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Red Cell Immunohematology Research Conducted in China

Ziyan Zhu, Yuli Ye, Qin Li, Hongwei Gao, Yinxia Tan, Wei Cai

PII: S0887-7963(16)30089-X DOI: doi: 10.1016/j.tmrv.2016.11.004 Reference: YTMRV 50494

To appear in: Reviews

Received date: 14 June 2016 Revised date: 15 November 2016 Accepted date: 15 November 2016

Please cite this article as: Zhu Ziyan, Ye Yuli, Li Qin, Gao Hongwei, Tan Yinxia, Cai Wei, Red Cell Immunohematology Research Conducted in China, Transfusion Medicine Reviews (2016), doi: 10.1016/j.tmrv.2016.11.004

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Red Cell Immunohematology Research Conducted in China

Ziyan Zhu1, Yuli Ye 1, Qin li1, Hongwei Gao2, Yinxia Tan2, Wei Cai3

1 Shanghai Blood Center, Shanghai, China 2 Beijing Institute of Transfusion Medicine, Beijing, China 3 The Johns Hopkins Hospital, Baltimore, USA

Corresponding Author: Ziyan Zhu E-mail: [email protected]

Abstract ABO subtypes and RhD variants are the most studied blood groups in China. Some of the polymorphisms in these two blood groups have direct clinical relevance. Molecular diagnosis of blood group polymorphisms is underway in China. In addition, research groups have developed methods such as screening for blood group mimetic peptides using phage display technology. New reagents, akin to antibodies directed against RhD and ABO, are being investigated using aptamer-based techniques. Progress is also being made in the development of synthetic exoglycosidases for conversion of group A and/or B antigens to group O. Development of methoxy-polyethylene-glycol modified red cells has been successful in vitro but has not reached clinical application. In this paper, we summarize red cell immunohematology research that has been conducted in China.

Keywords: blood groups; ABO; immunohematology.

ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 2

Blood groups are of great clinical importance in and in transplantation.

Many blood group antibodies have the potential to cause rapid destruction of transfused red cells bearing the corresponding antigen, resulting in either acute or delayed hemolytic transfusion reactions. Routine blood group testing continues to rely upon hemagglutination reactions between antigens and antibodies that are read and interpreted either manually or by automated means. Methods such as flow cytometry and analysis of antigens at the molecular level are often reserved for specialized testing or research. Here we summarize research in red cell immunohematology conducted in China, specifically focusing on ABO and RhD polymorphisms, the development of blood group mimetic peptides for ABO and

RhD antigens, and the modification of red blood cell surface antigens.

ABO subtypes

High genetic diversity has been found in blood group antigens in Chinese persons. In the past three years, more than 30 novel ABO subgroup have been reported [1-5]. Among them, 27 point mutations, five hybrid alleles, and one deletion-mutation have been found. In one proband, a nucleotide deletion in the ABO promoter region was discovered and its effect was further investigated using dual-luciferases assays to analyze ABO promoter activity [1]. The para-Bombay phenotype is a clinically relevant ABO subtype found in approximately 0.01% of Chinese persons. In a retrospective study, the 17 reported FUT1 and

FUT2 alleles in China were reviewed and summarized [6]. The results pointed out that four FUT1 alleles wereACCEPTED predominant in Chinese paraMANUSCRIPT-Bombay individuals and should be included in relevant genotyping methods. The results of another study suggested that mutations in the FUT1 allele including C35T (Ala12Val) or A682G (Met228Val), found either as a single mutation or combined mutations, might lead to the reduction of the activity of

α(1,2)fucosyltransferase [7].

Hyper-methylation of the ABO promoter also might affect the expression of ABO antigens.

In one healthy blood donor with the genotype B101/O01, B antigen expression was significantly decreased [8], although no abnormality was found in the coding regions, the adjacent introns, or the regulatory regions of the ABO transcripts. No anti-B was detected in ACCEPTED MANUSCRIPT 3 the serum. However, B glycosyltransferase activity in the serum was decreased, using the method developed by Nagai et al.[9,10] The ABO promoter methylation level in this sample was higher than that found in controls with normal B phenotypes. The authors speculated that

ABO promoter hyper-methylation might down-regulate the expression of B antigen. Similarly, in another study focusing on the expression of A and H antigens in patients with malignant hematological diseases, the researchers found that the process of H antigen conversion to A antigen was blocked with ABO promoter CpG island methylation [11]. Thus, CpG methylation within the ABO promoter region might be related to weakened ABO antigen expression in patients or healthy individuals. More detailed epigenetic studies will be necessary before any definite conclusions can be reached.

cis-AB is an ABO allele encoding a glycosyltransferase with dual A and B activity, consisting of cis-AB and B(A). Molecular genetic data derived from the RBC gene mutation database

(http://www.ncbi.nlm.nih.gov/gv/mhc/xslcgi.cgi?cmd=bgmut/home) have revealed that cis-AB and B(A) phenotypes are found in Chinese and Japanese. Sha confirmed 71 cases of cis-AB and B(A) genotype by serological and genomics methods from among 1,716,442 healthy donors[12]. Six known alleles cis-AB01, cis-AB02, cis-AB06, B(A)02, B(A)04, and B(A)06 have been identified in these individuals (Table 1). The prevalence of the cisAB and B(A) phenotype was estimated to be 8.3/100,000 in the Chinese population after correction for the occurrence of alleles that might be overlooked when present in a heterozygous combination with a normal A orACCEPTED B allele. The estimated MANUSCRIPTgene frequency without consideration of the above correction is shown in Table 2.

Rh variants

From 2007 to 2011, more than 10 novel RHD alleles were found in Chinese persons, mainly in blood donors with weak D or partial D phenotypes [13-14]. Most RhD variants fit the classic genetic definitions of weak D and partial D types. Weak D Type 15 and DVI Type 3 are the most predominant weak D and partial D alleles in the population. However, some rare mechanisms have been reported to result in abnormal RhD antigen expression. For example, a partial D phenotype was found to result from a three basepair deletion of RHD gene, and a ACCEPTED MANUSCRIPT 4 weak D phenotype was reported to result from intronic mutations. However, the molecular mechanisms under these unusual variants remain unclear.

DEL, historically D-elute, was first reported in the Japanese population [15] and is the weakest known D positive phenotype in the . DEL is relatively common in Eastern Asia. In China, about 10–30% of apparently D negative individuals are

DEL phenotypes [16-21]. The DEL phenotype derives from several mechanisms, including a splice-site mutation, missense mutation, frame shift mutation, and a long deletion of the RHD gene[16,22-28]. RHD (K409K) is the most frequent DEL allele in mainland China, occurring with a reported frequency of 1:110 [16]. The RHD (K409K) allele consists of a nucleotide change of exon 9 in RHD gene, RHD(1227G>A). Besides the RHD(K409K) allele, other DEL alleles, such as RHD(3G>A), RHD(R10W), RHD(L18P), RHD(L84P) and RHD(A137E) also exist in the Chinese population at low frequencies [18]. The RHD(K409K) is also found in several Chinese minority ethnic groups, such as Uigur and Hui, but the frequency is much lower than that in the Han population [29]. Zhu and colleagues [30] did quantitative analysis to determine the D antigen density of RHD(K409K) DEL in Chinese individuals. They estimated that D expression on DEL erythrocytes was <28 sites per cell. Shao and colleagues [31] determined the D antigen epitopes for DEL phenotype red cells. All detected epitopes (1.2, 2.1, 3.1, 4.1, 5.4, 6.2, 6.6, 8.2, and 9.1) were found in DEL individuals with

RHD(K409K) allele. The authors concluded that RHD(K409K) DEL might result from intact but reduced D antigenACCEPTED expression. MANUSCRIPT

A central question in research on the DEL phenotype is to determine how to clinically treat individuals with this phenotype. Three groups of investigators [32-34] studied apparently D negative individuals who produced anti-D. The three teams tested 20 individuals (4 healthy blood donors, 15 women who experienced fetal loss, and one patient), 45 pregnant women, and 104 pregnant women, respectively. The individuals who developed anti-D were truly RhD negative or were partial D, such as DVI. None of the detected samples showed the DEL phenotype. Because almost all Chinese examples of DEL are caused by the RHD(K409K) allele, Shao [34] suggested that persons with "Asia type" DEL can safely receive transfusions ACCEPTED MANUSCRIPT 5 from RhD-positive donors. Wang and colleagues [35] obtained different results. They investigated 142 pregnant women with DEL phenotypes, and 6 women were found to develop anti-D with the titer range from 4 to 128. However, each of the 6 DEL samples also contained RHD-CE-D hybrid alleles, and thus were not exclusively the "Asia type" DEL.

Several cases involving anti-D immunization by RHD(K409K) DEL red blood cells were reported in Japan and Korea [36-38], but have not been reported in the Chinese population.

In an interesting clinical experiment, Zhang et al [39] transfused blood from DEL persons to

10 true RhD-negative individuals. None of the recipients developed anti-D. These limited transfusion results suggest that RhD-negative recipients may have little risk of RhD alloimmunization following transfusion with RHD(K409K) DEL red blood cells in China.

Blood group mimetic antigens

An area of recent special interest in Chinese immunohematology is the development of cell independent blood group antigens, including recombinant blood group proteins, synthetic peptides, and aptamers expressing blood group mimotopes selected using phage display peptide libraries. [40]. These reagents are not dependent on the presence of viable red cells, but can still react in immunohematologic assays with antigenicity similar to viable cells.

Recombinant blood group antigens:

Transfected prokaryotic or eukaryotic cells can express recombinant proteins bearing clinically relevantACCEPTED blood group antigens. In MANUSCRIPTtheory such recombinant proteins could be utilized as immunogens to produce corresponding specific monoclonal antibodies. Membrane-bound antigens or soluble antigens expressed by transfected prokaryotic or eukaryotic cells may also be used to detect corresponding red blood antibodies. Chen et al

[41] have successfully constructed transgenic L929 cells that express blood group and have shown that these cells can be used either as targets to detect anti-glycophorin C antibodies or as immunogens for the preparation of glycophorin C monoclonal antibodies. The same research team also tried to amplify several corresponding sequences coding for glycophorin B, Kell-30c, Kidd and Diego antigens. The inserts have been expressed in prokaryotic cells and the gene products purified and used for ACCEPTED MANUSCRIPT 6 immunization in mice. Unfortunately, thus far, all immunized mice did not produce any monoclonal antibodies capable of causing noticeable hemagglutination [42].

Synthetic peptides:

Blood group antigen analogues or peptides can be chemically synthesized and demonstrated to react with specific antibodies or to inhibit agglutination between these antibodies and antigen bearing red cells. It is easier to obtain more highly purified antigens when synthetic peptides are utilized to produce blood group monoclonal antibodies. Since glycoproteins A and B have one trans-membrane domain, synthetic peptides of these structures could mimic membrane peptides. Guo et al reported a KLH-conjugated synthetic peptide corresponding to the sequence of the N-terminal position (17-36) of glycophorin B. The peptide was used to immunize BALB/c mice. One mouse successfully produced an anti-glycophorin B monoclonal antibody, but the antibody demonstrated comparatively low reactivity [43-44].

Aptamers:

Blood group antigens can be mimicked by production of mimotopes. A mimotope is a macromolecule, often a peptide, which mimics the structure of an antigenic epitope. Aptamers are an example of peptide mimotope. Peptide aptamers are small synthetic proteins which have been selected for their ability to bind to a target. Using a screening strategy from blood cell phage display libraries, aptamers can potentially be created for use as blood group mimotopes. AlthoughACCEPTED aptamers are different MANUSCRIPT in constitution and structure from natural blood group epitopes, they share some relevant characteristics. Hu et al took advantage of a phage display random peptide library, followed by ELISA screening with anti-A and anti-B monoclonal antibodies, and successfully isolated several peptides with mimicking epitopes of blood group A and B.[45-48] Using a similar strategy, Liu et al identified mimicking epitopes of blood group RhD.[49-50] Chen et al constructed a human Fab phage antibody library, and identified one anti-Fab antibody which could specifically aggregate RhD-positive RBC.[51]

Finally, Zhang et al analyzed single-stranded DNA from an aptamer library of RhD antibodies in an attempt to predict those of greatest reactivity.[52] Although cell free blood group antigens have not yet reached the level of clinical practice, they are an area of active ACCEPTED MANUSCRIPT 7 investigation in immunohematology and related fields.

Exoglycosidase conversion to group O RBCs

The preparation and application of universal group O donor red blood cells (RBC) has great potential in transfusion medicine. Methods to convert healthy donor group A and/or B blood to group O have been the subject of several investigations. Two basic methods can be used for that purpose: One is enzymatic cleavage of the terminal immunodominant sugars from carbohydrate chains on the membrane of group A or/and group B RBC, in order to produce so-called enzyme-converted group O (ECO) RBCs. Another is coating of low-immunogenic or non-immunogenic polymers, such as methoxy-polyethylene glycol (mPEG), to the surface of

RBCs. The second approach has the advantage that multiple blood groups can in principle be masked, providing a RBC with a functionally negative phenotype.

Enzymatic removal of blood group A and B antigens to develop a universal RBCs was originally explored more than 30 years ago by Goldstein and others [53-55]. The original procedure for conversion of group B RBCs with an α-galactosidase derived from green-roasted coffee beans (Coffea canephora) required as much as 1–2 g of enzyme per unit of group B red cells (～200mL packed RBCs) for adequate conversion at pH 5.5. Despite this, ECO RBCs prepared using the coffee-bean-derived enzyme successfully passed both phase I[56-58] and phase II clinical trials in which B-ECO RBCs were shown to be safe and functional[59]. In ACCEPTEDphase II clinical trials, the survivalMANUSCRIPT and recovery of B-ECO RBCs were comparable to ABO-matched RBCs as measured with 51Cr-labelling studies [59]. This clinical success occurred despite the fact that 20% of group A and 40% of group O sera agglutinated B-ECO RBCs when testing with a PEG-enhanced indirect antiglobulin test.

In China, Zhang and co-workers followed on these studies and did preliminary investigation of the enzymatic conversion of blood group B to O in 1998.[60-61] For conversion of group

B RBCs, they used a recombinant α-galactosidase derived from green Catimor coffee beans

(200mg enzyme for 60-minute; or 100mg enzyme for 120-minute per unit of group B

RBCs)[62-63]. The conversion process has little impact on physiologic and metabolic ACCEPTED MANUSCRIPT 8 parameters of B-ECO RBCs[62]. In 2007, approval for clinical investigation was granted by the CFDA (China Food and Drug Administration).[64] In contrast, research progress of A to

O conversion has been slow. In 2007, Liu et al reported two bacterial glycosidase gene families that provide enzymes capable of efficient removal of A and B antigens at neutral pH with low concentrations of recombinant enzymes[65]. The process has a projected requirement for 60mg (A-ECO) and 2mg (B-ECO) recombinant enzyme with 60-minute treatment per unit RBCs. This is 30 fold (A-ECO) and 1,000 fold less (B-ECO) enzyme usage than the conversion protocol developed for group B RBCs using the Coffee bean

α-galactosidase[65]. Thereafter, Yu et al successfully prepared A-ECO RBCs from both group A1 and A2 RBCs using a recombinant -N-acetylgalactosaminidase from

Elizabethkingia meningosepticum isolated from a domestic clinical sample (A-enzyme)[66].

Then, Gao et al prepared B-ECO and AB-ECO RBCs by using a recombinant

-galactosidase from Bacteroides fragilis (B-enzyme) and an existing A-enzyme from their laboratory[67]. The process has a projected requirement for 3mg A-enzyme for A cells, 2mg

B-enzyme for B cells, and 3mg A-enzyme and 2mg B-enzyme for AB cells with 60-minute treatment per unit of RBCs. In 2009, a phase I clinical trial of A-ECO RBCs was carried out by

ZymeQuest Corporation and the results indicated that small amounts of a person's own red cells, when treated with an enzyme used to make A-ECO RBCs, can be safely re-infused, even repeatedly[68]. So far, no clinical study for transfusion of A-ECO RBCs or AB-ECO

RBCs to volunteers of other blood groups has been reported. A recognized obstacles is the finding that A-ECOACCEPTED and AB-ECO RBCs are stillMANUSCRIPT agglutinated with some examples of group O and group B sera[69-70]. It is speculated that this agglutination results because small amounts of residual A antigen associated with type 3 H structures remain on the surface of

ECO RBCs[69-70].

Polyethylene glycol (PEG) coating of RBCs

Antigen ‘camouflage’ imparted by PEG treatment of RBCs is thought to result from the special physico-chemical nature of PEG: its molecular size, large exclusion volume, and extensive hydration. These characteristics may prevent the interaction of antibodies with

RBC surface antigens on PEG coated cells [71]. Because covalent binding of PEG to intact ACCEPTED MANUSCRIPT 9

RBC could produce antigenically silent RBC, several research groups have attempted the pegylation of RBCs with different commercially-available methoxy-PEG (mPEG) derived materials, such as cyanuric chloride, benzotriazole carbonate, N-hydroxylsuccinimide as well as succinimidyl propionate [71-77]. It was found that mPEG modification could significantly reduce the antigenicity of human RBCs, especially for non-ABO blood group antigens such as RhD, Duffy, Kell, Kidd, and MNSs[71,72,78]. Among these antigens, RhD antigen poses a significant clinical risk and antigen-negative blood is in short supply in China. Therefore, more importance and clinical significance has been assigned to the preparation

RhD(masked) RBCs using mPEG technology[79-81].

In China, the Zhang research group became engaged in pegylation research in 2000[82]. mPEG modified RBCs (mPEG-RBCs) were found to be morphologically and functionally normal in vitro. mPEG coating, at immunoprotective levels, produced RBCs with normal in vivo survival in mouse models[71,75-76]. mPEG-RBCs also survived normally in a rhesus monkey (unpublished data). In addition, pegylation is homogeneous on the red cell and mPEG did not appear to detach from membrane proteins after in vitro storage for 30 days[81].

Furthermore, mPEG-RBC could effectively block the binding of autoantibodies in a patient with autoimmune hemolytic anemia[83]. These findings suggest that the pegylation technology may provide a practical and effective way to address clinical problems with transfusion compatibility. Importantly, the in vivo function of mPEG-RBCs was investigated in previous studies ACCEPTEDwhich indicated that mPEG-RBCs MANUSCRIPT carry O2 and could improve some physiologic indexes of in a mouse model of exsanguination[84]. These studies suggest that mPEG technology may have potential utility in transfusion medicine, especially in situations where RhD negative blood is in short supply. However, it has recently been reported that

PEG is immunogenic in animals and humans and that antibodies to PEG can shorten the survival of PEG-RBCs in rabbits and pegylated proteins in humans[85-88], suggesting that an immune response by the recipient to PEG-RBCs may be an important obstacle for future clinical use of this technology. Although some progress in the field of universal RBCs has been made, a number of challenges still remain to be solved before these technologies can be used in clinical practice. Nevertheless, this avenue of research may provide solutions for ACCEPTED MANUSCRIPT 10 important problems in transfusion medicine including blood supply shortages, emergency blood transfusion, and transfusion safety for difficult-to-match transfusion recipients.

Future Perspectives

Although genotyping has contributed to the expansion of the rare donor program, routine serological methods remain the mainstay in China. ABO genotyping is currently recommended only for resolving specific cases of following stem cell transplantation. Blood group incompatibility remains an important contributor to adverse transfusion reactions and can be effectively prevented by high-performance antibody detection tests.

Immunohematology research in China is exploring potential future reagents that may be developed with mimetic peptides. Although modification of red cell surface antigens has been successfully accomplished in in vitro experiments, clinical application awaits further research.

Conflict of Interest Statement

None.

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Table 1. Distribution of cis-AB and B(A) genotypes in 71 cases from China. Data from reference 12.

Phenotype Genotype n

A2B cis-AB01/B 1 B(A)02/O 1 B(A)06/B 1

A2Bx cis-AB01/O 9

ABx B(A)02/A 2 cis-AB01/A 2 cis-AB02/A 2

AxB cis-AB06/O 1 cis-AB01/B 1 B(A)02/O 3 B(A)04/O 2 B(A)06/O 5 B(A)06/B 1

AintBx cis-AB01/A 1 cis-AB01/O 1 cis-AB02/O 1 B(A) B(A)02/B 1 B(A)02/O 9 B(A)04/O 25 B(A)04/B 1 B(A)06/B 1

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Table 2. Estimated cisAB/O and B(A)/O genotype frequency in Chinese population.

genotype frequency n （*10-5） B(A)02 0.78 13 B(A)04 1.60 27 B(A)06 0.30 5 cis-AB01 0.66 10 cis-AB02 0.06 1 cis-AB06 0.06 1

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Red Cell Immunohematology Research Conducted in China

* ABO and RhD variants are the most studied blood group in China. * New techniques, including phage display and aptamer technology, are being used for immunohematology research in China. * Progress is being made towards modification of red blood cell surface antigens from A and/or B to blood group O.

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