M. A. M. Overbeeke

Blood group terminology 2004: from the International Society of Blood Transfusion committee on terminology for red cell surface antigens

1 Bristol Institute for Transfusion Sciences, Bristol, UK 2 Growing your Knowledge, Spit Junction, NSW, Australia 3 American Red Cross Blood Services, Los Angeles-Orange Counties Region, Los Angeles, CA, USA 4 Biotechnology Research Centre, Auckland University of Technology, Auckland, New Zealand 5 Regional Blood Transfusion Center, Department of Clinical Immunology, University Hospital, Arhus N, Denmark 6 Department of Pathology, University Hospitals UH-2G332, Ann Arbor, Michigan, USA 7 Reference Laboratory for Immunohematology and Blood Groups, National Blood Services Centre, Tel Hashomer, Israel 8 New York Blood Center, New York, NY, USA 9 Ortho-Clinical Diagnostics, Raritan, NJ, USA 10 Drexel University College of Medicine, Philadelphia, PA, USA 11 Gamma Biologicals Inc (subsidiary of Immunocor Inc), Houston, TX, USA 12 Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, the Netherlands 13 Centre national de Reference pour les Groupes sanguines (CNTS), Paris, France 14 International Blood Group Reference Laboratory, Bristol, UK 15 Finnish Red Cross Blood Transfusion Service, Helsinki, Finland 16 South African National Blood Service, East Coast Region, Pinetown, South Africa 17 Osaka Red Cross Blood Center, Osaka, Japan 18 Blood Bank, Hospital Sirio-Libanes, Sao Paulo, Brazil 19 Rh Laboratory, University of Manitoba, Winnipeg, Manitoba, Canada

Blood Group Terminology 1995: ISBT Working Party on Terminology for Red Cell Surface Antigens

Since the first human blood groups were discovered almost a century ago, many hundreds of new red cell antigens have been identified. Because of the extended time period over which these antigens were discovered, a variety of different terminologies has been introduced. In some cases single capital letters were used (A, B, M, K), in some superscripts distinguished allelic products (Fy’, Fyh), and in some a numerical notation was introduced (Fy3). Some antigens were given different names in different laboratories, based on alternative genetic theories (D and Rho). In 1980 the International Society of Blood Transfusion (ISBT) established a Working Party to devise a genetically based numerical terminology for red cell surface antigens. In 1990 the Working Party published a monograph describing a numerical terminology for 242 red cell antigens [I], and brief updatings followed in 1991 [2] and 1993 [3]. In the 6 years since the 1990 report many amendments to the classification have been necessary: 18 new antigens have been identified and 6 others declared obsolete due to lack of suitable reagents; four new systems have been created (all from existing collections); the Auberger antigens joined the Lutheran system; the Gregory antigens and Jo” joined the Dombrock system; the Wright antigens joined the Diego system. Furthermore, since, 1990, many of the blood group genes have been isolated: of the genes controlling the 23 systems, only four (PI, JK, SC, DO) remain to be cloned. The purpose of this monograph is to describe the ISBT terminology for red cell surface antigens and to tabulate the complete 1995 version of the classification. In addition, an alternative ‘popular’ terminology is suggested in an attempt to reduce the number of different names used in publications on red cell antigens. Much of the information provided in the 1990 monograph [l] is reiterated here so that referral back will not generally be required, but only references after 1990 are provided.

International Society of Blood Transfusion Committee on Terminology for Red Cell Surface Antigens: Cape Town report.

The Committee met in Cape Town during the 2006 Inter-national Society of Blood Transfusion (ISBT) Congress (seeAppendix 1 for Committee members). Some changes to theclassification documented in Blood Group Terminology 2004[1] were agreed and are described below. The full updatedclassification can be found on the Blood Group Terminologywebsite at http://www.blood.co.uk/ibgrl. New antigens wereadded to the MNS, Kell, Scianna, Cromer, Indian, Knops,and JMH systems (Table 1). In line with convention, aminoacid positions are numbered with the translation-initiatingmethionine as 1, although the more traditional numberingfor glycophorin A, with number 1 representing the first aminoacid of the mature protein, is also provided.

SEQUENCE ANALYSIS OF CDNA DERIVED FROM RETICULOCYTE MRNAS CODING FOR RH POLYPEPTIDES AND DEMONSTRATION OF E/E AND C/C POLYMORPHISMS

RNA derived from enriched reticulocytes of Rh‐phenotyped donors was isolated, reversely transcribed into cDNA and amplified with Rh‐specific primers by polymerase chain reaction. Nucleotide sequence analyis of the entire coding region of the Rh cDNAs was carried out. Four types of cDNAs were identified, tentatively designated as RhSCI, RhSCII, RhSCIII and RhSCIV. Comparison of RhSCII with RhSCI (identical to the previously reported RhIXb/30A cDNA), showed a single base pair difference. Since RhSCI and RhSCII were found to be related to the presence of E or e antigen, respectively, the P226A amino acid polymorphism appears to be the genetic basis of the E/e polymorphism. RhSCIII was demonstrated to be a transcript derived from the RhD gene, with 35 amino acid substitutions as compared to RhSCI. RhSCIV was found to be present only in RhC‐positive individuals, indicating that RhSCIV encodes a polypeptide carrying the C antigen. Six nucleotide changes, resulting in four amino acid substitutions W16C, L60I, N68S and P103S, were observed between RhSCII and RhSCIV, probably representing the C/c polymorphism.

International Society of Blood Transfusion Committee on terminology for red blood cell surface antigens: Macao report

The committee met in Macao Special Administrative Region,China, during the 2008 International Society of Blood Trans-fusion (ISBT) Congress. Some changes to the classificationdocumented in Blood Group Terminology 2004 [1] and updatedin 2007 [2] were agreed and are described below. The fullupdated classification can be found on the blood groupterminology website at http://www.blood.co.uk/ibgrl. A newblood group system, the RHAG system, was established andnew antigens were added to the Rh, Kell, and Dombrocksystems (Table 1). A total of 308 antigens are now recognized,270 of which are clustered in 30 blood group systems.

The R0Har Rh:33 phenotype results from substitution of exon 5 of the RHCE gene by the corresponding exon of the RHD gene

The highly polymorphic Rh (Rhesus) system is encoded by two homologous genes, one encoding the D polypeptide and the other the CcEe polypeptides. Partial D antigens may be caused by gene rearrangements, deletions or point mutations. In this study the molecular basis of R0Har Rh:33, a Rh phenotype of low frequency, is described. The R0Har Rh:33 phenotype is characterized by partial expression of D, altered expression of e, absence of G and the presence of two antigens of low frequency: Rh33 and FPTT. Southern blot analysis, RHD typing by PCR and sequence analysis of Rh transcripts revealed that the RHD gene is absent in subjects with this phenotype. Apart from the expected RHCE transcripts, a new Rh transcript, RHc(D)(e), was identified in three unrelated individuals expressing R0Har Rh:33. The RHc(D)(e) transcript showed the same sequence as the RHce transcript, with the exception of exon 5, which was substituted by the corresponding exon of the RHD gene. A method for PCR‐based genotyping was developed to determine specifically the c(D)(e) haplotype. The c(D)(e) PCR proved to be a reliable alternative method for R0Har Rh:33 typing.

The genetic basis of a new partial D antigen: DDBT

The Rh system, the most polymorphic system on red cells, is genetically controlled by two different but highly homologous genes on chromosome 1. The RHCE gene encodes different RhCcEe polypeptides and the RHD gene encodes D antigens. It is well established that in D negative individuals the RHD gene is either absent or grossly deleted. The D antigen comprises at least nine serologically defined D epitopes. The D antigen can be divided into different partial D categories, reflecting a different pattern of specific D epitopes.

Quantitation of D Sites on Selected ‘Weak D’ and ‘Partial D’ Red Cells

Measurements have been made of the number of available sites on 10 examples of red cells in which the only abnormality appeared to be a quantitative reduction in the expression of D (weak D cells); these estimates were carried out using three monoclonal anti‐D antibodies, Fog‐1, Brad‐3 and Los‐2. The values varied with the monoclonal antibody that was used and fell within the range of 170–1,870 sites/cell. A further 3 examples of weak D cells which had brought about immunisation following transfusion were found to have between 390 and 1,470 sites per red cell. The implications of the D site density on the immunogenicity of weak D cells are discussed. The number of sites on red cells with structurally abnormal D (partial D cells) were also estimated, using the antibody Fog‐1. Four of the 5 examples of cells of category IVa (probable phenotype R0r) were found to have a high expression of D (range 29,300–41,300), but the available D sites of categories DVa, DVIa, and DVII were considerably reduced (<500, <500 and 2,400–7,500 sites/cell, respectively). As a working hypothesis, it is suggested that there are two types of genetic abnormality leading to an abnormal expression of D. First, a defect in genomic DNA leading only to a quantitative reduction in the number of available D sites; this genomic lesion should be termed ‘weak D’. Secondly, genomic defects leading to amino acid sequence abnormalities and structural change in the D polypeptide; these lesions should be collectively known as ‘partial D’.

International Society of Blood Transfusion working party on terminology for red cell surface antigens

G. L. Daniels (Chair), D. J. Anstee, J. P. Cartron, W. Dahr, A. Fletcher, G. Garratty, S. Henry, J. Jorgensen, W. J. Judd, L. K ornstad, C. Levene, M. Lin, C. Lomas-Francis, A. Lubenko, J. J. Moulds, J. M. Moulds, M. Moulds, M. Overbeeke, M. E. Reid, P. Rouger, M. Scott, P. Sistonen, E. Smart, Y. Tani, S. Wendel & T. Zelinski*

Section 1C: Assessment of the functional activity and IgG Fc receptor utilisation of 64 IgG Rh monoclonal antibodies. Coordinator’s report

Sixty-four IgG Rh monoclonal antibodies (Mabs) submitted to the Fourth International Workshop on Monoclonal Antibodies Against Human Red Blood Cells and Related Antigens were characterised and tested in quantitative functional assays at five laboratories. The biological assays measured the ability of anti-D to mediate phagocytosis or extracellular lysis of RBC by IgG Fc receptor (Fc gamma R)-bearing effector cells. Interactions of RBC pre-sensitised with anti-D (EA-IgG) with monocytes in chemiluminescence (CL) assays were found proportional to the amount of IgG anti-D on the RBC. Using antibodies to inhibit Fc gamma RI, Fc gamma RII or Fc gamma RIII, the only receptor utilised in the monocyte CL and ADCC assays for interactions with EA-IgG1 was found to be Fc gamma RI. In these assays, enhanced interactions were promoted by EA-IgG3 and additional Fc gamma receptors may have contributed. IgG2 anti-D was not reactive in these assays and EA-IgG4 promoted weak reactions through Fc gamma RI. A macrophage ADCC assay showed that haemolysis of EA-IgG3 was greater than that of EA-IgG1, mediated mainly through Fc gamma RIII. In ADCC assays using lymphocytes (NK cells) as effector cells and papainised RBC target cells, only a minority of IgG1 anti-D Mabs were shown to be able to mediate haemolysis in the presence of monomeric IgG (AB serum or IVIg). These interactions were mediated solely through Fc gamma RIII. Haemolysis via Fc gamma RIII may depend on the presence of certain sugars on the oligosaccharide moiety of IgG. Most Mabs (IgG1, IgG2, IgG3 and IgG4) elicited intermediate, low or no haemolysis in these assays. Blocking studies indicated that low activity IgG1 and IgG4 anti-D utilised only Fc gamma RI. Other IgG1 and IgG3 Mabs appeared to promote haemolysis through Fc gamma RI and Fc gamma RIII while IgG2 was inhibited by Mabs to both Fc gamma RII and Fc gamma RIII, suggesting a variety of Fc gamma R are utilised for anti-D of low haemolytic activity. Excellent agreement between the results of the lymphocyte ADCC assays and antibody quantitation was observed between the participating laboratories.