Nanodefects of membranes cause destruction of packed red blood cells during long-term storage

doi:10.1016/j.yexcr.2015.07.009

Experimental Cell Research

Volume 337, Issue 2, 1 October 2015, Pages 192-201

https://doi.org/10.1016/j.yexcr.2015.07.009 Get rights and content

Highlights

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Domains with “grains” are formed on membranes surface on 9–16 days of PRBC storage.
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The development of domains is the reason of irreversible changes of PRBC structure.
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The origin of domains is the consequence of alterations of spectrin cytoskeleton.
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Study of nanostructure may form basis of assessing the quality of the stored PRBC.

Abstract

Packed red blood cells (PRBC) are used for blood transfusion. PRBC were stored for 30 days under 4 °С in hermetic blood bags with CPD anticoagulant-preservative solution. Hematocrit was 50–55%. The distortions of PRBC membranes nanostructure and cells morphology during storage were studied by atomic force microscopy. Basic measurements were performed at the day 2, 6, 9, 16, 23 and 30 of storage and additionally 2–3 days after it.

Topological defects occurred on RBC membranes by day 9. They appeared as domains with grain-like structures (“grains”) sized up to 200 nm. These domains were appeared in almost all cells. Later these domains merged and formed large defects on cells. It was the formation of domains with the “grains” which was onset process leading eventually to destruction of PRBC. Possible mechanisms of transformation of PRBC and their membrane are related to the alterations of spectrin cytoskeleton. During this storage period potassium ions and lactat concentrations increased, pH decreased, intracellular concentration of reduced glutathione diminished in the preservative solution. Changes of PRBC morphology were detected within the entire period of PRBC storage. Discocytes predominated at the days 1 and 2. By day 30 PRBC transformed into irreversible echinocytes and spheroechinocytes. Study of defects of membranes nanostructure may form the basis of assessing the quality of the stored PRBC. This method may allow to work out the best recommendations for blood transfusion.

Introduction

Blood transfusion in acute and chronic anemia, in massive blood loss, in leukemia and other diseases implies utilization of packed red blood cells (PRBC). According to WHO guidelines [1] PRBC can be stored under 4 °C within 30–40 days. These PRBC are supposed to be acceptable for blood transfusion. But the storage environment of PRBC differs significantly from physiological. PRBC are stored in hemopreservative solution in hermetic bags. After blood transfusion at least 25% of transfused red blood cells (RBC) are eliminated from circulation during one day [2]. Preservation of the stability of PRBC is one of the key problems of transfusion medicine [3]. The final protocol including long-term storage of PRBC and the efficient blood transfusion is still under discussion [4].

Alterations of PRBC structure during storage are conventionally split into reversible and irreversible. The reversibility of the cell shape is inversely proportional to the storage time [4]. The quality of PRBC is mainly determined by the PRBC shape and the structure of their membranes [5]. Changes of PRBC shapes and alterations of their membrane structure [6] may lead to a decrease of RBC membrane deformability, deterioration of blood rheology and decrease of gas transporting function.

Changes of RBC morphology and their membrane structure may be caused by oxidative processes which develop in solution volume during prolonged storage and also by activation of free-radical processes in RBC themselves [7], [8].

One of the effective methods of RBC morphology and their membrane investigation is atomic force microscopy (AFM). This method requires no preliminary modification of the object, and its resolution is less than 1 nm, which provides us with a possibility to study the cell membrane structure in detail.

The aim of the investigation was to study with AFM the alterations of membrane nanostructure of PRBC during long-term storage.

Section snippets

Packed red blood cells

PRBC in hermetic blood bag (400 ml) with CPD preservative solution were obtained from 3 independent blood transfusion centers (Moscow, Russian Federation). In total 12 blood bag were used in study (blood groups O(I), A(II), B(III), AB (IV), three bags of each group). Blood at these stations was routinely screened for infections, centrifuged and filtered according to the National guidelines. Packed RBC hematocrit was 50–55%.

PRBC were preserved for 30 days under 4 °С in accordance with WHO

PRBC morphology

Alterations of RBC morphology were detected during the whole period of PRBC storage. Fig. 1, Fig. 2, Fig. 3, Fig. 4 shows AFM images of cells at different days of storage. Fig. 1A shows typical 3D AFM image of RBC in 120х120 µm² scale. At early stages of storage due to natural poikilocytosis various cell shapes were presented on monolayer.

On days 1–2 of storage discocytes made up to 68±8% of cells (Fig. 1B). By day 16 their percentage decreased to 11±1%, and by day 30 – down to 3±1% (Table 1).

Cytoskeleton

RBC morphology and nanostructure of their membranes is determined by the state of spectrin matrix. It lines the inside part of the plasma membrane of RBC. Matrix is a hexagonal network of spectrin tetramers. The connections of spectrin cytoskeleton to the membrane are organized by means of transmembrane protein complexes band 4.1 and ankyrin. The typical size of a cell of spectrin matrix is normally is 60–100 nm [16]. These dimensions have been shown in the analysis of the membranes

Conclusion

The distortions of membrane nanostructure and of morphology are arised during prolonged storage of packed RBC in hermetic bags with CPD preservative solution under 4 °С. 9–16 days after storage local topological defects in the form of domains with grain-like structures developed. The occurrence of domains with “grains” was the onset process that induced spheroechinocytes formation and PRBC destruction. Decrease of intracellular reduced glutathione, diminishing of pH, increase of potassium ions

Ethics review

The study was performed in accordance with the principles of the Declaration of Helsinki and was approved by the Ethics Committee of V.A. Negovsky Scientific Research Institute of General Reanimatology, Moscow, Russian Federation.

Competing financial interests

The authors declare no competing financial interests.

Acknowledgments

The authors thank S. Malakhova for her invaluable assistance in carrying out experiments.

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Additionally, analysis of the time distribution of erythrocyte shape variability during storage was performed (Figure S8). Such analysis agrees with previous reports and confirms mainly the decrease in the discocytes (20% of the healthy RBCs visible at the beginning of studies decreased to around 5% population at the 49th* day of storage) and an increase of 20% in the echinocytes with the time of storage.48 Moreover, at the 21st day (in the third week) of PRBC storage, groups of spheroechinocytes and spherocytes (irreversible cells) started to appear, and a concomitant decrease in discocytes was observed.54
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Research ArticleNanodefects of membranes cause destruction of packed red blood cells during long-term storage

Highlights

Abstract

Introduction

Section snippets

Packed red blood cells

PRBC morphology

Cytoskeleton

Conclusion

Ethics review

Competing financial interests

Acknowledgments

J. Crit. Care

Micron

Nitric Oxide

Blood

Transfus. Apher. Sci.

Chem. Biol. Interact.

Blood

Recommendations for the transfusion of red blood cells

Blood Transfus.

Red blood cell hemolysis during blood bank storage: using national quality management data to answer basic scientific questions

Transfusion

Red blood cell storage: the story so far

Blood Transfus.

Red blood cell deformability during storage: towards functional proteomics and metabolomics in the Blood Bank

Blood Transfus.

An AFM approach of RBC micro and nanoscale topographic features during storage (World Academy of Science, Engineering and Technology)

Int. J. Med. Health Biomed. Pharm. Eng.

Research Article
Nanodefects of membranes cause destruction of packed red blood cells during long-term storage