key: cord-0763134-8bqquww7
authors: Kanda, Yoshinobu; Inoue, Masami; Uchida, Naoyuki; Onishi, Yasushi; Kamata, Reiko; Kotaki, Mika; Kobayashi, Ryoji; Tanaka, Junji; Fukuda, Takahiro; Fujii, Nobuharu; Miyamura, Koichi; Mori, Shin-Ichiro; Mori, Yasuo; Morishima, Yasuo; Yabe, Hiromasa; Kodera, Yoshihisa
title: 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
date: 2021-05-05
journal: Transplant Cell Ther
DOI: 10.1016/j.jtct.2021.04.022
sha: 0d8edb5d1d61c79f032c4cc0872db2d714361059
doc_id: 763134
cord_uid: 8bqquww7

Background: During the COVID-19 pandemic, donor hematopoietic stem cell grafts are frequently cryopreserved to ensure the availability of graft before starting a conditioning regimen. However, the safety of cryopreservation has been controversial in unrelated hematopoietic stem cell transplantation, especially for bone marrow grafts. In addition, in unrelated HSCT, the effect of the time from harvest to cryopreservation of donor grafts required for the transportation of donor graft has not been fully clarified. Study design: In this study, we retrospectively analyzed the first 112 patients with available data who underwent cryopreserved unrelated blood and marrow transplantation through the Japan Marrow Donor Program during the COVID-19 pandemic. Results: There were 112 patients, including 83 who received bone marrow (BM) graft and 29 who received peripheral blood stem cell (PBSC) graft. The median time 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. 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 one achieved neutrophil engraftment within 60 days after HSCT. The time-to-neutrophil and time-to-platelet engraftment were shorter in PBSCT than in BMT, but these differences were not statistically significant (P=0.064 and P=0.18). Multivariate analysis among BM recipients revealed that a higher number of frozen nucleated cells 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. Conclusion: Unrelated donor BM and PBSC grafts can be safely 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.

We retrospectively analyzed the outcome of unrelated cryopreserved blood and marrow transplantation, and concluded that unrelated donor BM and PBSC grafts can be safely cryopreserved even after the transit time from the harvest center to the transplantation center.

Background: During the COVID-19 pandemic, donor hematopoietic stem cell grafts are frequently cryopreserved to ensure the availability of graft before starting a conditioning regimen. However, the safety of cryopreservation has been controversial in unrelated hematopoietic stem cell transplantation, especially for bone marrow grafts. In addition, in unrelated HSCT, the effect of the time from harvest to cryopreservation of donor grafts required for the transportation of donor graft has not been fully clarified.

Study design: In this study, we retrospectively analyzed the first 112 patients with available data who underwent cryopreserved unrelated blood and marrow transplantation through the Japan Marrow Donor Program during the COVID-19 pandemic.

Results: There were 112 patients, including 83 who received bone marrow (BM) graft and 29 who received peripheral blood stem cell (PBSC) graft. The median time 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. 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 one achieved neutrophil engraftment within 60 days after HSCT. The time-to-neutrophil and time-to-platelet engraftment were shorter in PBSCT than in BMT, but these differences were not statistically significant (P=0.064 and P=0.18). Multivariate analysis among BM recipients revealed that a higher number of frozen nucleated cells 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.

Conclusion: Unrelated donor BM and PBSC grafts can be safely 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.

Key words cryopreservation, COVID-19, bone marrow transplantation, peripheral blood stem cell transplantation, Japan Marrow Donor Program Introduction Hematopoietic stem cell transplantation (HCT) has been established as a curative treatment strategy for a variety of hematological disorders. Hematopoietic stem cell grafts are obtained from the patients themselves in autologous HCT, whereas they are obtained from healthy donors in allogeneic HCT. In autologous HCT, cryopreservation techniques are required, as patients should receive conditioning regimens between the stem 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 (HES) and a lower concentration of DMSO was also investigated for the cryopreservation of hematopoietic 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 HES 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 transplantation (PBSCT), donor grafts are often cryopreserved in order 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 transplantation (BMT) to avoid unnecessary mononuclear cell separation and cryopreservation (6) . In addition, cryopreservation is generally not permitted in unrelated BMT through the Japan Marrow Donor Program (JMDP), as the cryopreservation of donor grafts may increase the number of unused grafts.

In 2020, however, during the COVID-19 pandemic, JMDP introduced the cryopreservation of donor grafts as an exception, because donor cell collection may be practically impossible during this period. Although the cryopreservation of allogeneic donor grafts has been shown to be safe and effective in small case series, sufficient information is still not available, especially for the preservation of donor BM grafts (7, 8) .

In addition, in unrelated HCT, the effect of the time from harvest to cryopreservation of donor grafts required for the 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.

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 transplant centers under room temperature. All the stem cells were cryopreserved at the transplant centers upon receipt.

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 one patient without response to the questionnaire were included in this study. This study was approved by the ethics committee of the Japan Marrow Donor Program.

The primary endpoint was neutrophil engraftment, defined as the first of the three consecutive days with an absolute neutrophil count of at least 0.5 x 10 3 /μL, while platelet engraftment was defined as the first day with a platelet count exceeding 20 x 9 to compare categorical variables 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 the Fine-Gray proportional-hazard modelling based on an available-case analysis for missing data, including variables with P values less than 0.15 in univariate analysis as independent variables. Information about the use of granulocyte colony-stimulating factor (G-CSF) was not obtained, and therefore, background diseases were grouped into myeloid malignancies and others, and this was also included as an independent variable as a substitute for the use of (G-CSF), since the use of G-CSF was avoided for myeloid malignancies in some centers.

In addition, the patients who received cryopreserved BM graft were compared with those who received BM graft without cryopreservation between January 2016 and December 2018 with and without matching for 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 of the standard deviations.

All P values were two-sided and P values less than 0.05 were considered to indicate statistical significance. All statistical analyses were performed with EZR (version 1·54, Jichi Medical University Saitama Medical Center) (9) . Table 1 .

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, and among 109 patients excluding 3 patients who died early on 16, 23, and 35 days after HCT, respectively, all but one achieved neutrophil engraftment within 60 days after HCT. The incidence of platelet engraftment was 33.0% (95% CI 24.5%-41.8%) at day 28 and 72.3% (95% CI 62.9%-79.7%) at day 60 after HCT. The time-to-neutrophil and time-to-platelet engraftment were shorter in PBSCT than in BMT (16 days in median, range 14-18 days vs. 19 days in median, range 18-21 days for neutrophil engraftment, and 27 days in median, range 24-49 days vs. 35 days in median, range 32-39 days for platelet engraftment), but these differences were not statistically significant (P=0.064 and P=0.18, Figure 1 ).

Factors that predicted neutrophil engraftment after HCT were analyzed only in BMT, as the number of PBSCT recipients was limited. Continuous variables were equally divided into three groups and treated as categorical variables. Univariate 13 analysis revealed that the number of frozen nucleated cells, not harvested cells, and the presence of HLA mismatch were significantly associated with neutrophil engraftment (Table 2, Figure 2A, B) . In addition, time from stem cell harvest to cryopreservation was also significant, but, contrary to our expectations, the shortest duration was associated with a delayed neutrophil engraftment ( Figure 2C ).

Multivariate analysis revealed that a higher number of frozen nucleated cells and the presence of HLA mismatch were independently associated with neutrophil engraftment. Time from stem cell harvest to cryopreservation was not significant in the multivariate analysis.

We compared neutrophil engraftment of the 83 patients who received cryopreserved BM graft with 3105 patients who received BM graft without cryopreservation. The median days to engraftment were 18 and 19 days (P=0.32), and the engraftment curves were superimposing ( 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 was observed in the neutrophil engraftment ( Figure 3B , 18 and 19 days in median, P=0.32).

During the COVID-19 pandemic, donor grafts are more frequently cryopreserved to ensure the availability of graft before starting a conditioning regimen (10) . The safety of cryopreservation has been established in allogeneic PBSCT, and most allogeneic PBSCT has been safely performed using cryopreserved grafts (6) . In addition, a recent large retrospective study from Princess Margaret Cancer Centre showed no difference in engraftment or survival between cryopreserved and fresh PBSC grafts (11) . On the other hand, there has been less experience with allogeneic BMT using cryopreserved graft.

Hamadani et al. recently reported that graft cryopreservation did not affect the outcome of HCT using post-transplantation cyclophosphamide, but most recipients received PBSC graft (12) . In allogeneic HCT for severe aplastic anemia, the use of cryopreserved graft was associated with increased graft failure and inferior overall survival (13) . In this study, two thirds of the patients received BM graft. Therefore, the safety of cryopreservation of allogeneic BM grafts has not been established. In addition, most previous large studies analyzed HCT from a related donor, and thus, in unrelated HCT, in which the time from stem cell harvest to cryopreservation is longer than in related HCT, even the safety of the cryopreservation of PBSC graft is unclear. Lioznov et al.

suggested that PBSC grafts are more sensitive to cryopreservation after transport and storage(14). A recent analysis from Australia showed that a longer transit time before cryopreservation was associated with inferior post-thaw viability of CD34-positive cells(15).

In this study, however, when we excluded 3 patients with early death, all but one patient achieved neutrophil engraftment. Therefore, both unrelated BM and PBSC grafts appeared to be safely cryopreserved. The transit time before cryopreservation in the longest group was between 11.0 and 32.1 hours in BMT recipients and between 26.6 and 44.0 hours in PBSCT recipients, suggesting that a transit time of up to 24 hours may be acceptable before cryopreservation. An unexpected finding in this study was the relationship between the shorter time from stem cell harvest to cryopreservation and delayed engraftment in the univariate analysis. However, this relationship was not confirmed in the multivariate analysis. We considered that the coincidentally higher 

Successful engraftment of cryopreserved autologous bone marrow in patients with malignant lymphoma

The effects of a simplified method for cryopreservation and thawing procedures on peripheral blood stem cells

Comparative analysis of engraftment after cryopreservation of peripheral blood stem cell autografts by controlled-versus uncontrolled-rate methods

Unfractionated human marrow cell cryopreservation using dimethylsulfoxide and hydroxyethyl starch

A simplified method for cryopreservation of peripheral blood stem cells at -80 degrees C without rate-controlled freezing

Adverse events associated with infusion of hematopoietic stem cell products: A prospective and multicenter surveillance study

Use of cryopreserved bone marrow in unrelated allogeneic transplantation

Use of cryopreserved bone marrow in allogeneic bone marrow transplantation

Investigation of the freely available easy-to-use software 'EZR' for medical statistics

Transplantation of Allogeneic Cryopreserved Hematopoietic Cell Grafts during the COVID-19 Pandemic: a National Marrow Donor Program Perspective

Fresh vs. frozen allogeneic peripheral blood stem cell grafts: A successful timely option

Graft Cryopreservation Does Not Impact Overall Survival after Allogeneic Hematopoietic Cell Transplantation Using Post-Transplantation Cyclophosphamide for Graft-versus-Host Disease Prophylaxis

Cryopreserved Grafts for Severe Aplastic Anemia

This study was approved by the ethics committee of the Japan Marrow Donor Program.

The authors declare no conflicts of interest associated with this manuscript