Method of nucleic acid typing for selecting registered donors for cross-matching to transfusion recipients
View Patent ↗Disclosed are a method and an algorithm for genetic cross-matching based on the comparison of recipient and donor genotypes—and the underlying combinations of alleles and haplotypes. The method of the invention, rather than focusing on phenotype prediction, instead relies on a comparison of genetic variants identified in the recipient and available donors, whose information preferably will be compiled in a widely available donor registry, to maximize molecular compatibility. The genotypes can be matched based on the weighted clinical significance of a genotypic difference between donor and recipient, such that certain mismatches are more acceptable than others.
1. A method of selecting a transfusion donor by determination of compatibility to a recipient by comparing combinations of polymorphic markers in a set of such markers, of candidate donors and a recipient where said determination is performed following subjecting genomic material/DNA from candidate donors and the recipient to amplification to thereby generate amplified products; wherein the amplified products are further subjected to a hybridization assay or to a capture-mediated elongation assay, or both, wherein an assay signal is generated by the individual hybridization or elongation events, as applicable, by pairs of probes wherein pair members are complementary, in whole or in part, to the subsequences of the amplified products which are the same as (and/or which are complementary to) polymorphic markers in the set;
and an assay signal intensity pattern is generated from hybridization or elongation events such that each marker in the set is represented by a pair of particular assay signal intensities, the method comprising: determining the assay signal intensity pattern generated by said hybridization or elongation assay and forming, for each probe pair, a combination of signal intensities associated with the members of the pair;
generating, from said combinations of signal intensities, a series of values, each value in the series selected from one of three possible values, respectively indicating a state of homozygous-normal, heterozygous or homozygous-variant to thereby form a ternary reaction pattern;
determining the polymorphic marker combinations represented by said ternary reaction pattern; and
selecting from the candidate donors a donor having said polymorphic marker combination identical to that of the recipient.
2. The method of claim 1 wherein polymorphic sites within the markers are known to affect the transcription or expression of clinically relevant antigens.
3. The method of claim 1 wherein combination of signal intensities of members are represented such that one such intensity, i.sub.N, correlates with the amount of normal marker in the sample, and the other such intensity, i.sub.V, correlates with the amount of variant marker in the sample, and said intensities are combined to form a discrimination parameter .DELTA.=(i.sub.N−i.sub.V)/(i.sub.N+i.sub.V), which varies between −1 and 1.
4. The method of claim 3 wherein the ternary reaction pattern is generated by designating values of the discrimination parameter below a first threshold as a lower value, designating values of the discrimination parameter above a second threshold as an upper value, and designating values of the discrimination parameter below the second threshold but above the first threshold as an intermediate value.
5. The method of claim 4 wherein when the assay is a hybridization assay wherein the signal intensity pattern is converted to the ternary reaction pattern by forming ratios of signal intensities associated with a probe pair and designating the sample as homozygous for a marker for ratios above a first threshold, and designating the sample as homozygous for the marker for a variant allele for ratios below a second threshold, and designating the sample as heterozygous for the marker ratios below the first threshold and above the second threshold.
6. The method of claim 4 wherein the first threshold is a lower threshold and the second threshold is an upper threshold.
7. The method of claim 4 wherein the values of the discrimination parameter below the first threshold are designated by a −1, the values above second threshold are designated by a +1, and the values between the upper and lower thresholds are designated by a 0.
8. The method of claim 1 wherein, an ambiguity arises because more than one polymorphic marker combination is represented by the ternary reaction pattern, an additional assay step is performed on at least one of the amplified products including the subsequences (or the single nucleotide polymorphic sites) of the amplified products which are the same as and/or are complementary to at least one of said markers in said combination, to thereby resolve the ambiguity.
9. The method of claim 8 wherein the additional assay step is hybridization, where annealing of an oligonucleotide to a marker in an amplified product indicates the presence of one of the subsequences in the amplified product.
10. The method of claim 8 wherein the additional assay step is capture-mediated probe elongation, where elongation of an oligonucleotide following annealing to a subsequence in an amplified product indicates the presence of one of the markers in the amplified product.
11. The method of claim 10 wherein elongation products incorporate a labeled ddNTP or a labeled dNTP.
12. The method of claim 11 wherein the dNTPs or ddNTPs are labeled so as to generate optically detectable assay signals when incorporated into elongation products.
13. The method of claims 11 or 12 wherein the label is fluorescence.
14. The method of claim 1 wherein the polymorphic markers are included in the following blood groups, and associated with the corresponding phenotypes as listed:
Blood Group
Phenotype
Markers
Colton
Co a /Co b
134 C or T
Diego
Di b /Di a
2561 C or T
Duffy
Fy a /Fy b
125 G or A
Fy x [Fy(b+ w )]
265 C or T
GATA (Fy(a−b−)
−33 T or C
Dombrock
Do a /Do b
378 C or T
624 T or C
793 A or G
Hy+/Hy−
323 G or T
Jo(a+)/Jo(a−)
350 C or T
Kidd
Jk a /Jk b
838 G or A
Kell
K/k
698 T or C
Landsteiner-Wiener
LW a /LW b
308 A or G
Lutheran
Lu a /Lu b
230 A or G
MNS
GYPA (M/N)
59 C or T
GYPB (S/s)
143 T or C
Scianna
Sc1/Sc2
169 G or A
Rh
S68N (C/c)
203 A or G
Rh
A226P (E/e)
676 G or C
Hemoglobin S
HbS
173 A or T.
15. The method of claim 1 wherein the polymorphic markers are included in the blood groups RhCE and RhD and the markers are as listed:
Exon
Markers
1
48 G or C
2
178 C or A
203 A or G
307 C or T
3
455 A or C
4
37 bp dup.-ins.
5
667 T or G
5
676 G or C
5
697 G or C
5
733 G or C
7
1006 G or T.
16. The method of claim 1 wherein the amplified products are amplicons or transcripts.
17. The method of claim 1 wherein the amplified products are generated by performing polymerase chain reaction or reverse transcription on samples of genomic DNA or RNA, respectively.
18. The method of claim 17 further including the step of generating single-stranded amplified products.
19. The method of claim 18 wherein the single-stranded amplified products are generated by phosphorylating one of the target strands and then enzymatically digesting it.
20. The method of claim 1 wherein the assay signal intensities used in comparison are normalized intensity ratios.
21. The method of claim 1 wherein the probes are encoded by displaying them on encoded carriers that permit identification of the associated probe.
22. The method of claim 21 wherein the encoded carriers are microparticles.
23. The method of claim 21 wherein the encoding is with color.
24. The method of claim 1 wherein the polymorphic markers are associated with minor blood group types.
25. The method of claim 1 wherein donor constituent allele combinations are collated in a registry that may be queried electronically.