagnosis and conditioning regimen used
can be found in Table 1.
Hematopoietic cells were collected using P1YA kits on ComTec
(Fresenius kabi). A Cryomix solution made up of 10% DMSO, 20%
human serum albumin with Normal Saline was used for cryopreser-
vation of cells. An equal amount of Cryomix solution was mixed
to the hematopoietic cell product and transferred into Cryocyte
bags (CryoMacs) that were placed into a −80 ◦ C mechanical freezer
(REMI). Whole procedure was done under fully aseptic technique
using laminar airflow. PBHCs were thawed in 37 ◦ C water bath
and then infused within 15 min without washing. The criteria of
engraftment were to reach count of 20 × 109 /L for platelets and
0.5 × 109 /L for neutrophils.
Table 1
Details of patients with their diagnosis and conditioning regimen used.
S. No. Patient details Type of transplant
Case 1 25 yr Male Autologous
Case 2 38 yr Male Autologous
Case 3 28 yr Male Autologous
Case 4 56 yr Male Autologous
Case 5 41 yr Male Autologous
! The work is attributed to Department of transfusion medicine, Sparsh Hospital,
Yeshwantpur, Bangalore. The manuscript was not presented as part at a meeting,
the organisation, place, and exact date on which it was read.
https://doi.org/10.1016/j.tracli.2021.01.001
1246-7820/© 2021 Société française de transfusion sanguine (SFTS). Published by Elsevier Masson SAS. All rights reserved.
Please cite this article as: Bhasker B, Uncontrolled freezing of peripheral blood hematopoietic cell at −80 ◦ C: Our experience, Transfusion
Clinique et Biologique, https://doi.org/10.1016/j.tracli.2021.01.001
Average time duration of storage for cryopreserved hematopoi-
etic cells was 25 days (range 13–47 days). The initial yield of
the products before cryopreservation ranged from 2.8 × 106 to
8.6 × 106 per Kg of the patient (mean dose 5.6 × 106 per Kg), which
decreased to a mean of 3.92 × 106 per Kg post-thawing of frozen
hematopoietic cells. The hematopoietic cells viability after thaw-
ing ranged from 75 to 98% of total CD34+ cells. All the patients had
neutrophil and platelet engraftment before they were discharged.
Days taken for engraftment of neutrophil ranged from 10 to 14 days
(mean 11.2 days) whereas for platelets, it ranged from 15 to 35 days
(mean 21.2 days).
We used the standard 10% cryoprotectant DMSO, which pre-
vents freezing damage to living cells. Varying percentages of DMSO
has been used with success, but 10% DMSO has been found to be
the optimal one as higher percentage leads to more toxicity and
lower concentration leads to suboptimal storage [3].
It was Stiff et al. [4], in 1987, who first demonstrated the success-
ful engraftment with bone marrow stem cells stored at −80 ◦ C after
uncontrolled-rate freezing. Various studies have shown that there
is no adverse effect on storage of PBHCs at −80 ◦ C and have reported
no significant change in post-thawing recovery parameters includ-
ing cell viability, CD34+ cells, burst forming unit, erythroid or
colony forming unit-granulocyte macrophage.
To conclude, our results of cryopreserving PBHCs at −80 ◦ C after
uncontrolled rate freezing were satisfactory in terms of engraft-
ment. In settings where controlled rate freezers and liquid nitrogen
at −196 ◦ C facility for storage are not available,uncontrolled freez-
ing at −80 ◦ C can be considered as a safe alternative, more
convenient and cost effective for cryopreservation of PBHCs in the
emergency conditions and under close supervision of the trained
staff.
Diagnosis Conditioning regimen used
Relapse classical Hodgkins Lymphoma Carmustine, etoposide, cytarabine, and
– Ann Arbor stage 4 melphalan (BEAM) regimen
Philadelphia positive B Cell ALL-high Myeloablative conditioning with TBI and
risk CNS stage I Cyclophoaphamide
Relapse Hodgkins Lymphoma – Carmustine, etoposide, cytarabine, and
Nodular sclerosis stage IV melphalan (BEAM) regimen
Mantle cell Lymphoma–Blastoid R-BEAM (rituximab, carmustine, etoposide,
variant stage 4 with complex cytarabine, melphalan)
karyotype in CR1
Multiple Myeloma– Salmon Durie Melphalan
stage III
Bhasker B
Disclosure of interest
The author declares that he has no competing interest.
References
[1] Hirata Y, Kishino K, Onozaki F, Nakaki Y, Yamamoto C, Matsuyama T, et al. Use of
cryoprotectant-depleted allogeneic peripheral blood stem cells for transplanta-
tion. Hematology 2011;16:221–4.
[2] Detry G, et al. Impact of uncontrolled freezing and long-term storage of
peripheral blood stem cells at −80◦ C on haematopoietic recovery after autol-
ogous transplantation. Report from two centres. Bone Marrow Transplant
2014;49:780–5.
Transfusion Clinique et Biologique xxx (xxxx) xxx–xxx
[3] Galmes A, Besalduch J, Bargay J, et al. Long-term storage at −80◦ C of hematopoi-
etic progenitor cells with 5% dimethyl sulfoxide as the sole cryoprotectant.
Transfusion 1999;39:70–3.
[4] Stiff PJ, Koester AR, Weidner MK, Dvorak K, Fisher RI. Autologous bone
marrow transplantation using unfractionated cells cryopreserved in dimethyl-
sulfoxide and hydroxyethyl starch without controlled-rate freezing. Blood
1987;70:974–8.
B. Bhasker
Sparsh Hospital, Yeshwantpur, Bangalore, India
E-mail address: balag4g@gmail.com
Available online xxx