Clinical Hemorheology and Microcirculation 60 (2015) 437–449 DOI 10.3233/CH-141899 IOS Press

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Albumin reverses the echinocytic shape transformation of stored erythrocytes Sophie A. Reinharta , Thomas Schulzkib and Walter H. Reinharta,∗ a b

Department of Internal Medicine, Kantonsspital Graub¨unden, Chur, Switzerland Division of Transfusion Medicine, Kantonsspital Graub¨unden, Chur, Switzerland

Abstract. The storage of red blood cells (RBCs) leads to storage lesions, which have a negative impact on the clinical outcome after transfusion. A hallmark of storage lesions is echinocytosis. Albumin may reverse this shape transformation, which was the topic of this study. Echinocytosis was generated by incubation of blood for 48 h at room temperature or in RBC units stored 48 days at 5◦ C. Human serum albumin was diluted in phosphate-buffered saline. RBCs were fixed in 1% glutaraldehyde and examined by light and scanning electron microscopy. The degree of echinocytosis was quantified by calculating the morphological index. Incubation and storage of RBCs led to an echinocytic shape transformation, which was reversible upon incubation in albumin solutions. This process was time-, concentration- and hematocrit-dependent. Treating RBC units at the end of their shelf-life by adding 20% albumin or washing them in 0.2% albumin reversed all degrees of echinocytosis towards discocytosis. In conclusion, albumin has the capacity to reverse echinocytosis generated by RBC storage. This observation may improve the quality of RBC units stored for longer periods of time. Keywords: Albumin, blood storage, echinocyte, erythrocyte, transfusion

1. Introduction Considerable progress has been made in the preparation of RBC units before storage, primarily by removal of leukocytes and by adding a conservation medium. By that, the maximum storage duration has been prolonged to 6–7 weeks. However, even under the best storage conditions, the functional properties of RBCs deteriorate over time. These so called storage lesions are of biochemical, structural, and morphological origin [22, 48, 51]. Recently, transfusion medicine has come under scrutiny [3, 11] because the transfusion of RBCs stored for a longer period of time is associated with an increased morbidity and mortality [17, 24, 46]. The mechanism behind it is currently not entirely understood. Posttransfusional hemolysis has been implicated, the free hemoglobin binds nitric oxide [4] and by that vasodilation is decreased [1] and the microcirculation may be impaired. The most prominent feature of storage lesions is an echinocytic shape transformation of RBCs [18, 44, 52], which is caused by changes in the RBC membrane. Extracorporeal storage leads to membrane lipid peroxidation [21], a decrease of unesterified cholesterol [21], and phosopholipids within the RBC membrane [2] with an accumulation of thrombogenic phosphatidylserine on the membrane surface [36], ∗ Corresponding author: W.H. Reinhart, MD, Department of Internal Medicine, Kantonsspital Graub¨unden, CH 7000 Chur, Switzerland. Tel.: +41 81 256 63 05; Fax: +41 81 256 63 81; E-mail: [email protected].

1386-0291/15/$35.00 © 2015 – IOS Press and the authors. All rights reserved

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S.A. Reinhart et al. / Albumin reverses echinocytosis after storage

a clustering of the trans-membraneous anion channel protein-band-3 [21], and a degradation of the cytoplasmatic domain of this integral protein [38]. Echinocytosis, i.e. the formation of spicules extruding from the cell surface, is a first step in exovesiculation and the shedding of microparticles, which occurs after prolonged RBC storage [37] and initiates and propagates thrombin generation [40]. Echinocytes have a decreased membrane deformability as measured with micropipette aspiration [7], a decreased cell deformability [31, 36] and aggregability [16], and they increase whole blood viscosity [34, 44, 52], which impairs blood flow in the microcirculation [6]. We had recently observed that echinocytic RBCs from incubated blood regained a discocytic shape when they were suspended in an albumin solution [33, 34]. This observation may be of considerable interest for the ongoing attempt to optimize RBC storage [49]. In the present study we analysed kinetic and dynamic aspects of this albumin-induced shape recovery process and extended these studies to RBC units stored under standard conditions for transfusion purposes. 2. Materials and methods 2.1. Materials Human serum albumin (Albumin CSL, Behring, Bern, Switzerland) with a concentration of 20 g/l was used. When necessary, it was diluted in phosphate buffered saline (PBS: 122 mmol/l NaCl, 25 mmol/l Na2 PO4 xH2 0, 5 mmol/l K2 HPO4 , 2 g/l glucose). PBS was also used for the suspension of RBCs and the preparation of 1 or 2% glutaraldehyde for RBC fixation. 2.2. Methods of blood incubation and storage Blood was drawn from healthy volunteers using 1.8 mg/ml K2 EDTA as an anticoagulant (Becton Dickinson, Allschwil, Switzerland). The unopened tubes were incubated at room temperature (22 ± 1◦ C) for 48 h. Stored RBC units were obtained from the division of transfusion medicine of the hospital. They had been prepared according to standard operational procedures, using citrate-phosphate-dextrose (CPD: citrate 263 g/l, citric acid 3.27 g/l, glucose 25.5 g/l, NaH2 P04 2.51 g/l) as an anticoagulant, inline leukocyte filtration of whole blood, centrifugation to separate-out plasma, and the addition of the conservation medium PAGGSM (NaCl 4.21 g/l, adenine 0.194 g/l, glucose 9.4 g/l, guanosin 0.408 g/l, Na2 H(PO4 ) 1.432 g/l, NaH2 P04 1.225 g/l, mannitol 10 g/l). The RBC units were stored under standardized conditions at 4◦ C. 2.3. Assessment of RBC morphology For the assessment of the RBC morphology, 20 ␮l of the RBC suspensions were fixed in 1000 ␮l 1% glutaraldehyde. The specimens were analysed by light microscopy in wet preparations using a Neubauer counting chamber. In each sample, at least 200 RBCs were classified according to the nomenclature of Bessis [5]. Thereby, a discocyte has a score of 0, an echinocyte I (an irregularly contoured discocyte with up to 5 protrusions) has a score of +l, an echinocyte II (a flat RBC with several spicules) has a score of +2, an echinocyte III (ovaloid or spherical RBC with multiple spicules) has a score of +3, an echinocyte IV or sphero-echinocyte (a sphere with multiple short and thin spicules) has a score of +4, a stomatocyte I (convex-concave instead of biconcave RBC) has a score of −1, etc. The morphological index according to

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Ferrell and Huestis [13] was calculated as the sum of all scores divided by the number of analysed RBCs. Some glutaraldehyde-fixed samples were also prepared for scanning electron microscopy (P. Furlong, AO Research Institute, Davos, Switzerland). 2.4. Experiments with RBCs incubated at room temperature (22◦ C) for 48 h The role of the albumin concentration was studied on RBCs suspended in PBS containing 0.05, 0.l, 0.2, 0.4, 0.8, 1.6, or 3.2% albumin. The influence of the hematocrit was studied by suspending packed RBCs in PBS containing 0.2% albumin. The hematocrit was initially adjusted to 64%. By further dilutions with 0.2% albumin in PBS, hematocrit levels of 32, 16, 8, 4, 2, 1, and 0.5% were prepared. For both experiments, RBCs were fixed in 1% glutaraldehyde after 10 min. The time-dependence of the shape recovery process was analysed as follows. 20 ␮l of packed RBCs were suspended in 500 ␮l 0.2% albumin, which was immediately (after 1 s) followed by the addition of 500 ␮l 2% glutaraldehyde. The study of longer time periods was done by suspending packed RBCs in 0.2% albumin and retrieving and fixing specimens in 1% glutaraldehyde after 5 s, 10 s, 20 s, 60 s, 360 s (6 min), 2180 s (36 min), and 12960 s (3 h 36 min). 2.5. Experiments with RBCs stored for either 24 or 48 days under blood banking conditions The influence of the albumin concentration, hematocrit and suspension time was analysed as described above. Mixing experiments with stored and fresh RBCs, simulating a transfusion, were done as follows. Samples from RBC units stored for 7 weeks were diluted with saline to obtain a hematocrit of 40%. Blood was drawn from an individual with the same blood group and was adjusted to a hematocrit of 40%. Specimens of both were fixed in 1% glutaraldehyde. Then, 1:1 mixtures were prepared and specimens were fixed after 1 min and 1 h. The frequency distributions of RBC shapes were assessed in the mixture and compared with the values expected from the single components prior to the mixture. The possibility of preventing an echinocytic shape transformation during blood banking was studied by splitting RBC units during preparation on day 1 and adding either 0.2% albumin or PBS alone under sterile conditions. RBCs were analysed after 24 and 48 days. The possibility of a pretransfusional RBC shape recovery was tested as follows. The hematocrit of the RBC units was adjusted to a hematocrit of 40% by the addition of either 20%, 2%, or 0.2% albumin in PBS. Washing in 0.2% albumin was done by diluting the hematocrit to 5%, followed by an adjustment to 40%. The RBC morphology was assessed as described above. 2.6. Statistical analysis Statistical analysis was performed with STATISTICA for Windows, Version 9.1 (Statsoft Inc., Tulsa, OK, USA, www.statsoft.com). A one way analysis of variance (ANOVA) was used for the comparison of more than two groups, a paired t-test for two groups, as appropriate. The results are presented as mean values ± standard deviation (SD). A p-value of

Albumin reverses the echinocytic shape transformation of stored erythrocytes.

The storage of red blood cells (RBCs) leads to storage lesions, which have a negative impact on the clinical outcome after transfusion. A hallmark of ...
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