Effect of Cryopreservation of Perıpheral Blood-Derıved Cells on Transplant Outcomes in Allogeneic Hematopoietic Cell Transplantation

1. Introduction

Allogeneic hematopoietic cell transplantation (HCT) is a widely used procedure for treating hematologic diseases; it involves eliminating the patient’s own hematopoietic cells and replacing them with hematopoietic stem cells collected from a donor (1). According to data updated in 2023 by the Center for International Blood and Marrow Transplant Research (CIBMTR), 89,144 allogeneic HCT procedures were performed in the United States between 2012 and 2022 (2). As is the case globally, rates of allogeneic HCT are steadily increasing in Turkey.

Peripheral blood mobilized with granulocyte colony-stimulating factor (G-CSF) is the primary source of stem cells for HCT and is used in approximately 65% of allogeneic HCT procedures. Hematopoietic cells can be used either fresh or cryopreserved. Hematopoietic stem cells derived from bone marrow or peripheral blood are stored for use in a planned infusion following the patient’s treatment or in the event of a future relapse (3,4). Specific cryopreservative agents are used to store cells for extended periods while maintaining their viability. Dimethyl sulfoxide (DMSO) is the most commonly used of these agents. Stem cells intended for cryopreservation can be stored after being mixed with specific proportions of DMSO and a plasma product. Following the global SARS-CoV-2 pandemic in 2019, there has been a marked increase in the number of transplants utilizing cryopreserved cells (5,6).

The process by which donor-derived stem cells begin to produce healthy peripheral blood cells is defined as engraftment. When assessing the adequacy of engraftment, attention is paid to whether neutrophil and platelet engraftment has occurred. Graft failure is defined as pancytopenia and severely hypocellular bone marrow (cellularity <5%) persisting beyond 28 days after bone marrow-derived HSCT, 21 days after peripheral blood stem cell-derived HSCT, or 42 days after cord blood HSCT. Graft failure occurs in 5–10% of allogeneic HSCT cases involving unrelated donors and in 1% of cases involving matched related donors (7,8).

Following transplantation, donor cells are expected to develop immunity against foreign cells in the recipient’s body while developing tolerance toward host cells. In Graft-versus-Host Disease (GvHD), however, the transplanted cells perceive host cells as foreign and induce inflammation and fibrosis—primarily in the skin, but also in the liver, gastrointestinal tract, lungs, and other mucosal areas (9).

Cytomegalovirus (CMV) infection is common in immunosuppressed patients. Assessing the CMV serostatus of both the recipient and the donor prior to transplantation is crucial for preventing future CMV infections. Whenever possible, a seronegative donor should be selected for a CMV-seronegative recipient. Regardless of initial serostatus, reactivation can occur even in CMV IgG-positive patients due to the use of immunosuppressive drugs following HSCT. Therefore, frequent monitoring of CMV replication is required after HSCT (10). This study aimed to determine whether there were any differences—in terms of neutrophil engraftment time, platelet engraftment time, disease-free survival, overall survival, GvHD incidence, and transplant-related mortality rates—between patients over the age of 18 with hematological diseases undergoing allogeneic hematopoietic stem cell transplantation who received cryopreserved products and those who received fresh products.

2. Materials and Methods

In our study, we retrospectively compared neutrophil engraftment time, platelet engraftment time, GvHD incidence, and DFS, OS, and TRM rates between patient groups receiving cryopreserved products and those receiving fresh products following allogeneic hematopoietic stem cell transplantation (HSCT) for hematological diseases.

Patients aged 18 years and older who underwent allogeneic HSCT for hematological diseases at the University of Health Sciences Dr. Abdurrahman Yurtaslan Ankara Oncology Training and Research Hospital Hematology and Stem Cell Transplantation Clinic between January 1, 2018, and December 30, 2022, were included in this study. The study examined 127 patients who received fresh stem cells and 95 patients who received cryopreserved stem cells.

Approval for this study was obtained from the Clinical Research Ethics Committee of the University of Health Sciences Dr. Abdurrahman Yurtaslan Ankara Oncology Training and Research Hospital on February 6, 2025, under decision number 2025-02/25.

Patients who underwent HSCT due to solid tumors or autoimmune diseases, those under the age of 18, and those for whom bone marrow or cord blood was used as the stem cell source for HSCT were excluded from the study. The study evaluated patients’ demographic characteristics (age, gender, weight) alongside hematological disease subtypes, pre-HCT disease status, infused CD34+ stem cell dose, donor type, HLA compatibility, blood group incompatibility, conditioning regimen intensity, time to neutrophil and platelet engraftment, engraftment failure, Sorror comorbidity index, CMV seropositivity at day 100 post-HCT, incidence of GVHD, TRM, DFS, and OS.

The hematological diseases of the patients evaluated in the study were categorized as AML, ALL, Ph+ B-ALL, Ph- B-ALL, T-ALL, MDS, CML, PMF, HL, B-NHL, T-NHL, MM, AA, MPAL, and other hematological diseases.

Donors included in the study were classified based on HLA compatibility and kinship status as: 10/10 fully matched related donor (MRD), 10/10 fully matched unrelated donor (MUD), 9/10 matched related donor, 9/10 unrelated donor, and haploidentical (8/10 match or lower).

Pre-transplant disease status was categorized as complete remission (CR), partial remission (PR), active disease, and chemosensitive refractory disease.

Blood group incompatibility was evaluated in four categories: compatible, major incompatibility (recipient possesses antibodies against donor blood cells), minor incompatibility (donor possesses antibodies against recipient cells), and mixed-type incompatibility.

Pre-HCT conditioning regimens used in our study were analyzed in two categories: MAC and RIC. There were no patients receiving NMA regimens among the study cohort. Neutrophil engraftment was considered to have occurred on the first of three consecutive days during which the neutrophil count remained > 0.5 x 10⁹/L. Platelet engraftment was considered to have occurred on the first of seven consecutive days during which no platelet transfusions were required and the platelet count remained > 20 x 10⁹/L.

The Sorror index used in our study is a scoring system that predicts post-transplant survival in patients with hematological diseases undergoing hematopoietic stem cell transplantation (HSCT). This scoring system evaluates the presence of arrhythmias, cardiovascular disease, inflammatory bowel disease, diabetes, cerebrovascular disease, psychiatric disorders, hepatic dysfunction, infection, obesity, rheumatological diseases, peptic ulcer, renal dysfunction, pulmonary diseases, a history of malignancy, and valvular heart disease.

The time elapsed from HSCT until disease progression/relapse or death was assessed as disease-free survival (DFS), while the time from HSCT until death was assessed as overall survival (OS).

Transplant-related mortality was defined as treatment-related death occurring within 100 days post-HSCT in the absence of active hematological disease.

For patients with related donors, donors received subcutaneous granulocyte colony-stimulating factor (G-CSF) at a daily dose of 10 μg/kg (administered as two divided doses of 5 μg/kg every 12 hours) for 5 days to mobilize peripheral blood stem cells. To initiate the collection procedure, CD34-positive stem cell counts were performed via flow cytometry on peripheral blood samples from all donors on the fourth day of G-CSF administration. Donors were accepted for the procedure if the CD34 stem cell count was ≥50/μL. If the CD34-positive cell count was <50/μL on the fourth day, the attending physician evaluated the donor for the procedure, taking into account whether the white blood cell count in the donor’s hemogram exceeded 50,000/mL. Donors were invariably accepted for the collection procedure on the fifth day of granulocyte colony-stimulating factor administration. Upon completion of the collection process, the product bag was detached from the system using a tube sealer. The product was gently rocked between the hands 5–10 times to ensure a homogeneous distribution. The rubber port of the sample bag was cleaned with a sterile povidone-iodine (Betadine) swab, and a 4 cc sample was drawn. One cc of the collected sample was sent to the flow cytometry laboratory for CD34+ cell counting, while another 1 cc was used for a complete blood count (hemogram).

The remaining 2 cc sample was dispensed into aerobic and anaerobic culture tubes (1 cc each). The product collected in the hemogram tube was diluted with autologous plasma at a ratio of 1:8 to 1:16, and the hemogram count was performed on this dilution. The resulting product was placed inside a biosafety cabinet. Under sterile conditions within the airflow cabinet, the product was transferred into labeled cryobags positioned on a +4 °C surface amidst ice packs. Accessing the product bag via the fistula port, the product was drawn into 50 ml syringes. A volume equivalent to 50–60% of the total product was slowly transferred into a storage bag kept in a cold environment. The final mixture was adjusted to contain 10% DMSO and 30–40% autologous plasma, ensuring the total product volume did not exceed 100 ml. The prepared DMSO-autologous plasma mixture was added to the product while the bag was gently agitated. Prior to freezing, air bubbles were removed from the storage bag by gently tapping it while using a plasma extractor. The storage bag was heat-sealed at its end and placed into a labeled protective cassette. A final 2 cc sample was taken from the product to check for microbiological contamination via aerobic and anaerobic culture tests. Once the controlled-rate freezer reached +4 °C, the product bag was placed inside the device within cold cassettes, and the cooling process was initiated. After controlled freezing, the storage bags within the cassettes were transferred to a liquid nitrogen tank (-196 °C). Nitrogen levels in the nitrogen tank were recorded hourly in a computerized system and weekly in written logs. Data regarding the stored stem cells were maintained within the automation system and in separate files. For transplantation, the cryopreserved product was prepared by thawing it in a 37.5 °C distilled water bath for 3–4 minutes and was then infused into the patient. Products in the fresh group were infused without any additional modification.

Determining CMV copy numbers via quantitative real-time polymerase chain reaction (RT-qPCR) is the gold standard for assessing CMV-related complications in the post-transplantation period. CMV copy numbers for the patients included in the study were measured using the Cobas 6800 CMV PCR test. This test detects the CMV DNA polymerase region and can quantify viral loads ranging from 34.5 to 1×10⁷ IU/mL. The system detects CMV glycoprotein B genotypes 1–4. Patients in the study were monitored via CMV PCR twice weekly following hematopoietic stem cell transplantation (HSCT). Patients found to have a CMV viral load of ≥1000 IU/mL during follow-up were considered to have reactivation, and ganciclovir treatment was initiated.

Statistical Analyses

Statistical analyses were performed using IBM SPSS software (version 26; Armonk, NY). Demographic data were summarized using descriptive statistics. Numerical variables were presented as medians (minimum–maximum). The normality of the data distribution was assessed using the Mann-Whitney U test and the independent samples t-test. Categorical variables were expressed as proportions. The chi-square test was used to compare categorical variables. Variables with p-values less than 0.2 in the univariate analysis were subsequently included in a multivariate Cox regression analysis to further investigate factors affecting overall survival. A p-value of ≤0.05 was considered statistically significant, based on a 95% confidence interval and a 5% margin of error.

3. Results

This study compared data from fresh (n = 127) and cryopreserved (n = 95) hematopoietic cells from 222 individuals who received allogeneic HCT because of hematological disease.

Patient characteristics are summarized in Table 1. The median age of patients receiving cryopreserved hematopoietic cells was 42 ± 23 years, while the median age of patients receiving fresh hematopoietic cells was 36 ± 20 years (p=0.036). There were no significant differences between the groups in terms of gender and body weight (p>0.05 for both parameters).

When patients were evaluated in terms of hematological diseases, no significant difference was found between the cryopreserved and fresh product receiving groups in terms of hematological disease subtypes (p=0.092).

In the study, no significant difference was found in terms of pre-HCT disease status in either group (p=668). The number of CR, PR, active and chemosensitive refractory patients was similar between the groups receiving fresh product and cryopreserved product (Table 2).

When patients were evaluated according to HLA match, in the group receiving cryopreserved cells, the number of patients with 10/10 HLA match was 47 (49.5%), the number of patients with 9/10 HLA match was 45 (47.4%), and the number of haploidentical patients was 3 (3.2%), while in the group receiving fresh products, the number of patients with 10/10 HLA match was 95 (74.8%), the number of patients with 9/10 HLA match was 3 (2.4%), and the number of haploidentical patients was 29 (22.8%) (p<0.001).

In the cryopreserved cell group, the number of mismatch sibling donors (MSD) was 19 (20%), mismatch unrelated donors (MUD) were 28 (29.4%), 9/10 HLA-matched relatives were 2 (2.1%), 9/10 HLA-matched unrelated donors were 43 (45.3%), and haploidentical donors were 3 (3.2%). While in the fresh group, MSD was 93 (73.2%), MUD was 3 (2.4%), 9/10 HLA-matched relatives were 1 (0.8%), 9/10 HLA-matched unrelated donors were 1 (0.8%), and haploidentical donors were 29 (22.8%) (p<0.001) (Table 3).

Regarding blood group compatibility, no significant difference was found between the two groups in terms of major, minor, and mixed type incompatibility (p=0.247).

In terms of preparation regimen intensity, 7 patients (7.4%) in the cryopreserved cell group received RIC, while 88 patients (92.6%) received MAC, while in the fresh cell group, 19 patients (15%) received RIC, and 108 patients (85%) received MAC (p=0.082) (Table 4).

When evaluated in terms of the Sorror comorbidity index, no significant difference was found between the two groups in terms of Sorror comorbidity index score (p=0.943).

The median number of CD34+ stem cells received by patients receiving cryopreserved products was 6.56×10⁶ (1.50×10⁶ -9.4×10⁶), while the median number of CD34+ stem cells received by patients receiving fresh products was 7.44×10⁶ (1.80×10⁶ -10.38×10⁶) (p<0.001).

No significant difference was found between cryopreserved and fresh cell groups in terms of platelet engraftment time (p=0.665) and neutrophil engraftment time (0.897). Similarly, when evaluated in terms of engraftment failure rate, no significant difference was found between the two groups (p=0.708).

Table 5. Median CD34+ stem cell count, neutrophil and platelet engraftment time obtained in cryopreserved cell and fresh cell recipient patient groups.

Characteristics	Cryopreserved (n=95)	Fresh (n=127)	P value
CD34+ Stem Cell Count (×106 /kg)	6,56 (1,50-9,4)	7,44(1,80-10,38)	<0.001
Neutrophil Engraftment Time (day)	15(10-28)	15(11-24)	0.897
Platelet Engraftment Time (day)	13(7-57)	13(7-25)	0.665
Engraftment Failure (n/%)
Yes	9(9,5%)	14(11%)	0.708
No	86(90,5%)	113(89%)

Table 5. Median CD34+ stem cell count, neutrophil and platelet engraftment time obtained in cryopreserved cell and fresh cell recipient patient groups.

Characteristics	Cryopreserved (n=95)	Fresh (n=127)	P value
CD34+ Stem Cell Count (×106 /kg)	6,56 (1,50-9,4)	7,44(1,80-10,38)	<0.001
Neutrophil Engraftment Time (day)	15(10-28)	15(11-24)	0.897
Platelet Engraftment Time (day)	13(7-57)	13(7-25)	0.665
Engraftment Failure (n/%)
Yes	9(9,5%)	14(11%)	0.708
No	86(90,5%)	113(89%)

When examined in terms of GvHH development, it was found that GvHH developed in 35 patients in the cryopreserved stem cell group and in 52 patients in the fresh stem cell group, and there was no significant difference in GvHH development between the two groups (p=0.536).

In the group receiving fresh cell cultures, one patient developed chronic gastrointestinal GvHD, with 39 cases of skin involvement, 8 cases of gastrointestinal involvement, 14 cases of liver involvement, and 1 case of eye involvement. In the group receiving cryopreserved products, 6 cases of liver GvHD, 1 case of skin GvHD, and 2 cases of gastrointestinal GvHD were observed, along with 11 cases of liver GvHD, 24 cases of skin GvHD, 4 cases of gastrointestinal involvement, and 1 case of cardiac GvHD. When evaluating these data, it should be considered that some patients developed both acute and chronic GvHD, and that in some GvHD cases, multiple sites (e.g., skin and liver) were affected simultaneously.

Disease-free survival times were observed to be a median of 8.9 months in the cryopreserved cell group and a median of 13.7 months in the fresh stem cell group, with no significant difference between the two groups (p=0.081). Similarly, no significant difference was observed between the two groups in overall survival times (p=0.229).

When evaluated in terms of transplant-related mortality, TRM occurred in 8 patients in the cryopreserved cell group and 6 patients in the fresh cell group; no significant difference was observed between the two groups in terms of TRM (p=0.262).

Table 6. The development of graft-versus-host disease, disease-free survival, overall survival, and transplant-related death were compared between cryopreserved cell and fresh cell recipient groups.,.

Characteristics	Cryopreserved (n=95)	Fresh (n=127)	P value
GvHD Development
Yes	35(36.8%)	52 (40.9%)	0.536
No	60 (63.2%)	75 (59.1%)
Transplant-Related Death
Yes	8 (8,4%)	6 (4,7%)	0.262
No	87 (91,6%)	121 (95,3%)
Median Disease-Free Survival (months)	8,9 (0,07-54,73)	13,73 (0,27-50,8)	0.081
Median Overall Survival (months)	13,1 (0,07-54,73)	19,63 (0,27-53,50)	0.229

GvHD: Graft versus host disease.

Table 6. The development of graft-versus-host disease, disease-free survival, overall survival, and transplant-related death were compared between cryopreserved cell and fresh cell recipient groups.,.

Characteristics	Cryopreserved (n=95)	Fresh (n=127)	P value
GvHD Development
Yes	35(36.8%)	52 (40.9%)	0.536
No	60 (63.2%)	75 (59.1%)
Transplant-Related Death
Yes	8 (8,4%)	6 (4,7%)	0.262
No	87 (91,6%)	121 (95,3%)
Median Disease-Free Survival (months)	8,9 (0,07-54,73)	13,73 (0,27-50,8)	0.081
Median Overall Survival (months)	13,1 (0,07-54,73)	19,63 (0,27-53,50)	0.229

GvHD: Graft versus host disease.

When comparing the patient groups receiving cryopreserved cells and those receiving fresh cells in terms of the development of CMV reactivation or infection in the first 100 days after transplantation, no significant difference was observed (p=0.821).

Table 7. In both cryopreserved and fresh cell recipient patient groups, CMV reactivation or infection occurred within the first 100 days after HCT.

Characteristics	Cryopreserved (n=95)	Fresh (n=127)	P value
Patient CMV IgG Positivity	95 (100%)	127 (100%)	>0.005
Donor CMV IgG Positivity	89 (93,7%)	124 (97,6%)	0.139
CMV reactivation and/or infection within 100 days	6 (6,3%)	9 (7,1%)	0.821

CMV IgG: Cytomegalovirus immunoglobulin G.

Table 7. In both cryopreserved and fresh cell recipient patient groups, CMV reactivation or infection occurred within the first 100 days after HCT.

Characteristics	Cryopreserved (n=95)	Fresh (n=127)	P value
Patient CMV IgG Positivity	95 (100%)	127 (100%)	>0.005
Donor CMV IgG Positivity	89 (93,7%)	124 (97,6%)	0.139
CMV reactivation and/or infection within 100 days	6 (6,3%)	9 (7,1%)	0.821

CMV IgG: Cytomegalovirus immunoglobulin G.

4. Discussion

The use of cryopreserved hematopoietic cells in allogeneic hematopoietic stem cell transplantation (HSCT) has become widespread both globally and in our country, particularly following the SARS-CoV-2 pandemic. This trend has heightened the importance of understanding the impact of cryopreservation on engraftment, the incidence of transplant-related complications, and patient survival.

In our study, patients over the age of 18 who underwent allogeneic HSCT for hematological diseases were evaluated in two separate groups based on whether they received cryopreserved or fresh hematopoietic cells. Patients in both groups were assessed regarding the number of CD34+ stem cells infused, HLA compatibility, blood group compatibility, Sorror comorbidity index, intensity of the conditioning regimen, pre-transplant disease status, time to neutrophil and platelet engraftment, engraftment failure, development of graft-versus-host disease (GvHD), transplant-related mortality (TRM), overall survival (OS), and disease-free survival (DFS).

With the increasing prevalence of transplants utilizing cryopreserved hematopoietic cells, various studies have been published regarding the impact of stem cell cryopreservation on HSCT complications and survival outcomes.

In a study conducted by Facchin et al. in 2022, a total of 54 patients who underwent HSCT between January 2020 and July 2021—comprising 31 who received cryopreserved products and 23 who received fresh products—were evaluated; although the total number of CD34+ stem cells infused was lower in the cryopreserved group, no significant difference was observed between the two groups regarding engraftment adequacy (11). In a study conducted by Alotaibi et al. in 2020, data from a total of 958 patients—comprising 648 who received fresh products and 310 who received cryopreserved products—were evaluated; no significant prolongation in neutrophil and platelet engraftment times was observed in the group receiving cryopreserved products (12). In a study conducted by Guo et al. in 2022, a total of 55 patients (21 receiving fresh products and 34 receiving cryopreserved products) were evaluated, and similarly, no difference was observed between the two groups regarding neutrophil and platelet engraftment times (13). Likewise, in our study, when comparing the group receiving cryopreserved cells with the groups receiving fresh products, no significant difference was observed in neutrophil and platelet engraftment times. On the other hand, a study by Hamadani et al. in 2020 examined a total of 1,354 patients (1,080 receiving fresh products and 274 receiving cryopreserved products); while no difference was found between the groups regarding the number of CD34+ stem cells received, a significant prolongation in neutrophil and platelet engraftment times was observed in the cryopreserved product group—contrary to the findings of other studies and our own (14).

In the study conducted by Alotaibi et al., no significant difference regarding engraftment failure was demonstrated between the groups receiving cryopreserved products and fresh products (12). Similarly, our study found no significant difference in terms of engraftment failure between patients receiving cryopreserved cells and those receiving fresh cells. The effects of cryopreservation on engraftment failure—a critical factor in the treatment success of hematological diseases—were also evaluated in a 2023 study by Keyzner et al. The study examined engraftment times in 44 patients who received cryopreserved cells and 37 patients who received fresh product infusions. The results showed that the use of cryopreserved grafts did not have a significant adverse effect, even in patients with a high risk of post-transplant engraftment failure, such as those with myelofibrosis (15).

A review of the literature regarding the number of CD34+ stem cells administered reveals conflicting findings: while Alotaibi and Hamadani found no significant difference in the number of stem cells between the cryopreserved and fresh cell groups, studies by Guo and Facchin reported that patients in the fresh cell group received a statistically significantly higher number of CD34+ cells (70-73). In studies where the CD34+ stem cell count was found to be significantly lower in the cryopreserved group, this difference was attributed to cell loss occurring during the freezing and thawing processes. Similarly, in our study, a comparison of the CD34+ stem cell counts between the two groups revealed that patients who received the fresh product had received a significantly higher number of hematopoietic stem cells.

A review of the literature regarding cryopreservation and the development of GVHD reveals that a study by Guo et al. found no significant difference between the groups in terms of GVHD development (13). Similarly, a study by Keyzner et al. observed that cryopreservation did not lead to a significant difference regarding the development of GVHD (15). Consistent with these findings, our study also showed no significant difference in GVHD development between groups receiving cryopreserved versus fresh hematopoietic stem cells. In contrast, a study by Facchin et al. found no difference between the two groups regarding acute GVHD development, yet chronic GVHD occurred at a higher rate in the group receiving cryopreserved products (11). Likewise, a study by Alotaibi et al. concluded that hematopoietic stem cell transplantations (HSCT) involving cryopreservation were associated with a higher incidence of GVHD. The higher rate of chronic GVHD observed in cryopreserved HSCTs has been attributed to either greater T-cell depletion in the fresh stem cell group—thereby reducing the risk of abnormal immune reactions in the patient—or the impairment of existing T-cell function caused by the cryopreservation process. Conversely, a study by Hamadani et al. found that transplants using cryopreserved cells did not differ in terms of acute GVHD development but were associated with a lower incidence of chronic GVHD (14).

In the study conducted by Facchin et al., no significant difference in transplant-related mortality (TRM) was observed between patients receiving cryopreserved cells and those receiving fresh cells (11). Similarly, in our study, no statistically significant difference regarding TRM was found between the group receiving cryopreserved cells and the group receiving fresh stem cells.

A review of the literature regarding disease-free survival (DFS) reveals that studies by Guo, Hamadani, Facchin, and Alotaibi found no significant difference in DFS between patients receiving cryopreserved cells and those receiving fresh cells (11-14). In a 2023 study by Ersal et al. investigating the impact of cryopreservation on transplant outcomes, a total of 93 patients—37 receiving cryopreserved cells and 56 receiving fresh cells—were evaluated; monitoring of transplant outcomes showed no significant difference in DFS between the two groups (16). Likewise, our study found no statistically significant difference in DFS between the group receiving cryopreserved cells and the group receiving fresh stem cells.

Regarding overall survival (OS), although the median OS was longer in the group receiving fresh cells in our study, no statistically significant difference was found between the two groups. Similarly, studies by Hamadani and Alotaibi reported longer OS in the fresh-cell group, but the difference compared to the other group was not statistically significant (12, 14). Studies by Facchin and Ersal also found no statistically significant difference between the two groups; however, Guo’s study found OS to be significantly longer in the cryopreserved group (11, 13, 16).

Regarding CMV infection, studies by Facchin and Hamadani showed no significant difference in the rates of CMV infection and reactivation between patients receiving cryopreserved cells and those receiving fresh cells (11, 14). In our study, no significant difference was observed between the groups receiving cryopreserved and fresh stem cells regarding the rates of CMV infection and reactivation. CMV reactivation was also evaluated in the study by Guo; consistent with our findings, no statistically significant difference in reactivation rates was detected between the group receiving cryopreserved cells and the group receiving the fresh product (13).

5. Conclusions

The use of cryopreserved hematopoietic cells in allogeneic hematopoietic stem cell transplantation (HSCT) has become widespread globally and in our country, particularly following the SARS-CoV-2 pandemic. This trend has heightened the importance of understanding the impact of cryopreservation on engraftment, the incidence of transplant-related complications, and patient survival. With the increasing prevalence of transplants utilizing cryopreserved hematopoietic cells, various studies have been published regarding the effects of stem cell cryopreservation on HSCT complications and survival outcomes.

In this study, data were compared from a total of 222 patients undergoing allogeneic HSCT for hematological diseases: 127 who received fresh hematopoietic cells and 95 who received cryopreserved ones. Patients in both groups were evaluated based on the number of CD34+ stem cells infused, HLA compatibility, blood group compatibility, Sorror index, conditioning regimen intensity, pre-transplant disease response, neutrophil and platelet engraftment times, engraftment failure, development of graft-versus-host disease (GvHD), transplant-related mortality (TRM), overall survival (OS), and disease-free survival (DFS). No significant differences were found between the cryopreserved and fresh product groups regarding median age, gender, body weight, hematological disease subtype, pre-HSCT disease status (complete remission, partial remission, active disease, and chemosensitive refractory disease), Sorror comorbidity index, blood group compatibility, or conditioning regimen intensity. However, the number of haploidentical HSCTs was higher in the fresh cell group. The number of CD34+ stem cells infused in patients receiving cryopreserved products was lower than that in patients receiving fresh products. When cryopreservation and thawing processes are examined, the reduction in cell count is considered an expected outcome attributable to temperature fluctuations and the chemical effects of cryoprotectants. No significant differences were observed between the groups receiving cryopreserved and fresh products regarding platelet and neutrophil engraftment times, the rate of engraftment failure, the rate of CMV reactivation/infection within the first 100 days post-HSCT, the development of GvHD, TRM, DFS, or OS. As previously noted, although patients receiving cryopreserved products received a relatively lower number of CD34+ stem cells, this did not result in significant differences in neutrophil and platelet engraftment times. In cases of HSCT involving cryopreservation, acute GvHD may occur more frequently—potentially due to alterations in T-cell activity caused by cryoprotectants—whereas chronic GvHD may be observed less frequently.

The increasing availability of cellular therapy options for treating various diseases—particularly malignancies—has led to a rise in the cryopreservation and storage of cellular products. Cryopreservation is crucial for cell storage as it helps prevent adverse outcomes such as delays in the patient’s transplantation process due to donor-related issues, prolonged hospital stays, and associated complications; it also enhances the overall accessibility of cellular therapies. Outcomes of hematopoietic stem cell transplantations (HSCT) utilizing cells stored via appropriate cryopreservation methods can be just as successful as those using fresh cellular products.

The primary limitation of our study is the disparity in the number of haploidentical HSCTs between the fresh cell group and the cryopreserved cell group; the fresh cell group had a higher number of haploidentical transplants. This imbalance limits the ability to directly compare HSCT outcomes between the two groups. There remains a need for further studies involving larger patient cohorts and comparing groups with similar numbers of haploidentical HSCTs.