Transfusion and Apheresis Science xxx (2014) xxx–xxx
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Transfusion and Apheresis Science journal homepage: www.elsevier.com/locate/transci
Effects of hydroformylation treatment on the storage time and blood group antigen expressions of reagent red blood cells Yang Yu, Xiaolin Sun, Xiaozhen Guan, Xiaojuan Zhang, Chunya Ma, Linfeng Chen, Deqing Wang ⇑ Department of Blood Transfusion, The Center for Clinical Transfusion Medicine, Chinese PLA General Hospital, Beijing, China
a r t i c l e
i n f o
Article history: Received 29 March 2013 Received in revised form 12 January 2014 Accepted 24 February 2014 Available online xxxx Keywords: Hydroformylation Blood group antigen Reagent red blood cells Agglutination strength Hemolysis
a b s t r a c t Objective: To evaluate the effects of hydroformylation treatment on the storage time and blood group antigen expressions of reagent red blood cells (RBCs). Material and methods: RBCs from healthy donors were treated by using various final concentrations of paraformaldehyde (0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5% and 1.0%) and glutaraldehyde (0.01%, 0.025%, 0.05%, 0.1%, 0.2%, 0.5% and 1.0%), and one aliquot was used as control (untreated with aldehydes). Supernatant free hemoglobin (FHb) levels in all groups stored at 4 °C were detected every week, and the optimal procedure was selected. Expression of blood group antigens on RBCs treated by the optimal procedure was determined, and the total scores of blood group antigens were calculated. Results: 0.2%, 0.5% and 1.0% Glutaraldehyde groups were ruled out directly due to serious crosslinking and aggregation of RBCs. As the extension of time, FHb levels in other 11 groups gradually increased (p < 0.01 or p < 0.05). FHb level in 0.025% glutaraldehyde group was significantly lower than that in other groups after 13 weeks (p < 0.01), and the antigen strength of Fyb, Jkb, and Leb decreased slightly compared with those before treatment and storage (p < 0.05), and there was no significant change for antigen strength of A, B, D, C, E, c, e, M, N, S, s, k, P1, Fya, Jka, and Lea (p > 0.05). Conclusion: 0.025% Glutaraldehyde treatment can provide optimal protection for the membrane of RBCs and keep hemolysis at a low level after 13 weeks storage, and the majority of blood group antigen systems are not significantly affected, and the slight decline of Fyb, Jkb, and Leb antigen strength was acceptable for classical serological tests. Ó 2014 Published by Elsevier Ltd.
1. Introduction Reagent RBCs for transfusion compatibility testing are generally prepared from healthy donor’s RBCs which are commonly used in blood grouping, unexpected antibody screening and identification, as well as internal quality control and external quality assessment. Long-term effective preservation of reagent RBCs has always been a thorny ⇑ Corresponding author. Address: Department of Blood Transfusion, Chinese PLA General Hospital, No. 28 Fuxing Road, Haidian District, Beijing 100853, China. Tel./fax: +86 1066936508. E-mail address: [email protected] (D. Wang).
issue. Reagent RBCs usually play two roles: one is directly involved in the antigen–antibody reaction as a red blood cell antigen, the other is showing the results of hemagglutination test as an indicator cells. Sufficient strength of blood group antigens on the reagent RBCs needs to be maintained during storage to ensure the detection sensitivity of the corresponding blood group antibodies. Hemolysis of RBCs should be controlled at a low level because the integrity of the RBC membrane directly affects the accuracy of hemagglutination tests. Traditional reagent RBCs are stored referring to the experience from storage of RBCs used in human blood transfusion. Citrate, glucose, adenine, mannitol, and other
http://dx.doi.org/10.1016/j.transci.2014.02.019 1473-0502/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: Yu Y et al. Effects of hydroformylation treatment on the storage time and blood group antigen expressions of reagent red blood cells. Transf Apheres Sci (2014), http://dx.doi.org/10.1016/j.transci.2014.02.019
2
Y. Yu et al. / Transfusion and Apheresis Science xxx (2014) xxx–xxx
ingredients are added to the RBCs storage medium. Glucose and adenine can continue to provide a source of energy for the RBCs during storage, and maintain morphology and structure of the RBCs. Mannitol has some resistance to RBCs hemolysis and makes the membrane structure of RBCs remain relatively intact [1–4]. In addition, some antibiotics are added to inhibit bacterial growth. With the extension of the storage time in vitro, energy substances are exhausted, membrane damage of RBCs is aggravated and FHb levels in supernatant increase gradually. Thus, the shelf life of reagent RBCs could be at most 8–9 weeks by adding nutrients and antibiotics, which led to a limitation in practical application. At the same time, there was no systematic research in changes of blood group antigen strength on the reagent RBCs during longterm storage. Paraformaldehyde and glutaraldehyde are commonly used as cell-fixing agents in cell biology research, and aldehyde reagents can make the membrane protein and the intracellular protein be crosslinked, so that the tissue cells are fixed, and the storage time is extended, which make observational studies on morphology and structure of the cell easy [5–7]. However, widespread formation of protein crosslinking can also cause some of the antigens to be masked and even lead to the degeneration of blood group antigens. The objective of this study was to use various concentrations of paraformaldehyde and glutaraldehyde to treat reagent RBCs before preservation, and try to look for an optimal method of aldehyde treatment which can effectively extend the shelf life of reagent RBCs, and keep the strength of the main blood group antigens stable at the end of storage, and meet the requirements for blood group serological testing.
obtained from 10 participants. 5 Voluntary and regular blood donors who all met the requirements of blood donation in China were selected. 200 ml whole blood was collected from each donor using commercially available blood collection bags containing ACD-B anticoagulant, and centrifuged at 1400g for 10 min. Plasma was separated from whole blood, and the buffy coat was discarded, and packed red cell was then washed three times with the use of sterile normal saline. 2.2.2. Hydroformylation treatment of RBCs A portion of the washed RBCs was divided into fifteen aliquots (1 ml). Paraformaldehyde was added to the washed RBCs to achieve RBCs suspension with final concentrations of 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5% and 1.0%, respectively. Glutaraldehyde was added to the washed RBCs to achieve RBCs suspension with final concentrations of 0.01%, 0.025%, 0.05%, 0.1%, 0.2%, 0.5% and 1.0%, respectively. One aliquot was as control (untreated with aldehydes). The RBCs suspensions were incubated at room temperature (22 °C) for 5 min, and then washed three times with the use of sterile normal saline and resuspended in RBCs preservative solution and prepared 5% red cell suspensions. Sulfuric gentamicin was added to all specimens to obtain final concentration of 0.05 mg/ml. 2.2.3. Determination of FHb during storage All specimens in fifteen groups were stored at 4 °C and FHb in the supernatant was detected on 0, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84 and 91 days of storage in accordance with the FHb determination kit specification. 2.3. Assessment of RBC blood group antigens after optimal hydroformylation treatment
2. Materials and methods 2.1. Laboratory equipment and reagents DG SPIN gel cards centrifuge, DG-230 Incubator, DG Gel Coombs card and DG Gel Neutral card (Grifols, Spain), KA2200 serological centrifuge (Kubota, Japan), B-600A centrifuge (Baiyang, China), IgM anti-A, IgM anti-B, IgM anti-D, IgG anti-Fya, IgG anti-Fyb, and IgG anti-S (Immucor, USA), IgM anti-C, IgM anti-c, IgM anti-E, IgM anti-e, IgM antiLea, IgM anti-Leb, IgM anti-Jka, IgM anti-Jkb, IgM anti-P1, IgM anti-s, IgG anti-M, IgG anti-N and IgG anti-k (Millipore, UK), reagent RBCs for ABO serum grouping (A1, A2, B and O) and identification of unexpected red cell antibodies (Immucor, USA), Paraformaldehyde (Wako, Japan), 50% glutaraldehyde (Amresco, USA), RBCs preservative solution (Fresenius Kabi, China) and micro free hemoglobin determination kit (Jiancheng Technology, China) were the major equipment and reagents. 2.2. The selection of optimal hydroformylation treatment program 2.2.1. Blood samples This study was conducted in accordance with the declaration of Helsinki. Written informed consents were
2.3.1. Blood samples 10 ml of AB RhD positive whole blood with ACD-B anticoagulant was collected from 5 other healthy donors who met the requirements of blood donation in China, and centrifuged at 1400g for 10 min, and packed red cell was then washed three times with the use of sterile normal saline. 2.3.2. Hydroformylation treatment of RBC The optimal hydroformylation treatment procedure which can lightest damage RBCs was selected, and the processing steps were as the aforementioned. 2.3.3. Determination of the best dilution of serum antibodies The titers of all commercial IgM serum were detected using DG Gel Neutral card in accordance with specification, and A, B, D antigen-positive RBCs were selected from reagent RBCs for ABO serum grouping, and other homozygous positive antigen RBCs were selected from Panocell16. The activities of all commercial IgG serum were detected using DG Gel Coombs card in accordance with specification, and all homozygous positive antigen RBCs were selected from Panocell-16. Four consecutive appropriate dilution concentrations were selected for every antibody. All diluted antibodies
Please cite this article in press as: Yu Y et al. Effects of hydroformylation treatment on the storage time and blood group antigen expressions of reagent red blood cells. Transf Apheres Sci (2014), http://dx.doi.org/10.1016/j.transci.2014.02.019
Y. Yu et al. / Transfusion and Apheresis Science xxx (2014) xxx–xxx
were divided into aliquots and stored at thawed before use and cannot be reused.
20 °C and
2.3.4. Assessment of RBC blood group antigens Blood group antigens of all specimens were detected using various antibodies and various diluted concentrations on 0, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84 and 91 days of storage according to the manufacturer’s instruction. Agglutination of RBCs caused by various diluted concentrations was recorded by scoring method [8], and the strength of RBCs blood group antigens were described as total scores. 2.4. Calculations and statistics Free hemoglobin levels were recorded as mean ± standard deviation. Variance analyses within groups and among the groups were performed by SPSS Statistics 18.0 (SPSS Inc., USA). A p value 0.05) during 13 weeks preservation.
3. Results 4. Discussion 3.1. RBCs hemolysis after hydroformylation treatment during storage Glutaraldehyde 0.2%, 0.5% and 1.0% groups were ruled out directly due to serious crosslinking and aggregation of RBCs. The results showed (Fig. 1) that FHb levels in glutaraldehyde 0.2% and 0.5% groups were higher than that in control group after 1 week storage (p < 0.01). There was no significant difference for FHb levels between glutaraldehyde 0.01% group and control group (p > 0.05) at the different storage time. FHb level in glutaraldehyde 0.025% group was significantly lower than that in other groups (7 paraformaldehyde treatment groups, 3 glutaraldehyde
The strength of blood group antigens on the surface of reagent RBCs for blood transfusion compatibility testing should be stable within a certain period to ensure that the corresponding alloantibodies can be sensitively detected. Reagent RBCs are not only as blood group antigens component involved in the antigen–antibody reaction, but as an indicator cell which directly displayed results of the agglutination reaction. So the structural integrity of reagent RBCs is one of important performance indices. Hemolysis can directly manifest the destruction of structural integrity of reagent RBCs. On the one hand, hemolysis can make the supernatant become red and
Fig. 1. Changes of FHb levels in the different groups during 13-week preservation.
Please cite this article in press as: Yu Y et al. Effects of hydroformylation treatment on the storage time and blood group antigen expressions of reagent red blood cells. Transf Apheres Sci (2014), http://dx.doi.org/10.1016/j.transci.2014.02.019
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Y. Yu et al. / Transfusion and Apheresis Science xxx (2014) xxx–xxx
Table 1 The choice of 19 kinds of antibodies dilutions for assessment of blood group antigen strength. Type of antibody
IgM
IgG
Antibody name
Anti-Lea Anti-Leb Anti- e Anti-c Anti-E Anti-C Anti-Jka Anti-Jkb Anti-P1 Anti-s Anti-D Anti-A Anti-B Anti-M Anti-N Anti-Fya Anti-Fyb Anti-k Anti-S
Dilution multiple of antibodies 1:1
1:2
1:4
1:8
1:16
1:32
2+A 3+A 4+ 4+ 4+ 4+ 4+A 4+A 3+A 4+ 4+ 4+ 4+ 4+A 4+ 3+ 4+A 3+ 4+A
1+A 3+A 4+ 4+ 4+ 4+ 3+A 2+A 3+A 4+A 4+ 4+ 4+ 3+A 4+A 3+A 3+A 3+A 4+A
1+A 2+A 4+ 4+ 4+ 4+ 2+A 2+A 2+A 3+A 4+ 4+ 4+ 3+A 3+A 3+A 2+A 3+A 3+A
1+A 1+A 4+ 4+ 4+ 4+ 1+A 1+A 2+A 2+A 4+ 4+ 4+ 2+A 3+A 2+A 0 1+A 1+A
± ± 4+A 4+A 4+ 4+A ± 0 ± 1+A 4+A 4+ 4+ 1+ 1+A 1+A
0 0 4+A 4+A 4+A 3+A 0 0 0 3+A 4+A 4+A ± 1+ 0
1+A ±
1+ 0
1:64
1:128
1:256
1:512
1:1024
3+A 3+A 3+A 2+A
3+A 3+A 3+A 1+A
0 0 2+A ±
± 0
0
3+A 3+A 3+A 0 ±
1+A 2+A 2+A
± 1+A 1+A
0 ± ±
±
0
0 0
0
A 4 Diluted concentrations for18 kinds of antibodies and only 3 diluted concentrations for anti-Fyb were selected to evaluate the strength of blood group antigens.
Fig. 2. Changes of 19 blood group antigens on RBCs treated by 0.025% glutaraldehyde during 13-week preservation.
interfere with normal judgment on hemagglutination result, and produce debris of RBCs which will generate smearing affect in the the microcolumn gel reaction medium and impact on interpretation of results; on the other hand, hemolysis is also an important manifestation of incompatible reaction other than hemagglutination. Hemolysis of reagent RBCs itself cannot be distinguished from incompatible results. Therefore, effective control of hemolysis is a prerequisite for the storage of reagent RBCs. Reagent RBCs are generally prepared and stored by drawing in the experience of RBCs used in human blood
transfusion, and the usage of RBCs preservative solution can extend the shelf life of the reagent RBCs to 8–9 weeks, but under the conditions of global economic integration, this shelf life makes its global promotion be still limited. Paraformaldehyde can stabilize biological membranes, and widely used in a variety of cell scientific research. The appropriate concentration of glutaraldehyde as a cell cross-linking agent, can improve the strength of a cell membrane and not affected significantly the membrane skeletal matrix structure [9–11]. However, the cells treated by both chemical reagents are usually used for short-term
Please cite this article in press as: Yu Y et al. Effects of hydroformylation treatment on the storage time and blood group antigen expressions of reagent red blood cells. Transf Apheres Sci (2014), http://dx.doi.org/10.1016/j.transci.2014.02.019
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storage. Long-term storage of RBCs after hydroformylation treatment and the major blood group antigen expression are unknown. In the present studies seven concentrations of paraformaldehyde and seven concentrations of glutaraldehyde were used to treat reagent RBCs, respectively. The results showed that the high concentration (1.0%, 0.5%, 0.2%, 0.1% and 0.05% group) glutaraldehyde directly led to membrane crosslinking and RBCs agglutinated or serious membrane damage of RBCs which was effected by higher concentration of FHb than that in control group significantly (p < 0.01). There was no significant difference for the concentration of FHb between the low concentration glutaraldehyde group (0.01% group) and control group which showed the low concentration glutaraldehyde did not provide additional protective effect on the membrane of RBCs for long-term storage. The concentration of FHb in the 0.025% glutaraldehyde group had been maintained at a low level, and was significantly lower than that in other groups after 11 weeks. The shelf life of reagent RBCs in 0.025% glutaraldehyde group can be significantly extended to at least 13 weeks. The strength of blood group antigens on the surface of reagent RBCs is generally assessed by agglutination reaction with a corresponding standard blood group antibodies, but the activity of antibody also affects the agglutination strength in addition to the strength of blood group antigen. If the activity of antibody is too high, it will cover up the reduced blood group antigens on the RBCs; if the activity of antibody is too low, it will make normal RBCs be unable to express strong agglutination. In this study, four successive concentrations for each antibody were selected to eliminate the influence on agglutination strength caused by the difference of the antibody activity, and the antigen strength of RBCs blood group was reflected by calculating the total scores of RBCs agglutination from the four concentrations antibodies. At the same time, all the selected antibodies were packed and stored at 20 °C, to eliminate the potential activity decay of antibodies at 4 °C. The results showed that Fyb, Jkb, and Leb
5
antigen strength decreased slightly (p < 0.01), and there was no significant change for antigen strength of A, B, D, C, E, c, e, M, N, S, s, k, P1, Fya, Jka, and Lea (p > 0.05) on the surface of reagent RBCs treated by 0.025% glutaraldehyde during 13-week storage. In summary, 0.025% glutaraldehyde treatment can provide the optimal protection for the red cell membrane and keep hemolysis at lower level after 13-week storage, and the majority of blood group antigen systems have not been significantly affected, and the slight decline of Fyb, Jkb, and Leb antigen strength was acceptable for blood group serological tests. Reference [1] Mollison PL. The introduction of citrate as an anticoagulant and glucose as a red cell preservative. Brit J Haematol 2000;108:13–8. [2] Hess JR, Greenwalt TJ. Storage of red blood cells: new approaches. Transfus Med Rev 2002;16:283–95. [3] D’Angelo A, L iumbruno G, Grazzini G, Zolla L. Red blood cell storage: the story so far. Blood Transfus. 2010;8(2):82–8. [4] Yu Y, Ma CY, Fen Q, Chen X, Guan XZ, Zhang XJ, et al. Exp Ther Med 2013;5:1466–70. [5] Lanier LL, Warner NL. Paraformaldehyde fixation of hematopoietic cells for quantitative flow cytometry (FACS) analysis. J Immunol Methods 1981;47:25–30. [6] Zachary G, Jason G, Shramik S, Chang Hsueh-Chia. Bovine red blood cell starvation age discrimination through a glutaraldehyde amplified dielectrophoretic approach with buffer selection and membrane cross-linking. Electrophoresis 2008;29:2272–9. [7] Daisuke K, Masato N, Tatsuo U. A useful method for observing intracellular structures of free and cultured cells by scanning electron microscopy. J Electron Microsc 2012;61:105–11. [8] American association of blood banks. Technical manual (17th ed.). Maryland, USA; 2011. p. 874. [9] Girasole M, Cricenti A, Generosi R, Congiu-Castellano A, Boumis G, Amiconi G. Artificially induced unusual shape of erythrocytes: an atomic force microscopy study. J Microsc 2001;204(Pt 1):46–52. [10] Betz T, Bakowsky U, Müller M, Lehr C-M, Bernhardt I. Conformational change of membrane proteins leads to shape changes of red blood cells. Bioelectrochemistry 2007;70:122–6. [11] Kovit P, Egarit N, Surada L, BSc, Kasama S, MSc, et al. The use of glutaraldehyde-fixed chicken red blood cells as counting beads for performing affordable single-platform CD4+ T-lymphocyte count in HIV-1–infected patients. J Acq Immun Def Synd 2010;53:47–54.
Please cite this article in press as: Yu Y et al. Effects of hydroformylation treatment on the storage time and blood group antigen expressions of reagent red blood cells. Transf Apheres Sci (2014), http://dx.doi.org/10.1016/j.transci.2014.02.019
Contents lists available at ScienceDirect
Transfusion and Apheresis Science journal homepage: www.elsevier.com/locate/transci
Effects of hydroformylation treatment on the storage time and blood group antigen expressions of reagent red blood cells Yang Yu, Xiaolin Sun, Xiaozhen Guan, Xiaojuan Zhang, Chunya Ma, Linfeng Chen, Deqing Wang ⇑ Department of Blood Transfusion, The Center for Clinical Transfusion Medicine, Chinese PLA General Hospital, Beijing, China
a r t i c l e
i n f o
Article history: Received 29 March 2013 Received in revised form 12 January 2014 Accepted 24 February 2014 Available online xxxx Keywords: Hydroformylation Blood group antigen Reagent red blood cells Agglutination strength Hemolysis
a b s t r a c t Objective: To evaluate the effects of hydroformylation treatment on the storage time and blood group antigen expressions of reagent red blood cells (RBCs). Material and methods: RBCs from healthy donors were treated by using various final concentrations of paraformaldehyde (0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5% and 1.0%) and glutaraldehyde (0.01%, 0.025%, 0.05%, 0.1%, 0.2%, 0.5% and 1.0%), and one aliquot was used as control (untreated with aldehydes). Supernatant free hemoglobin (FHb) levels in all groups stored at 4 °C were detected every week, and the optimal procedure was selected. Expression of blood group antigens on RBCs treated by the optimal procedure was determined, and the total scores of blood group antigens were calculated. Results: 0.2%, 0.5% and 1.0% Glutaraldehyde groups were ruled out directly due to serious crosslinking and aggregation of RBCs. As the extension of time, FHb levels in other 11 groups gradually increased (p < 0.01 or p < 0.05). FHb level in 0.025% glutaraldehyde group was significantly lower than that in other groups after 13 weeks (p < 0.01), and the antigen strength of Fyb, Jkb, and Leb decreased slightly compared with those before treatment and storage (p < 0.05), and there was no significant change for antigen strength of A, B, D, C, E, c, e, M, N, S, s, k, P1, Fya, Jka, and Lea (p > 0.05). Conclusion: 0.025% Glutaraldehyde treatment can provide optimal protection for the membrane of RBCs and keep hemolysis at a low level after 13 weeks storage, and the majority of blood group antigen systems are not significantly affected, and the slight decline of Fyb, Jkb, and Leb antigen strength was acceptable for classical serological tests. Ó 2014 Published by Elsevier Ltd.
1. Introduction Reagent RBCs for transfusion compatibility testing are generally prepared from healthy donor’s RBCs which are commonly used in blood grouping, unexpected antibody screening and identification, as well as internal quality control and external quality assessment. Long-term effective preservation of reagent RBCs has always been a thorny ⇑ Corresponding author. Address: Department of Blood Transfusion, Chinese PLA General Hospital, No. 28 Fuxing Road, Haidian District, Beijing 100853, China. Tel./fax: +86 1066936508. E-mail address: [email protected] (D. Wang).
issue. Reagent RBCs usually play two roles: one is directly involved in the antigen–antibody reaction as a red blood cell antigen, the other is showing the results of hemagglutination test as an indicator cells. Sufficient strength of blood group antigens on the reagent RBCs needs to be maintained during storage to ensure the detection sensitivity of the corresponding blood group antibodies. Hemolysis of RBCs should be controlled at a low level because the integrity of the RBC membrane directly affects the accuracy of hemagglutination tests. Traditional reagent RBCs are stored referring to the experience from storage of RBCs used in human blood transfusion. Citrate, glucose, adenine, mannitol, and other
http://dx.doi.org/10.1016/j.transci.2014.02.019 1473-0502/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: Yu Y et al. Effects of hydroformylation treatment on the storage time and blood group antigen expressions of reagent red blood cells. Transf Apheres Sci (2014), http://dx.doi.org/10.1016/j.transci.2014.02.019
2
Y. Yu et al. / Transfusion and Apheresis Science xxx (2014) xxx–xxx
ingredients are added to the RBCs storage medium. Glucose and adenine can continue to provide a source of energy for the RBCs during storage, and maintain morphology and structure of the RBCs. Mannitol has some resistance to RBCs hemolysis and makes the membrane structure of RBCs remain relatively intact [1–4]. In addition, some antibiotics are added to inhibit bacterial growth. With the extension of the storage time in vitro, energy substances are exhausted, membrane damage of RBCs is aggravated and FHb levels in supernatant increase gradually. Thus, the shelf life of reagent RBCs could be at most 8–9 weeks by adding nutrients and antibiotics, which led to a limitation in practical application. At the same time, there was no systematic research in changes of blood group antigen strength on the reagent RBCs during longterm storage. Paraformaldehyde and glutaraldehyde are commonly used as cell-fixing agents in cell biology research, and aldehyde reagents can make the membrane protein and the intracellular protein be crosslinked, so that the tissue cells are fixed, and the storage time is extended, which make observational studies on morphology and structure of the cell easy [5–7]. However, widespread formation of protein crosslinking can also cause some of the antigens to be masked and even lead to the degeneration of blood group antigens. The objective of this study was to use various concentrations of paraformaldehyde and glutaraldehyde to treat reagent RBCs before preservation, and try to look for an optimal method of aldehyde treatment which can effectively extend the shelf life of reagent RBCs, and keep the strength of the main blood group antigens stable at the end of storage, and meet the requirements for blood group serological testing.
obtained from 10 participants. 5 Voluntary and regular blood donors who all met the requirements of blood donation in China were selected. 200 ml whole blood was collected from each donor using commercially available blood collection bags containing ACD-B anticoagulant, and centrifuged at 1400g for 10 min. Plasma was separated from whole blood, and the buffy coat was discarded, and packed red cell was then washed three times with the use of sterile normal saline. 2.2.2. Hydroformylation treatment of RBCs A portion of the washed RBCs was divided into fifteen aliquots (1 ml). Paraformaldehyde was added to the washed RBCs to achieve RBCs suspension with final concentrations of 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5% and 1.0%, respectively. Glutaraldehyde was added to the washed RBCs to achieve RBCs suspension with final concentrations of 0.01%, 0.025%, 0.05%, 0.1%, 0.2%, 0.5% and 1.0%, respectively. One aliquot was as control (untreated with aldehydes). The RBCs suspensions were incubated at room temperature (22 °C) for 5 min, and then washed three times with the use of sterile normal saline and resuspended in RBCs preservative solution and prepared 5% red cell suspensions. Sulfuric gentamicin was added to all specimens to obtain final concentration of 0.05 mg/ml. 2.2.3. Determination of FHb during storage All specimens in fifteen groups were stored at 4 °C and FHb in the supernatant was detected on 0, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84 and 91 days of storage in accordance with the FHb determination kit specification. 2.3. Assessment of RBC blood group antigens after optimal hydroformylation treatment
2. Materials and methods 2.1. Laboratory equipment and reagents DG SPIN gel cards centrifuge, DG-230 Incubator, DG Gel Coombs card and DG Gel Neutral card (Grifols, Spain), KA2200 serological centrifuge (Kubota, Japan), B-600A centrifuge (Baiyang, China), IgM anti-A, IgM anti-B, IgM anti-D, IgG anti-Fya, IgG anti-Fyb, and IgG anti-S (Immucor, USA), IgM anti-C, IgM anti-c, IgM anti-E, IgM anti-e, IgM antiLea, IgM anti-Leb, IgM anti-Jka, IgM anti-Jkb, IgM anti-P1, IgM anti-s, IgG anti-M, IgG anti-N and IgG anti-k (Millipore, UK), reagent RBCs for ABO serum grouping (A1, A2, B and O) and identification of unexpected red cell antibodies (Immucor, USA), Paraformaldehyde (Wako, Japan), 50% glutaraldehyde (Amresco, USA), RBCs preservative solution (Fresenius Kabi, China) and micro free hemoglobin determination kit (Jiancheng Technology, China) were the major equipment and reagents. 2.2. The selection of optimal hydroformylation treatment program 2.2.1. Blood samples This study was conducted in accordance with the declaration of Helsinki. Written informed consents were
2.3.1. Blood samples 10 ml of AB RhD positive whole blood with ACD-B anticoagulant was collected from 5 other healthy donors who met the requirements of blood donation in China, and centrifuged at 1400g for 10 min, and packed red cell was then washed three times with the use of sterile normal saline. 2.3.2. Hydroformylation treatment of RBC The optimal hydroformylation treatment procedure which can lightest damage RBCs was selected, and the processing steps were as the aforementioned. 2.3.3. Determination of the best dilution of serum antibodies The titers of all commercial IgM serum were detected using DG Gel Neutral card in accordance with specification, and A, B, D antigen-positive RBCs were selected from reagent RBCs for ABO serum grouping, and other homozygous positive antigen RBCs were selected from Panocell16. The activities of all commercial IgG serum were detected using DG Gel Coombs card in accordance with specification, and all homozygous positive antigen RBCs were selected from Panocell-16. Four consecutive appropriate dilution concentrations were selected for every antibody. All diluted antibodies
Please cite this article in press as: Yu Y et al. Effects of hydroformylation treatment on the storage time and blood group antigen expressions of reagent red blood cells. Transf Apheres Sci (2014), http://dx.doi.org/10.1016/j.transci.2014.02.019
Y. Yu et al. / Transfusion and Apheresis Science xxx (2014) xxx–xxx
were divided into aliquots and stored at thawed before use and cannot be reused.
20 °C and
2.3.4. Assessment of RBC blood group antigens Blood group antigens of all specimens were detected using various antibodies and various diluted concentrations on 0, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84 and 91 days of storage according to the manufacturer’s instruction. Agglutination of RBCs caused by various diluted concentrations was recorded by scoring method [8], and the strength of RBCs blood group antigens were described as total scores. 2.4. Calculations and statistics Free hemoglobin levels were recorded as mean ± standard deviation. Variance analyses within groups and among the groups were performed by SPSS Statistics 18.0 (SPSS Inc., USA). A p value 0.05) during 13 weeks preservation.
3. Results 4. Discussion 3.1. RBCs hemolysis after hydroformylation treatment during storage Glutaraldehyde 0.2%, 0.5% and 1.0% groups were ruled out directly due to serious crosslinking and aggregation of RBCs. The results showed (Fig. 1) that FHb levels in glutaraldehyde 0.2% and 0.5% groups were higher than that in control group after 1 week storage (p < 0.01). There was no significant difference for FHb levels between glutaraldehyde 0.01% group and control group (p > 0.05) at the different storage time. FHb level in glutaraldehyde 0.025% group was significantly lower than that in other groups (7 paraformaldehyde treatment groups, 3 glutaraldehyde
The strength of blood group antigens on the surface of reagent RBCs for blood transfusion compatibility testing should be stable within a certain period to ensure that the corresponding alloantibodies can be sensitively detected. Reagent RBCs are not only as blood group antigens component involved in the antigen–antibody reaction, but as an indicator cell which directly displayed results of the agglutination reaction. So the structural integrity of reagent RBCs is one of important performance indices. Hemolysis can directly manifest the destruction of structural integrity of reagent RBCs. On the one hand, hemolysis can make the supernatant become red and
Fig. 1. Changes of FHb levels in the different groups during 13-week preservation.
Please cite this article in press as: Yu Y et al. Effects of hydroformylation treatment on the storage time and blood group antigen expressions of reagent red blood cells. Transf Apheres Sci (2014), http://dx.doi.org/10.1016/j.transci.2014.02.019
4
Y. Yu et al. / Transfusion and Apheresis Science xxx (2014) xxx–xxx
Table 1 The choice of 19 kinds of antibodies dilutions for assessment of blood group antigen strength. Type of antibody
IgM
IgG
Antibody name
Anti-Lea Anti-Leb Anti- e Anti-c Anti-E Anti-C Anti-Jka Anti-Jkb Anti-P1 Anti-s Anti-D Anti-A Anti-B Anti-M Anti-N Anti-Fya Anti-Fyb Anti-k Anti-S
Dilution multiple of antibodies 1:1
1:2
1:4
1:8
1:16
1:32
2+A 3+A 4+ 4+ 4+ 4+ 4+A 4+A 3+A 4+ 4+ 4+ 4+ 4+A 4+ 3+ 4+A 3+ 4+A
1+A 3+A 4+ 4+ 4+ 4+ 3+A 2+A 3+A 4+A 4+ 4+ 4+ 3+A 4+A 3+A 3+A 3+A 4+A
1+A 2+A 4+ 4+ 4+ 4+ 2+A 2+A 2+A 3+A 4+ 4+ 4+ 3+A 3+A 3+A 2+A 3+A 3+A
1+A 1+A 4+ 4+ 4+ 4+ 1+A 1+A 2+A 2+A 4+ 4+ 4+ 2+A 3+A 2+A 0 1+A 1+A
± ± 4+A 4+A 4+ 4+A ± 0 ± 1+A 4+A 4+ 4+ 1+ 1+A 1+A
0 0 4+A 4+A 4+A 3+A 0 0 0 3+A 4+A 4+A ± 1+ 0
1+A ±
1+ 0
1:64
1:128
1:256
1:512
1:1024
3+A 3+A 3+A 2+A
3+A 3+A 3+A 1+A
0 0 2+A ±
± 0
0
3+A 3+A 3+A 0 ±
1+A 2+A 2+A
± 1+A 1+A
0 ± ±
±
0
0 0
0
A 4 Diluted concentrations for18 kinds of antibodies and only 3 diluted concentrations for anti-Fyb were selected to evaluate the strength of blood group antigens.
Fig. 2. Changes of 19 blood group antigens on RBCs treated by 0.025% glutaraldehyde during 13-week preservation.
interfere with normal judgment on hemagglutination result, and produce debris of RBCs which will generate smearing affect in the the microcolumn gel reaction medium and impact on interpretation of results; on the other hand, hemolysis is also an important manifestation of incompatible reaction other than hemagglutination. Hemolysis of reagent RBCs itself cannot be distinguished from incompatible results. Therefore, effective control of hemolysis is a prerequisite for the storage of reagent RBCs. Reagent RBCs are generally prepared and stored by drawing in the experience of RBCs used in human blood
transfusion, and the usage of RBCs preservative solution can extend the shelf life of the reagent RBCs to 8–9 weeks, but under the conditions of global economic integration, this shelf life makes its global promotion be still limited. Paraformaldehyde can stabilize biological membranes, and widely used in a variety of cell scientific research. The appropriate concentration of glutaraldehyde as a cell cross-linking agent, can improve the strength of a cell membrane and not affected significantly the membrane skeletal matrix structure [9–11]. However, the cells treated by both chemical reagents are usually used for short-term
Please cite this article in press as: Yu Y et al. Effects of hydroformylation treatment on the storage time and blood group antigen expressions of reagent red blood cells. Transf Apheres Sci (2014), http://dx.doi.org/10.1016/j.transci.2014.02.019
Y. Yu et al. / Transfusion and Apheresis Science xxx (2014) xxx–xxx
storage. Long-term storage of RBCs after hydroformylation treatment and the major blood group antigen expression are unknown. In the present studies seven concentrations of paraformaldehyde and seven concentrations of glutaraldehyde were used to treat reagent RBCs, respectively. The results showed that the high concentration (1.0%, 0.5%, 0.2%, 0.1% and 0.05% group) glutaraldehyde directly led to membrane crosslinking and RBCs agglutinated or serious membrane damage of RBCs which was effected by higher concentration of FHb than that in control group significantly (p < 0.01). There was no significant difference for the concentration of FHb between the low concentration glutaraldehyde group (0.01% group) and control group which showed the low concentration glutaraldehyde did not provide additional protective effect on the membrane of RBCs for long-term storage. The concentration of FHb in the 0.025% glutaraldehyde group had been maintained at a low level, and was significantly lower than that in other groups after 11 weeks. The shelf life of reagent RBCs in 0.025% glutaraldehyde group can be significantly extended to at least 13 weeks. The strength of blood group antigens on the surface of reagent RBCs is generally assessed by agglutination reaction with a corresponding standard blood group antibodies, but the activity of antibody also affects the agglutination strength in addition to the strength of blood group antigen. If the activity of antibody is too high, it will cover up the reduced blood group antigens on the RBCs; if the activity of antibody is too low, it will make normal RBCs be unable to express strong agglutination. In this study, four successive concentrations for each antibody were selected to eliminate the influence on agglutination strength caused by the difference of the antibody activity, and the antigen strength of RBCs blood group was reflected by calculating the total scores of RBCs agglutination from the four concentrations antibodies. At the same time, all the selected antibodies were packed and stored at 20 °C, to eliminate the potential activity decay of antibodies at 4 °C. The results showed that Fyb, Jkb, and Leb
5
antigen strength decreased slightly (p < 0.01), and there was no significant change for antigen strength of A, B, D, C, E, c, e, M, N, S, s, k, P1, Fya, Jka, and Lea (p > 0.05) on the surface of reagent RBCs treated by 0.025% glutaraldehyde during 13-week storage. In summary, 0.025% glutaraldehyde treatment can provide the optimal protection for the red cell membrane and keep hemolysis at lower level after 13-week storage, and the majority of blood group antigen systems have not been significantly affected, and the slight decline of Fyb, Jkb, and Leb antigen strength was acceptable for blood group serological tests. Reference [1] Mollison PL. The introduction of citrate as an anticoagulant and glucose as a red cell preservative. Brit J Haematol 2000;108:13–8. [2] Hess JR, Greenwalt TJ. Storage of red blood cells: new approaches. Transfus Med Rev 2002;16:283–95. [3] D’Angelo A, L iumbruno G, Grazzini G, Zolla L. Red blood cell storage: the story so far. Blood Transfus. 2010;8(2):82–8. [4] Yu Y, Ma CY, Fen Q, Chen X, Guan XZ, Zhang XJ, et al. Exp Ther Med 2013;5:1466–70. [5] Lanier LL, Warner NL. Paraformaldehyde fixation of hematopoietic cells for quantitative flow cytometry (FACS) analysis. J Immunol Methods 1981;47:25–30. [6] Zachary G, Jason G, Shramik S, Chang Hsueh-Chia. Bovine red blood cell starvation age discrimination through a glutaraldehyde amplified dielectrophoretic approach with buffer selection and membrane cross-linking. Electrophoresis 2008;29:2272–9. [7] Daisuke K, Masato N, Tatsuo U. A useful method for observing intracellular structures of free and cultured cells by scanning electron microscopy. J Electron Microsc 2012;61:105–11. [8] American association of blood banks. Technical manual (17th ed.). Maryland, USA; 2011. p. 874. [9] Girasole M, Cricenti A, Generosi R, Congiu-Castellano A, Boumis G, Amiconi G. Artificially induced unusual shape of erythrocytes: an atomic force microscopy study. J Microsc 2001;204(Pt 1):46–52. [10] Betz T, Bakowsky U, Müller M, Lehr C-M, Bernhardt I. Conformational change of membrane proteins leads to shape changes of red blood cells. Bioelectrochemistry 2007;70:122–6. [11] Kovit P, Egarit N, Surada L, BSc, Kasama S, MSc, et al. The use of glutaraldehyde-fixed chicken red blood cells as counting beads for performing affordable single-platform CD4+ T-lymphocyte count in HIV-1–infected patients. J Acq Immun Def Synd 2010;53:47–54.
Please cite this article in press as: Yu Y et al. Effects of hydroformylation treatment on the storage time and blood group antigen expressions of reagent red blood cells. Transf Apheres Sci (2014), http://dx.doi.org/10.1016/j.transci.2014.02.019
