Transplantation and Cellular Therapy 27 (2021) 664.e1 664.e6

Transplantation andCellular Therapy
journal homepage: www.tctjournal.org

Full Length Article
Allogeneic

Adult

Cryopreservation of Unrelated Hematopoietic Stem Cells from a
Blood and Marrow Donor Bank During the COVID-19 Pandemic:
A Nationwide Survey by the Japan Marrow Donor Program
Yoshinobu Kanda1,*, Masami Inoue2, Naoyuki Uchida3, Yasushi Onishi4, Reiko Kamata5, Mika Kotaki6,
Ryoji Kobayashi7, Junji Tanaka8, Takahiro Fukuda9, Nobuharu Fujii10, Koichi Miyamura11,
Shin-Ichiro Mori12, Yasuo Mori13, Yasuo Morishima14, Hiromasa Yabe15, Yoshihisa Kodera16
1

Division of Hematology, Department of Medicine, Jichi Medical University, Tochigi, Japan
Department of Hematology/Oncology, Osaka Women’s and Children’s Hospital, Osaka, Japan
Department of Hematology, Toranomon Hospital, Tokyo, Japan
4
Department of Hematology, Tohoku University Hospital, Sendai, Japan
5
Nonproﬁt Organization Blood Disorder Information and Support Service "Tsubasa", Tokyo, Japan
6
Hematology Division, Tokyo Metropolitan Cancer and Infectious Diseases Center, Komagome Hospital, Tokyo, Japan
7
Department of Hematology/Oncology for Children and Adolescents, Sapporo Hokuyu Hospital, Sapporo, Japan
8
Department of Hematology, Tokyo Women's Medical University, Tokyo, Japan
9
Department of Hematopoietic Stem Cell Transplantation, National Cancer Center Hospital, Tokyo, Japan
10
Division of Blood Transfusion, Okayama University Hospital, Okayama, Japan
11
Department of Hematology, Japanese Red Cross Nagoya First Hospital, Nagoya, Japan
12
Hematology Department, St Luke's International Hospital, Tokyo, Japan
13
Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan
14
Department of Promotion for Blood and Marrow Transplantation, Aichi Medical University School of Medicine, Nagakute, Japan
15
Department of Innovative Medical Science, Tokai University School of Medicine, Isehara, Japan
16
Japan Marrow Donor Program, Aichi Medical University School of Medicine, Nagakute, Japan
2
3

Article history:
Received 25 February 2021
Accepted 25 April 2021
Key Words:
Cryopreservation
COVID-19
Bone marrow transplantation
Peripheral blood stem celltransplantation
Japan Marrow Donor ProgramA B S T R A C T
During the COVID-19 pandemic, donor hematopoietic stem cell grafts are frequently cryopreserved to ensure theavailability of graft before starting a conditioning regimen. However, the safety of cryopreservation has been controversial in unrelated hematopoietic stem cell transplantation (HSCT), especially for bone marrow (BM) grafts. In
addition, in unrelated HSCT, the effect of the time from harvest to cryopreservation of donor grafts required for thetransportation of donor graft has not been fully clariﬁed. In this study, we retrospectively analyzed the ﬁrst 112
patients with available data who underwent cryopreserved unrelated blood and marrow transplantation throughthe Japan Marrow Donor Program during the COVID-19 pandemic. There were 112 patients, including 83 whoreceived BM grafts and 29 who received peripheral blood stem cell (PBSC) grafts. The median time from stem cell
harvest to cryopreservation was 9.9 hours (range, 2.6 to 44.0 hours), and the median time from cryopreservation
to infusion was 231.2 hours. The incidence of neutrophil engraftment at day 28 after HSCT was 91.1%, and among
109 patients (excluding 3 patients with early death), all but 1 patient achieved neutrophil engraftment within
60 days after HSCT. The time to neutrophil engraftment and time to platelet engraftment were shorter in PBSC
transplantation compared with BM transplantation (BMT), but the differences were not statistically signiﬁcant(P = .064 and .18). Multivariate analysis among BM recipients revealed that a higher number of frozen nucleatedcells and the absence of HLA mismatch were associated with faster neutrophil engraftment. The time to neutrophil
engraftment after unrelated cryopreserved BMT was not different from that after unrelated BMT without cryopreservation. Our ﬁndings suggest that unrelated donor BM and PBSC grafts can be safely cryopreserved even after
transit from the harvest center to the transplantation center. In the current COVID-19 pandemic, cryopreservation
can be considered as an option while balancing the risks and beneﬁts of the procedure.
© 2021 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Members of the Medical Committee of the Japan Marrow Donor Program: Yoshinobu Kanda, Masami Inoue, Naoyuki Uchida, Yasushi Onishi, Reiko Kamata, Mika
Kotaki, Ryoji Kobayashi, Junji Tanaka, Takahiro Fukuda, Nobuharu Fujii, Koichi Miyamura, Shin-Ichiro Mori, Yasuo Mori, Yasuo Morishima, and Hiromasa Yabe.
Financial disclosure: See Acknowledgments on page 664.e6.
*Correspondence and reprint requests: Yoshinobu Kanda, MD, PhD, Division of Hematology, Department of Medicine, Jichi Medical University, 3311-1 Yakushiji,
Shimotsuke-city, Tochigi 329-0498, Japan
E-mail address: ycanda-tky@umin.ac.jp (Y. Kanda).

https://doi.org/10.1016/j.jtct.2021.04.022
2666-6367/© 2021 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Y. Kanda et al. / Transplantation and Cellular Therapy 27 (2021) 664.e1 664.e6

INTRODUCTION
Hematopoietic stem cell transplantation (HCT) has been
established as a curative treatment strategy for a variety ofhematologic disorders. In autologous HCT, hematopoietic stem
cell grafts are obtained from the patients themselves, whereas
in allogeneic HCT. they are obtained from healthy donors. In
autologous HCT, cryopreservation techniques are required, as
patients should receive conditioning regimens between thestem cell harvest and stem cell infusion. Dimethyl sulfoxide(DMSO) has been successfully used as a cryoprotectant in the
cryopreservation of hematopoietic stem cells [1]. The combination of hydroxyethyl starch and a lower concentration of
DMSO was also investigated for the cryopreservation ofhematopoietic stem cells without rate-controlled freezing[2,3]. In Japan, CP-1 (Kyokutoseiyaku, Tokyo, Japan), a freezing
medium that enables cryopreservation with a ﬁnal concentration of hydroxyethyl starch at 6% and DMSO at 5%, is commercially available and widely used in HCT [4,5]. In contrast to
autologous HCT, cryopreservation is not necessary in allogeneic HCT; however, in allogeneic peripheral blood stem cell
(PBSC) transplantation (PBSCT), donor grafts are often cryopreserved to start a conditioning regimen after conﬁrming a sufﬁcient collection of stem cells [6]. On the other hand, donor
grafts are rarely cryopreserved in allogeneic bone marrow(BM) transplantation (BMT) to avoid unnecessary mononuclear cell separation and cryopreservation [6]. In addition,
cryopreservation is generally not permitted in unrelated BMT

Table 1
Patient characteristics according to type of stem cell graft664.e2through the Japan Marrow Donor Program (JMDP), because
the cryopreservation of donor grafts may increase the numberof unused grafts.
In 2020, however, during the COVID-19 pandemic, theJMDP introduced the cryopreservation of donor grafts as an
exception, because donor cell collection may be practically
impossible during this period. Although the cryopreservationof allogeneic donor grafts has been shown to be safe andeffective in small case series, sufﬁcient information is still notavailable, especially for the preservation of donor BM grafts[7,8]. In addition, in unrelated HCT, the effect of the timefrom harvest to cryopreservation of donor grafts required forthe transportation of donor graft has not been fully clariﬁed.
Therefore, in this study, we retrospectively analyzed the ﬁrst
112 patients with available data who underwent cryopreserved unrelated HCT through the JMDP during the COVID-19
pandemic.
METHODS
Patients
All the requests for the cryopreservation of stem cells were reviewed by
the JMDP Central Ofﬁce. After approval, stem cells were collected at harvest
centers and then shipped to transplantation centers under room temperature. All the stem cells were cryopreserved at the transplantation centers onreceipt.
Questionnaires were sent to transplantation centers to retrospectively
collect data on unrelated cryopreserved HCT performed through the JMDP
between April and November 2020, and all but 1 patient without response to
the questionnaire were included in this study. This study was approved by
the Ethics Committee of the JMDP.

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Y. Kanda et al. / Transplantation and Cellular Therapy 27 (2021) 664.e1 664.e6

Statistical Analysis
The study’s primary endpoint was neutrophil engraftment, deﬁned as
the ﬁrst of the 3 consecutive days with an absolute neutrophil count ofat least 0.5 £ 103/mL, and platelet engraftment, deﬁned as the ﬁrst day
with a platelet count exceeding 20 £ 103/mL without platelet transfusion
for at least 7 days. Fisher’s exact test was used to compare categoricalvariables, and the Mann Whitney U test was used to compare continuous variables. Time to engraftment data were analyzed while treating
death without engraftment as a competing risk and compared between
groups with Gray’s test. Multivariate analysis was performed using
Fine Gray proportional hazards modeling based on an available caseanalysis for missing data, including variables with P < .15 on univariate
analysis as independent variables. Information about the use of granulocyte colony-stimulating factor (G-CSF) was not obtained, and thus background diseases were grouped into myeloid malignancies and others.
This was also included as an independent variable as a substitute for theuse of G-CSF, because the use of G-CSF was avoided for myeloid malignancies in some centers.
In addition, the patients who received cryopreserved BM grafts werecompared with those who received BM grafts without cryopreservation
between January 2016 and December 2018 with and without matchingfor recipient age, donor age, background disease, background disease status, and HLA mismatch. Categorical variables were strictly matched,
whereas continuous variables were matched using caliper widths equal
to 0.2 SD.
All P values were 2-sided, and P<.05 was considered to indicate statistical signiﬁcance. All statistical analyses were performed with EZR version 1.54 (Saitama Medical Center, Jichi Medical University, Shimotsuke,
Japan) [9].

but the differences were not statistically signiﬁcant (P = .064
and .18, respectively) (Figure 1).

Predictive Factors of Neutrophil Engraftment in BMT
Factors that predicted neutrophil engraftment after HCT
were analyzed only in BMT, because the number of PBSCT
recipients was limited. Continuous variables were divided
equally into 3 groups and treated as categorical variables. Univariate analysis revealed that the number of frozen nucleatedcells, not harvested cells, and the presence of HLA mismatch
were signiﬁcantly associated with neutrophil engraftment(Table 2, Figure 2A, B). The time from stem cell harvest to cryopreservation was also signiﬁcant, but, contrary to our expectations, the shortest duration was associated with delayed
neutrophil engraftment (Figure 2C).
Multivariate analysis revealed that a higher number of frozen nucleated cells and the presence of HLA mismatch wereindependently associated with neutrophil engraftment. The

RESULTS
Patients and Stem Cell Grafts
The study cohort comprised 112 patients (64 males and 48
females) with a median age of 54 years (range, 12-69 years).
Eighty-three patients underwent BMT and 29 underwent
PBSCT. In this period, 456 BM grafts and 134 PBSC grafts weretransplanted through the JMDP, and thus 18.2% of the BM
grafts and 21.6% of the PBSC grafts had been cryopreserved.
The median age of the donors was 41.5 years (range, 20-54
years). The background disease was acute myelogenous leukemia in 32 patients, acute lymphoblastic leukemia in 18, chronic
myelogenous leukemia in 4, myelodysplastic syndrome in 30,
myeloproliferative neoplasms in 7, adult T cell leukemia/lymphoma in 7, lymphoma in 7, plasma cell neoplasms in 2, andnonmalignant diseases in 5. There was an HLA mismatch
between the donor and the recipient in 43 HCTs (38.3%). CP-1
was used for cryopreservation in 106 patients, and DMSO wasused in 6 patients. Frozen grafts were stored in a deep freezer
in 77 patients and in liquid nitrogen in 35 patients. The mediantime from stem cell harvest to cryopreservation was 9.9 hours(range, 2.6-44.0 hours), and the median time from cryopreservation to infusion was 231.2 hours (range, 36.0-664.7 hours).
The patient characteristics grouped according to stem cellsource are shown in Table 1.

Neutrophil and Platelet Engraftment
The incidence of neutrophil engraftment at day 28 after
HCT was 91.1% (95% conﬁdence interval [CI], 83.8%-95.2%),
treating death without engraftment as a competing risk, andamong 109 patients (excluding 3 patients who died early, at
16, 23, and 35 days after HCT), all but 1 patient achieved neutrophil engraftment within 60 days after HCT. The incidence ofplatelet engraftment was 33.0% (95% CI, 24.5%-41.8%) at day 28
post-HCT and 72.3% (95% CI, 62.9%-79.7%) at day 60 post-HCT.
The time to neutrophil engraftment and time to platelet
engraftment were shorter in PBSCT than in BMT (median,
16 days [range, 14-18 days] versus 19 days [range, 18-21 days]
for neutrophil engraftment and 27 days [range, 24-49 days]
versus 35 days [range, 32-39 days] for platelet engraftment),

Figure 1. Time to neutrophil (A) and platelet (B) engraftment according to thestem cell source.

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Table 2
Univariate and multivariate analyses for neutrophil engraftment after unrelated BMTtime from stem cell harvest to cryopreservation was not significant in the multivariate analysis.
Comparison of BMT with Cryopreservation and without
Cryopreservation
We compared neutrophil engraftment between the 83
patients who received cryopreserved BM grafts and 3105
patients who received BM grafts without cryopreservation.
The median day to engraftment was 18 in the cryopreserved
BM group and 19 days in the noncryopreserved BM group(P = .32), and the engraftment curves were superimposed(Figure 3A). After matching for recipient age, donor age, background disease, background disease status, and HLA mismatch,
55 pairs were matched, and no signiﬁcant difference in neutrophil engraftment was observed (median 18 and 19 days,
respectively; P = .32) (Figure 3B).
DISCUSSION
During the COVID-19 pandemic, donor grafts are more frequently cryopreserved to ensure graft availability before starting a conditioning regimen [10]. The safety of cryopreservation
has been established in allogeneic PBSCT, and most allogeneic

PBSCTs are safely performed using cryopreserved grafts [6]. In
addition, a recent large retrospective study from Princess Margaret Cancer Centre showed no difference in engraftment orsurvival between cryopreserved and fresh PBSC grafts [11]. On
the other hand, there has been less experience with allogeneic
BMT using cryopreserved grafts. Hamadani et al. [12] recently
reported that graft cryopreservation did not affect the outcomesof HCT using post-transplantation cyclophosphamide, but mostrecipients received PBSC grafts. In allogeneic HCT for severe
aplastic anemia, the use of cryopreserved graft was associatedwith increased graft failure and inferior overall survival [13]. In
this study, two-thirds of the patients received BM grafts. Therefore, the safety of cryopreservation of allogeneic BM grafts has
not been established. In addition, most previous large studies
analyzed HCT from related donors, and thus in unrelated HCT,in which the time from stem cell harvest to cryopreservation islonger than in related HCT, even the safety of the cryopreservation of PBSC grafts is unclear. Lioznov et al. [14] suggested that
PBSC grafts are more sensitive to cryopreservation after transport and storage. A recent analysis from Australia showed thata longer transit time before cryopreservation was associatedwith inferior post-thaw viability of CD34+ cells [15].

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Y. Kanda et al. / Transplantation and Cellular Therapy 27 (2021) 664.e1 664.e6

Figure 3. Time to neutrophil engraftment after unrelated BMT with or without cryopreservation, before (A) and after (B) matching for recipient age, donorage, background disease, background disease status, and HLA mismatch.

Figure 2. Time to neutrophil engraftment after unrelated BMT, according to
the number of cryopreserved nucleated cells (A), the presence of HLA mismatch (B), and the time from stem cell harvest to cryopreservation (C).

In this study, however, when we excluded 3 patients withearly death, all but 1 patient achieved neutrophil engraftment.
Therefore, both unrelated BM and PBSC grafts appeared to besafely cryopreserved. The transit time before cryopreservationin the longest group was between 11.0 and 32.1 hours in the
BMT recipients and between 26.6 and 44.0 hours in the PBSCT
recipients, suggesting that a transit time of up to 24 hours
before cryopreservation may be acceptable. An unexpectedﬁnding in this study was the relationship between the shortertime from stem cell harvest to cryopreservation and delayedengraftment in the univariate analysis. However, this relationship was not conﬁrmed in the multivariate analysis. We considered that the coincidentally higher proportion of patientswith nonremission diseases and the lower number of cryopreserved nucleated cells in the shortest transit time group,
although not statistically signiﬁcant, resulted in the signiﬁcantly delayed engraftment in the univariate analysis, but notin the multivariate analysis adjusted for these factors.
The cryopreserved nucleated cell number, rather than theharvested nucleated cell number, was associated with neutrophil engraftment after unrelated cryopreserved BMT. This may
reﬂect that the number of cryopreserved cells is more closely
related to the number of hematopoietic precursor cells, by

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condensing mononuclear cells before cryopreservation. The
number of cryopreserved mononuclear cells in the lowest group
ranged between 0.31 and 0.54 £ 1010. Although we did nothave information on patient body weight, if we assume an average body weight of 60 kg and consider that the median recovery
rate of mononuclear cells before cryopreservation was 0.50 inthis study, the number of cryopreserved mononuclear cells in
the lowest group corresponds to a range of 1.0 to 1.8 £ 108/kg
harvested nucleated cells. A limitation of this study is the lack ofdata on the viability of graft cells before and after cryopreservation, but the fact that the number of mononuclear cells before
cryopreservation was signiﬁcantly associated with neutrophil
engraftment suggests that the cryopreservation proceduremight not have strongly affected graft quality.
In conclusion, unrelated donor BM and PBSC grafts can besafely cryopreserved even after the transit time from the harvest center to the transplantation center. In the current
COVID-19 pandemic, cryopreservation can be considered as an
option while balancing the risks and beneﬁts of the procedure.
ACKNOWLEDGMENTS
Financial disclosure: Nothing to disclose.
Conﬂict of interest statement: There are no conﬂicts of interest to report.
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