ORIGINAL PAPER. Introduction

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1 ORIGINAL PAPER Vox Sanguinis (2011) ª 2011 The Author(s) Vox Sanguinis ª 2011 International Society of Blood Transfusion DOI: /j x Prevalence and specificity of red-blood-cell antibodies in a multiethnic South and East Asian patient population and influence of using novel MUT+Mur+ kodecytes on its detection V. S. Nadarajan, 1,2 A. A. Laing, 2 S. M. Saad 2 & M. Usin 2 1 Department of Pathology, Faculty of Medicine, University Malaya, Kuala Lumpur, Malaysia 2 Department of Transfusion Medicine, University Malaya Medical Center, Kuala Lumpur, Malaysia Received: 20 February 2011, revised 14 April 2011, accepted 18 April 2011 Background and Objectives Appropriate screening for irregular red-cell antibodies is essential for ensuring transfusion compatibility and for antenatal management of mothers at risk of haemolytic disease of the foetus and newborn. Screening for all relevant antibodies is, however, limited by screening cells that do not express antigens present in the patient and donor population. Technology to artificially incorporate antigens into red cells is currently available and may be an option for customizing screening cells. Materials and Methods We sought to identify retrospectively the changing patterns of alloantibody prevalence in our multiethnic population on change of screening cells. Antibody screening records of patients tested from 2004 to 2010 were retrieved and divided into two groups: period-1 ( ) and period-2 ( ). During period-1, standard screening cells were used while in period-2, MUT+Mur+ KODE Ô transformed red cells (kodecytes) were used. Results Four per cent of samples tested during period-2 were positive on antibody screening compared to 3Æ2% in period-1. Specific antibodies, excluding anti-d, were identified in 1Æ66% and 1Æ52% of patients in period-2 and -1, respectively. When confined to antibodies of clinical significance only, period-2 showed higher alloantibody prevalence of 1Æ16% as compared to 0Æ66% in period-1. Antibodies to glycophorin variants of MNS (vmns) were more commonly detected while antibodies to Lewis antigens declined during period-2. Conclusion Antibodies to vmns antigens are common in South and East Asian populations and are often missed when using standard screening cells. Use of specifically engineered screening cells to express red-cell antigens artificially is beneficial in detecting the diverse alloantibodies present in our population. Key words: blood groups, kodecytes, MUT+Mur, RBC antigens and antibodies, South-East Asia, vmns. Introduction Correspondence: Veera S. Nadarajan, Department of Pathology, Faculty of Medicine, Universiti Malaya, Lembah Pantai, Kuala Lumpur, Malaysia veera@um.edu.my Screening for the presence of irregular, non-abo red-cell antibodies is an important component of pre-transfusion testing as well as in antenatal screening for management of haemolytic disease of foetus and newborn (HDFN). 1

2 2 V. S. Nadarajan et al. The antibody screening test should be designed to ensure that all clinically significant antibodies are recognized so that steps can be taken to provide compatible blood to redcell recipients, and appropriate counselling and management can be provided to pregnant mothers at risk of HDFN [1]. The sensitivity for detection of clinically significant antibodies is, however, largely dependent on the antigenic composition of the screening red cells used and the methodology employed for its detection [2,3]. The range of potentially clinically significant antibodies that may develop within a certain patient group is determined by the immunogenicity and corresponding antigenic diversity and differences between donor and recipients within the population [4,5]. Various studies have shown that antigenic composition and red-cell alloantibody frequencies and specificities vary widely between populations [6 9]. In cosmopolitan, multi-ethnic environments, the degree of antigenic diversity is further amplified, and developing an effective antibody screening test necessitates the inclusion of all relevant antigens within the population into a limited number of screening cells without increasing cost and complexity. Most transfusion laboratories, especially in developing countries, elect to use commercially available screening cells rather than prepare panels sourced from their own population. The use of commercial panels may, however, not reflect the red-cell antigenic composition of the local population and risk missing antibodies that are more specific to certain populations. Our transfusion laboratory as in many other laboratories worldwide deals with a diverse group of patients from different ethnic backgrounds. The major ethnic groups of our patient population include individuals of Malay, Chinese and Indian descent, and it is a fair representation of the ethnic make-up in South and East Asia. We routinely perform antibody screening on all blood samples received, using a three-cell panel obtained commercially. Between 2004 and 2010, we have had several changes in our screening cell suppliers. The primary goal of this study was to retrospectively evaluate the performance and impact of changing antibody screening cells on the detection of irregular antibodies in our population as well as identify their prevalence. We believe that such information would be useful in designing red-cell antibody screening procedures in this region of the world. Materials and methods Patients Laboratory records of all antibody screening tests performed from 2004 to 2010 were retrieved and were divided into two groups. Tests performed from 2004 to 2008 and from 2009 to 2010 were classified into period-1 and period-2, respectively. Where a patient had multiple antibody screens performed during the same period and all of them were negative, only his first record was used. However, if any of the antibody screen was positive, the first record with that instance was included together with information on all antibodies identified in that patient throughout the period. This was to ensure that the same patient was not repeatedly included in the study, but at the same time, all instances of positive antibody screens and antibody identifications were captured. Antibody screening During the early part of period-1, spanning , samples for antibody screening were subjected to a threecell screening procedure by column agglutination technique (CAT) using 0Æ8% Surgiscreen Ò cells [Ortho Clinical Diagnostics (OCD), Raritan, NJ] on BioVue Ò anti-igg cassettes (OCD), performed by an AutoVue Ò immunohematology analyzer (OCD). In 2006, a Techno Twin Station Ò analyzer (Diamed AG, Cressier, Switzerland) was installed. Subsequent to that, samples for antibody screening either continued to be subjected to the previous method or alternatively were analysed using 0Æ8% ID-DiaCell-I-II-III Asia (Mia+) (Diamed AG) screening cells on Coombs anti-igg Diamed ID-cards (Diamed AG), on the Techno Twin Station Ò. The ID-DiaCell-I-II-III Asia (Mia+) screening panel included a third cell with Mi a phenotype. Supply of this screening panel was, however, disrupted in 2008 forcing us to switch to 0Æ8% ID-DiaCell-I-II-III panel that did not include a Mi a + cell. During period-2, antibody screening was solely performed using Abtectcell Ò III 0Æ8% (CSL Limited, Parkville, Victoria, Australia) screening cells using either of the previous two CAT. The Abtectcell Ò screening cells include kodecytes that are red cells modified using KODE Ô (KODE Biotech Ltd., Auckland, New Zealand) technology to artificially express the MUT and Mur peptides of variant glycophorin molecules (vmns) [10]. In this proprietary technology, synthetic constructs are synthesized by conjugating MUT and Mur representative peptides to lipid and spacer molecules that are then incorporated into the membrane of vmns-negative red blood cells. Antibody identification Samples found positive on antibody screening were subjected to antibody identification procedures utilizing one or more antibody identification panels that included Resolve Panel A (OCD), ID DiaPanel Ò (Diamed AG) and Phenocell Ò C 3% (CSL Limited). The Phenocell Ò C panel includes one cell expressing both MUT and Mur antigens added using

3 Red-cell antibodies among South-East Asians 3 KODEÔ technology. Prior to the introduction of this cell panel, known GP.Mur+ donor cells were used to confirm antibody specificities to vmns. Corresponding antigen typing of red-cell samples was also routinely performed to discriminate autoantibodies from alloantibodies. Specialized technicians familiar with immunohematology testing performed all procedures. Statistical analysis Descriptive and statistical analysis was performed using SPSS version 16.0 (SPSS Inc., Chicago, IL). Comparisons of proportions between groups were made with the chi-square test. A P-value < 0Æ05 was considered significant. Results Antibody screening was performed on a total of (period-1: , period-2: ) patients, of whom 3Æ4% had positive results. Samples screened during period- 2 had significantly higher positive screen rates as compared to those in period-1 (4Æ0% against 3Æ2%, P-value < 0Æ00), but had lower likelihood of antibody with a defined specificity identified [period-1: (78%), period-2: (74%)] (Table 1). Samples during period-2 were also more likely to have antibodies detected with undefined specificities or to have non-specific or pan-agglutinating autoantibodies. The proportion of samples showing negative reactions with all antibody identification cells although being positive on antibody screening was also higher during period-2. Despite the lower percentage of antibody screen positive samples that had antibodies of definable specificity detected in period-2, the frequency of patients in period-2 with one or more antibodies was still higher than during period-1 (1Æ93% vs. 1Æ73%) (Table 1). A total of 2955 irregular antibodies were identified from 2579 patients of whom 345 (13Æ4%) had multiple antibodies. Anti-D was the sole antibody identified in 336 subjects, of whom 249 were pregnant. We chose to exclude anti-d from further prevalence analysis as it was not possible to discriminate anti-d actively acquired through alloimmunization from that acquired passively through anti-d prophylaxis. No significant differences were observed between period-1 and period-2 in the type of antibody specificities identified except for antibodies to Lewis, vmns [Mi a (MNS7), Mur (MNS10), MUT (MNS35)] and Diego. During period-2, antibodies to vmns were the most common identified antibody while the detection of anti-le a b declined (Table 2). s had significantly higher prevalence of anti-c, anti-s, anti-le b and anti-jk a as compared to males who conversely had higher prevalence of antibodies to vmns (Table 3). Forty-seven per cent ( ) of all antibodies identified during the study period were deemed as naturally occurring or of low clinical significance. These include anti-le a, anti-le b, anti-m, anti-p1, anti-h, anti-i and anti-wr a. If these antibodies were excluded, males and females showed about equal prevalence for clinically significant antibodies (Table 4). Discussion Screening for irregular red-cell antibodies is an important component of pre-transfusion testing. Difficulties are however often encountered when designing a suitable panel of screening cells in ethnically heterogeneous populations Table 1 Results of antibody screening and identification performed during period-1 ( ) and period-2 ( ) Period 1 Period 2 No of subjects screened for antibodies (100Æ00) (100Æ00) (100Æ00) (29Æ01) (33Æ28) (30Æ44) During pregnancy (28Æ91) (22Æ42) (26Æ74) Outside pregnancy (42Æ07) (44Æ30) (42Æ82) Patients negative on antibody screen (96Æ82) (96Æ03) (96Æ56) Patients positive on antibody screen 3042 (3Æ18) 1900 (3Æ97) 4942 (3Æ44) Antibody identification performed* 2119 (69Æ66) 1253 (65Æ95) 3372 (68Æ23) Antibody identification omitted* 923 (30Æ34) 647 (34Æ05) 1570 (31Æ77) Antibody identification performed 2119 (2Æ22) 1253 (2Æ62) 3372 (2Æ35) Antibodies detected with defined specificity ies 1654 (1Æ73) 925 (1Æ93) 2579 (1Æ80) Antibodies detected but specificity undetermined 120 (0Æ13) 79 (0Æ16) 199 (0Æ14) Autoantibodies or pan-agglutinating antibodies 187 (0Æ20) 139 (0Æ29) 326 (0Æ23) No irregular antibodies detected 158 (0Æ17) 110 (0Æ23) 268 (0Æ19) Numbers in parentheses indicate percentage of patients to total number of patients tested except for items marked * where they refer to percentage of patients to number of patients positive on antibody screen.

4 4 V. S. Nadarajan et al. Table 2 Specificity, frequency and prevalence of red-cell alloantibodies identified during period-1 ( ) and period-2 ( ) Table 3 Specificity, frequency and prevalence of red-cell alloantibodies identified among males and females Antibody Period 1 (95 587) Period 2 (47 914) ( ) P-value (43 680) (99 821) P-value D* 234 (2Æ45) 135 (2Æ82) 369 (2Æ57) 0Æ132 C 21(0Æ22) 10 (0Æ21) 31 (0Æ22) 0Æ881 c 76(0Æ80) 46 (0Æ96) 122 (0Æ85) 0Æ298 E 345 (3Æ61) 166 (3Æ46) 511 (3Æ56) 0Æ231 e 11(0Æ12) 5 (0Æ10) 16 (0Æ11) 0Æ853 C w 6(0Æ06) 2 (0Æ04) 8 (0Æ06) 0Æ615 P1 42 (0Æ44) 11 (0Æ23) 53 (0Æ37) 0Æ051 M 79(0Æ83) 44 (0Æ92) 123 (0Æ86) 0Æ538 N 5(0Æ05) 0 (0Æ00) 5 (0Æ03) 0Æ113 S 33(0Æ35) 14 (0Æ29) 47 (0Æ33) 0Æ593 s 2(0Æ02) 0 (0Æ00) 2 (0Æ01) 0Æ317 vmns 90 (0Æ94) 279 (5Æ82) 369 (2Æ57) 0Æ000 Le a 642 (6Æ72) 153 (3Æ19) 795 (5Æ54) 0Æ000 Le b 241 (2Æ52) 80 (1Æ67) 321 (2Æ24) 0Æ001 Jk a 46 (0Æ48) 26 (0Æ54) 72 (0Æ50) 0Æ609 Jk b 9(0Æ09) 6 (0Æ13) 15 (0Æ10) 0Æ587 K 14(0Æ15) 8 (0Æ17) 22 (0Æ15) 0Æ765 Kp a 3(0Æ03) 0 (0Æ00) 3 (0Æ02) 0Æ220 Di a 0(0Æ00) 13 (0Æ27) 13 (0Æ09) 0Æ000 Lu a 1(0Æ01) 2 (0Æ04) 3 (0Æ02) 0Æ222 H 2(0Æ02) 0 (0Æ00) 2 (0Æ01) 0Æ317 Fy a 11 (0Æ12) 7 (0Æ15) 18 (0Æ13) 0Æ620 Fy b 20 (0Æ21) 12 (0Æ25) 32 (0Æ22) 0Æ619 Wr a 0(0Æ00) 2 (0Æ04) 2 (0Æ01) 0Æ046 I 0(0Æ00) 1 (0Æ02) 1 (0Æ01) 0Æ Numbers in parentheses indicate prevalence per 1000 patients; items marked in bold indicate antibodies with proportions significantly different between period-1 and period-2. *Identified anti-d may include both passively acquired anti-d from antenatal prophylaxis of RhD- mothers and actively acquired antibodies. Includes antibodies to Mi a, Mur, MUT. where knowledge of blood group polymorphisms and antibody prevalence is incomplete. Selection of the antibody screening cells has to be carefully made to include all clinically relevant red-cell antigens expressed by members of the population, represented at adequate dosage, to allow detection of all known clinically significant antibodies. Our overall antibody prevalence after exclusion of anti- D was 1Æ6%, a figure similar or higher than that quoted in many other studies involving a general patient population. It is to be cautioned, however, that a correct comparison between studies is often difficult because of the variation in the composition of the study population, methodology for antibody screening as well as which antibodies were included in calculation of the prevalence. Retrospective studies of general hospital-based patient populations have usually yielded prevalence rates of 1 1Æ5% [6,11 13]. C 10(0Æ23) 21 (0Æ21) 0Æ882 c 27(0Æ62) 95 (0Æ95) 0Æ046 E 144 (3Æ30) 367 (3Æ68) 0Æ191 e 4(0Æ09) 12 (0Æ12) 0Æ636 C w 4(0Æ09) 4 (0Æ04) 0Æ229 P1 16 (0Æ37) 37 (0Æ37) 0Æ931 M 30(0Æ69) 93 (0Æ93) 0Æ143 N 2(0Æ05) 3 (0Æ03) 0Æ642 S 5(0Æ11) 42 (0Æ42) 0Æ003 s 0(0Æ00) 2 (0Æ02) 0Æ350 vmns* 143 (3Æ27) 226 (2Æ26) 0Æ000 Le a 280 (6Æ41) 515 (5Æ16) 0Æ001 Le b 77 (1Æ76) 244 (2Æ44) 0Æ011 Jk a 13 (0Æ30) 59 (0Æ59) 0Æ022 Jk b 6(0Æ14) 9 (0Æ09) 0Æ421 K 8(0Æ18) 14 (0Æ14) 0Æ545 Kp a 0(0Æ00) 3 (0Æ03) 0Æ252 Di a 2(0Æ05) 11 (0Æ11) 0Æ238 Lu a 2(0Æ05) 1 (0Æ01) 0Æ173 H 1(0Æ02) 1 (0Æ01) 0Æ548 Fy a 5(0Æ11) 13 (0Æ13) 0Æ805 Fy b 10 (0Æ23) 22 (0Æ22) 0Æ912 Wr a 0(0Æ00) 2 (0Æ02) 0Æ350 I 1(0Æ02) 0 (0Æ00) 0Æ Numbers in parentheses indicate prevalence per 1000 patients; items marked in bold indicate antibodies with proportions significantly different between males and females. *Includes antibodies to Mi a, Mur, MUT. When comparing between the two periods of the study, the prevalence rate during period-2 (1Æ66%) was significantly higher as compared to period-1 (1Æ52%) despite the decline in detection of Lewis antibodies, which in period-1 was the most common antibody specificity identified. No studies have described antibody detection sensitivities of KODEÔ modified screening red cells for Lewis antibodies, and it is unclear as to why these cells should be less sensitive. It is possible that the incorporation of synthetic peptides into the red-cell membrane disrupts Lewis antigen expression on the cells, although this would require confirmation. When only clinically significant antibodies are considered, the alloantibody prevalence rate is 0Æ66% and 1Æ16% for period-1 and period-2 respectively. A near doubling of the prevalence during period-2 as compared to period-1 is attributed largely to the increased detection frequency of antibodies to vmns antigens. The vmns antigens result from genetic recombination events, producing hybrid glycophorin molecules [14]. The most common variant GP.Mur

5 Red-cell antibodies among South-East Asians 5 Table 4 Overall antibody prevalence based on postulated clinical significance of antibodies according to sex and period during which they were identified Period-1 Period-2 Grand total (27 733) (67 854) (95 587) (15 947) (31 967) (47 914) (43Æ680) (99 821) ( ) Patients with antibody ies identified* 455 (1Æ64) 994 (1Æ46) 1449 (1Æ52) 247 (1Æ66) 547 (1Æ71) 794 (1Æ66) 702 (1Æ61) 1541 (1Æ54) 2243 (1Æ56) Patients with only naturally occurring antibodies or antibodies of low clinical significance 273 (0Æ98) 544 (0Æ80) 817 (0Æ85) 71 (0Æ45) 169 (0Æ53) 240 (0Æ50) 344 (0Æ79) 713 (0Æ71) 1057 (0Æ74) Patients with antibodies of clinical significance 182 (0Æ66) 450 (0Æ66) 632 (0Æ66) 176 (1Æ10) 378 (1Æ18) 554 (1Æ16) 358 (0Æ82) 828 (0Æ83) 1186 (0Æ83) Numbers in parentheses indicate prevalence rates expressed as percentage of patients tested. *Excluding patients who only had anti-d detected. Includes anti-le a b, anti-m, anti-p1, anti-h, anti-i and anti-wr a. is capable of inducing antibodies to Mi a, MUT and Mur antigens. GP.Mur is detected rarely within a Caucasian population [15] but relatively common in East Asia where incidence of 5 6% has been described [16,17]. IgM antibodies reactive with GP.Mur red cells are more frequent than IgG antibodies and are likely to be clinically insignificant and naturally occurring [18,19]. Some of the antibodies to vmns identified during period-1 while using Mi a screening red cells could well be IgM only antibodies as although we were using Coombs anti-igg card, this card is not heavy chain specific and is capable of reacting with the light chains of IgA and IgM molecules in addition to IgG. However, the vmns antibodies identified during period-2 are more likely IgG antibodies, as MUT+Mur+ kodecytes do not appear to be capable of detecting IgM antibodies [10,17]. The prevalence rate of 5Æ8 per 1000 during period- 2 is considerably higher than that reported in any of the other large-scale studies [20 22] including a previous study within our population which showed a prevalence of only 2Æ28 per 1000 [23]. This may be reflective of higher sensitivity of both screening cells and CAT employed within our current setting. Considering that antibodies to GP.Mur have been reported to be responsible for both haemolytic transfusion reactions and HDFN [17,21,24], it is pertinent that employed antibody screening procedures are capable of detecting them especially if an indirect antiglobulin crossmatch (IAT-XM) is omitted from pre-transfusion testing. In our setting, an IAT-XM has been obligatory precisely for such reasons and that has circumvented potential transfusion events where the antibody screening would have been negative in the presence of vmns antibodies but the IAT- XM would be incompatible. Transfusion laboratories that handle patients of Asian background and perform electronic cross-matches, however, may need to be wary of the antibody screening cells employed. In addition to antibodies to vmns, we also identified during period-2 previously unrecognized anti-di a in our population as the screening panel routinely included a Di(a+) cell. Screening cells in Japan routinely include Di(a+) cells although this is not standard practice outside the region. The incidence of Di(a+) is 12% among Japanese and 5% among Chinese but is rare among White people. Facilities dependent on commercial screening cells sourced from predominantly white populations are likely to miss anti-di a antibodies in addition to vmns antibodies. Higher prevalence was observed in females among the clinically significant antibodies: anti-c, -S, -Jk a. Antibodies to vmns were, however, significantly more frequent in males, suggesting that unlike the other antigens, immunization via foeto-maternal red cell transfer may be less efficient compared to red-cell transfusions for vmns antigens. We however did not have reliable data on transfusion history to test this assumption. Another possible explanation for the difference may be that the degree of racial difference between partners in conception is markedly less than that expected through allogeneic transfusions, and thus, the chances of being exposed to an ethnically mismatched antigen through transfusion would be higher than that through pregnancy. The trade-off to the higher antibody detection rate using KODEÔ modified antibody screening cells during period-2 was a higher false-positive rate as well as increased detection of non-specific antibodies and autoantibodies. Tests with KODEÔ transformed cells had indicated occurrence of positive reactions in < 0Æ5% of expected negative sera although higher rates were reported with BioVue Ò cassettes as compared to DiaMed Ò cards [10]. This concurs with our experience, where about 9% of positive antibody screen samples (or 0Æ22% of all samples) failed to show reactions on antibody identification. A possible explanation for the false reactions would be the existence of cross-reacting antibodies that are only active against the peptide artificially expressed on the KODEÔ modified cells but not

6 6 V. S. Nadarajan et al. whole antigenic structures expressed on native cells. Weak and clinically insignificant autoantibodies were also more commonly detected while using the KODEÔ modified screening cells, especially among female patients, where up to 15% of samples positive on antibody screen either showed weak pan-agglutinating antibodies on the identification panel or a weak direct anti-globulin test. This could be owing to heightened sensitivity of the surface-altered screening cells to red-cell autoantibodies or cross-reacting antibodies. In conclusion, we have shown that alloantibody prevalence and their specificities are determined by the choice of antibody screening cells that are used. We have established that alloantibodies to the antigens of GP.Mur variant glycophorin of the MNS blood group system occur most commonly within our hospital setting and are often missed because appropriate screening cells expressing the antigen are not used. The identification of these antibodies is often complicated by the lack of readily characterized and commercially available screening cells and phenotyping reagents. Use of modified screening cells carrying synthetic epitopes or alternative technologies such as peptide enzyme-linked immunosorbent assay (ELISA) [17] may be able to address the problem of detecting clinically significant red-cell antibodies irrespective of ethnic compositions. Acknowledgements We thank the medical and laboratory staff of the Department of Transfusion Medicine, University Malaya Medical Centre for their diligent work, without whom the extensive data for this study would not have been generated. The data for this study have also been compiled in part for the All- IADP Alloimmunity to Antigen Diversity in Asian Populations project headed by Professor Akihiro Takeshita in Japan. Disclosures VSN has served as a member of the Transfusion Medicine Advisory Panel for Ortho Clinical Diagnostics. The remaining authors have no disclosures to make. Authorship VSN conceived the study, analysed data and wrote the manuscript, AAL compiled and analysed data, SMS supervised and performed the antibody identifications tests, and MUF supervised the performance of all laboratory tests. References 1 Spielmann W, Seidl S: Prevalence of irregular red cell antibodies and their significance in blood transfusion and antenatal care. Vox Sang 1974; 26: Garozzo G, Licitra V, Criscione R, et al.: A comparison of two automated methods for the detection and identification of red blood cell alloantibodies. Blood Transfu 2007; 5: Weisbach V, Kohnhäuser T, Zimmermann R, et al.: Comparison of the performance of microtube column systems and solid-phase systems and the tube low-ionic-strength solution additive indirect antiglobulin test in the detection of red cell alloantibodies. Transfus Med 2006; 16: Singer ST, Wu V, Mignacca R, et al.: Alloimmunization and erythrocyte autoimmunization in transfusion-dependent thalassemia patients of predominantly asian descent. Blood 2000; 96: Tormey CA, Stack G: Immunogenicity of blood group antigens: a mathematical model corrected for antibody evanescence with exclusion of naturally occurring and pregnancy-related antibodies. Blood 2009; 114: Cerdas-Quesada C: Specificity of 136 patient s antibodies to human red blood cells in Dr. Max Peralta J Hospital Blood Bank 2004-February Transf Apheres Sci 2010; 42: M baya B, Mfune T, Mogombo E, et al.: The prevalence of red cell antigens and antibodies in Malawi. Transfus Med 2009; 20: Ameen R, Al Shemmari S, Al-Bashir A: Red blood cell alloimmunization among sickle cell Kuwaiti Arab patients who received red blood cell transfusion. Transfusion 2009; 49: Natukunda B, Schonewille H, van de Watering L, et al.: Prevalence and specificities of red blood cell alloantibodies in transfused Ugandans with different diseases. Vox Sang 2010; 98: Heathcote D, Carroll T, Wang J-J, et al.: Novel antibody screening cells, MUT+ Mur kodecytes, created by attaching peptides onto red blood cells. Transfusion 2010; 50: Schonewille H, Brand A: Alloimmunization to red blood cell antigens after universal leucodepletion. A regional multicentre retrospective study. Br J Haematol 2005; 129: Winters JL, Pineda AA, Gorden LD, et al.: RBC alloantibody specificity and antigen potency in Olmsted County, Minnesota. Transfusion 2001; 41: Pujol M, Sancho J, Zarco M: The gel enzyme technique in pretransfusion antibody screening. Haematologica 2002; 87: Huang CH, Blumenfeld OO: Molecular genetics of human erythrocyte MiIII and MiVI glycophorins. Use of a pseudoexon in construction of two delta-alphadelta hybrid genes resulting in antigenic diversification. J Biol Chem 1991; 266: Tippett P, Reid ME, Poole J, et al.: The Miltenberger subsystem: is it obsolescent? Transfus Med Rev 1992; 6: Broadberry RE, Lin M: The incidence and significance of anti- Mia in Taiwan. Transfusion 1994; 34: Heathcote DJ, Carroll TE, Flower RL: Sixty years of antibodies to MNS system

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