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Method for the simultaneous determination of blood group and platelet antigen genotypes

Method for the simultaneous determination of blood group and platelet antigen genotypes description/claims

This invention relates to an ultra high throughput (UHT) multiplex PCR genotyping method. More specifically, the present invention relates to an automated method of determining a plurality of blood group and platelet antigen, preferably human platelet antigen (HPA), genotypes simultaneously from a single sample through the detection of single nucleotide polymorphisms (SNPs) for various blood group and platelet antigens.

At present, there are 29 blood group systems and 6 HPA systems recognized by the International Society of Blood Transfusion (ISBT), wherein, with a few exceptions, a blood group ‘system’ may be defined by a single gene at a given locus of the human genome (Daniels, G. L. et al. Vox Sang 2003; 84:244; Metcalfe P. et al., Vox Sang. 2003; 85:240). Most people know their ABO and Rh blood group. However, the ABO and Rh blood group systems expressed on red cells simply represent antigens from only two of the 29 blood group systems, and more systems are being discovered each year. Some examples of blood group systems are the ABO, Rh (D, C, c, E, e), P, Lutheran, Kell (K, k), Lewis, Duffy (Fya, Fyb), or Kidd (Jka, Jkb). Moreover, there are over 250 blood group and 12 human platelet antigens assigned to one of the blood group or HPA systems, respectively. A system is defined by a gene or group of genes at a specific locus of the human genome. The alleles or genotype of a person for each blood group or HPA system represent the unique nucleotide gene sequences that express specific blood group or platelet antigens (for a review see Denomme, G. et al., Approaches to Blood Group Molecular Genotyping and Its Applications: in Stowell, C. and Dzik W., editors; Emerging Technologies in Transfusion Medicine, AABB 2003, Ch 4).

A blood group or HPA system maps to a specific region of the human genome, termed a locus. Nearly all blood group or HPAs can be identified by the presence of its unique nucleotide sequence, termed an ‘allele’, at the locus of interest. Every person has two alleles for any given autosomal gene. Some individuals are homozygotes for a specific allele, i.e. they have two identical alleles, while others are heterozygotes for a specific allele, i.e. they have two different alleles. By definition, alleles that represent different blood group or HPAs differ by at least one nucleotide; sometimes they differ by several nucleotides. For example, a deoxythymidine (T) or a deoxycytidine (C) nucleotide can be found at cDNA position 196 of the glycoprotein IIIa (GP3A) gene that expresses the HPA-1 (Newman P. J. et al., J Clin Invest 1989; 83:1778). The allele containing the deoxthymidine nucleotide expresses the HPA-1a antigen and the allele containing the deoxycytidine nucleotide expresses the HPA-1b antigen. We refer to the T/C nucleotide difference between the two alleles as a single nucleotide polymorphism (SNP).

Blood group alleles for a given blood group system represent genetic variations of the same gene. For example, the ABO blood group system has 3 common alleles, that confer 6 genotypes within this blood group system. Moreover, many alleles within a blood group system express different blood group ‘antigens’, that is to say, dependent on the allelic genotype the corresponding antigenic phenotype is accordingly expressed. Alleles differ in their nucleotide sequence, and the difference between one allele and another, usually within a single blood group system, may be one single nucleotide variation. Therefore, two alleles can differ by one nucleotide, i.e. a SNP and represent a co-dominant bi-allelic system. Alternatively, alleles can differ by a few to several dispersed nucleotides, or by a stretch of nucleotides, any one of which can be used to identify the alleles. Regardless of whether the variations in the nucleotide are due to single or multiple nucleotide differences, the phenotype associated with a specific genotype (the specific nucleotide sequence) will result in the expression of a specific blood group or platelet antigen on the red cell or platelet surface, respectively.

Normally, all blood donations are blood grouped for ABO and RhD. However, sometimes a previously transfused recipient will require more blood that is antigen-matched with one of their own antigens because they have made antibodies to a different blood group or platelet antigen. The gold standard in the industry is to ‘phenotype’ blood for the presence of specific blood group and platelet antigens using government regulated antisera (antibodies) performed by single-test methods or by an automated platform, which is a cost ineffective method for a blood collection facility that routinely performs tests on a high volume basis.

Blood group phenotypes are presently determined using commercially available government-regulated serological reagents and human red cells. These known tests rely on the principle of antibody binding and red cell agglutination to identify clinically important blood group phenotypes. The presently known tests were originally devised some 60 years ago and today require the use of government regulated (for example, Health Canada) approved serological reagents. Some of the tests being employed today have been automated (for example, ABO and Rh typing) while some have been semi-automated (for example, RhC/c and RhE/e). However, many of the presently used tests are performed manually by highly-trained laboratory technologists and are done on a test-by-test basis. In other words, a technologist must perform four separate tests to determine, for example, the Fya, Fyb, Jka and Jkb phenotype of a single blood donation. Essentially, the current tests which employ government-approved reagents in a manual, single-test driven method are a very cost ineffective method for a blood collection facility that is often required to perform such tests on a high volume basis.

In an effort to reduce costs, a blood collection facility will often use non-regulated antisera to ‘screen’ blood donations for important blood group phenotypes and then confirm the phenotype with the regulated antisera. However, since much of the blood is sent to hospitals within 24-48 hours after collection, manual blood group phenotyping cannot meet the short turn-around time required to provide the end user with the information required before blood must be shipped. Therefore, hospital blood banks must perform their own tests on the blood that they have in their inventories. It would be advantageous to provide a cost effective blood screening method that would provide quick and reliable results relating to the clinically important blood group phenotypes.

The prior art uses two basic techniques to detect SNPs; polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) (Chaudhuri A., et al. 1995; 85:615), and sequence specific primer (SSP)-PCR (McFarland J. G. et al., Blood 1991; 78:2276). For PCR-RFLP analysis, restriction enzymes are used to digest PCR amplified genomic DNA fragments. In brief, DNA is extracted from nucleated blood cells manually for each blood sample to be analyzed. The PCR is set up manually; a separate PCR is performed on each sample for each SNP of interest. The PCR amplified fragments are digested with a specific restriction enzyme and the digested products are separated on a gel. The pattern of digested DNA fragments viewed from the gel predicts the presence or absence of either nucleotide of a SNP of interest. In SSP-PCR, two PCRs are set up in separate tubes for each SNP of interest. One tube contains a universal primer and a primer with a sequence that is specific to detect one nucleotide of a SNP. The other tube contains the same universal primer and a primer specific for the other nucleotide of a SNP. Prior art has used two pair or three pair PCR to analyze a nucleotide for a given SNP, with at least one pair acting as an internal control to ensure DNA is available for PCR amplification. The prior art does not provide the use of multiple DNA sequences as primer pairs that work simultaneously on a single sample. Moreover, the prior art does not employ novel DNA sequences to detect blood group SNPs in an automated high-throughput fashion.

St-Louis M., et al. (Transfusion 2003; 43:11126-32) have used allele-specific PCR-ELISA to detect blood group SNPs, wherein some of the PCR primers were publicly known and all primers were labeled with digoxigenin; SNPs were detected by oligonucleotide hybridization using solid-phase microplate wells coated with individual blood group-specific complementary oligonucleotides. An abstract by Buffleir E. et al. (Transfusion 2003; 43:92 A) outlines a combined HPA-1 and HPA-5 genotyping method that uses biotin labeled PCR-amplified targets and allele specific oligonucleotide probes arrayed on the bottom of 96 well microplates. Specific hybridization is detected with the use of an enzyme conjugate which produces a specific colourimetric signal. An array of several oligonucleotides reportedly can be used to detect HPA SNPs. The publications, cited above, do not use multiplex PCR primers, nor do they use extension probes, and rely on a less sensitive and more error-prone allele-specific hybridization to detect the SNPs. There are a few other publications that refer to the multiplex PCR amplification of the RHD gene alone, or together with sex determination, or with internal control primers designed to confirm the presence of DNA in various blood group PCR applications. U.S. Pat. No. 5,723,293 describes a diagnostic method and kit for determining Rh blood group genotypes, wherein there is provided a method for directly determining D and associated CcEe genotypes using restriction fragment length polymorphisms (RFLPs) for diagnosis. U.S. Pat. No. 5,804,379 describes a diagnostic method and kit for determining Kell blood group genotype, wherein there is provided a method for determining the K1/K2 genotype using RFLPs for diagnosis. U.S. Pat. No. 5,780,229 provides polynucleotides for determining the Pen polymorphism of human platelet membrane glycoprotein IIIa, and generally describes diagnostic and therapeutic uses relating to the “Pen” human platelet polymorphism (HPA-4) and differs from the teachings of the present invention. United States patent application 20020098528 describes methods and apparatus for blood typing with optical bio-disc, and essentially describes a method for determining the ABO blood cell type of an individual with optical bio-discs and a disc-reading apparatus.

In the SSP-PCR application by St. Louis et al. (Transfusion 2003; 43:1126), two PCR primer pairs are set up, each in a separate well, to detect the nucleotides of a SNP of interest. For example, one primer pair containing a universal primer and a sequence specific primer is set up in a tube to detect a nucleotide of a SNP. Another primer pair containing the same universal and another sequence specific primer is set up in another tube to detect the alternate nucleotide for the same SNP. In addition, each tube includes a primer pair that detects a universal sequence contained in all human DNA. Contained in the PCR tube is digoxigenin-dUTP that is incorporated into the amplified DNA fragment if the sequence specific primer detects the appropriate nucleotide of an SNP. For the detection phase, one of each primer pair contains the chemical tag biotin, which is used to capture the DNA amplified fragment in sets of microtitre wells containing streptavidin. An optical colorimetric assay is used to detect the presence of digoxigenin-dUTP in each of the wells; anti-digoxigenin peroxidase conjugated antibody detects the presence of digoxigenin dUTP and the peroxidase can convert a substrate added to the well into a colored end product. Therefore, the presence of a nucleotide of a SNP is detected by the presence of a color in the microtitre well. Such assays are routinely designed in a 96-well microtitre plate format to facilitate semi-automation. The calorimetric results are evaluated by the operator to determine the presence or absence of the nucleotides for a SNP. The deficiencies of these test systems are the use of a single PCR reaction for each nucleotide of a given nucleotide of each SNP, and the pooling of samples prior to the detection phase and manual post-analyte data analysis.

No prior art has used a multiple, or 12, primer pair multiplexed PCR that successfully works in a single tube, nor has prior art employed novel DNA sequences as probes to detect both nucleotides of a plurality of blood group and HPA genotypes simultaneously, such as the detection of all 12 blood group and HPA SNPs in these mixtures using an automated high-throughput platform.

Accordingly, there is a need for a high-throughput automated multiple blood-group associated SNP analysis of genomic DNA that is capable of rapidly and accurately determining the genotypes and associated phenotypes of a plurality of blood group systems in a single test sample.

The present invention provides a method of detecting the presence or absence of nucleotides relating to various SNPs for the determination of a specific genotype and accordingly the inferred phenotype. More specifically, the present invention allows for the detection of the presence or absence of two nucleotides of a plurality of different SNPs, and more preferably of the 12 SNPs in a preferred embodiment of the present invention.

The present invention accordingly provides an automated, or robotic, high-throughput ‘screening’ tool for blood group and platelet antigens by evaluating the alleles of the genes that express these antigens on red cells and platelets, respectively. This is done by identifying the unique nucleotides associated with the specific alleles that occupy the gene locus using a testing platform, which requires novel and specific compounds that we designed. Our robotic high-throughput platform provides important blood group and HPA genotype information within 24 hours from the start of the test. We identified the alleles of blood group antigens for; RhD, RhC, Rhc, RhE, Rhe, S, s, Duffy (Fy)a, Fyb, K, k, Kpa, Kpb, Diego (Di)a, Dib, Kidd (Jk)a, Jkb, and the platelet antigens, Human Platelet Antigen (HPA)-1a and HPA-1b, representing, but not limited to 19 of the most clinically important antigens in red cell and platelet transfusion. Additional genotyping tests for other clinically important blood group and platelet antigens may be developed, and are encompassed in the teachings of the present invention. When performed on all blood donations for all clinically important blood group and platelet antigens, our invention will provide a comprehensive database to select and confirm the antigens when required using government regulated antisera. The use of this platform as a screening tool will lessen the number of costly government regulated tests to be done by the collection facility and end user (the hospital blood bank), and meet the demand of antigen-matched blood for specific transfusion recipients.

The invention discloses a method for DNA-based blood group genotyping for clinically important blood group and platelet antigens. The technology uses an ultra high-throughput multiplex PCR design to detect specific SNPs that represent clinically important blood group antigens: RhD, RhC, Rhc, RhE, Rhe, S, s, Duffy (Fy)a, Fyb, K, k, Kpa, Kpb, Diego (Di)a, Dib, Kidd (Jk)a, Jkb, and the platelet antigens, Human Platelet Antigen (HPA)-1a and HPA-1b. It should be noted however that the present invention is not limited to the detection of SNPs for only the SNPs listed, but additionally comprises the detection of SNPs for all blood group and platelet antigens. The invention discloses novel DNA sequences of PCR primers that are specifically designed to avoid inter-primer pair cross-reactions and post-PCR probes that make multiple analyses possible. The invention represents a novel approach to screening multiple blood group and HPA genotypes at once and addresses a clear need in the art for novel, rapid, cost-effective and reliable genotyping. This additionally replaces the use of expensive and difficult-to-obtain serological reagents, which can be reserved for use to confirm only the donors identified by the screening process.

More specifically, the present invention analyzes the HPA-1 GP3A mutation incorporated into our SNP assay, and the other blood group antigen SNPs in a method according to the present invention.

The invention addresses the need for an automated, accurate, rapid and cost-effective approach to the identification of multiple blood group antigens. The multiplex SNP assay design and automated genotyping platform allows one trained research technician to identify a plurality of blood group alleles, and more specifically, 19 blood group alleles, overnight on 372 to 2232 individual blood samples. In one application of the present invention, the multiplex PCR and SNP detection platform analyzed the nucleotides of 12 SNPs overnight on 372 individual blood samples. The cost using current standard blood group serology for 372 samples is estimated at CDN$99,500, which reflects a reagent cost of CDN$54,000 (excluding new capital equipment investments) and an operator cost of CDN$45,500 to analyse each of the antigens by Gel Card technology (n=5), immediate spin tube test (n=2), indirect antiglobulin tube test (n=8), and platelet GTI® test (n=1). Approximate 10 to 15 fold cost savings are obtained in the simultaneous DNA-based determination of these blood group alleles. It should be noted that the present invention is not limited to the detection of only 12 SNPs, and may be optimally used for the detection a plurality of SNPs for potentially all blood group and platelet alleles. Accordingly, the products, methods, platform and teachings of the present invention can detect all blood group and HPA SNP variations on a great number of samples, such as 744 samples overnight, as further described below.

The present invention overcomes the deficiencies of the prior art because the entire test, i.e. all steps of the method of the present invention, from PCR to computation analyses can be automated and multiplexed so that the nucleotides of a plurality of SNPs, and more preferably, the 12 SNPs of the present invention, can be identified simultaneously. This automated multiplex high throughput analysis can meet the demand of testing hundreds of blood samples, and the turn-around time of less than 24 hours, to provide valuable information to a blood collection facility before blood is shipped to the end user. This platform has the advantage over existing technology in that it reduces operator handling error. In addition, there are significant cost reductions compared with the current government-regulated serological analysis. It should be noted that present prior art technologies relating to PCR-RFLP and SSP-PCR for blood group and platelet antigens are not routinely used since they are no more cost efficient than serology. The present invention overcomes the deficiencies of the prior art and fulfils an important need in the present art for the automated, accurate, rapid and cost-effective identification of multiple blood group and HPA SNPs.

The invention provides the opportunity to screen all blood donors to obtain a daily or ‘live’ repository of the genotypes or combinations of genotypes currently available for specific transfusion needs. Accordingly, the present invention fulfills a need relating to the collection and antigen screening of blood and blood products.

For convenience, some terms employed in the present specification are noted below. Unless defined otherwise, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the present art.

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