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Replacement of Hydroxyethyl Starch by Human Serum Albumin for Post-Thaw Washing of Cryopreserved Peripheral Blood Hematopoietic Progenitor Cell Products: A Technical Feasibility Study

Submitted:

13 October 2026

Posted:

14 October 2026

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Abstract
Background/Objectives: Post-thaw washing of cryopreserved hematopoietic progenitor cell (HPC) products is performed to remove dimethyl sulfoxide (DMSO) and reduce infusion-related toxicity. Hydroxyethyl starch (HES) has traditionally been used as an osmotic buffer in washing solutions; however, severe regulatory restrictions and market suspensions by the European Medicines Agency (EMA) have limited its availability in Europe. This pilot study evaluated the technical feasibility of utilizing a 4.0% human serum albumin (HSA)-based washing solution to replace HES without compromising post-thaw product quality parameters. Methods: Sixteen cryopreserved peripheral blood HPC bags from eight patients (cryopreserved between 2006 and 2015) were analyzed using a paired experimental design. For each patient, one bag was thawed and washed using the standard HES-HSA solution, while the paired bag was processed using an HSA-based solution. Total nucleated cell (TNC) recovery, TNC viability, viable CD34+ cell recovery, CD34+ viability, and clonogenic potential (CFU assay) were assessed immediately after washing and after 90 minutes of short-term post-thaw storage. Results: No statistically significant differences were observed between the HES-HSA and HSA washing solutions for any evaluated parameter. TNC recovery rates were 86.6 ± 6.6% and 88.9 ± 7.9% for the HES-HSA and HSA groups, respectively (p = 0.54). Post-thaw viability, viable CD34+ cell recovery, CD34+ viability, and clonogenic potential were stable and comparable between both processing strategies. Conclusions: An albumin-based washing solution provides cellular recovery, viability, and clonogenic performance comparable to conventional HES-containing formulations in a laboratory setting. Given current restrictions on HES due to clinical safety concerns, an albumin-based formulation represents a practical and logistically viable operational alternative for the post-thaw processing of peripheral blood HPC products.
Keywords: 
hematopoietic progenitor cells
;  
cryopreservation
;  
post-thaw washing
;  
human serum albumin
;  
hydroxyethyl starch
Subject: 
Medicine and Pharmacology  -   Hematology

1. Introduction

Hematopoietic progenitor cell (HPC) transplantation is an established treatment for a variety of hematologic malignancies and selected solid tumors [1,2,3,4]. Autologous HPC transplantation remains a standard therapeutic approach for patients with multiple myeloma and relapsed or refractory lymphomas [5,6].
Cryopreservation is an essential component of autologous transplantation and is increasingly used in the allogeneic setting when fresh graft infusion is not feasible [5,7,8,9,10,11]. Reliable cryopreservation methods are required to preserve sufficient numbers of viable hematopoietic stem cells (HSCs) for subsequent transplantation [12,13,14,15,16,17], even after prolonged storage periods. [18,19,20,21,22].
However, post-thaw handling practices remain a matter of considerable controversy and heterogeneity across clinical centers [6,14].
The conventional post-thaw procedure consists of rapid thawing in a 37 °C water bath followed by immediate, direct bedside infusion [16]. The clinical choice between performing a post-thaw wash or proceeding with direct infusion involves distinct operational trade-offs. The primary advantages of post-thaw washing include the substantial reduction of dimethyl sulfoxide (DMSO) dose and total fluid volume, the prevention of severe infusion-related reactions (such as nausea, vomiting, cardiovascular symptoms, dyspnea, and allergic events), and the thorough removal of cellular debris and granulocyte lysis products, which is particularly critical in pediatric or low-weight recipients [10,23,24,25,26,27,28,29,30,31,32]. Conversely, the disadvantages of washing encompass a prolonged cellular exposure to DMSO during processing before centrifugation, potential cell loss during cycles, temporary drops in cell viability due to physical manipulation, operational delays in the transplantation timeline, and increased financial and procedural costs per graft.
Consequently, many institutions advocate strategies to reduce DMSO exposure such as washing procedures, reducing cryoprotectant concentrations, and testing alternative cryoprotective agents [16,20,26,28,33,34,35,36,37,38,39,40,41,42,43].
When post-thaw washing is indicated, a macromolecular agent is required to act as a colloid osmotic buffer to minimize osmotic shock and cellular lysis during the rapid removal of intracellular cryoprotectants. The original washing methods described by Rubinstein et al. successfully employed dextran-albumin solutions [44]. Due to historical dextran shortages, many centers, including our own—substituted dextran with hydroxyethyl starch (HES) solutions, which maintained high processing efficiency and graft quality [45].
However, recent safety updates from the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have highlighted that HES solutions are associated with an increased risk of mortality and acute kidney injury in critically ill patients. Consequently, regulatory bodies have strictly limited or suspended its market authorization, causing severe supply chain deficits in European cell processing facilities [46].
No universally accepted standard currently exists for post-thaw processing of HPC products, and the reproducibility and effectiveness of washing procedures remain matters of debate [14,43,47,48]. While alternative automated cell washing platforms using proprietary albumin formulations have been described recently (such as the Lovo system) [42] , there is an urgent need to validate accessible, home-made HES-free formulations that can be readily integrated into existing laboratory workflows during material shortages. Therefore, the aim of this pilot study was to evaluate the technical feasibility of replacing HES with a 4.0% human serum albumin (HSA)-based washing solution for the post-thaw processing of cryopreserved peripheral blood HPC products, comparing laboratory cell recoveries, viability profiles, and clonogenic potential.

2. Material and Methods

2.1. Collection and Cryopreservation of HSC Grafts

A total of 16 peripheral blood HSC bags collected between 2006 and 2015 and designated for disposal following donor death, were utilized for this validation study. All patients had previously provided written informed consent authorizing the use of remaining cellular products for quality control, validation, and research purposes.
Grafts were collected via apheresis from patients mobilized with recombinant human G-CSF. Grafts were cryopreserved within 24 hours of collection at a final nucleated cell concentration of <3 x 108 cells/mL using a controlled-rate freezer (Kryo-10, Messer Griesheim). The final cryoprotectant mixture consisted of 30% Medium-199 (Sigma Aldrich), 50% of a 20% human serum albumin solution (Human Albumin Grifols® 20%), and 20% pure DMSO (CryoSure-DMSO, WAK-Chemie Medical GmbH), yielding a final DMSO concentration of 10%. Grafts were stored in liquid nitrogen vapor phase (-196 °C).

2.2. Thaw and Washing Procedure

Paired cryobags were thawed in a 37 °C water bath using a protective sterile overwrap. Under a class II biosafety cabinet, the product was slowly reconstituted by adding an equal volume of the designated chilled (1–6 °C) washing solution with continuous gentle agitation, followed by a 5-minute equilibration phase. The mixture was transferred to a standard processing set and placed into a semi-automated COBE 2991 Cell Washer (Terumo BCT, Lakewood, CO, USA). Centrifugation washing was conducted over three sequential automated cycles at 3000 rpm for 7 minutes at room temperature. Grafts were resuspended in an identical volume of Plasma-Lyte A supplemented according to the allocated study group.

2.3. Washing Solutions

The standard washing solution (HES-HSA) contains 6% HES (Volulyte 6%, Fresenius Kabi Deutschland GmbH; mean molecular weight 130,000 Da) and HSA diluted in Plasma-Lyte A (Baxter Health Corp.), to achieve final concentrations of 2.4% HES and 4.2% HSA, respectively. The test solution (HSA) was formulated using 20% human albumin (Human Albumin Grifols® 20%) and anticoagulant citrate dextrose solution formula A (ACD-A Grifols®) diluted in Plasma-Lyte A. The final targeted concentrations in this formulation were 4.0% HSA and 5.0% ACD-A.
The precise volumetric mixing proportions utilized to prepare the solutions are detailed in Table 1 and Table 2.:

2.4. Technical Evaluation and Sample Processing

An experimental paired-sample design was utilized (n = 8 patients, total of 16 individual bags). From each selected patient donor, two separate bags of identical collection characteristics and volume were evaluated. For each independent pair, one bag was randomly assigned to the standard HES-HSA protocol, and its twin bag was assigned to the HES-free HSA protocol. Thawing and washing were performed by a single technologist using identical equipment configurations to control for operational variability.

2.5. Cell Counting, Flow Cytometry, and Functional Assays

For each paired experiment, products were thawed at different times by the same technologist using the same equipment. Each final product was tested immediately after washing (sample 0’) and after 90 minutes (sample 90’) for TNC recovery, acridine orange viability [49,50], viable CD34+ enumeration [51] and clonogenicity (CFUassay) [52] .
Grafts were sampled at two specific experimental time points: immediately following the final wash cycle (0 minutes) and after short-term storage in a closed container at 1–6 °C (90 minutes) to simulate a clinical delivery window.
Total Nucleated Cell (TNC) counts were determined using an automated hematology analyzer (Sysmex XN-series, Kobe, Japan). Due to the presence of cellular debris and free background nuclei in post-thaw products, cellular viability was quantified using automated dual-fluorescence microscopy with acridine orange [49,50] and propidium iodide (AO/PI) nuclear staining to exclude non-cellular artifacts.
Enumeration of viable CD34+ hematopoietic progenitors was conducted via flow cytometry using an BD FACSCanto™ II flow cytometer adhering strictly to the standardized ISHAGE gating protocol. CD34+ cell specific viability was calculated via 7-aminoactinomycin D (7-AAD) exclusion staining [51]. The mathematical formula employed to calculate the absolute cellular recovery rate (%) was: Recovery Rate (%):{Total viable cells in the final washed product}*100/{Total viable cells in the pre-thaw product}.
Functional hematopoietic potency was assessed using colony-forming unit (CFU) assays. Samples were seeded in duplicate into methylcellulose-based media (MethoCult™, Stemcell Technologies) and incubated at 37 °C in a humidified 5% CO2 atmosphere for 14 days prior to colony scoring [52]. Due to volume and cellular constraints in three specific clinical bags, stability assessments at the 90-minute time point could not be performed completely across all parameters, resulting in reduced sample sizes (N=5 for TNC viability, N=7 for CD34+ enumeration) which are explicitly declared.

2.6. Statistical Analysis

Data were expressed as the mean ± standard deviation (SD). Continuous variables were analyzed using a paired-samples t-test following confirmation of normal data distribution.. All statistical tests were performed using IBM SPSS Statistics vs 25. Statistical significance was defined as a p value<0.05.

3. Results

A total of eight paired experiments (16 distinct products) were successfully processed and tracked. Post-thaw quality indices obtained for both the reference HES-HSA solution and the HES-free HSA-based washing solution are detailed in Table 3.
The final volume of the processed grafts was uniform between both processing arms, with mean volumes of 228.4 ± 44.8 mL for the reference HES-HSA solution and 214.9 ± 37.3 mL for the HSA solution (p = 0.52). The absolute recovery of TNC did not display significant variations, yielding 1.91 ± 0.95× 108 cells in the HES-HSA cohort compared to 1.99 ± 1.13 × 108 cells in the HSA cohort (p = 0.86). The calculated mean TNC recovery rate exceeded 85% in both study arms (86.6 ± 6.6% vs. 88.9 ± 7.9%, respectively; p = 0.54).
Cellular viability remained high immediately post-processing. TNC viability determined by automated AO/PI staining was 93.6 ± 4.5% for HES-HSA and 91.7 ± 8.1% for the HSA formulation (p = 0.67). Following a 90-minute storage window, TNC viability values were 90.0 ± 4.8% and 89.0 ± 7.0%, respectively, showing no statistical variance between treatments (p = 0.79).
The preservation of the CD34+ progenitor cell compartment was comparable between both procedures. Immediate post-wash viable CD34+ cell counts normalized to recipient weight were 0.37 ± 0.54 x 106/kg for the HES-HSA arm and 0.48 ± 0.58 x 106/kg for the HSA arm (p = 0.69). After 90 minutes of storage, viable CD34+ counts remained stable at 0.30 ± 0.34 x 106/kg and 0.30 ± 0.32 x 106/kg (p = 0.98). The final CD34+ cell recovery rates were 87.0 ± 29.8% for the reference group and 101.2 ± 24.0% for the HES-free albumin group (p=0.31). Specific progenitor cell viability determined via flow cytometric 7-AAD exclusion did not drop significantly between immediate and delayed tracking points (p=0.57 and p=0.74, respectively).
Functional clonogenic potential, measured through colony-forming unit outputs (CFU), was successfully maintained across both processing modalities. Grafts washed with the HES-HSA solution yielded a mean colony output of 18.2 ± 24.6 × 104/kg, while those treated with the albumin-based solution yielded 18.8 ± 28.2 × 104/kg (p=0.97).

4. Discussion

This technical feasibility study evaluated whether an alternative HES-free, 4.0% human serum albumin-based washing solution could effectively replace conventional HES formulations for post-thaw DMSO removal from peripheral blood HPC products. In our laboratory series, we observed no statistically significant deviations between both processing configurations regarding immediate cell recovery, viability profiles, or long-term clonogenic proliferation.
Our findings indicate that HES is not strictly required to maintain osmotic stability during washing, and that a formulation containing 4.0% human serum albumin can provide sufficient oncotic pressure and mechanical stabilization to prevent cell lysis during rapid cryoprotectant clearance. These outcomes align with recent work by Santurette et al. and Lachica et al., which demonstrated that closed, automated cell processing setups using distinct albumin-based crystalloid dilutions successfully preserve the viability and functional expansion profiles of cord blood stem cells [42,43]. Our study expands upon this evidence by confirming the performance of an albumin-based wash in a paired-sample series of cryopreserved peripheral blood HPC products.
From an administrative and safety standpoint, transitioning to an entirely HES-free washing method addresses a critical regulatory challenge currently facing European cell processing facilities. Following the definitive market suspensions enacted by the EMA and FDA due to adverse renal complications in critical care environments, HES solutions have become largely inaccessible. The use of human serum albumin—which has a well-documented safety profile, widespread clinical availability, and established regulatory clearance—provides a sustainable solution to this supply bottleneck. Furthermore, avoiding HES eliminates any downstream risk of exposing transplant recipients to starch-induced macromolecular storage artifacts.
A critical logistical point regarding our study’s methodology must be explicitly addressed. The post-thaw washing procedures described herein were performed using the semi-automated COBE 2991 Cell Washer. This equipment was officially discontinued by its manufacturer, and comprehensive technical and disposable kit support concluded within the EMEA region. Consequently, the direct clinical utility of this specific automated protocol is restricted to centers utilizing remaining support stocks.
Nevertheless, validating the chemical performance of this HES-free washing formulation remains highly relevant. The 4.0% albumin and 5.0% ACD-A mixture in Plasma-Lyte A can be directly adapted to manual centrifugation protocols or programmed into contemporary, fully supported automated cell washers (such as the Lovo or Sepax platforms).
Introducing an extra post-thaw washing step involves certain economic considerations. Incorporating human serum albumin, single-use processing kits, and additional technologist hands-on time increases the overall processing cost per graft compared to direct bedside infusion. However, these expenditures are counterbalanced by a reduced incidence of severe infusion-related toxicities, which can lower total patient monitoring costs and decrease the need for post-infusion medical interventions.
This validation study has several limitations. First, the total sample size was small (n = 8 paired donor products), which restricts our statistical power to demonstrate non-inferiority. Second, this evaluation relied strictly on laboratory and benchtop endpoints; clinical outcomes, such as time to neutrophil and platelet engraftment or precise adverse event tracking, were not evaluated. Third, the processed products had been stored in liquid nitrogen for an extended period, which could introduce historical cryopreservation variables.
While our laboratory data indicate that cell viability is maintained for up to 90 minutes post-wash, we recommend that washed HPC grafts be infused as soon as possible after processing to mitigate the risk of progressive functional decline over time. Future large-scale clinical registries will be required to confirm long-term engraftment kinetics following the implementation of this HES-free protocol.
In conclusion, our results demonstrate that replacing HES with a 4.0% human serum albumin-based washing solution for post-thaw processing of peripheral blood HPC products is technically feasible and maintains key laboratory quality indicators. This formulation offers a reliable alternative for cell processing laboratories navigating current regulatory restrictions and HES supply shortages.
[5,14,43,47,48]. Several studies have shown that DMSO contributes substantially to infusion-related adverse reactions, including nausea, vomiting, flushing, cardiovascular symptoms, dyspnea, neurological toxicity, and allergic reactions [10,23,24,25,26,27,28,29,30,31,32]. Consequently, many institutions advocate washing procedures, particularly when large amounts of DMSO are infused or in pediatric recipients [48,53,54] . In our institution, DMSO removal is considered standard practice because of its favorable safety profile and its capacity to reduce exposure to potentially toxic components of the cryopreserved product.
The original washing strategy developed by Rubinstein and colleagues employed dextran as an osmotic buffer to minimize osmotic injury during DMSO removal [44]. Subsequently, dextran-albumin solutions became widely used for cord blood and peripheral blood HPC products [55]. Due to dextran shortages, our group previously substituted dextran with HES while maintaining satisfactory processing outcomes [45].
However, recent recommendations by the European Medicines Agency regarding suspension of HES-containing solutions for clinical use have considerably reduced their availability in Europe [46]. This situation has created an urgent need for validated alternatives that can be readily incorporated into established processing protocols. The current study was designed in response to this practical challenge. Our data indicate that an albumin-based solution represents a suitable replacement for HES, allowing continuation of established washing procedures without negatively affecting product quality.
The results are in agreement with more recent reports describing albumin-containing solutions as effective post-thaw suspension media for hematopoietic cell products [43]. Albumin provides colloid osmotic support, is widely available in cell processing facilities, and has an extensive record of clinical safety. In addition, albumin-based solutions simplify supply chain management and may improve standardization among centers, especially in environments where HES is no longer readily accessible. Our findings support the use of albumin-based washing solutions as a practical alternative to HES-containing formulations. Similar conclusions have recently been reported by Lachica et al., who demonstrated that albumin-based suspension media effectively preserve viability, functionality, and proliferative capacity of thawed cord blood hematopoietic stem cells and may represent an optimal post-thaw environment for these products [43]. The preservation of viable CD34+ cells and clonogenic potential observed in our study is consistent with those observations and extends the evidence to thawed peripheral blood HPC products processed using an automated washing platform.
An additional observation of interest is the stability of washed products during the post-thaw holding period. Although cell viability remained largely preserved during the evaluation period, our previous experience and reports from other groups indicate that prolonged delays between washing and infusion may result in progressive losses in viability and clonogenic capacity, particularly when prolonged delays occur before infusion [43,56]. Our group has previously observed progressive losses in viability and clonogenicity beyond several hours of post-thaw storage, supporting the recommendation that washed products should be infused as soon as possible after processing [56].Consequently, washed HPC products should still be infused as soon as feasible after processing. Nevertheless, the maintenance of acceptable cellular characteristics during the studied interval provides a degree of operational flexibility that may be useful in routine clinical practice.
The increasing diversification of cellular therapies also highlights the importance of optimizing post-thaw manipulation strategies. Beyond conventional HPC transplantation, preservation of mononuclear cell populations has become increasingly relevant for advanced therapy medicinal products, including CAR-T cell manufacturing. Albumin-based solutions may offer advantages in these settings due to their favorable effects on cell recovery and their broad regulatory acceptance, although further studies are required to confirm this hypothesis.
The present study has several limitations. First, the sample size was relatively small, reflecting the limited availability of paired cryopreserved products suitable for research use. Second, the study evaluated laboratory endpoints rather than clinical transplantation outcomes such as neutrophil or platelet engraftment. Third, the products analyzed had been cryopreserved over an extended period, which could introduce variability unrelated to the washing solution itself. Nonetheless, the paired-study design minimized inter-product variability and strengthened the direct comparison between both washing solutions. Future studies should evaluate engraftment kinetics and infusion-related adverse events after clinical implementation of the HSA-based washing protocol.
In conclusion, replacement of HES-containing washing solutions with a 4.0% albumin-based formulation resulted in equivalent recovery, viability, and clonogenic performance of thawed peripheral blood HPC products. Given the reduced availability of HES and current regulatory concerns regarding its use, albumin-based washing solutions constitute a practical, safe, and effective alternative for post-thaw processing. Our results support the implementation of HES-free washing protocols while maintaining the quality standards required for HPC transplantation.

Author Contributions

Conceptualization, L.L.; methodology, L.L.; validation, B.V., MJ.V. and J.S.; formal analysis, B.V. and L.L.; investigation, MJ.V.; resources, MJ.V.; data curation, J.S.; writing—original draft preparation, L.L.; writing—review and editing, B.V. and MJ.V.; visualization, V.M.; supervision, C.A. All authors have read and agreed to the published version of the manuscript.”.

Funding

This research received no external funding.

Institutional Review Board Statement

Ethical review and approval were waived for this study because it was conducted as a validation of a cell-processing procedure and did not involve any intervention, change in treatment, or collection of additional samples from living patients. The study was performed exclusively using cryopreserved hematopoietic progenitor cell products that had been designated for disposal after the death of the donors. All patients had previously provided written informed consent authorizing the use of any remaining unused cryopreserved cellular products for research, quality control, and validation purposes. Therefore, the study posed no risk to patients and did not influence clinical management or transplantation outcomes. A formal waiver/exemption was granted by our local Ethics Committee/Institutional Review Board.

Data Availability Statement

The datasets generated and analyzed during the current study are not publicly available because they are derived from human cellular products and are subject to institutional privacy and ethical restrictions. However, anonymized data supporting the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgments

The authors would like to thank all the technical staff of the Centro de Transfusión de la Comunidad Valenciana (CTCV) for their valuable work, commitment, and collaboration in the processing, cryopreservation, and quality control of hematopoietic progenitor cell products.

Conflicts of Interest

The authors declare no conflict of interest.

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Table 1. Volumetric formulation for the reference HES-HSA option.
Table 1. Volumetric formulation for the reference HES-HSA option.
Final Target Volume 100 200 400 600 1000 2000
20% Albumin (mL) 21 42 84 126 210 420
6% HES Solution (mL)* 40 80 160 240 400 800
Plasma-Lyte A (mL) 39 78 156 234 390 780
*Note: The historical institutional reference table contained a secondary 0.6% HES allocation protocol restricted for severe clinical renal insufficiency; however, that formulation was not utilized in any experimental arm of the current validation study.
Table 2. Volumetric formulation for the test HSA option (HES-free).
Table 2. Volumetric formulation for the test HSA option (HES-free).
Final Target Volume 100 200 400 600 1000 2000
20% Albumin (mL) 20 40 80 120 200 400
ACD-A Solution (mL) 5 10 20 30 50 100
Plasma-Lyte A (mL) 75 150 300 450 750 1500
Table 3. Laboratory quality indicators of peripheral blood HPC products after post-thaw washing.
Table 3. Laboratory quality indicators of peripheral blood HPC products after post-thaw washing.
Measured Parameter Washing Solution N Mean Value Standard Deviation p-value
Product Volume (mL) HSA 8 214.87 37.29 0.52
HES-HSA 8 228.41 44.77
Total Nucleated Cells (TNC x108) HSA 8 1.99 1.13 0.86
HES-HSA 8 1.91 0.95
TNC Recovery Rate (%) HSA 8 88.91 7.89 0.54
HES-HSA 8 86.63 6.62
TNC Viability-0 min (%) HSA 8 91.74 8.11 0.67
HES-HSA 8 93.62 4.45
TNC Viability-90 min (%) HSA 5 89.00 6.96 0.79
HES-HSA 5 90.00 4.80
Viable CD34+ Cells-0 min (106/kg) HSA 8 0.48 0.58 0.69
HES-HSA 8 0.37 0.54
Viable CD34+ Cells-90 min (106/kg) HSA 7 0.30 0.32 0.98
HES-HSA 7 0.30 0.34
CD34+ Cell Recovery Rate (%) HSA 8 101.21 24.03 0.31
HES-HSA 8 87.01 29.75
CD34+ Progenitor Viability-0 min (%) HSA 8 91.62 6.45 0.57
HES-HSA 8 90.00 8.65
CD34+ Progenitor Viability-90 min (%) HSA 7 92.56 5.81 0.74
HES-HSA 7 93.52 4.83
Clonogenic Potential (CFU x 104/kg) HSA 8 18.83 28.15 0.97
HES-HSA 8 18.23 24.56
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