D J M ram 14 rog 20 E P CM
The Journal of Molecular Diagnostics, Vol. 16, No. 3, May 2014
jmd.amjpathol.org
Flexible Automated Platform for Blood Group Genotyping on DNA Microarrays Sandra Paris,* Dominique Rigal,* Valérie Barlet,* Martine Verdier,* Nicole Coudurier,y Pascal Bailly,z and Jean-Charles Brès*x From the Établissement Français du Sang Rhône Alpes,* Lyon; the Établissement Français du Sang,y La Plaine Saint Denis; the Établissement Français du Sang Alpes Méditerranée,z Marseille; and the Établissement Français du Sang Pyrénées Méditerranée,x Montpellier, France CME Accreditation Statement: This activity (“JMD 2014 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity (“JMD 2014 CME Program in Molecular Diagnostics”) for a maximum of 48 AMA PRA Category 1 Credit(s). Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.
Accepted for publication February 12, 2014. Address correspondence to Jean-Charles Brès, Ph.D., Etablissement Français du Sang Pyrénées Méditerranée site Pierre Cazal, Laboratoire TransDiag, 392 Ave. du Pr J.-L. Viala - CS 37381, 34184 Montpellier Cedex 4, France. E-mail: jean-charles. [email protected].
The poor suitability of standard hemagglutinationebased assay techniques for large-scale automated screening of red blood cell antigens severely limits the ability of blood banks to supply extensively phenotype-matched blood. With better understanding of the molecular basis of blood antigens, it is now possible to predict blood group phenotype by identifying single-nucleotide polymorphisms in genomic DNA. Development of DNA-typing assays for antigen screening in blood donation qualification laboratories promises to enable blood banks to provide optimally matched donations. We have designed an automated genotyping system using 96-well DNA microarrays for blood donation screening and a first panel of eight single-nucleotide polymorphisms to identify 16 alleles in four blood group systems (KEL, KIDD, DUFFY, and MNS). Our aim was to evaluate this system on 960 blood donor samples with known phenotype. Study data revealed a high concordance rate (99.92%; 95% CI, 99.77%e99.97%) between predicted and serologic phenotypes. These findings demonstrate that our assay using a simple protocol allows accurate, relatively low-cost phenotype prediction at the DNA level. This system could easily be configured with other blood group markers for identification of donors with rare blood types or blood units for IH panels or antigens from other systems. (J Mol Diagn 2014, 16: 335e342; http:// dx.doi.org/10.1016/j.jmoldx.2014.02.001)
The standard method of phenotyping for red blood cell (RBC) antigens is antibody-based agglutination. This serologic approach has two major disadvantages.1e3 The first involves the limited range of antigen testing. In the French Blood Service, the Etablissement Francais du Sang (EFS), blood donation qualification laboratories test all blood donations for ABO, Rhesus (RH1), and KEL (KEL1), but testing for other clinically significant antigens, including FY1, FY2, JK1, JK2, MNS3, and MNS4, is performed on only a fraction of donations (ie, 5% to 10%). The second disadvantage of antibodybased agglutination involves long procedure duration. For these reasons, conventional hemagglutination is ill suited to high-throughput blood group phenotyping. Copyright ª 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jmoldx.2014.02.001
Although blood transfusion is generally safe using serologic methods, alloimmunization remains a dreaded complication4 and may cause problems, ranging from delayed hemolytic transfusion reaction to difficulty in obtaining matched RBCs.5e7 The risk of alloimmunization is greatest when multiple antigen-negative RBC products are requested for patients with alloantibodies or sickle cell disease that requires long-term transfusion.8,9 In such cases, usual practice Supported by the Direction Scientifique and Direction de la Valorisation des Innovations of the Etablissement Français du Sang, Paris, France (all authors). Disclosures: None declared.
Paris et al Table 1
Primer Sequences Used for Blood Group Amplification in the Multiplex PCR
Alleley
Amino acid change
Nucleotide change Forward
KEL*01/KEL*02
Met193Thr
698TC
KEL*03/KEL*04
Trp281Arg
961T>C
JK*01/JK*02
Asp280Asn
838G>A
FY*01/FY*02 FY*02M.01(FY*X) FY*02M.02 (FY*Fy)
Gly42Asp 125G>A Arg89Cys 265G>T Gata wt/mt e33T>C
GYPA*01(MNS*01)/* Ser1Leu GYPA*02(MNS*02) GYPB*03(MNS*03) Met29Thr GYPB*04(MNS*04)
59C>T 143T>C
Reverse
50 -Cy3-GGTAAATGGACTTCC TTAAACTTTAACCGA-30 50 -Cy3-ACTCTTCCTTGTCAA TCTCCATCACTT-30 0 5 -Cy3-CTCAGTCTTTCAGCC CCATTTGA-30 50 -Cy3-ATGATTCCTTCCCAG ATGGAGACTATG-30 0 5 -Cy3-CCTGATGGCCCTCAT TAGTCCT-30 50 -Cy3-ATGTGAGGGAATTTG TCTTTTGCA-30 0 5 -GATTCCAAAATGATTTTTT TCTTTGCACATGT-30
50 -TGTGTCTTCGCCAGT GCATC-30 50 -CATGCCCACAGTCTT CTGGC-30 0 5 -AGCGCCATGAACATT CCTCC-30 50 -Cy3-GGGCAGAGCTG CCAGC-30 0 5 -CAGACAGTTCCCCAT GGCAC-30 50 -TTCAGAGGCAAGAAT TCCTCC-30 0 5 -Cy3-AGTGAAACGAT GGACAAGTTGTCC-30
Amplicon size (bp)
Concentration (mmol/L)
152
0.09
149
0.09
98
0.09
186
0.13
113
0.13
363
0.25
103
0.38
PCR primers were designed to flank the SNP site of each blood group. The use of 50 -cyanine 3 (Cy3)elabeled PCR primers to anneal close to the nucleotide change site on either the sense or antisense DNA strand allowed fluorescence detection of amplified blood groupecorresponding alleles after hybridization on DNA microarrays. Differing strand lengths specific to the amplified blood group system made it possible to distinguish the multiplex PCR amplicons by capillary electrophoresis. In the FY blood group system, co-amplification in the same fragment using one pair of 50 -Cy3econjugated primers were performed because of proximity of the specific polymorphisms position for the FY*01, FY*02, and FY*02M.01 (FY*X) alleles.29,30 y Alleles are represented using the International Society of Blood Transfusion nomenclature with corresponding transfusion medicineefriendly terms in brackets.
consists of performing extensive RBC phenotyping, but producing a sufficient quantity of extensively typed blood units will never be feasible using conventional serologic donor screening methods. Overcoming the limitations of serologic testing for routine screening will require a technological step change for RBC donors.2,10 DNA-based methods offer the most realistic option. Indeed, knowledge of the molecular basis of blood group expression has enabled prediction of phenotype based on identification of single-nucleotide polymorphisms (SNPs) in genomic DNA. This has generated great interest in developing rapid screening methods for large-scale detection of SNPs. This technology would have a significant effect on Table 2
pretransfusion testing by allowing blood banks not only to provide optimally matched donations to patients but also to improve management of extensively typed blood unit stocks. Many low-throughput methods allow point-by-point identification of SNPs. These include allele-specific PCR,11,12 PCR restriction fragment length polymorphism, real-time PCR,13,14 and primer extensionebased method.15 However, these methods are unsuitable for large-scale genotyping and thus for routine blood donor screening. Large-scale blood group genotyping is possible using several assays in various formats, including BeadChip HEA16e25 (BioArray SolutionseImmucor, Norcross, GA), BLOODchip21e24/IDCore/IDCoreþ (Progenika Biopharma/
Probe Sequences Used for Multiplex Blood Group Genotyping
Allele
Probe sequences
Temperature ( C)
KEL*01 KEL*02 KEL*03 KEL*04 JK*01 JK*02 FY*01 FY*02 C-wt FY*02M.01 (FY*X) FY*wt FY*02M.02 (FY*Fy) GYPA*01 (MNS*01) GYPA*02 (MNS*02) GYPB*03 (MNS*03) GYPB*04 (MNS*04)
50 -GTCTCAGCATTCGGTTAeC6NH2-30 50 -TCTCAGCGTTCGGTTAeC6NH2-30 50 -GGAACAGCCATGAAGTGAeC6NH2-30 50 -GAACAGCCGTGAAGTGAeC6NH2-30 50 -AGTAGATGTCCTCAAATGGeC6NH2-30 50 -AGTAGATGTTCTCAAATGGeC6NH2-30 50 -AGGTTGGCACCATAGTCTCeC6NH2-30 50 -GGTTGGCATCATAGTCTeC6NH2-30 50 -ACCTCTCTTCCGCTGGCAGeC6NH2-30 50 -ACCTCTCTTCTGCTGGCAGeC6NH2-30 50 -GCTTCCAAGATAAGAGCCAeC6NH2-30 50 -GCTTCCAAGGTAAGAGCCAeC6NH2-30 50 -AGTGGTACTTGATGCTGATATeC6NH2-30 50 -AGTGGTACTTAATGCTGATATeC6NH2-30 50 -ATAGGAGAAATGGGACAACTTeC6NH2-30 50 -TATAGGAGAAACGGGACAACTTGeC6NH2-30
47.8 48.7 51.2 51.2 47.8 45.9 52.9 47.2 58.2 55.5 51.1 53.6 50.4 48.0 50.4 54.0
Bold and underlined nucleotides represent the SNPs in the allele-specific probes; they were placed in central position to enhance allele discrimination.33,34
336
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Robotic Platform Blood Group Genotyping Grifols, Vizcaya, Spain), and LIFECODES RBC/RBC-R26 (Immucor). These commercially available assays allow simultaneous detection of a large number of SNPs in a single reaction but are not designed for fully automated use. Another major obstacle to use of these assays for routine purposes is high cost. Several years ago, the EFS launched a project to develop molecular-based blood groupetyping techniques for use in its immunohematology qualification laboratories. In addition to training employees in molecular biology procedures, the goal of this project was to develop a low-cost, DNA-based method capable of supplying extensively typed blood components on a large-scale basis. After preliminary work revealed the feasibility of genotyping human platelet antigens by combining a single PCR and DNA microarray hybridization,27 this technique was first adopted for multiplex determination of human erythroid antigens of the most clinically significant human erythroid antigens. Extended phenotyping was based on a panel of antithetical blood group antigens that resulted from one SNP for each couple, which was easy to implement for molecular genotyping. The purpose of this study was to set up and validate a flexible robotic platform using a 96-well DNA microarray for multiplex blood group genotyping. With workstations before and after PCR processing, this automated system using a simple protocol for extended genotyping is intended to meet the requirements of blood transfusion laboratories in terms of throughput and low cost of testing. A first SNP module was designed to allow simultaneous determination of KEL (KEL*01/KEL*02, KEL*03/KEL*04), KIDD (JK*01/ JK*02), DUFFY (FY*01/FY*02, FY*02M.01 or FY*X, and FY*02M.02 or FY*Fy), and MNS (GYPA*01/GYPA*02 or MNS*01/MNS*02, GYPB*03/GYPB*04 or MNS*03/ MNS*04) as a basis for identification of the most clinically significant blood group antigens after ABO and Rhesus. The results of a pilot study on 960 blood donor samples with known extended blood group phenotype are presented.
Materials and Methods Blood Sample Processing, Phenotyping, and DNA Extraction A total of 1132 EDTA-anticoagulated blood samples were collected by the EFS in Rhône Alpes in the center east area of France. Random donors, mostly white, were extensively phenotyped using standard serologic hemagglutination techniques in the Blood Donation Qualification Laboratory (Metz-Tessy, France). One hundred seventy-two samples were used to determine scoring criteria for predicting phenotype. The remaining 960 samples were used for validation of the 96-well DNA microarray system. Genomic DNA extraction from whole blood samples (200 mL) was performed using a MagNA Pure 96 system (Roche Diagnostics, Rotkreuz, Switzerland) and Viral NA Small Volume Kit (Roche Diagnostics) in a 96-well microarray plate according to the manufacturer instructions. After extraction, DNA was eluted in 50 mL of buffer solution and quantified using a NanoVue spectrophotometer (GE Healthcare, Little Chalfont, UK).
Design of PCR Primers and DNA Probes Synthetic oligonucleotides, primers, and probes were obtained from Eurogentec (Seriang, Belgium). Probes and primers were designed using Annhyb sequence manager software (http://bioinformatics.org/annhyb; version 4.946, 2012, last accessed June 18, 2013). Testing for potential primer-dimer conflicts and second hairpin structures was performed using OligoAnalyzer 3.1 (Integrated DNA Technologies, Coralville, IA).28 Each PCR primer set was designed to flank each blood group SNP, except for FY*01/ FY*02 and FY*02M.01 (FY*X) alleles, for which a single primer set allows the co-amplification of the two SNPs (125G>A and 265G>T) in the same fragment. The forward
Figure 1 Schematic representation of the workflow process for automated DNA-based blood group genotyping. Step 1: Collection of whole blood samples from donors for processing. Step 2: Genomic DNA extraction using a workstation that combined an automated liquid handling MagNA Starlet system (Hamilton Robotics) with the MagNA Pure 96 system (Roche Diagnostics). The MagNA Starlet system performs all pre-PCR processing, including sample distribution into processing plate and preparation of the PCR plate after nucleic acid extraction on the MagNA Pure system. Step 3: Multiplex PCR amplification on a Biometra thermal cycler. Inset: An electropherogram obtained after development of the multiplex PCR protocol. Step 4: Post-PCR processing performed on the Hamilton Starlet system (Hamilton Robotics). Hybridization of amplified products on the 96-well DNA microarrays is followed by washing. Step 5: Scanning of DNA microarrays on a fluorescence scanner LS200 (TECAN) to detect labeled multiplex PCR products. Inset: An image of a well obtained after array scanning. Step 6: After analysis of images using GenePix Pro software version 6.0 (Molecular Devices), genotypes assignment to each sample by comparing the calculated ratio for each SNP to the defined ratio limits. Phenotypes are predicted from genotypes.
The Journal of Molecular Diagnostics
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Paris et al Table 3
Ratio Limits Used for Blood Group Genotype Determination Genotype
Probe set
*A/*A
*A/*B
KEL*01/KEL*02 KEL*03/KEL*04 JK*01/JK*02 FY*01/FY*02 FY*Cwt/FY*02M.01 (FY*X ) FY*wt/FY*02M.02 (FY*Fy) GYPA*01 (MNS*1)/GYPA*02 (MNS*2) GYPB*03 (MNS*3)/GYPB*04 (MNS*4)
r > 0.59 Undeterminedy r > 0.33 r > 0.52 r > 0.33 r > 0.21 r > 0.41 r > 0.21
0.11 0.07 0.11 0.19 0.13 0.13 0.21 0.23
*B/*B < < < < < < <
The Journal of Molecular Diagnostics, Vol. 16, No. 3, May 2014
jmd.amjpathol.org
Flexible Automated Platform for Blood Group Genotyping on DNA Microarrays Sandra Paris,* Dominique Rigal,* Valérie Barlet,* Martine Verdier,* Nicole Coudurier,y Pascal Bailly,z and Jean-Charles Brès*x From the Établissement Français du Sang Rhône Alpes,* Lyon; the Établissement Français du Sang,y La Plaine Saint Denis; the Établissement Français du Sang Alpes Méditerranée,z Marseille; and the Établissement Français du Sang Pyrénées Méditerranée,x Montpellier, France CME Accreditation Statement: This activity (“JMD 2014 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity (“JMD 2014 CME Program in Molecular Diagnostics”) for a maximum of 48 AMA PRA Category 1 Credit(s). Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.
Accepted for publication February 12, 2014. Address correspondence to Jean-Charles Brès, Ph.D., Etablissement Français du Sang Pyrénées Méditerranée site Pierre Cazal, Laboratoire TransDiag, 392 Ave. du Pr J.-L. Viala - CS 37381, 34184 Montpellier Cedex 4, France. E-mail: jean-charles. [email protected].
The poor suitability of standard hemagglutinationebased assay techniques for large-scale automated screening of red blood cell antigens severely limits the ability of blood banks to supply extensively phenotype-matched blood. With better understanding of the molecular basis of blood antigens, it is now possible to predict blood group phenotype by identifying single-nucleotide polymorphisms in genomic DNA. Development of DNA-typing assays for antigen screening in blood donation qualification laboratories promises to enable blood banks to provide optimally matched donations. We have designed an automated genotyping system using 96-well DNA microarrays for blood donation screening and a first panel of eight single-nucleotide polymorphisms to identify 16 alleles in four blood group systems (KEL, KIDD, DUFFY, and MNS). Our aim was to evaluate this system on 960 blood donor samples with known phenotype. Study data revealed a high concordance rate (99.92%; 95% CI, 99.77%e99.97%) between predicted and serologic phenotypes. These findings demonstrate that our assay using a simple protocol allows accurate, relatively low-cost phenotype prediction at the DNA level. This system could easily be configured with other blood group markers for identification of donors with rare blood types or blood units for IH panels or antigens from other systems. (J Mol Diagn 2014, 16: 335e342; http:// dx.doi.org/10.1016/j.jmoldx.2014.02.001)
The standard method of phenotyping for red blood cell (RBC) antigens is antibody-based agglutination. This serologic approach has two major disadvantages.1e3 The first involves the limited range of antigen testing. In the French Blood Service, the Etablissement Francais du Sang (EFS), blood donation qualification laboratories test all blood donations for ABO, Rhesus (RH1), and KEL (KEL1), but testing for other clinically significant antigens, including FY1, FY2, JK1, JK2, MNS3, and MNS4, is performed on only a fraction of donations (ie, 5% to 10%). The second disadvantage of antibodybased agglutination involves long procedure duration. For these reasons, conventional hemagglutination is ill suited to high-throughput blood group phenotyping. Copyright ª 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jmoldx.2014.02.001
Although blood transfusion is generally safe using serologic methods, alloimmunization remains a dreaded complication4 and may cause problems, ranging from delayed hemolytic transfusion reaction to difficulty in obtaining matched RBCs.5e7 The risk of alloimmunization is greatest when multiple antigen-negative RBC products are requested for patients with alloantibodies or sickle cell disease that requires long-term transfusion.8,9 In such cases, usual practice Supported by the Direction Scientifique and Direction de la Valorisation des Innovations of the Etablissement Français du Sang, Paris, France (all authors). Disclosures: None declared.
Paris et al Table 1
Primer Sequences Used for Blood Group Amplification in the Multiplex PCR
Alleley
Amino acid change
Nucleotide change Forward
KEL*01/KEL*02
Met193Thr
698TC
KEL*03/KEL*04
Trp281Arg
961T>C
JK*01/JK*02
Asp280Asn
838G>A
FY*01/FY*02 FY*02M.01(FY*X) FY*02M.02 (FY*Fy)
Gly42Asp 125G>A Arg89Cys 265G>T Gata wt/mt e33T>C
GYPA*01(MNS*01)/* Ser1Leu GYPA*02(MNS*02) GYPB*03(MNS*03) Met29Thr GYPB*04(MNS*04)
59C>T 143T>C
Reverse
50 -Cy3-GGTAAATGGACTTCC TTAAACTTTAACCGA-30 50 -Cy3-ACTCTTCCTTGTCAA TCTCCATCACTT-30 0 5 -Cy3-CTCAGTCTTTCAGCC CCATTTGA-30 50 -Cy3-ATGATTCCTTCCCAG ATGGAGACTATG-30 0 5 -Cy3-CCTGATGGCCCTCAT TAGTCCT-30 50 -Cy3-ATGTGAGGGAATTTG TCTTTTGCA-30 0 5 -GATTCCAAAATGATTTTTT TCTTTGCACATGT-30
50 -TGTGTCTTCGCCAGT GCATC-30 50 -CATGCCCACAGTCTT CTGGC-30 0 5 -AGCGCCATGAACATT CCTCC-30 50 -Cy3-GGGCAGAGCTG CCAGC-30 0 5 -CAGACAGTTCCCCAT GGCAC-30 50 -TTCAGAGGCAAGAAT TCCTCC-30 0 5 -Cy3-AGTGAAACGAT GGACAAGTTGTCC-30
Amplicon size (bp)
Concentration (mmol/L)
152
0.09
149
0.09
98
0.09
186
0.13
113
0.13
363
0.25
103
0.38
PCR primers were designed to flank the SNP site of each blood group. The use of 50 -cyanine 3 (Cy3)elabeled PCR primers to anneal close to the nucleotide change site on either the sense or antisense DNA strand allowed fluorescence detection of amplified blood groupecorresponding alleles after hybridization on DNA microarrays. Differing strand lengths specific to the amplified blood group system made it possible to distinguish the multiplex PCR amplicons by capillary electrophoresis. In the FY blood group system, co-amplification in the same fragment using one pair of 50 -Cy3econjugated primers were performed because of proximity of the specific polymorphisms position for the FY*01, FY*02, and FY*02M.01 (FY*X) alleles.29,30 y Alleles are represented using the International Society of Blood Transfusion nomenclature with corresponding transfusion medicineefriendly terms in brackets.
consists of performing extensive RBC phenotyping, but producing a sufficient quantity of extensively typed blood units will never be feasible using conventional serologic donor screening methods. Overcoming the limitations of serologic testing for routine screening will require a technological step change for RBC donors.2,10 DNA-based methods offer the most realistic option. Indeed, knowledge of the molecular basis of blood group expression has enabled prediction of phenotype based on identification of single-nucleotide polymorphisms (SNPs) in genomic DNA. This has generated great interest in developing rapid screening methods for large-scale detection of SNPs. This technology would have a significant effect on Table 2
pretransfusion testing by allowing blood banks not only to provide optimally matched donations to patients but also to improve management of extensively typed blood unit stocks. Many low-throughput methods allow point-by-point identification of SNPs. These include allele-specific PCR,11,12 PCR restriction fragment length polymorphism, real-time PCR,13,14 and primer extensionebased method.15 However, these methods are unsuitable for large-scale genotyping and thus for routine blood donor screening. Large-scale blood group genotyping is possible using several assays in various formats, including BeadChip HEA16e25 (BioArray SolutionseImmucor, Norcross, GA), BLOODchip21e24/IDCore/IDCoreþ (Progenika Biopharma/
Probe Sequences Used for Multiplex Blood Group Genotyping
Allele
Probe sequences
Temperature ( C)
KEL*01 KEL*02 KEL*03 KEL*04 JK*01 JK*02 FY*01 FY*02 C-wt FY*02M.01 (FY*X) FY*wt FY*02M.02 (FY*Fy) GYPA*01 (MNS*01) GYPA*02 (MNS*02) GYPB*03 (MNS*03) GYPB*04 (MNS*04)
50 -GTCTCAGCATTCGGTTAeC6NH2-30 50 -TCTCAGCGTTCGGTTAeC6NH2-30 50 -GGAACAGCCATGAAGTGAeC6NH2-30 50 -GAACAGCCGTGAAGTGAeC6NH2-30 50 -AGTAGATGTCCTCAAATGGeC6NH2-30 50 -AGTAGATGTTCTCAAATGGeC6NH2-30 50 -AGGTTGGCACCATAGTCTCeC6NH2-30 50 -GGTTGGCATCATAGTCTeC6NH2-30 50 -ACCTCTCTTCCGCTGGCAGeC6NH2-30 50 -ACCTCTCTTCTGCTGGCAGeC6NH2-30 50 -GCTTCCAAGATAAGAGCCAeC6NH2-30 50 -GCTTCCAAGGTAAGAGCCAeC6NH2-30 50 -AGTGGTACTTGATGCTGATATeC6NH2-30 50 -AGTGGTACTTAATGCTGATATeC6NH2-30 50 -ATAGGAGAAATGGGACAACTTeC6NH2-30 50 -TATAGGAGAAACGGGACAACTTGeC6NH2-30
47.8 48.7 51.2 51.2 47.8 45.9 52.9 47.2 58.2 55.5 51.1 53.6 50.4 48.0 50.4 54.0
Bold and underlined nucleotides represent the SNPs in the allele-specific probes; they were placed in central position to enhance allele discrimination.33,34
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The Journal of Molecular Diagnostics
Robotic Platform Blood Group Genotyping Grifols, Vizcaya, Spain), and LIFECODES RBC/RBC-R26 (Immucor). These commercially available assays allow simultaneous detection of a large number of SNPs in a single reaction but are not designed for fully automated use. Another major obstacle to use of these assays for routine purposes is high cost. Several years ago, the EFS launched a project to develop molecular-based blood groupetyping techniques for use in its immunohematology qualification laboratories. In addition to training employees in molecular biology procedures, the goal of this project was to develop a low-cost, DNA-based method capable of supplying extensively typed blood components on a large-scale basis. After preliminary work revealed the feasibility of genotyping human platelet antigens by combining a single PCR and DNA microarray hybridization,27 this technique was first adopted for multiplex determination of human erythroid antigens of the most clinically significant human erythroid antigens. Extended phenotyping was based on a panel of antithetical blood group antigens that resulted from one SNP for each couple, which was easy to implement for molecular genotyping. The purpose of this study was to set up and validate a flexible robotic platform using a 96-well DNA microarray for multiplex blood group genotyping. With workstations before and after PCR processing, this automated system using a simple protocol for extended genotyping is intended to meet the requirements of blood transfusion laboratories in terms of throughput and low cost of testing. A first SNP module was designed to allow simultaneous determination of KEL (KEL*01/KEL*02, KEL*03/KEL*04), KIDD (JK*01/ JK*02), DUFFY (FY*01/FY*02, FY*02M.01 or FY*X, and FY*02M.02 or FY*Fy), and MNS (GYPA*01/GYPA*02 or MNS*01/MNS*02, GYPB*03/GYPB*04 or MNS*03/ MNS*04) as a basis for identification of the most clinically significant blood group antigens after ABO and Rhesus. The results of a pilot study on 960 blood donor samples with known extended blood group phenotype are presented.
Materials and Methods Blood Sample Processing, Phenotyping, and DNA Extraction A total of 1132 EDTA-anticoagulated blood samples were collected by the EFS in Rhône Alpes in the center east area of France. Random donors, mostly white, were extensively phenotyped using standard serologic hemagglutination techniques in the Blood Donation Qualification Laboratory (Metz-Tessy, France). One hundred seventy-two samples were used to determine scoring criteria for predicting phenotype. The remaining 960 samples were used for validation of the 96-well DNA microarray system. Genomic DNA extraction from whole blood samples (200 mL) was performed using a MagNA Pure 96 system (Roche Diagnostics, Rotkreuz, Switzerland) and Viral NA Small Volume Kit (Roche Diagnostics) in a 96-well microarray plate according to the manufacturer instructions. After extraction, DNA was eluted in 50 mL of buffer solution and quantified using a NanoVue spectrophotometer (GE Healthcare, Little Chalfont, UK).
Design of PCR Primers and DNA Probes Synthetic oligonucleotides, primers, and probes were obtained from Eurogentec (Seriang, Belgium). Probes and primers were designed using Annhyb sequence manager software (http://bioinformatics.org/annhyb; version 4.946, 2012, last accessed June 18, 2013). Testing for potential primer-dimer conflicts and second hairpin structures was performed using OligoAnalyzer 3.1 (Integrated DNA Technologies, Coralville, IA).28 Each PCR primer set was designed to flank each blood group SNP, except for FY*01/ FY*02 and FY*02M.01 (FY*X) alleles, for which a single primer set allows the co-amplification of the two SNPs (125G>A and 265G>T) in the same fragment. The forward
Figure 1 Schematic representation of the workflow process for automated DNA-based blood group genotyping. Step 1: Collection of whole blood samples from donors for processing. Step 2: Genomic DNA extraction using a workstation that combined an automated liquid handling MagNA Starlet system (Hamilton Robotics) with the MagNA Pure 96 system (Roche Diagnostics). The MagNA Starlet system performs all pre-PCR processing, including sample distribution into processing plate and preparation of the PCR plate after nucleic acid extraction on the MagNA Pure system. Step 3: Multiplex PCR amplification on a Biometra thermal cycler. Inset: An electropherogram obtained after development of the multiplex PCR protocol. Step 4: Post-PCR processing performed on the Hamilton Starlet system (Hamilton Robotics). Hybridization of amplified products on the 96-well DNA microarrays is followed by washing. Step 5: Scanning of DNA microarrays on a fluorescence scanner LS200 (TECAN) to detect labeled multiplex PCR products. Inset: An image of a well obtained after array scanning. Step 6: After analysis of images using GenePix Pro software version 6.0 (Molecular Devices), genotypes assignment to each sample by comparing the calculated ratio for each SNP to the defined ratio limits. Phenotypes are predicted from genotypes.
The Journal of Molecular Diagnostics
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Paris et al Table 3
Ratio Limits Used for Blood Group Genotype Determination Genotype
Probe set
*A/*A
*A/*B
KEL*01/KEL*02 KEL*03/KEL*04 JK*01/JK*02 FY*01/FY*02 FY*Cwt/FY*02M.01 (FY*X ) FY*wt/FY*02M.02 (FY*Fy) GYPA*01 (MNS*1)/GYPA*02 (MNS*2) GYPB*03 (MNS*3)/GYPB*04 (MNS*4)
r > 0.59 Undeterminedy r > 0.33 r > 0.52 r > 0.33 r > 0.21 r > 0.41 r > 0.21
0.11 0.07 0.11 0.19 0.13 0.13 0.21 0.23
*B/*B < < < < < < <
