The Egyptian Journal of Haematology

: 2016  |  Volume : 41  |  Issue : 4  |  Page : 200--205

Leukoreduced red blood cell: storage-related complement regulatory proteins and CD47

Manal H Farahat1, Mohammad A Sharaf2,  
1 Department of Clinical Pathology, Faculty of Medicine, Zagazig University Hospitals, Zagazig, Egypt
2 Department of Biochemistry, Zagazig University Hospitals, Zagazig, Egypt

Correspondence Address:
MD Manal H Farahat
Department of Clinical Pathology, Faculty of Medicine, Zagazig University Hospitals, Zagazig, 11111


Background Despite progress in red blood cell (RBC) storage, storage lesions still occur, which lead to complement activation and removal of RBCs from circulation. The aim of this study was to assess the surface concentration of complement regulatory proteins, as well as CD47, on leukoreduced RBCs collected on the atreus device and stored in sodium–adenine–glucose–mannitol medium for 42 days. Materials and methods We studied the surface expression of leukoreduced RBC C3d, CD35, CD55, CD59, and CD47 using flow cytometry on weekly intervals from day 1 to day 42 of storage. Results We observed a nonsignificant increase of surface C3d levels (P=0.146) and a decrease in CD47 overall storage time (P=0.196). There was a significant decrease demonstrated in CD35 (P=0.025), and a strong significant decrease in CD55 in their expression over storage time (P=0.000). Last, a nonsignificant alteration of CD59 was observed (P=0.353). Conclusion The study of atreus-processed leukoreduced RBC units stored in sodium–adenine–glucose–mannitol medium for surface expression of complement regulatory proteins showed its inhibition until toward the end of storage time, and also showed a good preservation of CD47, which was thought to be important in transfused RBC survival. However, more research is potentially needed in areas of complement evaluation during component processing.

How to cite this article:
Farahat MH, Sharaf MA. Leukoreduced red blood cell: storage-related complement regulatory proteins and CD47.Egypt J Haematol 2016;41:200-205

How to cite this URL:
Farahat MH, Sharaf MA. Leukoreduced red blood cell: storage-related complement regulatory proteins and CD47. Egypt J Haematol [serial online] 2016 [cited 2022 Aug 8 ];41:200-205
Available from:

Full Text


The red blood cell (RBC) storage duration is the time needed for at least 70–75% of the RBCs transfused to survive 24 h after transfusion [1]. To improve RBC storage quality and minimize storage-induced damage, the in-line leukocyte filters were used before storage [2], and to prolong their shelf-life additive solutions were used such as slightly hypertonic SAGM (sodium–adenine–glucose–mannitol) most often for 42 days, or 50 days for isotonic PAGGSM (phosphate–adenine–glucose–guanosin–saline–mannitol) [1],[3].

However, little is known about the product changes during processing before storage, which varies to a great extent on a daily basis [4]. It was shown that initiators of complement activation during storage might contribute to storage lesion toxicity [5], and also it had a role in adverse effects of blood transfusion such as hemolytic reaction, anaphylactic reaction, and transfusion-related acute lung injury [6]. The complement activation may be initiated from foreign materials such as plastic surface, storage in plastic bags, process of component separation, and also during leukoreduction [5]. However, in contrast, others recommended leukoreduction to prevent complement activation during RBC storage [7].

The presence of major complement regulators on stored RBC suggests that they might be resistant to complement lysis in the circulation, such as CD35 [complement receptor type 1 (CR1); C3b/C4b receptor], which prevents excessive complement activation, CD55 (decay accelerating factor), which inhibits C3 and C5 cleavage, and CD59 (membrane inhibitor of reactive lysis; protectin), which inhibits membrane attack complex formation [6],[8].

Integrin-associated protein (CD47) is expressed on all blood cells; it is known as a marker of self [9]. It is suggested to function as a molecular switch for erythrophagocytosis [10]. Therefore, the decline of CD47 during storage might result in rapidly cleared RBCs after transfusion [11].

The aim of this study was to assess the RBC surface expression concentration of the complement regulatory proteins CD35, CD55, and CD59, as well as the activation marker C3d and CD47 alteration occurring in leukoreduced RBCs collected on the atreus (Terumo BCT) device and stored in SAGM medium for 42 days under standard blood banking storage conditions.

 Materials and methods

Blood collection and blood component preparation

Twelve whole blood (WB) units (450±45 ml) were obtained from healthy donors (three female and nine male) at our blood bank of University Hospitals after meeting institution’s standard donation criteria and after obtaining written informed consent; their mean age was 29.3±8.7 years and were of all blood group types (three A type, three B type, three O type, and three AB type). The study was approved by our institutional ethics committee. All units were collected into atreus three-component integrated processing sets (Terumo BCT, Lakewood, Colorado, USA) containing 63 ml of citrate–phosphate–dextrose (CPD) anticoagulant, stored for 14–24 h at room temperature, and processed into the atreus WB processing system, which automatically produced RBC, plasma, and an interim platelet unit [12]. This technology is self-contained, and it integrates all needed manual steps such as weighing, centrifugation, expression, sealing, volume determination, and management of data [13],[14].

Leukoreduced red blood cell preparation

RBC units (n=12) were processed into leukoreduced RBCs, by using a gravity drain filtration system through fitted in-line WB–leukocyte depletion filter; resuspended in a 100 ml solution of SAGM; and stored under standard blood banking conditions at 2–6°C. Aliquots were aseptically obtained from each leukoreduced RBC unit on days 1, 7, 14, 21, 28, 35, and 42.

Blood cultures were done for all units at the end of storage time to control sterility.

Flowcytometric analysis of C3d, CD35, CD55, CD59, and CD47 was performed as follows

The monoclonal antibodies (moAbs) used were C3d antibody–FITC clone (BGRL11) (American Research Products, Waltham, Massachusetts, USA), anti-CD35–APC clone (E11) (Miltenyi Biotec, Lindbergh, California, USA), anti-CD55–PE clone (143–30) and anti-CD59–FITC clone (OV9A2) (e-Bioscience, San Diego, California, USA), and anti-CD47–PerCP/Cy5.5 clone (CC2C6) (BioLegend, San Diego, California, USA).

RBC samples were directly fluorescence stained with the moAbs against RBC surface C3d, CD35, CD55, CD59, and CD47 on days 1–42 on a weekly interval. They were washed three times with PBS, 100 μl of suspended cells were incubated with 10 μl of moAbs at room temperature for 20 min, and then washed twice by adding PBS with 0.1% Na-azid, centrifuged at 1200 rpm for 10 min at 18°C, and then 500 μl of PBS was added. Samples were run on an FACSCalibur and data were analyzed with cellquest software (BD Biosciences, San Jose, California, USA). For analysis, a gate was set around intact population as defined by forward and side scatter with the logarithmic amplification, and at least 10 000 events were analyzed for each antibody. The percentage of positive cells of total marker-expressing cells above that of background (negative control), as well as the mean fluorescence intensity, was recorded. Daily controls of optics and fluorescence intensity were performed using standardized beads (CaliBite; BD Bioscience).

Statistical analysis

Statistical analysis was performed using the SPSS computer software version 20 (SPSS Inc., Chicago, Illinois, USA). Variance analysis was performed using analysis of variance (ANOVA) for repeated measurements. The paired t-test was applied to determine statistically significant differences between different time points relative to baseline value; P values less than 0.05 were considered statistically significant.


In this study, the atreus-processed leukoreduced RBCs in SAGM storage medium over a period of 42 days under standard blood banking storage conditions showed the following results.

C3d, CD35, CD55, and CD59 expression during storage

The surface C3d levels showed nonsignificant increase from day 1 to day 42 (0.146); the significant difference compared with day 1 was observed on day 42 of storage (P=0.006). A significant decrease in CD35 (0.025) and a strong significant decrease in expression of CD55 compared with baseline over all storage times were reported (P<0.000). A nonsignificant alteration in CD59 was noticed during all storage periods compared with baseline levels (P=0.353) ([Table 1]).{Table 1}

CD47 ‘marker of self-expression’ during storage

A nonsignificant decrease in CD47 expression during storage time was observed. The strongest significant decrease in expression was observed within the first 21 days of storage (P<0.000) ([Table 1]).

The mean±SD difference and its percentage in relation to values of day 1 of C3d, CD35, CD55, CD59, and CD47 showed higher expression loss (29.9%) of CD55 when compared with CD35 (8.5%) and CD59 (1.9%) over all storage times ([Table 2]).{Table 2}


Three major factors affect in-vitro RBC quality − donor properties, component processing, and storage time − so it is time to consider to standardize RBC processing methods to better influence transfusion outcome and help provide more consistent and safer products for transfusion [15].

However, leukocyte filtration had a better effect on RBC storage lesion, by improving both RBC hemolysis and its post-transfusion recovery; in contrast, another study reported that it might occasionally cause complement activation, which is affected by type and charge of filter used and could be aggravated by citrate concentration decline during storage, as citrate is believed to play a membrane-protective role [15],[16]. Therefore, regarding the effects of different component preparation methods, there is a need to better understand the complement-mediated alterations in RBC storage lesion progression, especially after the addition of leukocyte depletion, as well as additive storage solution [17].

In our study, the surface C3d levels showed a nonsignificant increase from day 1 to day 42 (P=0.146). The significance was observed only on day 42 compared with day 1 (P=0.006). A study conducted by Kamhieh-Milz et al. [17] on leukocyte-depleted RBCs with CPD/PAGGSM found a weak and significant increase in C3d expression over the storage duration (P=0.0103); significance was observed on day 35 of storage (P=0.0328). In addition, regarding the role of citrate on complement activation, it has been demonstrated from studies on RBCs stored in CPD–SAGM as anticoagulant that complement activation occurred in 25 mm CPD but was inhibited in 50 mm [18]. Thus, regarding our storage medium, 100 mm CPD–SAGM, we agreed with the study of Kamhieh-Milz et al. [17] who used 100 mm of CPD/PAGGSM and found that complement activation and C3d expression were inhibited until toward the end of storage time, but we achieved better inhibited activation until day 42 of storage. In contrast, we disagreed with their study regarding the difference in C3d levels, which was not significant in our study at all storage times, may be of the different processed components and storage medium we used, considering that there were other factors affecting C3d expression, such as progressed citrate reduction and ATP decline level, which occurred over all storage times [19]; this could explain the significantly increased expression of C3d.

In the present study, we observed a significant decrease in expression in CD35 compared with baseline at all storage times (P=0.025); the loss in percentage of expression started at 0.7% on day 7 and reached 8.5% on day 42 of storage, which was comparable to the study of Kamhieh-Milz et al. [17], who found a significant surface expression loss of CD35 of 4% during storage time on day 7 and increased to 10% on day 42. Thus, although there was a significant loss in both studies, it seemed that our results achieved better storage CD35 expression, which appeared clear from the very beginning at day 7 even until the end of storage time.

In our study, a strong significant decrease in CD55 expression was reported at all storage times compared with baseline levels (P<0.000). The percentage loss was 8.8% after 7 days and 18.5% on 14 days, and on day 42 of storage it was about 30%, which was comparable to the study by Kamhieh-Milz et al. [17] regarding the same storage time evaluated, who found a strong significant decrease in CD55 expression at only first 14 storage days studied (P<0.0001), with a percentage loss of 10 and 18 on days 7 and 14, respectively. Many studies demonstrated also strongly decreased CD55 expression at all storage times of senescent RBCs [20],[21].

Our results showed that although a nonsignificant alteration in CD59 expression during storage period compared with baseline levels was found (P=0.353), a progressive loss was noted until day 21, and then improved expression was reported in the last 3 weeks of storage time, with 1.9% expression loss on 42 days. Therefore, we could agree with the explanation of Antonelou et al. [22], who studied the effect of leukocyte depletion on CD59 levels and explained that a proportion of the storage lesion seen in the leukodepleted RBCs may be related to the effect of donor leukocyte that disintegrates within 24 h, and the released material is passed through a filter and it contributes to RBC storage damage, which is reported in our results by expression loss in early storage days, and they found that significantly reduced expression of CD59 in non-leukoreduced RBCs at 42 days of storage might be due to the RBC membrane vesiculation. Our results were comparable to those of Kamhieh-Milz et al. [17], who found no significant decrease for CD59 but a reported decrease was observed using percentage values during storage time. However, although the decrease in expression was noticed in both studies, the ANOVA test revealed a significant decrease in their study (P=0.0006), but in our study ANOVA test reported a nonsignificant difference (P=0.353). As CD59 is a complement regulatory protein that protects blood cells from complement attack, it plays a role in in-vivo survival of transfused RBCs; therefore, the better clinical relevance is clearly noticed in our study.

In our study, CD55 showed a higher expression loss (29.9%) when compared with CD35 (8.5%) and CD59 (1.9%) at all storage times. Kamhieh-Milz et al. [17] calculated a loss of CD55, evaluated until day 14 of storage only, of 18% and a loss of CD35, evaluated at all 42 days of storage time, of 10%, which was in agreement with our results: 18.5 and 8.5%, respectively.

We might explain that such higher CD55 loss allover storage time when compared with other markers in our study by that CD55 was more enriched in shedded vesicles which followed vesiculation and secretion of microparticles that associated RBC aging than CD35 [23] and CD59 [17], an alternative explanation may be that CD55 lost through proteolytic cleavage from residual proteases which might be present in RBC [20], considered that CD35, CD55 found to differ in their sensitivities to proteases [20],[24],[25],[26], and that CD59 protease resistant [7],[17]. Thus, the loss of CD55 may be mediated by both vesiculation and proteolysis, whereas the loss of CD35 may be caused by proteolysis [26], and the last likely mechanism was storage-induced ATP depletion [23].

Aging of stored RBCs is caused by storage-associated stresses; CD47 loss [27], and thus preservation of CD47 on RBCs, during storage still appears to be important in survival of transfused RBCs in vivo [22].

We observed a nonsignificant decrease in CD47 expression during all storage times (P=0.196). The strongest significantly decreased expression was observed within the first 21 days of storage (P<0.000), with higher expression loss reported to be 7–8%; then an increase in expression was noticed toward the end of storage until day 42 reached a percentage loss of about only 0.7% compared with the baseline value. Anniss and Sparrow [28], Antonelou et al. [22], and Kamhieh-Milz et al. [17] studied the CD47 expression, and they observed a small significant progressive decline in CD47 expression from 6 to 9% loss during storage, which were almost closer to our finding. It was also demonstrated that the degree of CD47 decline on stored red cells was reported to be between 10 and 65% depending on the type of product and type of assay used [29]. As a decrease of more than 50% in CD47 expression on stored RBCs results in increased susceptibility to phagocytosis [10],[22], our higher expression loss reported was 7–8%, which is highly unlikely to have a deleterious effect.

We observed in our results the lowest level of CD47 expression occurred on days 7–14, then a paradoxical increase in CD47 expression was noticed toward the end of storage time. Kamhieh-Milz et al. [17] had also reported an almost similar finding as they found the lowest CD47 expression level on days 21–28 and increased CD47 antigen at day 31, and Karon et al. [30] found that CD47 increased after day 24 of storage and they explained that by expressed cytoskeletal-bound molecules which are not prone to release, they also suggested that the remaining CD47 undergoes oxidation, proteolytic cleavage and conformational changes, which leading to exposure of extra CD47 antigen toward the end of the storage period.


As these complement regulatory proteins protect blood cells from complement attack, it plays a role in transfused RBC in-vivo survival; therefore, the clinical relevance of the observed changes noticed of the cell markers studied are not thought to be large enough to cause harmful effect of processed leukoreduced RBCs, as the progressive loss of markers; deformability of RBC had a potential impact on the overall storage lesion [11]. Considering that there was no study done before in this area of marker evaluation of the automated WB processing method used, in addition, the extent of stress influencing RBC vesiculation and storage lesion may occur during preparation steps could be affected by different procedures used and donor-related factors [27]. However, a more advanced complement evaluation study was potentially needed during component processing.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


1Lion N, Crettaz D, Rubin O, Tissot J. Stored red blood cells: a changing universe waiting for its map(s). J Proteomics 2010; 73:374–385.
2Barshtein G, Gural A, Manny N, Zelig O, Yedgar S, Arbell D. Storage-induced damage to red blood cell mechanical properties can be only partially reversed by rejuvenation. Transfus Med Hemother 2014; 41:197–204.
3Holme S. Current issues related to the quality of stored RBCs. Transfus Apher Sci 2005; 33:55–61.
4Meer P, de Korte D. The effect of holding times of whole blood and its components during processing on in vitro and in vivo quality. Transfus Med Rev 2015; 29:24–34.
5Hyllner M, Arnestad J, Bengtson J, Rydberg L, Bengtsson A. Complement activation during storage of whole blood, red cells, plasma, and buffy coat. Transfusion 1997; 37:264–268.
6Antoneloua M, Seghatchian J. Update on extracellular vesicles inside red blood cell storage units: adjust the sails closer to the new wind. Transfus Apher Sci 2016; 55:92–104.
7Hu X, Patel R, Weinberg J, Marques M, Ramos T, Barnum S. Membrane attack complex generation increases as a function of time in stored blood. Transfu Med 2014; 24:114–116.
8Khan R, Maduray K, Moodley J, Naicker T. Activation of CD35 and CD55 in HIV associated normal 3 and pre-eclamptic pregnant women. Eur J Obstet Gynecol Reprod Biol 2016; 204:51–56.
9Olsson M, Nilsson A, Oldenborg P. Dose-dependent inhibitory effect of CD47 in macrophage uptake of IgG-opsonized murine erythrocytes. Biochem Biophys Res Commun 2007; 352:193–197.
10Burger P, Hilarius-Stokman P, de Korte D, van den Berg T, van Bruggen R. CD47 functions as a molecular switch for erythrocyte phagocytosis. Blood 2012; 119:5512–5521.
11Bessos H, Seghatchian J. Red cell storage lesion: the potential impact of storage-induced CD47 decline on immunomodulation and the survival of leucofiltered red cells. Transfus Apher Sci 2005; 32:227–232.
12Sandgren P, Waeg G, Verheggen C, Sjödin A, Gulliksson H. Storage of interim platelet units for 18 to 24 hours before pooling: in vitro study. Transfusion 2011; 51:1213–1219.
13Cid J, Magnano L, Lozano M. Automation of blood component preparation from whole blood collections. Vox Sanguinis 2014; 107:10–18.
14Jurado M, Algora M, Garcia-Sanchez F, Vico S, Rodriguez E, Perez S et al. Automated processing of whole blood units: operational value and in vitro quality of final blood components. Blood Transfus 2012; 10:63–71.
15Hansen A, Kurach J, Turner T, Jenkins C, Busch M, Norris P et al. The effect of processing method on the in vitro characteristics of red blood cell products. Vox Sanguinis 2015; 108:350–358.
16Jordan A, Chen D, Yi Q, Kanias T, Gladwin M, Acker J. Assessing the influence of component processing and donor characteristics on quality of red cell concentrates using quality control data. Vox Sanguinis 2016; 111:8–15.
17Kamhieh-Milz J, Bartl B, Sterzer V, Kamhieh-Milz S, Salama A. Storage of RBCs results in an increased susceptibility for complement-mediated degradation. Transfus Med 2014; 24:392–399.
18Kobayashi E, Kitano E, Kondo H, Kitamura H. Complement activation in citrate plasma − inhibitory effect of anticoagulants on serum complement activation. Rinsho Byori 1999; 47:160–164.
19Gevi F, D’Alessandro A, Rinalducci S, Zolla L. Alterations of red blood cell metabolome during cold liquid storage of erythrocyte concentrates in CPD-SAGM. J Proteomics 2012; 76:168–180.
20Pascual M, Danielsson C, Steiger G, Schifferli J. Proteolytic cleavage of CR1 on human erythrocytes in vivo: evidence for enhanced cleavage in AIDS. Eur J Immunol 1994 24:702–708.
21Miot S, Marfurt J, Lach-Trifilieff E, Gonzalez-Rubio C, Lopez-Trascasa M, Sadallah S et al. The mechanism of loss of CR1 during maturation of erythrocytes is different between factor I deficient patients and healthy donors. Blood Cells Mol Dis 2002; 29:200–212.
22Antonelou M, Tzounakas V, Velentzas A, Stamoulis K, Kriebardis A, Papassideri I. Effects of pre-storage leukoreduction on stored red blood cells signaling: a time-course evaluation from shape to proteome. J Proteomics 2012; 76:220–238.
23Pascual M, Lutz H, Steiger G, Stammler P, Schifferli J. Release of vesicles enriched in complement receptor 1 from human erythrocytes. J Immunol 1993; 151:397–404.
24Willekens F, Werre J, Groenen-Dopp Y, Roerdinkholder-Stoelwinder B, Pauw B, Bosman G. Erythrocyte vesiculation: a self-protective mechanism? Br J Haematol 2008; 141:549–556.
25Rubin O, Canellini G, Delobel J, Lion N, Tissot J. Red blood cell microparticles: clinical relevance. Transfus Med Hemother 2012; 39:342–347.
26Ripoche J, Sim R. Loss of complement receptor type 1 (CR1) on ageing of erythrocytes studies of proteolytic release of the receptor. Biochem J 1986; 235:815–821.
27Solheim B, Flesland O, Brosstad F, Mollnes T, Seghatchian J. Improved preservation of coagulation factors after pre-storage leukocyte depletion of whole blood. Transfus Apher Sci 2003; 29:133–139.
28Anniss A, Sparrow R. Expression of CD47 (integrin-associated protein) decreases on red blood cells during storage. Transfus Apher Sci 2002; 27:233–238.
29De Watering L. Red cell storage and prognosis. Vox Sang 2011; 100:36–45.
30Karon B, Van C, Jaben E, Hoyer J, Thomas D. Temporal sequence of major biochemical events during blood bank storage of packed red blood cells. Blood Transfus 2012; 10:453–461.