High resolution HLA-C typing by PCR-SSP: identification of allelic frequencies and linkage disequilibria in 604 unrelated random UK Caucasoids and a comparison with serology

Recent evidence indicates that HLA-C molecules are biologically relevant by eliciting T-cell responses and exerting control over NK cell function. In addition, HLA-C is associated with susceptibility to various diseases, notably psoriasis vulgaris. Clarification of the full biological roles for HLA-C has however proved difficult because detection of HLA-C antigens by complement mediated cytotoxicity using alloantisera is inefficient. Up to 50% of individuals in every race have serologically undetectable HLA-C locus antigens due to a combination of relatively low expression, lack of serological reagents and a lack of information about the distribution of the HLA-C blank alleles. Recently, amplification of DNA using sequence-specific primers (PCR-SSP) has proved a reliable, accurate and rapid method for medium resolution HLA-C typing. We have now developed high resolution HLA-C typing by PCR-SSP utilizing allele and group-specific PCR-SSP reactions which can identify all HLA-C alleles (except non-coding change alleles) in most heterozygous combinations. Using this system we have typed 604 unrelated United Kingdom Caucasoids to generate accurate frequency and linkage disequilibrium data. To assess the validity of serology for HLA-C, PCR-SSP typings for 527 out of the 604 individuals were compared to serology. We find that the frequency of many HLA-C antigens has been underestimated by serology and some antigens such as Cw6 are consistently assigned incorrectly by serology. The overall discrepancy rate between serology and SSP was high at 37% (195/527). High-resolution HLA-C typing of 112 International Histocompatibility Workshop cell lines has also been performed     

Rapid DNA typing for HLA-C using sequence-specific primers (PCR-SSP): Identification of serological and non-serologically defined HLA-C alleles including several new alleles

Abstract: Detection of HLA-C antigens by complement mediated cytotoxicity using human alloantisera is often difficult. Between 20 to 40% of individuals in every race have undectectable HLA-C locus antigens and 9 out of the 29 sequenced HLA-C alleles so far published encode serologically undetected antigens. In addition, HLA-C molecules are expressed at the cell surface at about 10% of the levels of HLA-A and HLA-B. Recently, amplification of DNA using sequence-specific primers (PCR-SSP) has proved a reliable and rapid method for typing HLA-DR, HLA-DQA and HLA-DQB genes. PCR-SSP takes two hours to perform and is therefore suitable for the genotyping of cadaveric donors. We have designed a set of primers which will positively identify the HLA-C alleles corresponding to the serologically defined series HLA-Cw1, Cw2, Cw3, Cw4, Cw5, Cw6, Cw7 and Cw8. The serologically undetectable alleles have also been detected in groups according to sequence homology. In addition, three new unsequenced variants have been identified. DNA samples from 56 International Histocompatibility Workshop reference cell lines and 103 control individuals have been typed by the HLA-C PCR-SSP technique. 4/56 cell line types and 11/103 normal control individuals types were discrepant with the reported serological types. All combinations of serologically detectable and most of the serologically blank HLA-C antigens can be readily identified. DNA typing for HLA-Cw by PCR-SSP can take as little as 130 minutes from start to finish, including DNA preparation.     

Comprehensive, serologically equivalent DNA typing for HLA-B by PCR using sequence-specific primers (PCR-SSP)

Abstract: Polymorphic products of HLA class I genes from the human major histocompatibility complex (MHC) are traditionally assigned by serology with additional heterogeneity detectable using one-dimensional isoelectric focusing (1D-IEF). With the increased availability of HLA class I DNA sequence information it has become feasible to genotype for class I by polymerase chain reaction utilising sequence-specific primers (PCR-SSP). We describe here a comprehensive HLA-B PCR-SSP typing system based on available HLA nucleotide sequences which can detect all serologically defined antigens in most heterozygous combination in 48 one-step PCR reactions. In addition, four new unsequenced variants have been identified. DNA samples from 57 International Histocompatibility Workshop reference cell lines and 160 control individuals have been typed by the HLA-B PCR-SSP technique. 3/57 cell line types and 12/160 normal control individuals types were discrepant with the reported serological types. The SSP system has been designed to be higher resolution than serology but is not a complete allele-specific PCR although many single alleles can be identified. The system is entirely complementary to previous published PCR-SSP systems for HLA-Class II and HLA-Class I in that the same PCR conditions and controls are used which allows us to do one step PCR-SSP for all relevant HLA loci in under 3 hours in a system suitable for the typing of cadaver donors.     

A reliable and efficient high resolution typing method for HLA-C using sequence-based typing

Abstract: Serological typing of HLA-C has been poor and almost half of its alleles are serologically undetectable blanks in most populations. Therefore, DNA typing techniques have been used to identify and type for the HLA-C gene. Sequence-based typing (SBT) has proven a major typing strategy for highly polymorphic HLA genes. The technique enables direct identification of all sequence motifs without the need to continuously adjust primers. Here we describe a reliable solid-phase SBT strategy for HLA-C which can be used to distinguish all currently known HLA-C alleles without prior knowledge gained by low resolution typing. Exons 2 and 3 were amplified and sequenced and if necessary sequences of exons 1 and 5 were determined. A total of 257 individuals were typed for HLA-C using this protocol and 30 of the 42 known HLA-C alleles were detected. All heterozygous combinations found in this study were unambiguously discriminated.
One hundred and forty-four individuals from the Dutch population were typed randomly. In this group Cw*0701 and *0702 were the most frequently detected alleles. Of the serological Cw blank alleles Cw*1203 was found to have the highest frequency (16%). From the total group 212 individuals were typed serologically and 106 were retyped with 97 selected antisera to further compare serological and molecular defined phenorypes. Discrepancies between serological typing and SBT are mainly attributable to the serologically Cw blank alleles Cw*12–18.
The high resolution SBT protocol described will be a valuable tool for the identification of HLA-C alleles and the determination of the role of HLA-C in marrow and organ transplantation.     

Comparison of serological and molecular typing for HLA-A and -B on cord blood lymphocytes

Abstract: HLA class I typing by standard microcytotoxicity testing has been unsatisfactory for 14.5% of 1644 cord blood samples. In this study, we evaluated the capacity of PCR-SSP in solving problems in HLA-A,B typing with serological methods. With this aim we have compared serology with PCR-SSP in 100 cord blood samples with doubtful or unreliable HLA-A,B typing. PCR-SSP was successful in amplifying HLA-A,B alleles in all 100 cord blood samples. Forty-six typings gave discrepant results with the 2 methods (serology and PCR-SSP). Typings were considered discrepant also in the case of inability to define a split. For 19 specimens, no serological conclusion was drawn due to high mortality of the cell suspension, while PCR-SSP allowed the definition of a clear typing. In 6 cases it was necessary to infer information from serology to define the current typing. Finally, in 3 other cases it was impossible to exclude or attribute the antigen/allele B67 or B4802. PCR-SSP for HLA-A,B can improve the overall reliability of HLA-A,B typing requiring a small amount of blood although, with the set of sequence specific primers adopted, a number of alleles are still poorly defined.     

Error rate for HLA-B antigen assignment by serology: implications for proficiency testing and utilization of DNA-based typing methods

Until recently, the majority of HLA class I typing has been performed by serology. Expensive commercial typing trays are frequently used for testing non-Caucasian subjects and new strategies using DNA-based methods have been adopted for improving clinical histocompatibility testing results and adapted as supplements in proficiency testing. A double-blind comparison of the typing of HLA-B specificities in 40 samples was carried out between serology and two polymerase chain reaction (PCR) methods, PCR amplification with sequence-specific primers (PCR-SSP) and PCR amplification and subsequent hybridization with sequence-specific oligonucleotide probes (PCR-SSOP). The results demonstrated 22.5% misassignments of HLA-B antigens by serology. There was complete concordance between the results obtained with the two PCR based typing methods. A second panel of 20 donor samples with incomplete or ambiguous serologic results was analyzed by PCR-SSP and SSOP. Both PCR methods identified correctly the HLA-B antigens. Our results suggest that more accurate typing results can be achieved by complementing serologic testing with DNA-based typing techniques. The level of resolution for HLA-B antigen assignment can be obtained by this combination of serology and limited DNA-based typing is equivalent to the HLA-B specificities defined by the WHO-HLA Committee. This level of resolution cannot routinely be achieved in clinical histocompatibility testing or in proficiency testing using serologic reagents only.     

HLA-C high resolution typing: analysis of exons 2 and 3 by sequence based typing and detection of polymorphisms in exons 1–5 by sequence specific primers

Abstract: HLA-C high resolution sequence based typing developed in this study involves a unique DNA amplification encompassing exon 1 to intron 3 and four fluorescent sequencing reactions covering exon 2 and 3. Both dye primer and dye terminator sequencing techniques were performed and results compared. This approach allowed the identification of all of the 50 HLA-C allelic variants so far described, except for two allele pairs that are distinguished by non-coding nucleotide changes (Cw*12021 = 12022, Cw*15051 = 15052) and three allele pairs (Cw*0701=0706, Cw*1701 = 1702 and Cw*1801 = 1802) that share the same nucleotide sequence in exon 2 and 3. For complete subtyping of these allelic variants, an amplification based on sequence specific primers (PCR-SSP) was used. No ambiguous heterozygous combinations of alleles were detected in our panel so far. HLA-C typing data obtained by this method were compared with data from serological and low resolution PCR-SSP typing, which had been performed previously on the samples sequenced.     

Molecular diversity of the HLA-C gene identified in a caucasian population

A DNA typing procedure, based on a two stage polymerase chain reaction-sequence-specific oligonucleotide probe (PCR-SSOP) typing strategy, has been developed and applied to DNA from 1000 healthy individuals from the Northern Ireland region. The two-stage procedure involves human leukocyte antigen (HLA-C) identification through the use of a medium resolution PCR-SSOP system, followed by four secondary group specific PCR-SSOP systems, to enable allele resolution. The PCR-SSOP systems were designed for the identification of HLA-Cw alleles with possible discrimination within exons 2 and 3 of the HLA-C gene, i.e., HLA-Cw*01-Cw*16. PCR-SSP tests were designed for the resolution of HLA-Cw*17 and -Cw*18 alleles. The systems can also be used independently of each other if selective allele resolution is required. HLA-Cw allele frequencies occurring within the Northern Ireland population have been compiled, along with estimations of HLA-B/Cw haplotype frequencies.     

Validation of DNA-based HLA-A and HLA-B testing of volunteers for a bone marrow registry through parallel testing with serology

A total of 42,160 individuals were typed for HLA-A and HLA-B by both serology and PCR-based typing. The HLA assignments included all of the known serological equivalents. The majority of the individuals (99.9%) were from U.S. minority population groups. The serologic typing was performed between 1993 and 1997 at the time of recruitment for the National Bone Marrow Program (NMDP) registry. The polymerase chain reaction (PCR)-based typing was carried out in two phases. In phase I, DNA typing was performed by PCR using sequence-specific oligonucleotide probes (PCR-SSOP) or PCR using sequence-specific primers (PCR-SSP) without knowledge of the serologic assignments. Discrepancies were identified between the serologic and DNA assignments in 24% of the volunteers (8% of volunteers differed for only HLA-A assignments, 13% for HLA-B, and 3% for both HLA-A and -B) and a potential explanation was assigned each discrepant serology/DNA pair. In phase II, a random sampling scheme was used to select a statistically significant number of individuals for repeat DNA typing from each of these categories. The categories included antigens missed by serology, nonexpressed (null) alleles, PCR amplification failures, misassignment of antigens and nomenclature issues. Only a single individual was found to carry a null allele. DNA-based testing correctly typed nearly 99% of the donors at HLA-A, more than 98% at HLA-B, and more than 97% at both HLA-A and -B validating this methodology for registry typing.     

Serology versus PCR-SSP in typing for HLA-DR and HLA-DQ: a practical evaluation

Abstract: In this study, serological HLA-DR and -DQ typing results were compared to typing results obtained with sequence-specific primers in the polymerase chain reaction (PCR-SSP). HLA-DR typing was performed on a random Caucasian population consisting of 31 patients and 73 healthy individuals. Considering HLA-DR1-10, differences in typing results were found in 3 out of 73 healthy individuals and 8 out of 31 patients. When HLA-DR1-16 alleles were taken into account, differences in typing results were found in 11 out of 31 patients and 14 out of 73 healthy individuals. Typing results of PCR-SSP, different from that of serology, were all confirmed by sequencing-based typing of HLA-DRB1 alleles. HLA-DQ 1–3 typings were performed on 40 individuals consisting of 17 patients and 23 healthy individuals. Differences in typing results were found in 5 out of 17 patients and 1 out of 23 healthy individuals. From the results of this study it can be concluded that serology is a reliable technique, when restricted to identification of HLA-DR1-10 and HLA-DQ1-3 antigens in healthy individuals. By PCR-SSP, however, reliable HLA-DR1-16 and -DQ1-3 typings can be obtained both in patients and healthy individuals.     

Emerging new alleles suggest high diversity of HLA-C locus

Human leukocyte antigen (HLA)-C has only recently emerged as an important transplantation antigen and as a receptor for natural killer cells. Over the last few years, sequence-based typing (SBT) revealed the true diversity of HLA-C locus; however, the frequency at which new alleles are detected still remains high. During routine SBT of 3500 samples for the National Marrow Donor Program, we have identified 20 new HLA-C alleles reported in this article in 26 individuals. New variants have been characterized by direct sequencing of polymerase chain reaction product obtained by allele-specific amplification of potential new alleles. Most of the new alleles carry coding substitutions of residues located within the antigen-binding groove. The substitutions are predominantly located in the alpha2-helix which is consistent with the unique to HLA-C conservation of alpha1-helix. Seven new alleles, or 35%, have been identified in African Americans, two of them in three and four individuals each, suggesting that these alleles may not be rare. This observation reflects the fact that the minority groups, previously under-represented in the HLA research pools subjected to SBT, now begin to emerge as a main source of new HLA-C alleles. This study further confirms that HLA-C locus is at least as polymorphic as HLA-A and HLA-B.     

中国北方汉族HLA基因C位点遗传多态性研究

目的：研究北方汉族人群HLA(人类白细胞抗原)基因c位点的遗传特征。方法：采用序列特异性引物扩增(Polymerase Chain Reaction-Sequence-Specific Primers PCR-SSP)方法对205个样本进行HLA-C及-A、-B基因特异性分析。结果：检出C位点特异性14个，其中Cw*01、*07基因表现频率最高，分别为30．73％和27．80％；血清学方法检测不出的HLA-Cw*12、*14、*15表现频率分别为13．17％、4．88％、8．78％。统计显示显著性连锁不平衡的HLA-B-C单倍型24个，HLAs-A-C单倍型5个。结论：作为中国北方人HLA遗传多态性群体调查，提供了一套比较完整准确的基因频率和连锁不平衡参数，为人类学及疾病相关性等研究提供了较为完整的正常参考数据。     

Molecular characterization of the HLA-Cw*0409N allele

Various molecular methods in conjunction with serologic assays are used for clinical human leukocyte antigen (HLA) typing. Although serologic reagents detect most HLA-A and -B allospecificities, serologic HLA-C typing is hampered by the lack of informative antisera for many of the known HLA-C gene products. HLA antigens not detected by serology, but detected by molecular methods, are often referred to as "blank" antigens. Their lack of reactivity with antibodies in serological assays often reflects the presence of null alleles. The present study has characterized an HLA-Cw*04 allele (Cw*0409N) detected by DNA typing but not by serology. In cultured B-lymphoid 13W09501 cells carrying this Cw*04 null allele, isoelectric focusing analysis could not detect any component with a pattern compatible with that of the product of the HLA-Cw*0401 allele, but detected components reacting with an anti-HLA-Cw4 and Cw6 monoclonal antibody. Sequencing analysis of the full length HLA-Cw4 cDNA amplified from the cell line 13W095-01 revealed a base deletion at codon 365 in exon 7, resulting in a reading frame shift that added 32 amino acids at the C-terminal of the HLA-Cw4 heavy chain. These results indicate that the HLA-Cw*0409N allele may produce a putative long HLA-Cw4 heavy chain that is not expressed on the cell surface.     

High-resolution HLA typing for the DQB1 gene by sequence-based typing

Abstract: The ideal high-resolution typing strategy for polymorphic genes is sequence-based typing. SBT of genomic DNA has been developed for the HLA class H genes DRB1, DRB3/4/5 and DPB1. For the DQB1 gene the sequence-based typing method was shown to cause a number of problems. To resolve those problems, different primers to amplify and sequence exon 2 of DQB1 were designed and tested. With several primer combinations, preferential amplification was observed in individuals heterozygous for DQB1*02/*03 and DQB1*02/*04. The preference was for DQB1*02 in many instances but could also be demonstrated for DQB1*03 or *04 and resulted occasionally in allelic drop-out. The best primer combination was selected and successfully used to type individuals heterozygous for DQB1*02, *03 and*04. To distinguish DQB1*0201 and *0202, primers for amplification and sequencing of exon 3 were developed and correct subtyping was obtained. The ambiguous typing DQB1*0301/*0302 and DQB1*0303/*0304 was resolved by allele-specifk amplification and sequencing. A total of 258 individuals were fully typed for their DQB1 subtypes. All samples had been previously typed by PCR-SSP and serology. Concordant typing results were obtained for all individuals tested. The DQB1 alleles detected included *0501, *0502, *0503, *0601, *0602, *0603, *0604, *0609, *0201, *0202, *0301, *0302, *0303, *0304, *0401 and *0402. Sequence-based typing of the DQB1 gene proved a reliable typing strategy for assignment of the different DQB1 alleles after intensive selection of primers and test conditions.     

Characterization of the HLA class I genotypes of a Venezuelan Amerindian group by molecular methods

Abstract: We have characterized the HLA class I genotypes of the Yucpa, a tribe of Venezuelan Amerindians, using molecular methods. The study was carried out on DNA extracted from unrelated individuals using low resolution ARMS-SSP typing with sequence-specific primers, high resolution typing using reference strand conformation analysis (RSCA), and for some samples sequence-based typing (SBT). The following class I alleles were found to be present in this tribe: for the HLA-A locus A*0204, A*0212, A*0213, A *2402, A*3101 and A*6801; for the B locus B *1522, B *3512, B*3905, B *3909, B*4004 and B*52012, and for C locus Cw *0102, Cw*0302/4, Cw*0401, Cw*0702 and Cw*1503. This is the first rime these alleles have been described in this group, although all of them have previously been reported as being present in other Amerindian tribes. The study confirmed the high frequency of HLA-B39 which was previously observed in serologi-cal analysis of this tribe, and indicated that two different B*39 alleles were present in this population. The identification of the class I alleles by molecular methods for this ethnic group confirms the restricted polymorphism of the MHC molecules previously obtained by serology and has allowed a more accurate definition of the different alleles present in this population.     

Comparison of HLA-A antigen typing by serology with two polymerase chain reaction based DNA typing methods: implications for proficiency testing

Serology has been routinely used for class I HLA typing for the selection of donors for allotransplantation. However, serology is not adequate for the assignment of all class I specificities especially when testing non-Caucasians subjects and it is necessary to adopt new strategies for routine testing. At the present time the extent of incorrect serologic HLA-A assignments in clinical testing is not known. The polymerase chain reaction (PCR) based techniques have become useful standard clinical typing methods of HLA class II alleles but most laboratories still use serology for class I typing. In this report we have compared two PCR based techniques, PCR amplification with sequencespecific primers (PCR-SSP) and PCR amplification and subsequent hybridization with sequence-specific oligonucleotide probes (PCR-SSOP), for the assignment of HLA-A specificities in 56 blood samples from patients and families serologically typed for HLA-A. This side-by-side comparison of PCR methods showed 100% correlation between them. However, serology showed 7.1% misassignments and, in an additional panel of 19 cells where serology produced equivocal results, the PCR-SSP and SSOP methods identified the correct HLA-A specificity. Our results emphasize the need to complement routine serologic testing of HLA specificities with a small number of primers designed to test HLA-A34, A36, A43, A66, A74 and A80, that are not detected with high precision by serology. We concluded that the PCR-SSP and -SSOP methods can be used in routine HLA-A typing of patients and donors for transplantation with a greater precision than serology.     

Sequencing based typing for HLA-C. Identification of three new alleles: Cw*0307, Cw*0502 and Cw*0504

HLA-C has been described as a transplantation locus in the unrelated bone marrow transplantation setting, and noticeably the number of mismatches between HLA-A,-B,-DRB1 compatible pairs is considerably high. Sequencing based typing (SBT) is an accurate and efficient methodology utilised in the HLA class I and II allele level of resolution. SBT for HLA-C locus was applied on a sample of 40 HLA-A,B,DRB1,DRB3/4/5,DQB1-compatible bone marrow recipient-donor pairs, and 3 new HLA-C alleles have been found. Cw*0307, well defined by serology as Cw3, showed two amino acid changes at the NK motif 77-80 regarding all described Cw*03 alleles, N77K80 instead of S77N80. Two new Cw*05 alleles were described, Cw*0502 properly typed by serology, and Cw*0504 that behaves as a short antigen. Cw*0502 differed from Cw*0501 by only one nucleotide at exon 3, that generated an amino acid replacement at codon 177, K to E. Cw*0504 differs from Cw*0501 by two clustered amino acid positions (114 and 116) placed at the peptide binding site. The rate of new HLA-C alleles found in this small series evidences a high grade of hidden HLA-C diversity in the Spanish population, particularly in the well-defined serologic specificities.     

Identification of a new HLA-B*08 variant, HLA-B*0804

The HLA-B locus is the most polymorphic locus known with currently over 100 different alleles described. Many of these alleles encode variants of the serologically-defined tissue transplantation antigens. This high level of diversity makes accurate tissue typing difficult. Here we present the sequence of a new HLA-B *08 variant, HLA-B *0804. found in Caucasian siblings JH and PF serologically typed as HLA-B51/B59 and HLA-B59/B60, respectively. Additionally, DNA-based typing by the polymerase chain reaction using sequence-specific primers (PCR-SSP) identified HLA-B *51 in JH and HLA-B *4001 in PF. However, PCR-SSP failed to identify a second allele in either of these individuals. The unusual finding of a B59 antigen in a Caucasian and the discrepant molecular typing results suggested that these individuals might express novel HLA molecules. Using denaturing gradient gel electrophoresis (DGGE) followed by direct sequencing, we characterized a novel HLA-B *08 variant, HLA-B *0804. The presence of this allele was confirmed by cloning and sequencing. HLA-B *0804 differed from HLA-B *0801 by only one nucleotide substitution resulting in an amino acid replacement of phenylalanine by serine at position 67. Incidentally, this single nucleotide difference was sufficient to prevent amplification by PCR-SSP. This striking difference between both the serologically typed antigen and the PCR-SSP-identified allele compared to the sequenced allele supports the use of sequence-based typing for the analysis of HLA class I locus alleles.     

There is more to HLA-C than exons 2 and 3: sequencing of exons 1, 4 and 5

HLA-C was shown to be a highly polymorphic gene which can be accurately typed for by sequencing methodologies. Most HLA-C sequence-based typing protocols described so far are based on analysis of sequence data of exons 2 and 3. Nonetheless, exons 1, 4 and 5 also contain nucleotide substitutions which contribute to the polymorphisms of the HLA-C locus. Ten alleles contain polymorphic positions in exons 1, 4 and 5, Cw*0701/06, Cw*1202112, Cw*15051/2, Cw*1701/02, and Cw*1801/02. Here we describe a reliable solid-phase sequence-based typing strategy for sequencing exons 1, 4 and 5, which is an extended protocol of our previous HLA-C study. A panel of 16 individuals, carrying 27 different Cw-alleles, was typed for exons 1, 4 and 5 to check the newly designed primers. No allelic dropout or preferential amplification was noticed in these individuals. The panel was also sequenced in order to check the known polymorphisms present in exons 1, 4 and 5. For exon 5 the sequences of the alleles Cw*0302, *0501 and *07011 did not correspond with the published data. In addition, exons 1, 4 and 5 were sequence-based typing typed in 28, 17 and 59 individuals, respectively. Two new alleles were detected which contain polymorphic positions outside exons 2 and 3, Cw*07012 and Cw*1703. The unknown sequence data of exons 1, 4 and 5 of the alleles Cw*02024, *0308, *1506 and *16041 were elucidated. The described high-resolution sequence-based typing protocol for sequencing exons 1, 4 and 5 will be a valuable tool to study the HLA-C locus for polymorphisms outside exons 2 and 3 and for identification of the presently known HLA-C alleles with polymorphic positions in these exons.     

Technologies involved in molecular blood group genotyping

Background Until the 1990s, blood screening, typing and diagnostics depended entirely on serological techniques. For over a century, agglutination has been the gold standard for red blood cell (RBC) antigen detection used in all blood services. However, haemagglutination has certain limitations, such as, the difficulty to phenotype recently and multi-transfused patients or direct antiglobulin test (DAT)-positive patients. The haemagglutination test provides only indirect indications of risk or severity of haemolytic disease of the new born. In part, to overcome these limitations, nucleic acid-based technologies have been used in immunohematology reference laboratories. Methods There are many molecular methods available for red cell genotyping: PCR, PCR-RFLP, PCR-SSP or PCR-ASP, real time PCR, DNA sequencing and pirosequencing and methods with microarrays-based systems. Other molecular techniques that are under development and may be available for red cell genotyping in the next decade include fluidic or open microarrays; matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS); and mini-sequencing. Conclusions Although serology may be superior for some blood group typing, genotyping assays offer a good alternative for problems encountered by serology. In many laboratories, blood group genotyping is already used at a low-throughput level for diagnostics in cases of problematic serology. Especially in case of weak expression of antigens, the presence of rare antigens or auto-antibodies or after multiple transfusions, genotyping is superior. The non-invasive determination of the foetal RHD analysis in maternal plasma by real-time PCR is well established and already offered as a clinical service in a number of countries. The recent availability of automated, high throughput, DNA-array platforms, allows to introduce into the hospital and donor centres this DNA-based typing methodology. The evolution of molecular methods combined with automation and high-quality standards will make large-scale screening a cost-effective reality.