Introduction
In cases of massive blood loss, the time to start treatment is crucial. In such situations, the physician is faced with the need to make quick decisions, and any delay in starting transfusion therapy can be critical for the patient's life [1–3].
In recent years, there has been renewed interest in using whole blood type O with low titers of anti-A and anti-B antibodies (LTOWB) as a universal and balanced transfusion medium for the initial correction of traumatic and non-traumatic massive blood loss in patients with any ABO and Rh blood group [3–8].
Many authors present LTOWB as an alternative to transfusions of individual blood components at a ratio of 1:1:1:1. It contains all essential elements (erythrocytes, coagulation factors, and cold-stored platelets) required for the correction of anemia, coagulopathy, and stopping bleeding, and the minimal content of preservative and additive solutions reduces the risk of hemodilution in hemorrhagic shock [9, 10].
There is positive experience with the use of LTOWB as part of a massive transfusion protocol in prehospital and early hospital periods. A reduction in 24-hour and 30-day mortality has been demonstrated in patients with multiple trauma and massive blood loss [11–16]. The use of LTOWB in obstetrics and pediatrics is also actively discussed [17–21].
An unresolved issue in the storage of blood is the transfusion of large numbers of donor leukocytes to the recipient. It is well established that the use of non-leukoreduced blood components significantly increases transfusion-related risks and may lead to febrile non-hemolytic transfusion reactions, transmission of cytomegalovirus and human T-cell lymphotropic virus (HTLV), as well as HLA alloimmunization [22–28]. The benefits of leukoreduction in various patient groups may be associated with a reduction in post-transfusion immunomodulation, cardiopulmonary complications, length of hospital stay, and mortality. Universal leukodepletion can also improve the infectious and immunological safety of LTOWB, however, in this case, the effectiveness of transfusion in correcting coagulopathy and controlling bleeding becomes questionable, since platelets are also removed when a leukofilter is used [28, 29].
Thus, despite the promising potential of preserved blood for the management of massive blood loss, its widespread application remains primarily limited by immunological risks [30]. Currently, no universal technological approaches have been developed that enhance safety while maintaining both erythrocyte quality and the hemostatic properties of preserved blood.
It is known that pathogen reduction of plasma and platelet concentrates significantly reduces both infectious and immunological risks associated with transfusions of these blood components. One such method is the Mirasol system, which employs UV-B radiation (280–360 nm) in combination with riboflavin to inactivate pathogens and leukocytes. The resulting photochemical reaction damages nucleic acids of microorganisms, induces DNA or RNA strand breaks, and prevents replication. At the same time, lymphocyte proliferative activity is completely suppressed. The Mirasol technology is also approved for pathogen reduction in stored whole blood units [31–34].
In the Russian regulatory framework, the use of preserved blood and leukoreduced preserved blood is permitted, but only when taking into account the ABO and Rh compatibility of recipients [35].
Our study proposes modified technologies for the preparation of preserved blood: partial leukoreduction with platelet preservation and pathogen reduction using riboflavin and UV-B. Thus, the working hypothesis is that the sequential application of these modifications may provide effective transfusion with minimal risk to the recipient under conditions of massive blood loss.
Objective
The study aims to investigate in vitro the effects of modified processing methods on the functional properties of preserved blood and to assess its suitability for emergency transfusion therapy under conditions of massive blood loss.
Materials and methods
In vitro experiments were conducted using preserved blood obtained from 44 group O donors (34 men and 10 women). Written informed consent was obtained from all donors. All donor blood samples were screened for transfusion-transmissible infections.
Whole blood in volumes of 450 ± 10 ml was collected in plastic blood containers with 63 ml of citrate solution (CPD). Preserved blood was stored at +4–6 °C for 7 days.
Four groups were identified: control (Control), leukoreduced (LR), preserved blood collected using modified technology (PLR), and preserved blood collected using a modified method with subsequent pathogen inactivation (PLR + PR).
Control group (n = 12) — preserved blood that had not undergone leukoreduction or other modifications.
LR group (n = 12) — preserved blood leukoreduced using the Leucoflex LXT leukofilter (Macopharma, France).
PLR group (n = 12) — preserved blood obtained using a modified protocol with an off-label set of Reveos LR blood bags and the Reveos automatic whole blood processing system (Therumo BCT, USA), providing partial leukoreduction while preserving platelets.
PLR + PR group (n = 8) — preserved blood prepared using the Reveos system (as in the PLR group) followed by pathogen reduction using the Mirasol system (Terumo BCT, USA). The preserved blood was transferred into the Mirasol illumination container. Following the process technology, 35 ml of riboflavin with a concentration of 500 μmol/l was added. After mixing, erythrocytes were irradiated with UV-B light at a dose of 80 J/mL, after which the blood container was stored at +4–6 °C.
On days 1 and 7 of the experiment, 3–5 ml samples were taken from each container of donor blood under aseptic conditions using a sterile TSCD II connector.
The following parameters were assessed: erythrocyte morphology, cellular elastic modulus, alterations in cytoskeletal structure, hematological parameters, percentage of erythrocyte hemolysis, coagulation factors, and viscoelastic properties.
After completion of the experiment on day 7, but no later than 168 hours after donation, all preserved blood units were transferred to the cryobank for fractionation. The separated erythrocytes were cryopreserved using standard glycerolization technology on an ACP 215 system (Haemonetics, USA), transferred into MACO BIOTEC containers (Macopharma, France), and stored in liquid nitrogen.
Images were obtained and analyzed using NTEGRA Prima and NTEGRA BIO atomic force microscopes (NT-MDT SI, Russia), and the elastic modulus of cells was evaluated. Cell morphology and cytoskeletal integrity were assessed by obtaining AFM images in semi-contact mode. For this purpose, a NSG01 cantilever with a tip radius of 10 nm (NT-MDT SI, Russia) was used. FemtoScan Online software (Centre for Advanced Technologies, Russia) [36–39] was used for image processing. AFM images of the cytoskeleton were further analyzed using ImageJ software [40]. To assess the elastic properties of erythrocytes, force curves were recorded with an SD-R150-T3L450B-10 cantilever (Nanosensors, Switzerland) with a probe radius of 150 nm and a spring constant of 1 N/m. Next, the Young's modulus (E) was calculated using the Hertz model for each sample on 100 cells.
A Sysmex XN-350 hematology analyzer (Sysmex Corporation, Japan) was used to measure complete blood count parameters: hemoglobin, hematocrit, leukocytes, and platelets.
A ROTEM delta thromboelastometer (Tem Innovations GmbH, Germany) was used for integrated assessment of the coagulation system in preserved blood samples. The study was performed using the NATEM assay, which is based on contact activation of coagulation, with CaCl2 as a recalcification agent, no additional activator. The following parameters were recorded: clotting time, clot formation time, alpha angle, amplitude recorded after 20 minutes (A20), maximum clot firmness, and lysis index after 30 minutes (LI30).
An ACL TOP 750 automated coagulometer (Instrumentation Laboratory Company (Werfen), USA) measured fibrinogen, factor VIII, factor XIII, and von Willebrand factor. The percentage of erythrocyte hemolysis in the samples was also determined.
The study was approved by the local ethics committee of the N.V. Sklifosovsky Research Institute for Emergency Medicine, Moscow Healthcare Department (Protocol No. N4/2024, dated August 27, 2024), and by the Ethics Committee of the Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitology (Protocol No. 2/20, dated June 10, 2020). The study was conducted in accordance with ethical standards and in compliance with all requirements of the Declaration of Helsinki of the World Medical Association.
Statistical analysis was performed using Origin Pro 2019 software (OriginLab Corporation, USA). The distribution of the data was assessed using the Shapiro—Wilk test. Variables with a normal distribution were expressed as mean ± standard deviation (SD), whereas non-normally distributed data were presented as median (Me) and interquartile range (Q1; Q3). For comparisons between independent samples with non-normal distributions, the Kruskal—Wallis test (for multiple groups) and the Mann–Whitney U-test (for two groups) were applied. Paired samples were analyzed using the Wilcoxon signed-rank test. To account for multiple comparisons, the Bonferroni correction was applied. A p-value < 0.05 (two-tailed) was considered statistically significant.
Results
In the first stage, such parameters as the morphology of erythrocytes, their elastic properties, and cytoskeletal integrity were examined using AFM to evaluate the impact of leukoreduction and pathogen reduction on the quality of donor blood.
It is known that erythrocytes undergo morphological alterations during storage. Cells transform from discocytes to echinocytes. However, the effect of pathogen reduction on these changes has not previously been investigated using AFM.
Figure 1A presents 3D AFM images of 100 × 100 μm2 demonstrating the effect of pathogen reduction and storage time on erythrocyte morphology.
Fig. 1. Change in the shape of red blood cells before and after pathogen reduction Note: Before PR D0 — before pathogen reduction on day 0 of storage; After PR D0 — after pathogen reduction on day 0 of storage; After PR D1 — after pathogen reduction on day 1 of storage; After PR D7 — after pathogen reduction on day 7 of storage.
Four different forms of erythrocytes were identified in the samples studied: discocytes, stomatocytes, echinocytes, and others (Figure 1C). Before pathogen reduction on day 0 of storage, 91 ± 6 % of discocytes were observed (Figure 1B), which is a typical form of erythrocytes. Directly after pathogen reduction, the proportion of discocytes was 87 ± 6 %. On day 1 after pathogen reduction, an increase in the echinocyte fraction to 12 ± 5 % was observed, while the discocyte fraction decreased to 81 ± 6 %. By day 7 of storage, the echinocyte fraction had increased to 16 ± 4 %, while the discocyte fraction had decreased to 78 ± 7 %. The changes in cell morphology observed in the PLR + PR group were comparable to those in the other three groups (Control, LR, and PLR) and have been described previously [41–43]. In this study, no significant effect of pathogen reduction on erythrocyte morphology or the emergence of other cell forms was detected.
However, potential alterations may occur at the cytoskeleton level, which plays a key role in maintaining cell shape and their ability to deform, properties essential for erythrocyte passage through narrow capillaries [44]. To evaluate the condition of the cytoskeleton, its parameters were analyzed within a 2.5 × 2.5 μm2 area. For instance, Figure 2A compares the control sample on day 0 with samples from the control and pathogen-reduced groups on day 7 of storage.
Fig. 2. Cytoskeleton alterations and Young's modulus in control and pathogen-reduced erythrocytes Note: Control D0 — control on day 0 of storage; Control D7 — control on day 7 of storage; PLR + PR D7 — after leukoreduction and pathogen reduction on day 7 of storage.
As a result, it was established that neither storage time, nor leukoreduction, nor pathogen reduction had a significant effect on cytoskeletal reorganization. The pore area remained unchanged across all groups after 7 days of storage, and the number of pores did not differ from the control sample at day 0. The average pore size also showed no increase. However, by day 7 of storage, it was noted that the number of filament breaks had changed in all samples: in the control group, their number increased from 35 ± 8 on day 0 to 43 ± 10 on day 7, in the LR group — from 38 ± 7 to 45 ± 7, in the PLR group — from 36 ± 9 to 44 ±8, and in the PLR + PR group — from 35 ± 8 to 42 ± 9.
At the same time, assessment of the viscoelastic properties of erythrocytes, it was found that in both the control group and the experimental groups, the Young's modulus E did not change significantly and remained at 5.5 ± 2 kPa (Figure 2B). No statistically significant differences were found between the groups at all stages of the study (p > 0.01).
To assess the suitability of erythrocytes for replacement and correction of coagulopathy in potential transfusions to patients with massive blood loss, it was essential to perform a comprehensive in vitro evaluation of the balance of the composition of preserved blood collected using modified technologies in comparison with classical preparation methods. For this purpose, in the second part of the study, hematological parameters, viscoelastic tests, and coagulation factors were analyzed in samples obtained from blood bags (Figure 3). In Figure 3, reference values are indicated in gray.
Fig. 3. Changes in various hematological and coagulation parameters Note: Control — non-leukoreduced, unmodified stored blood; LR — leukoreduced preserved blood; PLR — preserved blood prepared by modification (partial leukoreduction); PLR + PR — after partial leukoreduction and pathogen reduction; D1 — on day 1 of storage; D7 — on day 7 of storage.
The study showed that hemoglobin concentration did not change during storage, remaining at 123 ± 5 g/L, 125 ± 11 g/L, and 118 ± 13 g/L in the Control, LR, and PLR groups, respectively, which corresponded to hemoglobin concentrations of 63 ± 3, 64 ± 6, and 61 ± 7 grams per unit of preserved blood. In the PLR + PR group, this parameter was equal to 112 ± 9 g/L or 57 ± 5 grams per unit (Figure 3A). The observed differences were associated with insignificant losses during additional processing in the PLR + PR group. Hematocrit levels also did not change significantly in any of the groups during storage (Figure 3B). The hemolysis rate did not exceed 0.8 % of erythrocytes [45] in any of the samples studied (Figure 3C).
The use of the Reveos system for partial leukocyte removal (outside of approved indications) reduced the detectable leukocyte level to 0.36 (0.25; 0.37) × 109/L in the PLR group after 1 day of storage, while in the PLR + PR group, the leukocyte level was 0.18 (0.14; 0.31) × 109/L, as shown in Figure 3D. The values did not differ between these groups, but a tendency toward a decrease in leukocytes (p = 0.016) was observed in the PLR + PR group by the 7th day of storage. In the LR group, leukocytes were not detected, as their number was below the resolution of the Sysmex XN-350 analyzer, which was expected following effective leukoreduction. In addition, the aim of the study was not to determine the exact number of residual leukocytes after leukoreduction. In the control group, which was not subjected to leukoreduction, the leukocyte level remained at 4.5 (3.8; 5.2) × 109/L at the beginning and 4.5 (3.8; 4.7) × 109/L after 7 days of storage.
Platelet counts at the beginning and end of storage were as follows: Control — 132 (86; 150) × 109/L and 84 (49; 141) × 109/L, PLR — 139 (102; 164) × 109/L and 86 (43; 107) × 109/L, PLR + PR — 114(94; 142) × 109/L and 70 (55; 96) × 109/L (Figure 3E). In the LR group, platelet counts were extremely low, as platelets were effectively retained by the leukofilter, and amounted to1.5 (1; 2) × 109/L (with no changes observed during storage) (Figure 3E).
In all groups, factor XIII levels remained stable over 7 days of storage (Figure 3F). In the PLR + PR group, the levels of fibrinogen, factor VIII, and von Willebrand factor decreased significantly compared with the other groups (Figure 3G, H, I).
Data obtained from the ROTEM delta analysis enabled assessment of the viscoelastic properties of preserved blood (Table 1).
| Parameters | Reference range |
Storage day |
Groups | The Kruskal—Wallis test | |||
|---|---|---|---|---|---|---|---|
| Control (n = 12) | LR (n = 12) | PLR (n = 12) | PLR + PR (n = 8) | ||||
| Clotting time, s | 300–750 | D1 | 520 (435; 553) |
860 (694; 922) |
545 (328; 668) |
575 (556; 583) |
H = 14.207 р = 0.003 |
| D7 | 639 (570; 695) |
788 (741; 893) |
588 (566; 633) |
568 (517; 636) |
H = 25.981 р < 0.001 |
||
| р = 0.0031 | р = 0.7331 | р = 0.3011 | р = 0.7421 | ||||
| Clot formation time, s | 150–700 | D1 | 185 (169; 231) |
NA | 215 (150; 277) |
269 (248; 290) |
H = 6.922 р = 0.031 |
| D7 | 260 (248; 316) |
NA | 283 (250; 326) |
353 (295; 479) |
H = 4.829 р = 0.089 |
||
| р = 0.0521 | NA | р = 0.0411 | р = 0.0151 | ||||
| Alpha angle, ° | 30–70 | D1 | 56 (50; 59) |
NA | 52 (45; 61) |
45 (43; 49) |
H = 6.381 р = 0.041 |
| D7 | 47 (41; 48) |
NA | 44 (41; 48) |
39 (31; 44) |
H = 4.114 р = 0.128 |
||
| р = 0.0491 | NA | р = 0.0321 | р = 0.0311 | ||||
| A20, mm | D1 | 51 (45; 55) |
7 (7; 9) |
49 (42; 52) |
44 (41; 45) |
H = 28.524 р < 0.001 |
|
| D7 | 45 (41; 45) |
8 (7; 9) |
42 (40; 43) |
37 (34; 41) |
H = 31.163 р < 0.001 |
||
| р = 0.0821 | р = 0.2851 | р = 0.0741 | р = 0.0071 | ||||
| Maximum clot firmness, mm | 40–65 | D1 | 54 (48; 58) |
8 (7; 8) |
52 (46; 56) |
49 (47; 50) |
H = 27.499 р < 0.001 |
| D7 | 51 (48; 53) |
8 (7; 10) |
47 (45; 51) |
45 (41; 48) |
H = 29.664 р < 0.001 |
||
| р = 0.2731 | р = 0.0551 | р = 0.0471 | р = 0.0231 | ||||
| LI30, % | 94–100 | D1 | 100 (100; 100) |
100 (100; 100) |
100 (100; 100) |
100 (100; 100) |
H = 2.666 р = 0.445 |
| D7 | 100 (100; 100) |
100 (100; 100) |
100 (100; 100) |
100 (100; 100) |
H = 0 р = 1 |
||
| р = 11 | р = 11 | р = 11 | р = 11 | ||||
The group with pathogen reduction (PLR + PR) showed longer clotting time and clot formation time than the control group after 1 day of storage. By day 7 of storage, mean values of the alpha angle and A20 parameters in groups Control, PLR, and PLR + PR had decreased. Similarly, a reduction in maximum clot firmness (MCF) was observed as a function of storage time and as a result of leukoreduction and pathogen reduction. Nevertheless, MCF values in the PLR + PR group remained within reference range and were comparable to those of the Control group.
The coagulation properties data for the LR group differed significantly from those of the other groups studied and fell outside the reference range due to impaired clot formation in the absence of platelets. This demonstrates the questionable potency of using preserved leukoreduced blood in cases of massive blood loss. Reference values were based on the range recommended by the analyzer manufacturer.
Discussion
There is an ongoing debate regarding the impact of preparation methods and additional processing of LTOWB on this transfusion medium's beneficial properties and safety in cases of massive blood loss. Standard leukofiltration removes both leukocytes and platelets, thereby depriving the transfusion medium of one of the key components required to control non-compressible bleeding [29]. In our study, we also demonstrated that the absence of platelets in leukoreduced preserved blood prevents the formation of a fully developed clot. On the other hand, the absence of leukodepletion is associated with the negative effects of such transfusions on patient recovery [46, 47]. Implementing special platelet-saving filters for leukocytes, which was initially considered a possible solution to this problem, significantly reduces platelet functional activity and, consequently, reduces the efficiency of LTOWB [34, 48, 49]. Moreover, these systems are not registered and cannot be imported into the Russian Federation.
Previously, Weisbach V. et al. demonstrated that cytokine levels were higher in non-leukoreduced preserved blood, while the erythrocyte suspension with reduced leukocyte number (after removal of the leukocyte layer) showed cytokine levels comparable to in erythrocyte suspension leukoreduced using a leukofilter [50]. Thus, this demonstrates that technology corresponding to partial leukoreduction also increases the safety of the transfusion medium.
Inactivation of pathogens using riboflavin and UV radiation has proven effective against various pathogens, including bacteria and viruses, in preclinical studies on platelets and plasma [51–53], and studies of pathogen reduction in preserved blood have further demonstrated the effectiveness of this method in damaging lymphocyte DNA, thereby making the restoration of their proliferative activity unlikely [54, 55].
In 2013, Pidcoke H.F. et al. evaluated the hemostatic potential and, for the first time, considered the possibility of using preserved blood subjected to pathogen reduction for treatment of massive blood loss [56].
Later, Thomas K.A. et al. also examined the impact of platelet-sparing leukoreduction and pathogen reduction with riboflavin on the hemostatic properties of preserved blood [34]. Both leukoreduction and pathogen reduction were shown to affect platelet function, with a notable decrease in platelet count, clot density, and fibrinogen levels observed in the group where preserved blood underwent leukoreduction followed by pathogen reduction. These findings align with our study; however, due to the use of modified blood collection technology, we achieved significant leukoreduction to 0.36 (0.25; 0.37) × 109/L and preserved platelet levels at 139 (102; 164) × 109/L without employing a leukofilter. Platelets that do not contact the filter membrane may be more resistant to the damaging effects of pathogen reduction, as evidenced by improved clot stability at the end of storage and insignificant differences in platelet count and MCF parameter between the PLR and PLR + PR groups, in contrast to the pronounced differences reported by our colleagues.
According to the literature, preserving blood at 4 °C keeps most hemostasis proteins steady [57,58]. Our study demonstrates that pathogen reduction results in decreased levels of fibrinogen, factor VIII, and von Willebrand factor. It is assumed that such an effect can be caused by damage or changes in the structure of proteins caused by processing methods, including ultraviolet radiation and chemical agents[59]. We also examined the stability of coagulation factor XIII, which demonstrated consistent concentration levels during 7 days of storage, regardless of the method used for additional processing of the preserved blood.
Analysis of viscoelastic properties using ROTEM confirmed that clotting time and clot formation time increased in the group with pathogen reduction of preserved blood. The A20 parameters and maximum clot firmness were also lower. However, they remained within the reference range, confirming the maintenance of platelet functional activity. Together with fibrinogen and factor XIII, platelets ensure clot formation, which is critical for the correction of traumatic coagulopathy and emergency hemostasis.
According to studies performed by other authors, using of pathogen reduction and leukoreduction technologies does not lead to significant changes in erythrocytes morphology [60, 61]. These changes are probably related to oxidative processes occurring during storage, but not directly caused by processing procedures. These findings are consistent with previous reports [42, 43, 62], showing that morphological changes in erythrocytes during storage are mainly associated with the accumulation of metabolites and oxidative stress rather than with blood processing methods. Examination of the cytoskeleton showed that its structure remained stable over a 7-day storage period, despite a slight increase in filament breaks. This indicates that the stability of cytoskeletal proteins such as spectrin and actin is not disrupted, and the elastic properties of erythrocytes are preserved [63].
Thus, the studies discussed previously confirm our operating hypothesis that partial leukoreduction and pathogen reduction can increase the safety of the transfusion environment without significantly damaging erythrocytes. The main feature of our study is the use of AFM to evaluate erythrocytes during additional processing and after 7 days of storage, confirming their preservation. These findings suggest that such blood can be used safely and effectively to restore gas transport function in patients with severe post-hemorrhagic anemia after massive blood loss.
Our study's limitation was the storage time of samples up to 7 days. In accordance with regulatory recommendations, subsequent fractionation and cryopreservation of these samples are permitted no later than 168 hours after preparation. In addition, our ongoing research will focus on preventing the disposal of pathogen-reduced preserved blood after the 7-day storage period and exploring the possibility of its further use as thawed, washed erythrocytes.
The results obtained in vitro require further investigation, as they cannot accurately predict in vivo results.
Conclusion
In this study, AFM was employed to assess the preservation of red blood cells in donor blood, enabling a detailed examination of their morphology, cytoskeletal structure, and mechanical properties. In addition to quantitative hematological measurements, the NATEM test was used to evaluate coagulation factors and clot density, thereby assessing hemostatic potential. The data obtained provide a comprehensive evaluation of the quality and safety of preserved donor blood prepared using modified technology. For the first time, it has been demonstrated that partial leukoreduction significantly decreases the number of residual leukocytes while preserving sufficient platelet counts, and that pathogen-reduced blood has potential applicability in patients with massive blood loss. Nevertheless, clinical studies are required to validate this hypothesis.
Disclosure. The authors declare no competing interests.
Author contribution. All authors according to the ICMJE criteria participated in the development of the concept of the article, obtaining and analyzing factual data, writing and editing the text of the article, checking and approving the text of the article.
Ethics approval. The study was approved by the local Ethical Committee of the N.V. Sklifosovsky Research Institute for Emergency Medicine, Moscow Healthcare Department (Protocol No. N4/2024, dated August 27, 2024), and by the Ethics Committee of the Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitology (Protocol No. 2/20, dated June 10, 2020).
Funding source.This study was supported the state assignment of the Ministry of Health of the Russian Federation No. 075-00479-24-04.
Data Availability Statement. The data that support the findings of this study are available from the corresponding author upon reasonable request.
