Molecular testing for blood group antigens: tools and applications Geoff Daniels International Blood Group Reference Laboratory NHS Blood and Transplant, UK Blood and Transplant
347 antigens 308 belong to 36 systems 41 genes
Molecular bases for almost all blood group polymorphisms and variants are known It is possible to predict blood group phenotype from DNA Why would we want to do this? No suitable red cell sample More or better information than serological testing More efficient or more cost-effective
Applications of blood grouping from DNA Fetal genotyping for prediction of risk of HDFN
Applications of blood grouping from DNA Transfused patients (transfusion-dependent patients) Helps in providing matched red cells
Applications of blood grouping from DNA Red cells with positive DAT Patients with AIHA: helps to identify underlying alloantibodies
Applications of blood grouping from DNA Defining RhD variants Defining RhCcEe variants SCD patients
Applications of blood grouping from DNA Screening serologically D donors for RHD For detecting very weak variants of D e.g. DEL Only done in a few parts of Europe
Applications of blood grouping from DNA Determining RHD zygosity fathers of fetuses at risk of HDFN
Applications of blood grouping from DNA Assisting serological reference laboratory in solving difficult problems
Applications of blood grouping from DNA Testing large numbers of donors & patients for multiple clinically significant polymorphisms
Some blood donors currently screened for multiple blood groups by serology Being replaced by DNA testing Why? DNA tests: more suitable for high-throughput more accurate indentifies more phenotypes cheaper Easier to find donor blood for transfusiondependent patients & patients with antibodies
Transfusion, ahead of print Normal matched: ABO & D Extended matched: ABO & D + c, C, E, K, Fy a, Jk a, S Extended matching for reduced alloimmunisation risk by 64% in surgical patients
Fetal genotyping for RHD D-negative pregnant woman with anti-d Valuable to know D type of fetus
Rh haplotypes RHD RHCE D+ D+ D
RhD genotyping tests detect presence or absence of RHD Complexities Rh variants Test for more than one region of RHD
Cell-free fetal DNA in maternal plasma 10 20 weeks: 10 15% cell-free DNA = fetal Range: 3 30% >21 weeks: increases by ~1% per week Excellent source of fetal DNA for fetal RHD testing in D-negative pregnant women
Fetal DNA in maternal plasma DNA isolated from maternal plasma 85-90% maternal DNA No RHD (mother D-neg) 10-15% fetal DNA (fetal fraction) RHD present if fetus D-pos No RHD if fetus D-neg
IBGRL Fetal typing RHD exons 4, 5, 7, 10
Real-time quantitative PCR Taqman chemistry Measures quantity of product at every cycle
Real time PCR with Taqman probe 3 5 Forward primer 5 5 Reverse primer 5 3
Real time PCR with Taqman probe 3 5 quencher reporter R 5 probe 3 5 5 5 3
Real time PCR with Taqman probe 3 5 5 quencher reporter R 5 probe 3 taq 5 3 5
Real time PCR with Taqman probe R 3 5 5 5 5 3
Advantages of real-time quantitative PCR Highly sensitive Quantitative ensures testing fetal, not maternal, DNA Closed system reducing risk of contamination
Fetal RHD genotyping by real-time PCR using maternal plasma as a source of fetal DNA RHD positive fetus (blue / green) RHD negative fetus (red / yellow) RHD exon 4
IBGRL, Bristol Blood and Transplant Service provided for D women with anti-d (>4 IU/ml) or with history of fetal/neonatal haemolysis Standard of care in England WHO collaborating centre WHO collaborating centre
Other tests on fetal DNA in maternal plasma K KEL T698 exon 6 Rh c RHCE C307 exon 2 Rh E RHCE C676 exon 5 Rh C RHCE insert intron 2 RQ-PCR with an allele-specific primer
Routine antenatal anti-d prophylaxis 1 or 2 doses of anti-d Ig given at around 28-34 weeks To prevent antenatal immunisation
Routine antenatal anti-d prophylaxis Anti-D Ig must be given to all D-negative pregnant women 40% will have a D-negative fetus (Caucasian population) Beneficial to test all D-negative pregnant women for fetal D type
Robotic DNA isolation Real-time PCR detecting RHD exons 5 & 7
A very high level of sensitivity & specificity from 11 weeks gestation If our results had been applied as a guide to treatment only 2% women would have received anti-d Ig unnecessarily compared to 38% without genotyping
Fetal RhD testing in all D pregnant women Provided as service in: Denmark The Netherlands Finland Some parts of England
Advantages Cost effective? Anti-D Ig in short supply Eliminates unnecessary treatment of pregnant women with blood products
Indian population Assuming 5% of pregnant women D Assuming antenatal anti-d prophylaxis provided 5% pregnant women require test 22% saved from unnecessary treatment
What technology is being used for blood grouping from DNA derived from peripheral blood from donors and patients?
Methods for blood group-associated SNP typing Traditional methods Restriction analysis of PCR products PCR with allele-specific primers Usually not high-throughput Often require gel electrophoresis Direct (Sanger) sequencing Low-throughput & expensive
Methods for blood group-associated SNP typing Direct sequencing of PCR products
Allelic discrimination by quantitative PCR with Taqman technology
Allelic discrimination by quantitative PCR with Taqman technology Pair of fluorescent probes each specific for an allele each carrying a different reporter dye Only fluoresce under laser when released from DNA during PCR Relative quantities of PCR product compared by computer
Allelic discrimination Fy b /Fy b Fy a /Fy b Fy a /Fy a Fy a -specific probe FAM reporter Fy b -specific probe VIC reporter
Allelic discrimination Results called automatically 384-well plates Medium throughput ~200 samples per day for all clinically significant blood groups except ABO & RhD Higher throughput automated adaptations with reactions in very small holes in a plate developed commercially QuantStudio
RBC-FluoGene Primers & Taqman probes dried in a well Compares fluorescence for each allele tested with internal control (HGH)
DNA array analysis Multiplex PCR on DNA + dye hybridised Microarray of oligonucleotides Array of oligonucleotides on coloured beads
Microarray analysis Jk(a b+) patient A A A A C C C C T T T T Jk a Jk b
Microarray analysis Jk(a b+) patient C C C C Jk a Jk b
Microarray analysis Jk(a+b+) patient A G A G C C C C T T T T Jk a Jk b
Microarray analysis Jk(a b+) patient Jk a Jk b
Microarray analysis Scan Analyse Microarray of oligonucleotides
BLOODchip Developed by Bloodgen, produced by Progenika First comprehensive blood group genotyping system
BLOODchip ABO O, A, B, AB, weak A 58 Rh D+, D, DEL, weak & partial D 115 C, c, E, e, C w, C x, VS, r s 9 Kell K, k, Kp a, Kp b, Kp c, Js a, Js b, Kmod 9 Kidd Jk a, Jk b, Jk-null 4 Duffy Fy a, Fy b, Fy-null 4 MNS M, N, S, s, Mi a (GP.Mur) 8 Diego Di a, Di b 2 Dombrock Do a, Do b 2 Colton Co a, Co b 2 + 24 PLT genotypes
BLOODchip Accuracy Comparing results with serologically defined phenotypes, then checking non-concordancies by repeats and DNA sequencing: BLOODchip: 99.8% (excluding ABO) Serology: 95.5% Not high-throughput
Collaboration between AXO Science & EFS
Microspheres of 100 different colours coated with different oligonuleotide probes Target DNA from PCR labelled with fluorescent dye and incubated with microspheres Flow cytometry: Red laser identifies the microspheres Green laser identifies whether target DNA has annealed
Luminex Rh C c E e V VS hr s hr B + other variants Kell K k Duffy Fy a Fy b Fy-null Kidd Jk a Jk b Jk-null MNS M N S s U Mi a Dombrock Do a Do b Colton Co a Co b Cartwright Yt a Yt b Lutheran Lu a Lu b Not ABO, not RhD
TM BEADCHIP
BEADCHIP TM Application of microbeads of different colours carrying different oligonucleotide probes immobilised on a matrix Rh C c E e VS V K k Kp a Kp b Js a Js b Jk a Jk b Fy a Fy b Fy-null M N S s U Di a, Di b Do a, Do b, Hy, Jo a Co a, Co b Lu a, Lu b LW a, LW b Sc1, Sc2 HbS Not ABO, not RhD
MALDI TOF MS Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry MassARRAY Technology
MALDI TOF MS nanosecond laser pulse ionises biomolecules sample electric field Detector Molecules separated according to mass:charge Reach detector at different times DNA fragments differing by single nt distinguished
Transfusion Medicine Reviews 2013;27:2-9.
MassARRAY Technology 6 modules RH x 2 KEL-JK-FY MNSs Kell + others HPA-HNA D (D-null), C, c, E, e, C w Selection of D variants MNS, Mt a, Vw, Hut Kell, Lu, Di, Co, Knops, Do, LW, Sc, Cromer, In Suitable for automation & high-throughput testing
What s around the corner? Next generation sequencing?
Next generation sequencing or massively parallel sequencing One sequencing procedure and one operator can generate as much data as several hundred Sanger-type capillary sequencers Next generation sequencing Sanger sequencing
Next generation sequencing Capacity to sequence the whole genome of 10 people in one run Capacity to sequence limited regions of genome of many individuals in one run Potential for testing all required blood group polymorphisms for many donors, in one sequencing run Electronic matching patient and donors
Next generation sequencing can be applied to fetal genotyping Mother Father Fetus Maternal blood Now possible to determine sequence of fetal genome from 5 ml of maternal blood Need to prepare for the ethical, legal, and practical implications
Next generation sequencing Potential for all genetic diagnostics, including blood grouping Proof of principle Not ready yet for blood grouping Too expensive Takes too long
If we were able to test all donors and patients for all clinically important blood groups, rapidly and at low cost. Electronic matching will be possible Reduce alloimmunisation HTRs serological investigations This will be possible in the future But will it be logistically possible? And will the benefits justify the cost?
ABO? Not currently feasible, & probably not in foreseeable future ABO is simple serologically and highly reliable ABO is complex genetically 100% accuracy cannot be guaranteed For ABO only 100% accuracy is acceptable
What will be the application of blood group serology in 10 years time? ABO RhD (in patients) antibody screening & ID and little else
Bristol