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. 2022 Aug 1;32(3):10708.
doi: 10.4081/ejtm.2022.10708.

The potential effect of leukocyte filtration methods on erythrocyte-derived microvesicles: One step forward

Fateme Roshanzamir  1 , Sedigheh Amini-Kafiabad  2 , Mahin Nikougoftar Zarif  3 , Ali Arabkhazaeli  4 , Mahshid Mohammadipour  5
Affiliations

The potential effect of leukocyte filtration methods on erythrocyte-derived microvesicles: One step forward

Fateme Roshanzamir et al. Eur J Transl Myol. .

Abstract

By harmonizing the pre-preparation conditions and also removing some donors' variations, the current study took one step forward to investigate whether different leukocyte filtration sets influence the quality of RBCs throughout the storage time. Twelve whole blood units were collected, and each unit was split into three equal parts. Thirty-six divided bags were filtered using three different leukocyte-filtration sets including Red Cell and Whole Blood Filters (12 units per filter). The prepared RBCs were refrigerated for up to 42 days and assessed for microvesicle count and size, clotting- and prothrombin time, hemolysis index, and biochemical parameters. A significant increment in erythrocytes microvesicle count (EMVs/μL) was observed during the time in the three filtration sets. The number of EMVs in WBF-RBCs was higher (~1.6 fold) than in F-RCF on day 42 (p=0.035). Interestingly the median fluorescence intensity of EMVs decreased during the storage. The size of MVs rose during the time without any significant differences among the filters. Coagulation time decreased in RBCs over the storage, with no significant differences among the filters. Hemolysis index and lactate concentration increased while glucose level decreased significantly throughout the time. The changes in WBF-RBCs were more drastic rather than RCF-RBCs. The only significant difference in the count of EMVs was between WBF and F-RCF components on day 42. Though the changes in WBF products were more drastic, all the values fell within the standard limits. Accordingly, all three filtration sets can be considered.

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As a common life-saving therapeutic approach, Red Blood Cell components (RBCs) are the most transfused component worldwide. However, the repercussions related to components' preparation and storage have remained the most significant challenge facing transfusion medicine., Accordingly, quality assurance possesses a crucial dignity in all products' collection, preparation, and storage level. Until now, the accomplishment of several processing strategies including leukocyte depletion has improved the quality of the components. Above all, blood components’ leukocyte-filtration is a momentous achievement that reduces the risk of leukocyte-associated complications such as febrile and febrile non-hemolytic reactions, HLA alloimmunization, and cytomegalovirus transmission.

During storage, erythrocytes undergo a variety of detrimental oxidative, biochemical, and biomechanical alterations collectively denoted as “hypothermal storage lesions”. Exposure to various stressors makes erythrocytes susceptible to destruction, hemolysis, and several membrane alterations, which end up with the formation of microvesicles (MVs).,MVs are submicron membrane-covered particles heterogeneous in size (50 nm-1 μm) and originated from different cells under various circumstances in health and disease. They carry their parents' molecular contents and cell-specific markers by which they are characterized. As a permanent feature of the aging process, vesiculation occurs in both in and ex vivo conditions. Actually, in the context of blood transfusion erythrocytes-derived microvesicles (EMVs) are assumed as storage lesions.

Growing documents have signified they are involved in a broad spectrum of physiological and pathological mechanisms and may boost some pathways, including coagulation and inflammation.,,, In addition to storage conditions, variability of donors, pre-processing conditions, and blood processing techniques are referred to as sources of RBCs quality variations associated with undesired clinical consequences and poor outcomes.,,, As regards, the issued RBCs produced through different preparation procedures are not equally processed, even though the whole process is tightly supervised, elucidating the role of blood preparation methods in the quality of the components is crucially important., At the Iranian Blood Transfusion Organization (IBTO), the leukocyte-filtration process began in 2005, and today about one-third of the RBCs components are produced using two different leukocyte filtration procedures including whole blood- (WBF) and red cell (RCF) filtration. Regarding the emphasis on using leukocyte-filtered products, the current experimental study was established to investigate the possible effects of three different leukocyte-depletion filters (which are currently used at IBTO) on the EMVs formation in RBCs components and also the quality of the components by harmonizing the preparation conditions and eliminating some confounding variables.

Figures

Fig 1.
Fig 1.
The illustration of flow cytometry gating strategy (A-D, Flowjo v10) and size distribution of MVs using dynamic light scattering (E). Plot A depicts two populations of MVs and beads (as the size guide) on the FSC vs SSC channel (logarithmic scale). Plots B, C, and D demonstrate the populations of beads, CD235a+MVs (FL2, log scale) and Ann V+MVs (FL1, log scale), respectively. The size distribution (diameter) of MVs is shown in plot E. F-RCF: Fresenius Kabi Red cell filtration, M-RCF; Macopharma Red cell filtration, WBF: Whole Blood filtration.
Fig 2.
Fig 2.
Assessment of coagulation time in stored RBCs during storage (simple and repeated contrasts). (A) Prothrombin time, (B) Clotting time, (C) data are presented as mean ± standard deviation, (D) reduction of PT vs the increasing amount of MVs (serial dilutions). * Significant differences at the level of 0.05. † The time point when corresponds to the most decrement among the days. δ Time points when did not change significantly compared to the immediate before time (repeated contrast). F-RCF: Fresenius Kabi Red Cell Filtration; M-RCF: Macopharma Red Cell Filtration; WBF: Whole Blood Filtration.
Fig 3.
Fig 3.
Bivariate correlation analysis (Pearson coefficient). (A and B) Negative relationships between the counts of EMVs & PT (p<0.001) and EMVs & CT (p<0.001), (C and D) Negative relationships between the counts of PS+MVs & PT (p<0.001) and PS+MVs & CT (p<0.001). (E) Positive correlation between the counts of EMVs and hemolysis index (p<0.001). (F) Negative correlation between glucose and hemolysis index (p<0.001). Significant correlations are considered at the level of 0.01. F-RCF: Fresenius Kabi Red Cell Filtration; M-RCF: Macopharma Red Cell Filtration; WBF: Whole Blood Filtration.
Fig 4.
Fig 4.
Evaluation of hemolysis index and biochemical parameters of RBCs over the storage time (simple and repeated contrasts). (A) Hemolysis index, (B) lactate concentration, (C) glucose concentration, and (D) Data are presented as mean ± standard deviation. # Significant differences between WBF and F-RCF/ WBF and M-RCF. * Significant differences at the level of 0.05. † The time point when corresponds to the most increment and decrement among the days. δ Time points when did not change significantly compared to the immediate before time. F-RCF: Fresenius Kabi Red Cell Filtration; M-RCF: Macopharma Red Cell Filtration; WBF: Whole Blood Filtration.
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