Cryopreserved Versus Fresh Peripheral Blood Allogeneic Stem Cell Transplantation Outcomes in Patients Receiving Post-transplant Cyclophosphamide for Graft-versus-host Prophylaxis during the COVID-19 Pandemic: A Single Center Experience

Abstract

Background/objectiveCryopreservation of grafts is not common practice in allogeneic hematopoietic stem cell transplant (HSCT) recipients. However, our center had to use cryopreserved cells for allogeneic HSCT during the COVID-19 pandemic to avoid delays in transplantation due to uncertainty regarding patient and donor exposures.Study designWe retrospectively evaluated post-transplant engraftment and survival outcomes of adult patients who received cryopreserved versus fresh allografts during the COVID-19 pandemic.ResultsFifty-five patients with hematologic malignancies received either cryopreserved (n = 34) or fresh (n = 21) allogeneic HSCT using peripheral blood stem cells between January 2020 and December 2020. At a median follow-up time of 15 months, cryopreserved allograft recipients had significantly lower overall survival (OS) (p = 0.02). They also experienced significantly delayed neutrophil (p = 0.01) and platelet engraftments (p < 0.0001), as well as higher red blood cell transfusion-dependence after day + 60 (67.6% vs. 28.6%; p = 0.01). Significantly more cryopreserved allograft recipients received donor lymphocyte infusion than fresh allograft recipients (35.3% vs. 4.8%, p = 0.01). Neither relapse-free survival nor non-relapse mortality differed significantly between the two groups.Conclusion
Cryopreservation of allografts in combination with post-transplant cyclophosphamide may negatively affect engraftment and OS outcomes in HSCT recipients.

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Vol.:(0123456789)
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International Journal of Hematology
https://doi.org/10.1007/s12185-022-03493-8
ORIGINAL ARTICLE
Cryopreserved versusfresh peripheral blood allogeneic stem cell
transplantation outcomes inpatients receiving post‑transplant
cyclophosphamide forgraft‑versus‑host prophylaxis
duringtheCOVID‑19 pandemic: asingle center experience
MengniGuo1· JieyingLiu1· PamelaClark2· SarfrazAhmad3· RushangPatel2· JuanCarlosVarela2· ShahramMori2
Received: 21 April 2022 / Revised: 7 November 2022 / Accepted: 7 November 2022
© Japanese Society of Hematology 2022
Abstract
Background/objective Cryopreservation of grafts is not common practice in allogeneic hematopoietic stem cell transplant
(HSCT) recipients. However, our center had to use cryopreserved cells for allogeneic HSCT during the COVID-19 pandemic
to avoid delays in transplantation due to uncertainty regarding patient and donor exposures.
Study design We retrospectively evaluated post-transplant engraftment and survival outcomes of adult patients who received
cryopreserved versus fresh allografts during the COVID-19 pandemic.
Results Fifty-five patients with hematologic malignancies received either cryopreserved (n = 34) or fresh (n = 21) alloge-
neic HSCT using peripheral blood stem cells between January 2020 and December 2020. At a median follow-up time of
15months, cryopreserved allograft recipients had significantly lower overall survival (OS) (p = 0.02). They also experienced
significantly delayed neutrophil (p = 0.01) and platelet engraftments (p < 0.0001), as well as higher red blood cell transfusion-
dependence after day + 60 (67.6% vs. 28.6%; p = 0.01). Significantly more cryopreserved allograft recipients received donor
lymphocyte infusion than fresh allograft recipients (35.3% vs. 4.8%, p = 0.01). Neither relapse-free survival nor non-relapse
mortality differed significantly between the two groups.
Conclusion Cryopreservation of allografts in combination with post-transplant cyclophosphamide may negatively affect
engraftment and OS outcomes in HSCT recipients.
Keywords Cryopreservation· Engraftment· Allogeneic· Transplantation· COVID-19
Introduction
Cryopreservation of hematopoietic progenitor cells (HPCs)
in autologous and cord blood transplantation is a common
practice. On the other hand, fresh allograft infusion is stand-
ard in allogeneic hematopoietic stem cell transplantation
(allo-HSCT) [1].
In the pre-COVID-19 era, cryopreserved allo-HSCT was
typically reserved for donor accommodation, and unantici-
pated changes in patient status. Transplanting cryopreserved
HPCs provides several advantages. It offers the ability to
evaluate HPC quality while ensuring an adequate amount is
collected prior to the initiation of conditioning regimen, and
it provides a solution to logistical challenges such as flex-
ibility of international transportation and other unforeseen
obstacles [2]. These benefits are offset by decreased viability
of cryopreserved cells, the toxic effect of longer term cell
exposure to dimethyl sulfoxide (DMSO), added cost, and
more graft manipulation [3, 4].
Although there is plenty of evidence on the safety and
efficacy of cryopreserved autograft [5], data on the outcomes
of cryopreserved allo-HSCT has been mixed and limited
[2–14]. Some studies showed increased risk of graft failure
* Shahram Mori
shahram.mori@HCAhealthcare.com
1 Department ofInternal Medicine AdventHealth, Orlando,
FL32804, USA
2 Blood andMarrow Transplant Center, AdventHealth Cancer
Institute, AHMG2415 N. Orange Ave., Suite 601, Orlando,
FL32804, USA
3 Gynecologic Oncology Program, AdventHealth Cancer
Institute, Orlando, FL32804, USA

M.Guo et al.
1 3
and higher mortality rate, while others showed no impact on
survival or engraftment.
Due to travel restrictions imposed by the COVID-19 pan-
demic, transplant centers were faced with the uncertainty
of graft and/or donor transport. To ensure allo-HSCT graft
availability at the scheduled infusion time, the National
Marrow Donor Program® (NMDP) required all unrelated
donor (URD) products to be cryopreserved prior to initia-
tion of conditioning regimen starting on March 30, 2020.
This requirement was relaxed to allow fresh allografts to
be infused for collections scheduled on or after August 10,
2020 [15]. Although cryopreservation was not required for
related donor products, concern over COVID-19 infections
forced many transplant centers to cryopreserve these prod-
ucts as well.
We retrospectively examined the effect of cryopreserva-
tion on the outcomes of allo-HSCT in 2020 at our institu-
tion. The outcomes in 55 adult patients with hematologic
malignancies who underwent either cryopreserved or fresh
allo-HSCT using peripheral blood stem cells (PBSC) as the
graft source between January 2020 and December 2020 were
analyzed.
Materials andmethods
Study population
A single center, retrospective cohort study was conducted.
All adult patients undergoing allo-HSCT for hematologic
malignancies between January 1, 2020 and December 31,
2020 at our transplant center were included in this analy-
sis. All patients received PBSC grafts and were censored at
last documented follow-up visit, with a minimum follow-
up period of 12months in surviving patients. Donors were
tested for SARS-CoV-2 by RT-PCR prior to graft collec-
tion, and recipients were tested for SARS-CoV-2 by RT-PCR
before starting the conditioning regimen. This study was
approved by the AdventHealth Institutional Review Board.
All patients provided written informed consent.
HPC product collection, cryopreservation
procedure, analysis, andinfusion
All allogeneic HPC products were collected via peripheral
blood apheresis from granulocyte-colony stimulating factor
(G-CSF) mobilized donors. Collections took place at our
institution or at other NMDP centers. At our institution,
all collections were conducted on Spectra Optia apheresis
instruments (Terumo BCT, Lakewood CO).The HPC prod-
ucts were then processed (plasma depleted, if required) and
either infused fresh or cryopreserved and thawed prior to
infusion in a 37°C water bath using gentle agitation to a
slushy consistency. All cryopreserved products were mixed
with a freezing media containing Plasmalyte, 5% human
serum albumin (HSA) (Baxter, Deerfield, IL) or concur-
rent plasma and DMSO to achieve a final concentration of
10% DMSO and frozen in a controlled rate freezer (Pla-
nar, Kryo 560, Middlesex, UK) to approximately -90°C,
then transferred to a liquid nitrogen storage freezer (less
than -165°C).All HPC products were frozen within 24h
of collection if collected on-site, within 48h if collected
domestically, and within 72h if collected internationally.
All cryopreserved products were thawed either in the cell
therapy facility or at bedside and were infused within 60min
of thawing. All HPC products were tested for CD34+ and
CD3+, then were calculated and reported as cells × 106/L and
cells × 106/kg of recipient body weight. CD34+concentration
(per µL), frequency (among CD45+events) and viability [fre-
quency of 7-amino-actinomycin D (7-AAD)] were assessed
using the Stem Cell Enumeration (SCE) kit (Becton–Dick-
inson, Heidelberg, Germany) as a single-platform.
Endpoints Definition
Engraftment kinetics, overall survival (OS), and relapse-free
survival (RFS) were evaluated. OS was defined as the time
from transplantation until death. RFS was defined as the time
from transplantation until disease relapse. Other specific sec-
ondary outcomes included neutrophil engraftment, platelet
engraftment, graft failure, poor graft function, cumulative
incidence of acute GVHD (aGVHD) and chronic GVHD
(cGVHD), and non-relapse mortality (NRM). Neutrophil
engraftment was defined as the first of three consecutive
days with an absolute neutrophil count (ANC) > 0.5 × 109/L,
and platelet engraftment was defined as the first of three
consecutive days with platelet count > 20 × 109/L without
transfusions in the prior 7days. Primary graft failure was
defined as never having achieved an ANC ≥ 0.5 × 109/L
in the absence of disease relapse. Secondary graft failure
was defined as loss of a previously functioning graft asso-
ciated with loss of full donor chimerism. Poor graft func-
tion was defined as two or three cytopenias for > 2weeks,
after day + 28 in the presence of donor chimerism > 5%
[16]. Acute and chronic GVHD were diagnosed and graded
according to standard criteria [17, 18]. Complete remission
(CR) was defined by standard morphological criteria. Liquid
transit time was defined as the time from completion of stem
cell collection to start of stem cell cryopreservation. Time
to infusion was defined as the time from completion of stem
cell collection to completion of stem cell infusion.
Statistical analysis
We used the Kaplan–Meier analysis to compare the OS, RFS,
and NRM of the patients transplanted with cryopreserved or

Cryopreserved versusfresh peripheral blood allogeneic stem cell transplantation outcomes…
1 3
fresh stem cells. We plotted accumulated incidence for the
time to neutrophil engraftment and platelet engraftment.
Univariate linear regression was used to evaluate the corre-
lation between liquid transit time and engraftment. We used
log-rank test for OS, RFS, NRM, and time to engraftment.
Censor date was set at March 20, 2022. The Cox propor-
tional hazards model was used for the multivariant analysis
for the OS, RFS, and NRM. The multivariant analysis of
OS, RFS, and NRM included type of transplantation (cryo-
preserved vs. fresh), age, disease status at the time of trans-
plant (CR vs. non-CR), moderate to severe cGVHD, and
CMV reactivation by day 100. We used the Mann–Whitney
U test to compare the continuous variables. Chi square test
and Fisher’s exact test were used to compare the categorical
variables between patients who had cryopreserved or fresh
transplantation. All analyses were done in R version 4.0.
Results
Patient andgraft baseline characteristics
A total of 55 patients who underwent allo-HSCT in the year
of 2020 were analyzed in the study. Thirty-four patients
(61.8%) received cryopreserved HPC products while 21
(38.2%) patients received fresh HPC products. All patients
received post-transplant cyclophosphamide on day + 3 and
day + 4, as well as tacrolimus and mycophenolate mofetil for
GVHD prophylaxis. The median duration of follow-up was
15months (range: 3–24) for the entire cohort, 14months
(range: 3–22) for the cryopreserved cohort, and 15months
(range: 8–24) for the fresh cohort. Baseline characteristics
are summarized in Table1.
There were no statistically significant differences between
the two groups in terms of age, gender, race, graft source,
ECOG score, distribution of hematologic diagnosis, disease
risk index, ABO compatibility, disease status or MRD status
prior to transplantation. Conditioning intensity and regimen
were comparable between the two groups. Among the AML
patients, there was a statistically significant difference in the
AML cytogenetics risk, with a higher proportion of patients
with favorable and adverse risk in the cryopreserved allo-
graft group, while more patients in the fresh allograft group
had intermediate risk (p < 0.03). Table2 shows graft char-
acteristics, the median CD34+, CD3+, total nucleated cell
(TNC) and percent viability of cells infused.
Hematopoietic recovery
We observed two graft failures in the recipients of cryo-
preserved grafts. One patient suffered primary graft failure
while the other experienced secondary graft failure. The two
patient’s baseline characteristics and outcomes are shown in
Table3. In patients who achieved engraftment, a statistically
significant longer time to neutrophil and platelet engraft-
ments were observed in the cryopreserved as compared
to the fresh group (p = 0.01 and p < 0.0001, respectively)
(Fig.1a and Fig.1b). In the cryopreserved group, no correla-
tion was observed between delayed engraftment and longer
liquid transit time (p = 0.42, and p = 0.14, respectively)
(Table4).
Poor graft function occurred in 44.1% of cryopreserved
allografts, while only 28.6% of fresh allografts had poor
graft function (p = 0.27). We observed a significant differ-
ence in the cumulative incidence of red blood cell (RBC)
transfusions after day + 60 between the two groups (67.6%
in the cryopreserved group vs. 28.6% in the fresh group,
p = 0.01). The difference in the cumulative incidence of
donor lymphocyte infusion (DLI) between the two groups
was also significant (p = 0.03). Of the 12 patients who
required DLI in the cryopreserved group, six (50%) were
due to poor chimerism or poor graft function, while the rest
(50%) were due to relapse. Only one patient (4.8%) in the
fresh group received DLI for relapse treatment. No differ-
ence was found in terms of cumulative incidence of second
allo-HSCT (p = 1.00). Four patients (11.8%) in the cryopre-
served allograft group underwent a second allo-HSCT, two
(50%) as a result of relapse and two (50%) due to graft fail-
ure. In the fresh allograft group, two patients (9.6%) under-
went second allo-HSCT due to relapse.
GVHD
There were no significant differences in the cumulative inci-
dence of aGVHD or cGVHD between the two groups. The
cumulative incidence of aGVHD grade II-IV was 58.8%
in the cryopreserved group and 42.9% in the fresh group
(p = 0.28). Grade III-IV aGVHD was observed in four
(11.7%) and two (9.5%) patients (p = 1.00), respectively.
The cumulative incidence of cGVHD was 64.7 and 76.2%
(p = 0.55), and the cumulative incidence of moderate to
severe cGVHD was 49.4% and 9.5% (p = 0.10) in the two
groups, respectively (Table5).
NRM
The cumulative incidence of NRM at 1-year was 12.8%
in the cryopreserved allograft group and 6.3% in the fresh
allograft group, which did not reach statistical significance
[hazard ratio (HR) = 0.94 with 95% confidence interval (CI)
0.29–34.18, p = 0.35] (Fig.2). In the cryopreserved allo-
graft group, three patients died from infection and one died
from cardiac arrest, while both patients in the fresh allograft
group died from infection. The incidence of cytomegalovirus
(CMV) reactivation by day + 100 was not significantly dif-
ferent between the two groups (p = 0.46).

M.Guo et al.
1 3
Table 1 Baseline characteristics
of patient population Characteristic Cryopreserved
n = 34
Fresh
n = 21
p-Value
Median age, years (range) 55 (21–74) 53 (19–70) 0.70
Gender, n (%) 0.16
Male 22 (64.7) 9 (42.9)
Female 12 (35.3) 12 (57.1)
Race, n (%) 1.00
White 25 (73.5) 15 (71.4)
African American 4 (11.8) 2 (9.5)
Hispanic 1 (2.9) 0 (0.0)
Other 4 (11.8) 4 (19.0)
Graft source, n (%) 0.33
MRD 4 (11.8) 1 (4.8)
MUD 18 (52.9) 8 (38.1)
MMRD 0 (0.0) 1 (4.8)
Haploidentical 12 (35.3) 11 (52.3)
ABO compatibility, n (%) 0.16
Matched 12 (35.3) 13 (61.9)
Minor mismatch 11 (32.4) 4 (19.0)
Major mismatch 11 (32.4) 4 (19.0)
Bidirectional 0 (0.0) 0 (0.0)
Disease risk index, n (%) 0.09
Low 8 (23.5) 3 (14.3)
Intermediate 9 (26.5) 12 (57.1)
High 17 (50.0) 6 (28.6)
Disease, n (%) 0.49
AML 21 (61.8) 10 (47.6)
MDS 5 (14.7) 3 (14.3)
ALL 2 (5.9) 5 (23.8)
Other 6 (17.6) 3 (14.3)
Disease status, n (%) 0.75
CR 25 (73.5) 17 (81.0)
Non-CR 9 (26.5) 4 (19.0)
Minimal residual disease prior to HSCT 0.44
No 19/25 (76.0) 15/17 (88.2)
Yes 6/25 (24.0) 2/17 (11.8)
AML cytogenetics 0.03
Favorable 8/21 (38.1) 1/10 (10.0)
Intermediate 4/21 (19.0) 5/10 (50.0)
Adverse 9/21 (42.9) 2/10 (20.0)
Unknown 0/21 (0.0) 2/10 (20.0)
ECOG, n (%) 0.52
 < 2 32 (94.1) 21 (100.0)
 ≥ 2 2 (5.9) 0 (0.0)
Conditioning intensity, n (%) 1.00
Myeloablative 27 (79.4) 17 (81.0)
RIC 7 (20.6) 4 (19.0)
Conditioning regimen 0.13

Cryopreserved versusfresh peripheral blood allogeneic stem cell transplantation outcomes…
1 3
Overall andrelapse‑free survivals
With a median follow-up of 15months for the entire cohort,
there was a statistically significant difference in OS favoring
the fresh allograft recipients (p = 0.02) (Fig.3a). The effect
of cryopreservation remained significant after multivari-
ate adjustment for age, disease status at time of transplant,
moderate to severe cGVHD and CMV infection by day 100
(HR 2.27 with 95% CI 1.23–16.51, p = 0.02). By day + 100,
94.1 and 100% of patients were alive, while at 1-year post-
transplant, 67.6% and 90.4% of patients were alive in the
cryopreserved and fresh allograft groups, respectively.
No significant difference in RFS was noted (p = 0.11,
Fig.3b; after multivariate adjustment, HR 1.61 with 95%
CI 0.84–6.07, p = 0.11). During the study period, relapse
occurred in 15 patients (44.1%) who received cryopreserved
cells with a median time to relapse of 5-months (range:
1–12), while six patients (28.6%) in the fresh group relapsed
with a median time to relapse of 7-months (range: 1–11).
By the censor date, 11 patients had died from relapse in the
cryopreserved allograft group, while two patients receiving
fresh allografts died due to relapse. Although the cumula-
tive incidence of relapse was higher in the cryopreserved
allograft group, this did not reach statistical significance
(p = 0.25, Table5).
Discussion
Cryopreservation of PBSC is not a common practice in
allogenic transplantation apart from umbilical cord blood
transplantation [11], and concerns regarding cryopreserved
products adversely affecting cell viability and effector cell
AML acute myelogenous leukemia, ALL acute lymphoblastic leukemia, CR complete remission, ECOG
eastern cooperative oncology group, MDS myelodysplastic syndrome, MMRD mismatched related donor,
MRD matched related donor, MUD matched-unrelated donor, RIC reduced intensity conditioning, Flu/Bu4
fludarabine and 4days of busulfan, Flu/Bu3 fludarabine and 3days of busulfan, Flu/Bu2 fludarabine and
2days of busulfan, Flu/TBI fludarabine and total body irradiation, Flu/Cy/TBI fludarabine, cyclophospha-
mide, and total body irradiation
Table 1 (continued) Characteristic Cryopreserved
n = 34
Fresh
n = 21
p-Value
Flu/Bu4 20 (58.8) 6 (28.6)
Flu/Bu3 4 (11.8) 3 (14.3)
Flu/Bu2 2 (5.9) 1 (4.8)
Flu/TBI 3 (8.8) 8 (38.1)
Flu/Cy/TBI 4 (11.8) 3 (14.3)
Other 1 (2.9) 0 (0.0)
Liquid transit time, hours, median (range) 22.3
(1.75–84.03)
NA NA
Time to infusion, hours, median (range) 261.86
(53.33–2447.77)
24.35
(2.57–215.13)
< 0.0001
Table 2 Graft characteristics
of fresh and cryopreserved
peripheral blood stem cell
products
NA not applicable, TNC total nucleated cell
Characteristic Cryopreserved
n = 34
Fresh
n = 21
p-Value
Pre-freeze CD34+ cell dose, × 106/kg,
median (range)
5.05 (2.71–10.19) NA NA
Infused CD34+ cell dose, × 106/kg,
median (range)
5.05 (2.11–8.56) 6.36 (5.00–12.27) 0.01
Infused CD3+ cell dose, × 108/kg,
median (range)
1.34 (0.50–3.73) 1.24 (0.51–3.49) 0.53
Infused TNC dose, × 108/kg,
median (range)
5.48 (2.28–12.66) 5.00 (2.88–12.05) 0.03
Infused TNC Viability, %,
median (range)
83 (69–94) 100 (100–100) < 0.001

M.Guo et al.
1 3
function has been the main reason for performing fresh allo-
graft infusions in these patients [14].
We retrospectively assessed the impact of cryopreserva-
tion on patient outcomes during the COVID-19 pandemic
in the year 2020. Our findings suggested that cryopreser-
vation was associated with a negative impact on allogeneic
PBSC transplant. We observed a longer time to neutro-
phil engraftment and a significantly longer time to platelet
engraftment. While some studies have suggested no differ-
ence in either neutrophil or platelet engraftment between
cryopreserved and fresh allografts [2–10], our findings
are supported by others who have shown outcomes con-
sistent with our observations. A large retrospective study
conducted by Hsu etal. analyzed outcomes from 1051
frozen PBSC allografts derived from related donors and
another 678 allografts derived from matched-unrelated
donors [5]. This study showed delayed platelet engraft-
ment in cryopreserved-related donor PBSC grafts and
Table 3 Summary of cases with graft failure in cryopreserved group
AML acute myelogenous leukemia, CR complete remission, Flu/Bu4 fludarabine and 4days of busulfan, Flu/Bu3 fludarabine and 3days of busulfan, Haplo haploidentical, MUD matched-unre-
lated donor, s/p status post
Gender/Age, year Disease AML cytogenetics Remission
status at trans-
plant
Condi-
tioning
regimen
Donor type CD34+ cell dose
pre-frozen, × 106/
kg
CD34+ cell dose
infused, × 106/kg
Total nucleated cell
dose infused, × 108/
kg
Graft failure Outcomes
Male/54 AML Favorable CR Flu/Bu3 Haplo 5.43 4.34 8.54 Primary Alive s/p 2nd transplant
Female/62 AML Favorable CR Flu/Bu4 MUD 10.19 8.56 3.69 Secondary Alive s/p 2nd transplant
Fig. 1 a Cumulative incidence of neutrophil engraftment; b Cumula-
tive incidence of platelet engraftment

Cryopreserved versusfresh peripheral blood allogeneic stem cell transplantation outcomes…
1 3
Table 4 Univariate linear
regression analysis of liquid
transit time with neutrophil
and platelet engraftment in
cryopreserved patients
CI confidence interval
Neutrophil engraftment Platelet engraftment
r2 Regression coefficient
(95% CI)
p-Value r2 Regression coefficient
(95% CI)
p-Value
Liquid transit time 0.02 −27.254
(−95.68–41.20)
0.42 0.07 −1.24
(-2.91–0.42)
0.14
Table 5 Transplant outcomes
RBC red blood cells, aGVHD acute graft-versus-host disease, cGVHD chronic graft-versus-host disease,
CMV cytomegalovirus
*Two patients in the cryopreserved group were excluded from time to neutrophil engraftment: one patient
who experienced primary graft failure and one patient who experienced early relapse (within one month of
transplant).
**Four patients in the cryopreserved group did not achieve platelet engraftment and were excluded from
time to platelet engraftment: one patient who experienced primary graft failure, one patient who experi-
enced secondary graft failure, and two patients who experienced relapse.
Cryopreserved
n = 34
Fresh
n = 21
p-Value
Median time to neutrophil engraftment, days (range) 21 (13–34)* 18 (15–27) 0.05
Median time to platelet engraftment, days, (range) 39.5 (18–140)** 20 (14–382) < 0.0001
Graft function, n (%)
Graft failure 2 (5.9) 0 (0.0) 0.52
Poor graft function 15 (44.1) 6 (28.6) 0.27
Donor lymphocyte infusion, n (%) 0.03
Yes, for relapse 7 (20.6) 1 (4.8)
Yes, for poor chimerism 5 (14.7) 0 (0.0)
No 22 (64.7) 20 (95.2)
RBC transfusion after day + 60, n (%) 0.01
Yes 23 (67.6) 6 (28.6)
No 11 (32.4) 15 (71.4)
aGVHD grade II-IV, n (%) 0.28
No 20 (58.8) 9 (42.9)
Yes 14 (41.2) 12 (57.1)
aGVHD grade III-IV, n (%) 4 (11.7) 2 (9.5) 1.00
cGVHD, total, n (%) 0.55
No 22 (64.7) 16 (76.2)
Yes 12 (35.3) 5 (23.8)
cGVHD, moderate to severe, n (%) 10 (49.4) 2 (9.5) 0.10
CMV reactivation by day + 100, n (%) 0.46
No 27 (79.4) 19 (90.5)
Yes 7 (20.6) 2 (9.5)
Second stem cell transplant, n (%) 1.00
Yes 4 (11.8) 2 (9.6)
No 30 (88.2) 19 (90.5)
Relapse, n (%) 0.25
Yes 15 (44.1) 6 (28.6)
No 19 (55.9) 15 (71.4)
Death, n (%) 0.06
Yes 15 (44.1) 4 (19.0)
No 19 (55.9) 17 (81.0)

M.Guo et al.
1 3
delayed engraftment of both neutrophils and platelets in
cryopreserved unrelated donor PBSC grafts. Dagdas etal.
also showed longer neutrophil engraftment kinetics using
frozen products [11].
Two graft failures were observed in the cryopreserved
group while none were observed in the fresh group. Impor-
tantly, poor graft function occurred in 44.1% of cryopre-
served allografts as compared to 28.6% of fresh allografts.
Twice as many cryopreserved recipients required prolonged
RBC transfusions after day + 60 (67.6 vs. 28.6%, respec-
tively). Additionally, more cryopreserved recipients required
DLI than recipients of fresh stem cells. Consistent with our
data, Lioznov etal. observed more graft failures in cryo-
preserved allograft recipients compared to fresh allograft
recipients (27 vs 1.4%, respectively) [13].
With a median follow-up of 15months, a significant
decrease in OS was noted in the cryopreserved group com-
pared with the fresh group, while NRM and RFS did not
reach statistical significance. Hsu etal. found minimal
impact of cryopreservation in related PBSC allografts in
terms of NRM, relapse, OS and RFS, but noted increased
relapse as well as decreased RFS and OS in cryopreserved
unrelated PBSC allografts [5]. The authors suspected a
potentially longer transit time of unrelated donor grafts prior
to cryopreservation, the decline in post-thaw colony forming
units, and other patient-related reasons for cryopreservation
may have played a role in the worse outcomes noted in their
study [5]. In our study, although the cryopreserved cells had
a slightly lower number of CD34+ cell infused, as expected
due to the freeze-thawing process, this cannot explain our
findings. Prior data have shown that the minimum acceptable
dose of CD34+ cells for successful transplantation is above
2.0 × 106 cells/kg [19]. Even though all of our post-thawed
products met this criteria, the transplant outcomes differed
significantly between the cryopreserved and fresh groups,
suggesting in cryopreserved allografts, other factors besides
CD34+ cell dose may play a role. The cryo-protective agent
used in the cryopreservation process could result in adverse
reactions and potentially have a negative impact on survival
outcomes [20]. It is important to note that all patients in
our study received post-transplant cyclophosphamide as part
of GVHD prophylaxis. As mentioned above, Linzov etal.,
demonstrated that graft failures in the cryopreserved cohort
appeared to occur more commonly in patients who received
allografts with low aldehyde dehydrogenase bright CD34+
cells [13]. Given the effect of aldehyde dehydrogenase on
the metabolism of cyclophosphamide, it is plausible that a
combination of cryopreservation and post-transplant cyclo-
phosphamide may adversely affect engraftment. Further
investigation is needed to test this hypothesis.
Published data for the incidence of aGVHD in cryopre-
served allo-HSCT are conflicting. Parody etal. reported a
higher incidence and earlier onset of aGVHD in patients
receiving cryopreserved PBSC while the difference in the
incidence of cGVHD was not significant [12]. Medd etal.
reported comparable incidence of aGVHD but a non-sig-
nificant increase in extensive cGVHD in the cryopreserved
Fig. 2 Non-relapse mortality (NRM)
Fig. 3 a Overall survival; b Relapse-free survival

Cryopreserved versusfresh peripheral blood allogeneic stem cell transplantation outcomes…
1 3
PBSC group [14]. Alotaibi etal. reported no difference in
grade II-IV aGVHD between cryopreserved versus fresh
allografts, but did note a higher incidence of moderate/
severe cGVHD in the cryopreserved group [2]. Our study
demonstrated no statistically significant differences in grade
II-IV aGVHD or cGVHD.
While our study population was relatively homogenous
and the rational for cryopreservation was predominantly due
to the impact of COVID-19, as opposed to heterogenous rea-
sons requiring cryopreservation, there are several limitations
to our work. Our study is limited by its retrospective nature,
small sample size, and relatively short duration of follow-
up. Additionally, the use of post-transplant cyclophospha-
mide prevents us from generalizing our conclusions to other
GVHD prophylaxis regimens.
In summary, our study showed decreased OS, delayed
engraftment, and a higher need for DLI and RBC trans-
fusions in patients undergoing cryopreserved PBSC allo-
HSCT with post-transplant cyclophosphamide. We suggest
cryopreserved allogeneic transplantation to be performed
with caution. Further prospective studies are required to fur-
ther investigate the impact of cryopreservation in allogeneic
PBSC transplantation.
Acknowledgements This research did not receive any specific grant
from funding agencies in the public, commercial, or not-for-profit
sectors.
Author contributions MG: data collection, methodology, investigation,
writing, review. JL: data collection, methodology, investigation, writ-
ing, review. PC: data collection, methodology, investigation, writing,
review. SA: writing, review. RDP: writing, review. SM: conceptualiza-
tion, methodology, supervision, writing, review and editing.
Data availability statement The data us accurate and available upon
request.
Declarations
Conflict of interest The authors declare that they have no conflict of
interest.
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