# Patent

## Title
Method for the genomic typing of erythrocyte systems, oligonucleotide probes and relative diagnostic kits

## Abstract

The invention relates to a method for the genomic typing of erythrocyte systems, oligonucleotide probes and relative diagnostic kits.

## Background
The present invention relates to a method for the genomic typing of erythrocyte systems, oligonucleotide probes and relative diagnostic kits.
Blood group typing has been traditionally performed with agglutination techniques using various methods such as slides, tubes, columns and solid/liquid phase microplate technology with both polyclonal and monoclonal commercial antisera.
The various agglutination techniques which can be applied in all competent laboratories have a sensitivity and appropriate specificity in clinical use for most cases.
However, due to limitations in hemagglutination, it is now customary in reference laboratories, to complement and support serologic blood group typing with molecular techniques and in many cases are the sole alternative method capable of solving complex problems.
There are various applications in transfusion medicine practice.
Most clinical applications appropriately respond to the demand for having a correct blood group typing of the patient in a short time and relate to multi-immunized subjects with autoimmune pathologies, to patients transfused immediately prior to blood group testing and/or transfusion-dependent patients such as thalassemic patients (ref. 1 Castilho L. et al. 2002; ref. 2 Montalvo L. et al. 2004). In these cases, typing with classical methods could be difficult to apply. For the first category of patients, difficulties arise due to the presence of antibodies adhering to the erythrocytes which require additional analyses and different typing methods on the part of the laboratory for immunohematological analyses, considerably prolonging the analysis time which is precious in emergency situations. In the second category of patients transfused immediately prior to blood group testing, the problem is due to the presence of massive quantities of transfused erythrocytes of the donor in the patient's circulation rendering it impossible to apply classical methods. In this case therefore, a correct typing of the patient's RH phenotype and of other common red blood cell antigens (for example, K/k; Fya/Fyb; Jka/Jkb; S/s) against which the development of antibodies may have a relevant clinical meaning, is extremely useful for confirming the nature of the antibodies identified both in the serum and adhering to the erythrocytes and consequently for providing the best possible transfusion support for the patient.
There are other interesting applications of the molecular typing of erythrocyte systems. These include the confirmation and at times the only resolution source in cases of antigens with weak expression such as the D antigen (RH system) or FyX antigen (Duffy system); the characterization of null forms; the determination of D-zygosity not otherwise possible and resolution in cases of ABO variants.
Another important application also relates to the possibility of confirming, with molecular techniques, the rare erythrocyte typing of patients or blood donors who are negative for high incidence antigens. A person having a rare phenotype can become immunized against the missing antigen following transfusion, pregnancy and to a lesser extent organ transplant. Immunization against a high incidence antigen can also complicate considerably the detection of additional blood group antibodies. The presence of antibodies having different specificities makes the identification process laborious and complicated and the finding of compatible blood units extremely problematical.
The possibility of having frozen typed blood units at the moment of need considerably facilitates patient management, without having to resort to the random typing of a high number of donors under emergency conditions, also with the risk of not finding the compatible unit. Rare blood units could be frozen and isolated for patients at risk. Furthermore, it should also be taken into consideration that ethnic differences between donor and patient could create greater problems, especially if the patient requires a long-term transfusion regime.
For this purpose, the use of molecular techniques will solve the problem of the high costs of rare antisera and at times, for some specificities, it also overcomes the problem of both the lack of and weak reactivity of these easily perishable antisera such as the specific antisera for the Dombrock system (ref. 3 Reid et al. 2002).
An important advantage of DNA methods consists in the possibility of obtaining a useful DNA quantity from both peripheral blood, even from minimum quantities, and other biological sources. Furthermore, if the DNA samples are appropriately preserved, they are stable over a long period of time. Working with DNA in transfusion medicine has also the considerable advantage of not being limited by the fact that the sample must be processed immediately as required by classical serology.
Various techniques applied in the field of transfusion medicine have been developed for all these potential applications. In particular, for blood group genotyping, the most common techniques used in immunohematology laboratories are PCR-RFLP (Restriction Fragment Length Polymorphism) and PCR-SSP (Sequence-Specific Primers).
New methods have been recently developed such as PCR-ELISA, real time PCR, SNP minisequencing analyses (ref. 4 Ferri E G et al., 2006) and microarray technology (ref. 5 Denomme G. et al., 2005). This latter technology in particular arose from the necessity to type a greater number of samples with respect to other available techniques which were low-throughput.
The principle of this technique is certainly not entirely new. The Southern blot techniques, for example, provides for the analysis of a large number of samples by hybridization of DNA fragments but by means of electrophoresis. The main difference lies in the type of material used as hybridization support; porous hybridization membranes have been replaced with non-porous glass or silicon support or fluorescently-labeled microspheres (ref. 6 Petrik J. 2001). These changes have allowed reagent volumes to be considerably reduced, improving the hybridization kinetics, miniaturizing the whole process, increasing throughput and allowing the possibility to test for several analytes contemporaneously in a single reaction. All these revolutionary changes reduce considerably operator-time, laboriousness and costs.
A variety of applications of the microarray technology have been developed in recent years. This technology is applied in both genetic analysis and serology.
The microarray technology, as applied in this case, is characterized by an amplification phase of the target DNA region, followed by denaturation, hybridization with specific probes complementary to the target polymorphism and fluorescence detection and data analysis by means of flow cytometry after suitable marking with phycoerythrin-streptavidin. With microarray technology using a solid hybridization support, it is possible to type antigens from the ABO and RH systems as well as clinically significant and high incidence antigens. This technology has also been applied to the genomic typing of platelet antigens (ref. 7 Beiboer S. et al., 2005). Furthermore, the use of agglutination techniques involves high costs in the case of mass screening for high incidence erythrocyte antigens in order to obtain negative donors, as the availability of commercial typing reagents is extremely limited, also making typing with antisera problematic due to poor reliability.
One of the main advantages of techniques based on DNA is the substitution of typing sera by oligonucleotides synthesized at low cost.
The new technologies currently seem to aim at automation and simplification and the new instruments are modified to accelerate the process. This latter concept is descriptive of dosages of multiplex flow cytometry based on microspheres. By the coupling of various purified antibodies or oligonucleotide probes to distinct sets of fluorescent microspheres, it is possible to obtain extremely efficient analysis systems which allow numerous analytes to be captured from a single sample. The quantification exploits the multiparametric resolutive potential of flow cytometry and the capacity of the processing systems of the digital signals which process the thousands of fluorescent signals generated by the microspheres (ref. 8 Kellar K L et al., 2003; ref. 9 Kettman J R et al., 1998).
More specifically, the microspheres consist of synthetic polymers and each microsphere set is characterized by a specific fluorescence intensity. Various commercial sources of fluorescent microspheres are available such as Bangs Laboratories (Fishers, Ind.), Duke Scientific (Palo Alto, Calif.), Luminex Corporation (Austin, Tex.), Polysciences (Warrington, Pa.), Seradyn (Indianapolis, Ind.) and Sperotech (Libertyville, Ill.) which offer microspheres with different dimensions and fluorescence characteristics. Luminex Corporation, for example, produces 100 microspheres differing in fluorescence intensity created by the incorporation of different ratios of two fluorochromes which emit at different wavelengths (ref. 10 Fulton R F et al., 1997). A compact flow cytometer (Luminex 100) with two projected laser sources is used for the detection of the microspheres and quantification of the fluorescence. An array of 100 microspheres has been produced with dyes which emit at 658 and 712 nm after stimulation with a 635 nm red diode laser to complement the laser system of the cytometer (ref. 11 Earley M C et al. 2002). This Multiple Analyte Profiling system (LabMAPTM) was used for the multiplex analysis of various single nucleotide polymorphisms (SNPs) (ref. 12 Colinas et al., 2000; ref. 13 Dunbar S A et al., 2000). SNPs are the most abundant variability source in the human genome, important for the identification of particular pathologies or for clarifying the predisposition for developing a particular illness or responding to a certain pharmacological therapy (ref. 8 Kellar K L, 2003). SNPs also represent the molecular basis of the polymorphisms of numerous antigen systems.
The authors have now set up a molecular blood group typing method which uses specific oligonucleotide probes which, when suitably modified, are coupled to an array of fluorescent microspheres, which does not have the disadvantages of the known typing techniques.
By using the method according to the invention, there is a considerable reduction in reagent costs and operator time.
From an applicative point of view, the method is particularly advantageous for the large-scale typing of blood samples and can facilitate the identification of a rare phenotype for alloimmunized patients and subjects belonging to ethnic minorities. More specifically, in the present invention, after identifying the polymorphism Xa and Xb relating to each of the systems subject of the study, the authors designed oligonucleotide probes capable of hybridizing, at a certain temperature, in a highly specific manner, at the polymorphic site of interest. These probes have given excellent results in terms of specificity and efficiency of the hybridization process (selected probe length/polymorphism/hybridization temperature).
The characteristics and advantages of the method and the experimental phases set up by the authors of the present invention and used in the present typing method are the following:

    
    
        application of the Luminex suspension array method to the genotyping of blood group systems.
        identification of pairs of specific primers for amplifying the genomic region containing the single nucleotide polymorphism of interest (see Table 1).
        identification of PCR conditions: same quantity and concentration ratios for both the primers and buffers used and same amplification cycles for all the systems studied.
        design of the oligonucleotide probes: designed complementary to the sequence amplified by the biotinylated primer, with localization of the polymorphism of interest at or near the centre of the probe (the polymorphic position is marked in bold in Table 2). The changes with respect to the central position are determined by the addition and/or removal of nucleotides from the 5′- and/or 3′-end of the probe to increase the hybridization efficiency and obtain a better probe-target match or to increase the specificity (ref. 14 Dunbar S A et al., 2005; ref. 15 Dunbar S A. Et al., 2006).
        direct hybridization of the designed probes with the PCR product, containing the polymorphism object of the study, at a specific hybridization temperature range defined for each system studied, wherein the optimal specific hybridization temperature is shown in parenthesis (see Table 2).
    
    













TABLE 1






Target
Name of




System
alleles
primer
Sequence (5′-3′)







KELL
K/k
K5F
TTTAgTCCTCACTCCCATgCTTCC




PCR1
ref. 16
(SEQ ID NO: 1)




K6R
TATCACACAggTgTCCTCTCTTCC




ref. 16
(SEQ ID NO: 2)



Kpa/Kpb
KpF
TgAggCCAggAgAAAAgCA (SEQ ID NO: 3)



PCR2
KpR
TgACCATCTggAAgAgCTTgC (SEQ ID NO: 4)



Jsa/Jsb
JsF
AACTTTgCCATgCTCCTgg (SEQ ID NO: 5)



PCR2
JsR
GCCTTgACACTTgCATACCT (SEQ ID NO: 6)


 


LUTHERAN
Lua/Lub
Lu91mF
CTgAggAgCgCTgggACACCCgg (SEQ ID NO: 7)



PCR3
Lu92R
CCCCgggTgTCgTgCATT (SEQ ID NO: 8)




ref. 18


 


MNS
S/s
SsF
AAgACTgACACATTACCTCA (SEQ ID NO: 9)



PCR4
ref. 17




SsR
AACATACCTggTACAgTgAA (SEQ ID NO: 10)


 


COLTON
Coa/Cob
CoF3
TATAAATAggCCCAgCCCAg (SEQ ID NO: 11)



PCR5
CoR3
CCAgCgACACCTTCACgTT (SEQ ID NO: 12)


 


DUFFY
Fya/Fyb
Duffy-F2
CTTCCggTgTAACTCTgATgg (SEQ ID NO: 13)



PCR6
Duffy-R3
CATCCAgCAggTTACAggAgT (SEQ ID NO: 14)


 


KIDD
Jka/Jkb
JK-781
CATgCTgCCATAggATCATTgC



PCR7
F3 ref. 19
(SEQ ID NO: 15)




JK-943
gAgCCAggAggTgggTTTgC




R3 ref. 19
(SEQ ID NO: 16)





ref. 16 Lee, 1997


ref. 17 Hashmi, 2005


ref. 18 El Nemer, 1997


ref. 19 Irshaid, 1998.

















TABLE 2








Microspheres



Target


Specificity/


alleles
Probe AmC12-5′
THYB RANGE
Region N.




















K/k
TTAACCgAACgCTgAgAC (SEQ ID NO 17)
45-50°
C.
K-088




TTAACCgAATgCTgAgAC (SEQ ID NO 18)
(45°
C.)
k-089



CTATCCCAAAgCTAAggC (SEQ ID NO 19)


NC-086


 


Kpa/
ATCACTTCACggCTGTTCCA (SEQ ID NO 20)
52-56°
C.
Kpa-072


Kpb
TCACTTCATggCTgTTCCAg (SEQ ID NO 21)
(54°
C.)
Kpb-073



AACTCTACAgggCTCTTCgA (SEQ ID NO 22)


NC-051


 


Jsa/
GgCTgCCTCgCCTgTgACAA (SEQ ID NO 23)
52-56°
C.
Jsa-053


Jsb
GgCTgCCCCgCCTgTgACAA (SEQ ID NO 24)
(54°
C.)
Jsb-055



GCCAgCCACgCgTgTCACTA (SEQ ID NO 25)


NC-064


 


Lua/
TCgCCCCCgCCTAgCCTC (SEQ ID NO 26)
43-47°
C.
Lua-063


Lub
TCgCCCCCACCTAgCCTC (SEQ ID NO 27)
(45°
C.)
Lub-065



TAgCCTCCTCCAAgACTA (SEQ ID NO 28)


NC-064


 


S/s
TAggAgAAACgggACAACTT (SEQ ID NO 29)
50-54°
C.
S-084



AggAgAAATgggACAACTTg (SEQ ID NO 30)
(54°
C.)
s-085



TCggATAAAAgAgACCACTg (SEQ ID NO 31)


NC-087


 


Coa/
AACCAgACggCggTCCAggA (SEQ ID NO 32)
62-66°
C.
Coa-074


Cob
CAACCAgACggTggTCCAgg (SEQ ID NO 33)
(64°
C.)
Cob-078



AgCCACACTggggACCTggA (SEQ ID NO 34)


NC-080


 


Fya/
GAgACTATggTgCCAACCTg (SEQ ID NO 35)
52-56°
C.
Fya-066


Fyb
TggAgACTATgATgCCAACC (SEQ ID NO 36)
(54°
C.)
Fyb-067



GAggCTATCCTgACAAgCTT (SEQ ID NO 37)


NC-069


 


Jka/Jkb
AgTAgATgTCCTCAAATg (SEQ ID NO 38)
37°-40°
C.
Jka-064



AgTAgATgTTCTCAAATg (SEQ ID NO 39)
(37°
C.)
Jkb-076



CgTggATTTCTTCAgAgg (SEQ ID NO 40)


NC-073









The erythrocyte systems and the relative alleles encoding common, rare and high incidence antigens, analyzed by the authors of the present invention are indicated in Table 1.
The authors then applied the Luminex Xmap technology using an array of microspheres in suspension for determining the polymorphisms relating to erythrocyte antigens in order to apply, in this field of research, the potentialities of a versatile method which provides a rapid, accurate and efficient instrument especially for the management of mass-screening. This method avails of the hybridization process between synthetic oligonucleotide capture probes coupled to fluorescent microspheres and the target DNA amplified by PCR, using specific primers which allow the genomic locus containing the nucleotide polymorphism of interest to be amplified.
The method according to the present invention was set up and tested with DNA samples of known genotype and/or phenotype (homozygote or heterozygote for the erythrocyte antigens of interest); the typing for the low incidence antigens (such as Kpa, Jsa, Lua and Cob) carried out with serologic agglutination techniques and/or molecular techniques, such as PCR-SSP, was not known for all the samples tested. The method is robust in its capacity of identifying with accuracy, on a genomic level, the polymorphism for the erythrocyte systems tested and is tolerant with respect to the quantity, quality and source of the material to be typed. Tables 3-10 indicate the values of the allelic ratios for each system studied of all the samples tested.
After DNA extraction, it is not necessary to determine DNA concentration on the spectrophotometer, thus considerably reducing operator time.
Unlike other microarray methods applied to the typing of erythrocyte or platelet systems, the specific hybridization process takes place in suspension.
From a study of recent literature, it has emerged that the specific method in question is applied in various research fields such as genotyping in the field of microbiology and virology (ref. 20 Deregt D. et al. 2006; ref. 21 Schmitt et al., 2006; ref. 22 Diaz M. et al., 2005). With respect to the microarray format using a solid support, the advantage of the array technology in suspension relates to the rapidity of data acquisition, good sensitivity and specificity and the possibility of multiplexing.
An object of the present invention therefore relates to sets of oligonucleotide probes amino-modified at the 5-end, characterized in that they have a sequence length ranging from 18 to 20 nucleotides and containing the specific SNP for each of the target alleles belonging to the genomic locus X, selected from K/k, Kpa/Kpb, Jsa/Jsb, Lua/Lub, S/s, Coa/Cob, Fya/Fyb and Jka/Jkb at or near the centre of said probe, capable of specifically hybridizing to each of said alleles; said probes being characterized in that they are coupled to a microparticle labeled with at least one fluorescent substance and that they comprise or consist of at least one set of oligonucleotide sequences indicated in the following table:














Probe




set


Probe
Probe set
number







k
TTAACCgAACgCTgAgAC (SEQ ID NO: 17)
1


K
TTAACCgAATgCTgAgAC (SEQ ID NO: 18)


NC
CTATCCCAAAgCTAAggC (SEQ ID NO: 19)


 


Kpb
ATCACTTCACggCTgTTCCA (SEQ ID NO: 20)
2


Kpa
TCACTTCATggCTgTTCCAg (SEQ ID NO: 21)


NC
AACTCTACAgggCTCTTCgA (SEQ ID NO: 22)


 


Jsb
ggCTgCCTCgCCTgTgACAA (SEQ ID NO: 23)
3


Jsa
ggCTgCCCCgCCTgTgACAA (SEQ ID NO: 24)


NC
gCCAgCCACgCgTgTCACTA (SEQ ID NO: 25)


 


Lua
TCgCCCCCgCCTAgCCTC (SEQ ID NO: 26)
4


Lub
TCgCCCCCACCTAgCCTC (SEQ ID NO: 27)


NC
TAgCCTCCTCCAAgACTA (SEQ ID NO: 28)


 


s
TAggAgAAACgggACAACTT (SEQ ID NO: 29)
5


S
AggAgAAATgggACAACTTg (SEQ ID NO: 30)


NC
TCggATAAAAgAgACCACTg (SEQ ID NO: 31)


 


Coa
AACCAgACggCggTCCAggA (SEQ ID NO: 32)
6


Cob
CAACCAgACggTggTCCAgg (SEQ ID NO: 33)


NC
AgCCACACTggggACCTggA (SEQ ID NO: 34)


 


Fya
GAgACTATggTgCCAACCTg (SEQ ID NO: 35)
7


Fyb
TggAgACTATgATgCCAACC (SEQ ID NO: 36)


NC
gAggCTATCCTgACAAgCTT (SEQ ID NO: 37)


 


Jka
AGTAGATGTCCTCAAATG (SEQ ID NO: 38)









Said probes are preferably conjugated with Aminolinker C12 modification at the 5′-end.
The invention relates to the use of at least one set of oligonucleotide probes as defined in the previous table, for the identification and typing of at least one SNP of the following allelic pair X selected from K/k, Kpa/Kpb, Jsa/Jsb, Lua/Lub, S/s, Coa/Cob, Fya/Fyb, Jka/Jkb.
According to alternative embodiments of the invention, it is possible to use one or more of the oligonucleotide probe sets according to the invention in the same hybridization mixture (e.g. the sets of oligonucleotide probes for the alleles Kpa/Kpb and Jsa/Jsb or all the probe sets together).
In the present embodiment, the use of the sets of oligonucleotide probes is performed at specific hybridization temperature ranges indicated in the following Table:











Set number
THYBRIDIZATION RANGE







1
45-50° C., preferably 45° C.


2
52-56° C., preferably 54° C.


3
52-56° C., preferably 54° C.


4
43-47° C., preferably 45° C.


5
50-54° C., preferably 54° C.


6
62-66° C., preferably 64° C.


7
52-56° C., preferably 54° C.


8
37-40° C., preferably 37° C.









The invention also, relates to microparticles labeled with at least one fluorescent substance having carboxylic groups on the surface, characterized in that they are coupled with at least one set of probes as defined above.
A further object of the present invention relates to a method for the identification and typing of at least one single nucleotide polymorphism (SNP) of the erythrocyte system X in heterozygote and homozygote individuals, comprising the following phases:
a) DNA extraction from a biological sample;
b) PCR amplification of the genomic locus comprising the SNP of the erythrocyte system of interest, by means of at least one specific pair of primers for a target allele selected from:











Target



alleles
Primer sequence (5′-3′)







K/k
Fw: TTTAgTCCTCACTCCCATgCTTCC



(SEQ ID NO: 1)



Rw: TATCACACAggTgTCCTCTCTTCC



(SEQ ID NO: 2)


 


Kpa/Kpb
Fw: TgAggCCAggAgAAAAgCA (SEQ ID NO: 3)



Rw: TgACCATCTggAAgAgCTTgC (SEQ ID NO: 4)


 


Jsa/Jsb
Fw: AACTTTgCCATgCTCCTgg (SEQ ID NO: 5)



Rw: gCCCTTgACACTTgCATACCT (SEQ ID NO: 6)


 


Lua/Lub
Fw: CTgAggAgCgCTgggACACCCgg (SEQ ID NO: 7)



Rw: CCCCgggTgTCgTgCATT (SEQ ID NO: 8)


 


S/s
Fw: AAgACTgACACATTACCTCA (SEQ ID NO: 9)



Rw: AACATACCTggTACAgTgAA (SEQ ID NO: 10)


 


Coa/Cob
Fw: TATAAATAggCCCAgCCCAg (SEQ ID NO: 11)



Rw: CCAgCgACACCTTCACgTT (SEQ ID NO: 12)


 


Fya/Fyb
Fw: CTTCCggTgTAACTCTgATgg (SEQ ID NO: 13)



Rw: CATCCAgCAggTTACAggAgT (SEQ ID NO: 14)


 


Jka/Jkb
Fw: CATgCTgCCATAggATCATTgC (SEQ ID NO: 15)



Rw: gAgCCAggAggTgggTTTgC (SEQ ID NO: 16)









wherein at least one primer (Fw or Rw) is marked at the 5′-end with biotin to obtain biotinylated PCR products; the oligonucleotide probes are complementary to the DNA sequence amplified by the biotinylated primer;

c) hybridization of the biotinylated PCR products obtained in phase b) with at least one set of oligonucleotide probes as described above and labeling with streptavidin-phycoerythrin at the specific hybridization temperature range for each system as illustrated below:
















THYBRIDIZATION



Probe
Probe set
Range



















k
TTAACCgAACgCTgAgAC (SEQ ID NO: 17)
45-50°
C.



K
TTAACCgAATgCTgAgAC (SEQ ID NO: 18)
preferably 45°
C.


NC
CTATCCCAAAgCTAAggC (SEQ ID NO: 19)


 


Kpb
ATCACTTCACggCTgTTCCA (SEQ ID NO: 20)
52-56°
C.


Kpa
TCACTTCATggCTgTTCCAg (SEQ ID NO: 21)
preferably 54°
C.


NC
AACTCTACGgggCTCTTCgA (SEQ ID NO: 22)


 


Jsb
ggCTgCCTCgCCTgTgACAA (SEQ ID NO: 23)
52-56°
C.


Jsa
ggCTgCCCCgCCTgTgACAA (SEQ ID NO: 24)
preferably 54°
C.


NC
gCCAgCCACgCgTgTCACTA (SEQ ID NO: 25)


 


Lua
TCgCCCCCgCCTAgCCTC (SEQ ID NO: 26)
43-47°
C.


Lub
TCgCCCCCACCTAgCCTC (SEQ ID NO: 27)
preferably 45°
C.


NC
TAgCCTCCTCCAAgACTA (SEQ ID NO: 28)


 


s
TAggAgAAACgggACAACTT (SEQ ID NO: 29)
50-54°
C.


S
AggAgAAATgggACAACTTg (SEQ ID NO: 30)
preferably 54°
C.


NC
TCggATAAAAgAgACCACTg (SEQ ID NO: 31)


 


Coa
AACCAgACggCggTCCAggA (SEQ ID NO: 32)
62-66°
C.


Cob
CAACCAgACggTggTCCAgg (SEQ ID NO: 33)
preferably 64°
C.


NC
AgCCACACTggggACCTggA (SEQ ID NO: 34)


 


Fya
GAgACTATggTgCCAACCTg (SEQ ID NO: 35)
52-56°
C.


Fyb
TggAgACTATgATgCCAACC (SEQ ID NO: 36)
preferably 54°
C.


NC
gAggCTATCCTgACAAgCTT (SEQ ID NO: 37)


 


Jka
AgTAgATgTCCTCAAATg (SEQ ID NO: 38)
37-40°
C.


Jkb
AgTAgATgTTCTCAAATg (SEQ ID NO: 39)
preferably 37°
C.


NC
CgTggATTTCTTCAgAgg (SEQ ID NO: 40)









d) fluorescence detection with a flow cytometry-based instrument, by detecting the fluorescence emitted by the specific microspheres preferably using a Luminex 100 instrument. FIG. 1 shows an example of the instrument software after fluorescence analysis of the samples.

The method adopted avails of the Luminex XmapTM system as it uses an array of fluorescent microspheres covalently coupled in the laboratory with the specific complementary probes for the analysis of the polymorphisms of the above erythrocyte systems and flow-cytometer Luminex 100 (Luminex Corporation). The amplification of phase b) in the case of polymorphisms of the alleles Kpa/Kpb and Jsa/Jsb of the KELL system is preferably carried out by multiplex PCR.
The invention relates to a diagnostic kit for the identification and typing of at least one SNP of the erythrocyte systems, subject of the study, to identify the heterozygote and homozygote asset of samples, comprising the following components:
a) one or more pairs of primers for PCR amplification of the genomic locus comprising the SNP of the pair X selected from K/k, Kpa/Kpb, Jsa/Jsb, Lua/Lub, S/s, Coa/Cob, Fya/Fyb, Jka/Jkb, said pair of primers being selected from:











Target



alleles
Primer sequence (5′-3′)







K/k
Fw: TTTAgTCCTCACTCCCATgCTTCC



(SEQ ID NO: 1)



Rw: TATCACACAggTgTCCTCTCTTCC



(SEQ ID NO: 2)


 


Kpa/Kpb
Fw: TgAggCCAggAgAAAAgCA (SEQ ID NO: 3)



Rw: TgACCATCTggAAgAgCTTgC (SEQ ID NO: 4)


 


Jsa/Jsb
Fw: AACTTTgCCATgCTCCTgg (SEQ ID NO: 5)



Rw: gCCCTTgACACTTgCATACCT (SEQ ID NO: 6)


 


Lua/Lub
Fw: CTgAggAgCgCTgggACACCCgg (SEQ ID NO: 7)



Rw: CCCCgggTgTCgTgCATT (SEQ ID NO: 8)


 


S/s
Fw: AAgACTgACACATTACCTCA (SEQ ID NO: 9)



Rw: AACATACCTggTACAgTgAA (SEQ ID NO: 10)


 


Coa/Cob
Fw: TATAAATAggCCCAgCCCAg (SEQ ID NO: 11)



Rw: CCAgCgACACCTTCACgTT (SEQ ID NO: 12)


 


Fya/Fyb
Fw: CTTCCggTgTAACTCTgATgg (SEQ ID NO: 13)



Rw: CATCCAgCAGGTTACAggAgT (SEQ ID NO: 14)


 


Jka/Jkb
Fw: CATgCTgCCATAggATCATTgC (SEQ ID NO: 15)









b) at least one set of oligonucleotide probes as defined above, said probes being capable of hybridizing to said SNP.

Preferably, the set of primers as above defined used in multiplex PCR reactions according to the present invention are:

    
    
        K5F/K6R, SsF/SsR, Duffy-F2/Duffy-R3 and JK-781-F3/JK-943-R3;
        KpF/KpR, JsF/JsR, Lu91mF/Lu92R and CoF3/CoR3.
    
    


The present invention will now be described for illustrative and non-limiting purposes according to its preferred embodiments, with particular reference to the tables and enclosed FIGURE in which:
FIG. 1 shows the analysis of the Colton system where the fluorescence of the three microspheres of interest is analyzed (microspheres 74, 78, 80); the identifying codes of the samples are shown (column “sample”); the value obtained for each microsphere is the value of fluorescence emitted from the microsphere in turn coupled with the relative probes according to the invention; the column “events” refers to the number of total microspheres so that a minimum of 100 events (microspheres) are analyzed for each microsphere classification.


EXAMPLE
Genomic Typing of the Erythrocyte System X by Means of the Microarray System in Suspension which Uses Oligonucleotide Probes Complementary to the Specific SNP Coupled to an Array of Fluorescently-Labeled Microspheres
Materials and Methods
Samples
7 mL of peripheral blood of the sample to be analyzed was collected in test-tubes containing the solution of EDTA as anticoagulant. The samples are preserved at −20° C. until the moment of testing. Aliquots of 200 μl of whole blood were used for DNA extraction with a commercial kit (QIAamp, Qiagen, Mississauga, Ontario, Canada), according to the instructions of the producer.
The samples tested are indicated in the relative tables (Tables 3-10).
Reagents
The polystyrene COOH xMAP Multi-Analyte microspheres were purchased from Luminex Corporation (Carboxylated Microspheres, L100-C1XX-01-Austin, Tex., USA).
The microspheres (5.6 μm in diameter) have carboxylic functional surface groups for the covalent bond with different analytes which, for the purposes of the present invention, are oligodeoxyribonucleotide probes amino-modified (AmC12) at the 5′-end. The polystyrene microspheres (commercially available) were classified by the producer by means of flow cytometry on account of the emission profile in the red/infrared wavelength of each microsphere classification.
100 microspheres are available as each specific region incorporates two fluorophores in a precise intensity ratio with each other which emit at different wavelengths (red and infrared) allowing them to be distinguished. Each distinct microsphere classification in fact has unique spectral characteristics and its own fluorescence intensity distribution which can be analyzed by the analysis instrument. Various regions were used in this study: see Table 2. All the different regions of microspheres numbered from 1 to 100 derive from the same starting material and differ only in terms of the quantities of red/infrared dyes.
2-N-morpholine ethanesulfonic acid (MES), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), SAPE (100× stock 0.5 mg/ml Streptavidin-phycoerythrin) were obtained by Sigma, Pierce and One Lambda, Inc. respectively. The following buffers of One Lambda Inc, LABType wash buffer and LABType sape buffer, were used respectively for the wash step after the hybridization phase and to dilute the SAPE stock.
Probe Design
All the oligonucleotides used for the covalent coupling with the microspheres were modified at the 5′-end during the synthesis, by means of Amino-Modifier (AmC12). The polymorphism of the various systems studied in the design of the probes was preferably localized at or near the centre of the probe (specific polymorphism position—Table 2).
The probe length varies from 18 to 20 nucleotides and they are selected complementary to the sequence amplified by the biotinylated primer, on the basis of the genomic sequences deposited.
A set of probes is used for each system to be analyzed, comprising: two probes specific for the alleles of the system object of the study; one non-specific probe used as negative control (NC), as it has been specifically designed by changing, with respect to the sequence of the specific probe, six nucleotides so as not to have the possibility of matching with the target DNA. This probe is only used for evaluating the background fluorescence signal, controlling that all the wash steps have been carried out correctly and indirectly confirming the positive or negative signal of the specific probes:

    
    
        Xa probe and Xb probe: from 18 to 20 nucleotides with AmC12 modification at the 5′-end: these are the specific probes for the polymorphism implied; the polymorphism of the various systems studied was preferably localized at or near the centre of the probe (specific polymorphism position—Table 2).
        negative control probe (NC): from 18 to 20 nt with AmC12 modification at the 5′-end differing from the allele-specific probe by six nucleotide modifications so as to obtain a probe which can not hybridize to the specific polymorphism (ref. 13 Dunbar et al., 2000).
    
    


Various problems arose during the identification of these probes, which not all the hybridization temperature/PCR product/probe length combinations and polymorphism positions were able to overcome.
On the basis of the specific polymorphism of the alleles K/k, Kpa/Kpb, Jsa/Jsb, of the Kell system, each characterized by a single nucleotide change and by the relative genomic sequence deposited, in preliminary experiments, it was possible to identify the complementary probes of 18 nt with the specific polymorphism in a central position and couple them to the development fluorescent microspheres (L100-CDEV1-01 (Luminex)). The hybridization temperature used for the preliminary experiments was 45° C.
The following results were obtained:
K/k: the 18 nt probes, central polymorphism and PCR product obtained from a pair of primers described in literature (Lee, 1997) gave good specificity results in the typing of the samples tested with known serological typing, effected at a temperature of 45° C.

Kpa/Kpb: the 18 nt probes, central polymorphism and PCR product obtained from a pair of primers described in literature (ref. 16 Lee, 1997) gave no hybridization signal at a temperature of 45° C.

Further hybridization experiments were then effected varying only the hybridization temperature (between 50° C. and 54° C.) and maintaining the same probes and the same PCR product. Even the temperature of 37° C. did not allow the specific alleles to be distinguished.
At this point the PCR product was modified by designing, with the programs available (Primer Express, Applied Biosystems; OligoAnalyzer 3.0, Integrated DNA Technologies) new pairs of primers to shorten the final amplified product, thus favoring the hybridization phase (ref. 14 Dunbar et al., 2005). 20 nt probes were used with the position of the polymorphism adjusted, i.e. no longer located only at the center of the probe sequence.
The results show a specific signal which can be obtained by changing both the PCR product and the length of the probes at a certain temperature (54° C.).
Once the suitable combination for obtaining a high specificity had been found, the probes were coupled to the xMAP® Multi-Analyte microspheres (L100-C1XX01 COOH). Duffy system (Fya/Fyb) and Colton system (Coa/Cob): the 20 nt probes, polymorphism not only in a central position and PCR product obtained from a pair of primers designed directly with computerized programs did not give good results in terms of specificity in the hybridization phase carried out at temperatures ranging from 45° C. to 54° C.
In this case the PCR product was amplified using the primer pairs according to the invention, i.e. different primers were designed.
In order to distinguish the allele Coa, two 20 nt probes were used with the polymorphism of interest situated in a different position. After various tests at different temperatures, specific results were obtained with the sequence indicated in Table 2.
MNS system (S/s): of the pair of primers for the amplification, only the specific sequence of the Forward primer (SsF) was obtained from literature (ref. 17 Hashmi et al., 2005). The Reverse primer was designed ex novo with the help of computerized programs, as described above. 20 nt probes were obtained, with the polymorphism at the center of the specific probes; in addition, for the allele s, 18 nt, 19 nt and 21 nt probes were also tested at various temperatures.
A specific distinguishing signal was obtained in the hybridization phase with the 20 nt probe at a temperature of 54° C.
Lutheran System (Lua/Lub): Only the Reverse primer sequence described in literature was used for the specific amplification phase (Elnemer et al., 1997). The Forward primer was decided ex novo. 18 nt and 20 nt probes were used with the polymorphism at the center.
A specific signal was obtained in the hybridization phase at a temperature of 45° C. with 18 nt probes.
In order to obtain a specific hybridization at a temperature of 54° C. we also tried to use 20 nt probes but without any results.
Coupling of the Oligonucleotide Probes to the Fluorescently-Labeled Microspheres
The various oligonucleotide probes modified at the 5′-end were conjugated, in separate reactions, with different classifications of carboxylated microspheres, according to the coupling protocol suggested by Luminex Corporation (Oligonucleotide Coupling Protocol).
An aliquot of each specific region containing 5×106 microspheres was microcentrifuged at 10,000 rpm for 2 minutes, the supernatant removed and the pellet resuspended in 50 μl of MES buffer 0.1 M, at pH 4.5. 0.2 nanomoles of amino-modified oligonucleotide probes were then added to the mixture.
An aqueous solution of 1-ethyl-3-(3-dimethylaminopropyl-carbodiimide HCl (EDC; 10 mg/ml) was then added to the mixture of microspheres/oligonucleotides and the resulting mixture was incubated at room temperature for 30 minutes in the dark. The addition of EDC and the incubation were repeated one more time. After a total incubation of 1 hour, the microspheres were washed with 1 ml of Tween-20 at 0.02%. The wash solution was removed by centrifugation, the wash step was repeated with 1 ml of SDS at 0.1% and the final mixture was resuspended in 100 μl of TE (Elution Buffer-QIAGEN), at pH 8 and preserved in the dark at 4° C. Before use, the microspheres were brought to room temperature for 5 minutes. The coupled microspheres, thus obtained, have an expected theoretic concentration of 50,000 microspheres/μl.
Amplification of Target DNA
The primers used for the amplification of all the systems in question are described in Table 1. The primers were used for the amplification of the specific locus under examination.
At least one primer of each primer pair was synthesized with Biotin TEG modification at the 5′-end, to label the target strand of the amplicon and detect hybridization of the specific probe with the target DNA, according to the indications of Luminex Corporation (synthesis and purification and modification of primers and probes by Primm).
The PCR was carried out with 0.5 μM of primer, 2-0.5 μL of genomic DNA (25-100 ng), 0.2 mM of dNTP, 1 mM of MgCl2 (from 25 mM Applied Biosystem), 1×PCR Buffer (from 10× Applied Biosystem) and 0.5 U of Taq (GoTaq Promega). The final reaction volume is equal to 20 μl.
Mastercycler epgradient S (Eppendorf) was used for the thermal cycles using the following parameters: 2 minutes of initial DNA denaturation at 94° C., followed by 35 cycles at 94° C. for 20 seconds, 60° C. for 20 seconds, 72° C. for 30 seconds, with a final elongation phase at 72° C. for 5 minutes. The amplification products obtained can be visualized by electrophoresis on agarose gel at 2%.
Multiplex PCR Reactions
The authors set up 2 multiplex PCR reactions, in order to minimize laboriousness and hands-on time. PCRs were divided in two separate multiplex reactions: PCR (I) and PCR (II) (see Table below) on the basis of the frequency of use in the Laboratory and, therefore, the utility of the reactions, i.e. PCR (I) amplifies the systems for which samples are tested for on a routine basis; PCR (II) amplifies those systems that are tested for less frequently.














Target alleles
Primer sequence (5′-3′)









K/k
Fw: TTTAgTCCTCACTCCCATgCTTCC



PCR (I)
Rw: TATCACACAggTgTCCTCTCTTCC



 



Kpa/Kpb
Fw: TgAggCCAggAgAAAAgCA



PCR (II)
Rw: TgACCATCTggAAgAgCTTgC



 



Jsa/Jsb
Fw: AACTTTgCCATgCTCCTgg



PCR (II)
Rw: gCCCTTgACACTTgCATACCT



 



Lua/Lub
Fw: CTgAggAgCgCTgggACACCCgg



PCR (II)
Rw: CCCCgggTgTCgTgCATT



 



S/s
Fw: AAgACTgACACATTACCTCA



PCR (I)
Rw: AACATACCTggTACAgTgAA



 



Coa/Cob
Fw: TATAAATAggCCCAgCCCAg



PCR (II)
Rw: CCAgCgACACCTTCACgTT



 



Fya/Fyb
Fw: CTTCCggTgTAACTCTgATgg



PCR (I)
Rw: CATCCAgCAggTTACAggAgT



 



Jka/Jkb
Fw: CATgCTgCCATAggATCATTgC



PCR (I)
Rw: gAgCCAggAggTgggTTTgC










The PCR is carried out with 0.3 μM of each primer, 4 μL of genomic DNA (100-400 ng), 0.2 mM of dNTP, 1.5 mM of MgCl2 (from 25 mM Applied Biosystem), 1.5×PCR Buffer (from 10× Applied Biosystem) and 4 U of Taq (GoTaq Promega). The final reaction volume is equal to 50 μl. PCR parameters remain the same as for the single PCR reactions.
Hybridization
After the DNA amplification, 4 μl from each amplification reaction were transferred to 96-well microplates and diluted with 13 μl of TE Buffer. They were then sealed with adhesive film and heat denatured at 99° C. for 10 minutes with the use of a preheated thermal cycler.
The hybridization of the PCR products with the three probes for each system object of the study (two specific probes and a negative control) is effected by diluting the single probes, in the hybridization buffer supplied by One Lambda, Inc. LABType hybridization buffer), at a final concentration of 150 microspheres per microliter. The coupled microspheres, as described above, have a theoretical recovery of 50,000 microspheres per microliter.
After denaturation of the PCR products, 33 μl of microspheres diluted in hybridization solution, are added to each sample.
The samples were mixed and the microplate rapidly transferred to the thermal cycler preheated to the specific optimal hybridization temperature for each system as indicated in Table 2.
The hybridization is carried out for 15 minutes and immediately afterwards 100 μl of wash buffer are added (LABType wash buffer—One lambda Inc.).
The wash steps were carried out at room temperature by centrifugation (2,800 rpm for 5 minutes) with elimination of the supernatant by manual inversion of the plate. The samples are washed for a total of three times.
The samples are subsequently incubated for 5 minutes, at the same hybridization temperature, with 50 μl of a freshly prepared solution of 1×SAPE (0.5 mg/l streptavidin-R-phycoerythrin) in a dilution buffer supplied by One Lambda Inc. (LABType SAPE-Buffer).
At the end of the incubation, 100 μl of LABType wash buffer were rapidly added to each well (One Lambda, Inc). The microspheres were re-pelleted by centrifugation and the supernatant removed by inversion. Each sample was then resuspended in 80 μl of Sheath Fluid buffer supplied by Luminex. The plate was ready to be analyzed at the flow-cytometer-based instrument.
If it is not possible to analyze the samples immediately, the analysis plate can be preserved at +4° C. in the dark, up to a maximum of 24 hours.
Data Acquisition and Analysis
The samples were analyzed using a LAB ScanTM100 (Luminex Corporation, Austin, Tex.).
The instrument is equipped with two laser sources of which one is a 635-nm red diode laser which excites the red and infrared fluorochromes and the other a 532-nm laser which excites the phycoerythrin (PE) reporter fluorochrome.
Each microsphere has a unique spectral address which can be identified by the instrument.
Two parameters, the count and median fluorescence intensity (MFI) are monitored for each data acquisition.
The count for each microsphere (single specific region) should be at least 100. The median fluorescence intensity (MFI) represents the average reporter fluorescence for the counted spheres, as previously described.
Allelic Ratio Determination:
The fluorescence intensity, generated by Luminex software, represents the MFI of each microsphere (or probe coupled with the microsphere) for each sample.
For each system studied, the allelic ratio was calculated in order to obtain a numerical value which, when analyzed on the basis of the reference threshold value, allows to distinguish between homozygote samples for each allele or heterozygote samples as indicated in Tables 3-10 (ref. 7 Beiboer et al. 2005).
In order to define the allelic ratio for each system, samples with a known typing obtained with hemagglutination and/or PCR-SSP, were tested.
The ratio value is obtained for each system from the ratio between the median fluorescence intensity (MFI) of the probe Xa, intended as being the most frequent allele in the Caucasian population, and the MFI sum of both alleles (Xa and Xb) of the system implied, as indicated in the following formula:

(MFIa−MFINC)/(MFIa−MFINC+MFIb−MFINC)

The allele-specific MFI values minus the MFI value generated by the negative control (NC) probe are used in the formula for each sample. On the basis of the samples tested, it was possible to define an allelic ratio for each system. The data obtained are indicated in Tables 3-10 and in the cumulative Table 11 hereunder.
The raw fluorescence data registered by the instrument are then processed. In this case, in the analysis program created in Excel, the necessary mathematical formulae were established for automatically obtaining, for each specific probe (allele Xa and allele Xb), the fluorescence values minus the negative control value (for example, MFI probe Xa-MFI NC probe). This corrected MFI value is then used for calculating the allelic ratio of each single sample as previously described. An example of the spreadsheet form prepared in Excel for the data analysis is provided hereunder.











Worksheet:
Date:












MFI
MFIAllele-
Allele















S
s
NC
MFINC
ratio

















ID
084
085
087
S
s
s/(S + s)
Genotyping













1A
144597



1B
144596


1C
144595


1D
144594


1E
144591


1F
144590


1G
144589


1H
144588









In formulating the table, the automatic conclusion of the typing was based on the reference allelic ratios. The typing cannot be automatically concluded if the allelic ratio obtained does not fall within the cutoff ranges established. In this case an automatic warning message appears.
The results obtained for each sample are also confirmed only if the fluorescence produced by the negative control probe does not exceed the value of 100 and if the sum of the fluorescence values of the specific probes is higher than four times the negative control value of the sample (MFIa+MFIb>4×MFINC). These formulae were also included in Excel.
The data analysis is easy, rapid and does not require complicated application software.
A list of the tables (3-10) of the allelic ratios obtained from the single samples tested is provided below, whereas Table 11 indicates the allelic ratio values used as reference (cutoff) range, obtained from the average of the ratios of the single samples plus and/or minus two standard deviations.
The allelic ratios of the single samples:











TABLE 3








Allelic ratio




Nr.
a/(a + b)
Genotype





















1
1.000
Coa/Coa
Coa/Coa > 0.941



2
1.000
Coa/Coa



3
0.995
Coa/Coa



4
0.992
Coa/Coa



5
0.990
Coa/Coa



6
0.989
Coa/Coa



7
0.984
Coa/Coa



8
0.984
Coa/Coa



9
0.984
Coa/Coa



10
0.984
Coa/Coa



11
0.984
Coa/Coa



12
0.980
Coa/Coa



13
0.980
Coa/Coa



14
0.979
Coa/Coa



15
0.978
Coa/Coa



16
0.976
Coa/Coa



17
0.976
Coa/Coa



18
0.976
Coa/Coa



19
0.975
Coa/Coa



20
0.973
Coa/Coa



21
0.973
Coa/Coa



22
0.972
Coa/Coa



23
0.971
Coa/Coa



24
0.971
Coa/Coa



25
0.970
Coa/Coa



26
0.970
Coa/Coa



27
0.969
Coa/Coa



28
0.968
Coa/Coa



29
0.968
Coa/Coa



30
0.967
Coa/Coa



31
0.967
Coa/Coa



32
0.966
Coa/Coa



33
0.966
Coa/Coa



34
0.966
Coa/Coa



35
0.965
Coa/Coa



36
0.965
Coa/Coa



37
0.965
Coa/Coa



38
0.964
Coa/Coa



39
0.964
Coa/Coa



40
0.963
Coa/Coa



41
0.963
Coa/Coa



42
0.963
Coa/Coa



43
0.962
Coa/Coa



44
0.962
Coa/Coa



45
0.962
Coa/Coa



46
0.961
Coa/Coa



47
0.961
Coa/Coa



48
0.960
Coa/Coa



49
0.960
Coa/Coa



50
0.959
Coa/Coa



51
0.958
Coa/Coa



52
0.958
Coa/Coa



53
0.958
Coa/Coa



54
0.958
Coa/Coa



55
0.958
Coa/Coa



56
0.956
Coa/Coa



57
0.954
Coa/Coa



58
0.954
Coa/Coa



59
0.950
Coa/Coa



60
0.948
Coa/Coa



61
0.946
Coa/Coa



62
0.944
Coa/Coa



63
0.923
Coa/Coa



64
0.747
Coa/Cob
0.652 < Coa/Cob < 0.754



65
0.723
Coa/Cob



66
0.717
Coa/Cob



67
0.712
Coa/Cob



68
0.704
Coa/Cob



69
0.701
Coa/Cob



70
0.690
Coa/Cob



71
0.675
Coa/Cob



72
0.663
Coa/Cob



73
0.106
Cob/Cob
Cob/Cob < 0.116



74
0.009
Cob/Cob



75
0.007
Cob/Cob



76
0.002
Cob/Cob



77
0.002
Cob/Cob





















TABLE 4








Allelic ratio




Nr.
b/(a + b)
Genotype





















1
0.996
Fyb/Fyb
Fyb/Fyb > 0.931



2
0.996
Fyb/Fyb



3
0.996
Fyb/Fyb



4
0.994
Fyb/Fyb



5
0.993
Fyb/Fyb



6
0.992
Fyb/Fyb



7
0.992
Fyb/Fyb



8
0.991
Fyb/Fyb



9
0.990
Fyb/Fyb



10
0.986
Fyb/Fyb



11
0.985
Fyb/Fyb



12
0.984
Fyb/Fyb



13
0.984
Fyb/Fyb



14
0.983
Fyb/Fyb



15
0.981
Fyb/Fyb



16
0.981
Fyb/Fyb



17
0.981
Fyb/Fyb



18
0.980
Fyb/Fyb



19
0.978
Fyb/Fyb



20
0.976
Fyb/Fyb



21
0.975
Fyb/Fyb



22
0.974
Fyb/Fyb



23
0.974
Fyb/Fyb



24
0.974
Fyb/Fyb



25
0.972
Fyb/Fyb



26
0.971
Fyb/Fyb



27
0.971
Fyb/Fyb



28
0.970
Fyb/Fyb



29
0.969
Fyb/Fyb



30
0.968
Fyb/Fyb



31
0.968
Fyb/Fyb



32
0.967
Fyb/Fyb



33
0.966
Fyb/Fyb



34
0.965
Fyb/Fyb



35
0.965
Fyb/Fyb



36
0.965
Fyb/Fyb



37
0.963
Fyb/Fyb



38
0.960
Fyb/Fyb



39
0.959
Fyb/Fyb



40
0.958
Fyb/Fyb



41
0.958
Fyb/Fyb



42
0.956
Fyb/Fyb



43
0.953
Fyb/Fyb



44
0.949
Fyb/Fyb



45
0.948
Fyb/Fyb



46
0.946
Fyb/Fyb



47
0.945
Fyb/Fyb



48
0.944
Fyb/Fyb



49
0.939
Fyb/Fyb



50
0.935
Fyb/Fyb



51
0.930
Fyb/Fyb



52
0.929
Fyb/Fyb



53
0.920
Fyb/Fyb



54
0.888
Fya/Fyb
0.752 < Fya/Fyb < 0.902



55
0.888
Fya/Fyb



56
0.871
Fya/Fyb



57
0.868
Fya/Fyb



58
0.862
Fya/Fyb



59
0.862
Fya/Fyb



60
0.860
Fya/Fyb



61
0.854
Fya/Fyb



62
0.851
Fya/Fyb



63
0.841
Fya/Fyb



64
0.831
Fya/Fyb



65
0.819
Fya/Fyb



66
0.817
Fya/Fyb



67
0.816
Fya/Fyb



68
0.812
Fya/Fyb



69
0.808
Fya/Fyb



70
0.792
Fya/Fyb



71
0.788
Fya/Fyb



72
0.787
Fya/Fyb



73
0.787
Fya/Fyb



74
0.779
Fya/Fyb



75
0.772
Fya/Fyb



76
0.772
Fya/Fyb



77
0.070
Fya/Fya
Fya/Fya < 0.050



78
0.049
Fya/Fya



79
0.041
Fya/Fya



80
0.040
Fya/Fya



81
0.034
Fya/Fya



82
0.032
Fya/Fya



83
0.029
Fya/Fya



84
0.025
Fya/Fya



85
0.025
Fya/Fya



86
0.024
Fya/Fya



87
0.024
Fya/Fya



88
0.018
Fya/Fya



89
0.017
Fya/Fya



90
0.016
Fya/Fya



91
0.016
Fya/Fya



92
0.016
Fya/Fya



93
0.016
Fya/Fya



94
0.015
Fya/Fya



95
0.014
Fya/Fya



96
0.013
Fya/Fya



97
0.012
Fya/Fya



98
0.010
Fya/Fya



99
0.009
Fya/Fya



100
0.007
Fya/Fya



101
0.007
Fya/Fya



102
0.006
Fya/Fya



103
0.006
Fya/Fya



104
0.002
Fya/Fya



105
0.002
Fya/Fya



106
0.000
Fya/Fya



107
0.000
Fya/Fya



108
0.000
Fya/Fya





















TABLE 5








Allelic ratio




Nr.
a/(a + b)
Genotype





















1
1.000
Jka/Jka
Jka/Jka > 0.870



2
1.000
Jka/Jka



3
1.000
Jka/Jka



4
0.988
Jka/Jka



5
0.975
Jka/Jka



6
0.974
Jka/Jka



7
0.960
Jka/Jka



8
0.954
Jka/Jka



9
0.942
Jka/Jka



10
0.942
Jka/Jka



11
0.939
Jka/Jka



12
0.938
Jka/Jka



13
0.934
Jka/Jka



14
0.934
Jka/Jka



15
0.930
Jka/Jka



16
0.930
Jka/Jka



17
0.924
Jka/Jka



18
0.924
Jka/Jka



19
0.923
Jka/Jka



20
0.921
Jka/Jka



21
0.921
Jka/Jka



22
0.920
Jka/Jka



23
0.920
Jka/Jka



24
0.919
Jka/Jka



25
0.917
Jka/Jka



26
0.913
Jka/Jka



27
0.913
Jka/Jka



28
0.911
Jka/Jka



29
0.910
Jka/Jka



30
0.910
Jka/Jka



31
0.909
Jka/Jka



32
0.909
Jka/Jka



33
0.907
Jka/Jka



34
0.907
Jka/Jka



35
0.906
Jka/Jka



36
0.906
Jka/Jka



37
0.902
Jka/Jka



38
0.895
Jka/Jka



39
0.873
Jka/Jka



40
0.269
Jka/Jkb
0.175 < Jka/Jkb < 0.260



41
0.256
Jka/Jkb



42
0.243
Jka/Jkb



43
0.240
Jka/Jkb



44
0.239
Jka/Jkb



45
0.233
Jka/Jkb



46
0.221
Jka/Jkb



47
0.221
Jka/Jkb



48
0.218
Jka/Jkb



49
0.215
Jka/Jkb



50
0.212
Jka/Jkb



51
0.212
Jka/Jkb



52
0.210
Jka/Jkb



53
0.209
Jka/Jkb



54
0.206
Jka/Jkb



55
0.204
Jka/Jkb



56
0.203
Jka/Jkb



57
0.203
Jka/Jkb



58
0.200
Jka/Jkb



59
0.197
Jka/Jkb



60
0.193
Jka/Jkb



61
0.184
Jka/Jkb



62
0.016
Jkb/Jkb
Jkb/Jkb < 0.016



63
0.014
Jkb/Jkb



64
0.014
Jkb/Jkb



65
0.014
Jkb/Jkb



66
0.013
Jkb/Jkb



67
0.011
Jkb/Jkb



68
0.010
Jkb/Jkb



69
0.009
Jkb/Jkb



70
0.009
Jkb/Jkb



71
0.009
Jkb/Jkb



72
0.009
Jkb/Jkb



73
0.009
Jkb/Jkb



74
0.008
Jkb/Jkb



75
0.008
Jkb/Jkb



76
0.007
Jkb/Jkb



77
0.007
Jkb/Jkb



78
0.004
Jkb/Jkb



79
0.004
Jkb/Jkb



80
0.001
Jkb/Jkb



81
0.001
Jkb/Jkb



82
0.001
Jkb/Jkb



83
0.000
Jkb/Jkb



84
0.000
Jkb/Jkb



85
0.000
Jkb/Jkb



86
0.000
Jkb/Jkb



87
0.000
Jkb/Jkb



88
0.000
Jkb/Jkb



89
0.000
Jkb/Jkb



90
0.000
Jkb/Jkb



91
0.000
Jkb/Jkb



92
0.000
Jkb/Jkb



93
0.000
Jkb/Jkb



94
0.000
Jkb/Jkb




















TABLE 6






Allelic ratio




Nr.
b/(a + b)
Genotype


















1
1.000
Jsb/Jsb
Jsb/Jsb > 0.831


2
1.000
Jsb/Jsb


3
1.000
Jsb/Jsb


4
1.000
Jsb/Jsb


5
1.000
Jsb/Jsb


6
1.000
Jsb/Jsb


7
1.000
Jsb/Jsb


8
1.000
Jsb/Jsb


9
1.000
Jsb/Jsb


10
1.000
Jsb/Jsb


11
1.000
Jsb/Jsb


12
0.993
Jsb/Jsb


13
0.993
Jsb/Jsb


14
0.991
Jsb/Jsb


15
0.990
Jsb/Jsb


16
0.988
Jsb/Jsb


17
0.987
Jsb/Jsb


18
0.986
Jsb/Jsb


19
0.984
Jsb/Jsb


20
0.983
Jsb/Jsb


21
0.983
Jsb/Jsb


22
0.979
Jsb/Jsb


23
0.979
Jsb/Jsb


24
0.978
Jsb/Jsb


25
0.973
Jsb/Jsb


26
0.973
Jsb/Jsb


27
0.972
Jsb/Jsb


28
0.972
Jsb/Jsb


29
0.971
Jsb/Jsb


30
0.967
Jsb/Jsb


31
0.966
Jsb/Jsb


32
0.964
Jsb/Jsb


33
0.962
Jsb/Jsb


34
0.960
Jsb/Jsb


35
0.955
Jsb/Jsb


36
0.953
Jsb/Jsb


37
0.953
Jsb/Jsb


38
0.949
Jsb/Jsb


39
0.949
Jsb/Jsb


40
0.949
Jsb/Jsb


41
0.948
Jsb/Jsb


42
0.946
Jsb/Jsb


43
0.944
Jsb/Jsb


44
0.940
Jsb/Jsb


45
0.932
Jsb/Jsb


46
0.931
Jsb/Jsb


47
0.930
Jsb/Jsb


48
0.929
Jsb/Jsb


49
0.929
Jsb/Jsb


50
0.923
Jsb/Jsb


51
0.916
Jsb/Jsb


52
0.908
Jsb/Jsb


53
0.900
Jsb/Jsb


54
0.899
Jsb/Jsb


55
0.899
Jsb/Jsb


56
0.897
Jsb/Jsb


57
0.896
Jsb/Jsb


58
0.894
Jsb/Jsb


59
0.893
Jsb/Jsb


60
0.893
Jsb/Jsb


61
0.893
Jsb/Jsb


62
0.891
Jsb/Jsb


63
0.891
Jsb/Jsb


64
0.887
Jsb/Jsb


65
0.886
Jsb/Jsb


66
0.885
Jsb/Jsb


67
0.879
Jsb/Jsb


68
0.877
Jsb/Jsb


69
0.877
Jsb/Jsb


70
0.875
Jsb/Jsb


71
0.868
Jsb/Jsb


72
0.857
Jsb/Jsb


73
0.853
Jsb/Jsb


74
0.852
Jsb/Jsb


75
0.852
Jsb/Jsb


76
0.845
Jsb/Jsb


77
0.838
Jsb/Jsb


78
0.835
Jsb/Jsb


79
0.830
Jsb/Jsb


80
0.828
Jsb/Jsb


81
0.545
Jsa/Jsb
0.509 < Jsa/Jsb < 0.562


82
0.526
Jsa/Jsb



















TABLE 7






Allelic ratio




Nr.
k/(K + k)
Genotype


















1
0.830
kk



2
0.828
kk


3
0.807
kk


4
0.807
kk


5
0.792
kk


6
0.790
kk


7
0.787
kk


8
0.783
kk


9
0.775
kk


10
0.773
kk


11
0.772
kk


12
0.772
kk


13
0.771
kk


14
0.770
kk


15
0.769
kk


16
0.767
kk


17
0.764
kk


18
0.763
kk


19
0.763
kk


20
0.759
kk


21
0.759
kk


22
0.759
kk


23
0.757
kk


24
0.756
kk


25
0.754
kk


26
0.754
kk


27
0.752
kk


28
0.748
kk


29
0.748
kk


30
0.748
kk


31
0.744
kk


32
0.743
kk


33
0.742
kk


34
0.741
kk


35
0.737
kk


36
0.735
kk


37
0.732
kk


38
0.731
kk


39
0.729
kk


40
0.729
kk


41
0.726
kk


42
0.726
kk


43
0.726
kk


44
0.724
kk


45
0.723
kk


46
0.722
kk


47
0.721
kk


48
0.710
kk


49
0.710
kk


50
0.710
kk


51
0.709
kk


52
0.709
kk


53
0.708
kk


54
0.708
kk


55
0.706
kk


56
0.704
kk


57
0.699
kk


58
0.697
kk


59
0.697
kk


60
0.696
kk


61
0.695
kk


62
0.695
kk


63
0.692
kk


64
0.692
kk


65
0.691
kk


66
0.690
kk


67
0.683
kk


68
0.681
kk


69
0.680
kk


70
0.679
kk


71
0.673
kk


72
0.673
kk


73
0.672
kk


74
0.669
kk


75
0.668
kk


76
0.665
kk


77
0.664
kk


78
0.656
kk


79
0.653
kk


80
0.549
kK
0.502 < K/k < 0.550


81
0.537
kK


82
0.535
kK


83
0.531
kK


84
0.531
kK


85
0.527
kK


86
0.521
kK


87
0.520
kK


88
0.518
kK


89
0.517
kK


90
0.504
kK


91
0.025
KK
K/K < 0.036


92
0.022
KK


93
0.006
KK


94
0.004
KK



















TABLE 8






Allelic ratio




Nr.
b/(b + a)
Genotype


















1
0.953
Kpb/Kpb
Kpb/Kpb > 0.867


2
0.951
Kpb/Kpb


3
0.950
Kpb/Kpb


4
0.947
Kpb/Kpb


5
0.946
Kpb/Kpb


6
0.945
Kpb/Kpb


7
0.943
Kpb/Kpb


8
0.943
Kpb/Kpb


9
0.942
Kpb/Kpb


10
0.942
Kpb/Kpb


11
0.940
Kpb/Kpb


12
0.939
Kpb/Kpb


13
0.937
Kpb/Kpb


14
0.934
Kpb/Kpb


15
0.933
Kpb/Kpb


16
0.932
Kpb/Kpb


17
0.930
Kpb/Kpb


18
0.929
Kpb/Kpb


19
0.928
Kpb/Kpb


20
0.928
Kpb/Kpb


21
0.927
Kpb/Kpb


22
0.926
Kpb/Kpb


23
0.925
Kpb/Kpb


24
0.925
Kpb/Kpb


25
0.924
Kpb/Kpb


26
0.923
Kpb/Kpb


27
0.921
Kpb/Kpb


28
0.921
Kpb/Kpb


29
0.921
Kpb/Kpb


30
0.919
Kpb/Kpb


31
0.918
Kpb/Kpb


32
0.916
Kpb/Kpb


33
0.915
Kpb/Kpb


34
0.915
Kpb/Kpb


35
0.915
Kpb/Kpb


36
0.914
Kpb/Kpb


37
0.912
Kpb/Kpb


38
0.912
Kpb/Kpb


39
0.912
Kpb/Kpb


40
0.911
Kpb/Kpb


41
0.911
Kpb/Kpb


42
0.911
Kpb/Kpb


43
0.909
Kpb/Kpb


44
0.909
Kpb/Kpb


45
0.908
Kpb/Kpb


46
0.908
Kpb/Kpb


47
0.908
Kpb/Kpb


48
0.905
Kpb/Kpb


49
0.905
Kpb/Kpb


50
0.905
Kpb/Kpb


51
0.900
Kpb/Kpb


52
0.897
Kpb/Kpb


53
0.893
Kpb/Kpb


54
0.893
Kpb/Kpb


55
0.890
Kpb/Kpb


56
0.890
Kpb/Kpb


57
0.889
Kpb/Kpb


58
0.888
Kpb/Kpb


59
0.888
Kpb/Kpb


60
0.888
Kpb/Kpb


61
0.86
Kpb/Kpb


62
0.882
Kpb/Kpb


63
0.882
Kpb/Kpb


64
0.882
Kpb/Kpb


65
0.880
Kpb/Kpb


66
0.872
Kpb/Kpb


67
0.862
Kpb/Kpb


68
0.862
Kpb/Kpb


69
0.853
Kpb/Kpb


70
0.357
Kpa/Kpb
0.342 < Kpa/Kpb < 0.364


71
0.349
Kpa/Kpb


72
0.025
Kpa/Kpa
Kpa/Kpa < 0.031


73
0.005
Kpa/Kpa


74
0.004
Kpa/Kpa


75
0.003
Kpa/Kpa



















TABLE 9






Allelic





ratio


Nr.
s/(S + s)
Genotype


















1
1.000
ss
s/s > 0.860


2
0.992
ss


3
0.990
ss


4
0.989
ss


5
0.981
ss


6
0.979
ss


7
0.979
ss


8
0.978
ss


9
0.977
ss


10
0.976
ss


11
0.972
ss


12
0.967
ss


13
0.964
ss


14
0.961
ss


15
0.958
ss


16
0.956
ss


17
0.955
ss


18
0.954
ss


19
0.951
ss


20
0.948
ss


21
0.947
ss


22
0.946
ss


23
0.945
ss


24
0.944
ss


25
0.944
ss


26
0.943
ss


27
0.942
ss


28
0.941
ss


29
0.939
ss


30
0.938
ss


31
0.936
ss


32
0.936
ss


33
0.932
ss


34
0.929
ss


35
0.928
ss


36
0.925
ss


37
0.922
ss


38
0.920
ss


39
0.918
ss


40
0.909
ss


41
0.909
ss


42
0.908
ss


43
0.895
ss


44
0.887
ss


45
0.885
ss


46
0.883
ss


47
0.879
ss


48
0.879
ss


49
0.878
ss


50
0.878
ss


51
0.880
ss


52
0.073
sS
0.014 < S/s < 0.059


53
0.052
sS


54
0.050
sS


55
0.047
sS


56
0.045
sS


57
0.038
sS


58
0.036
sS


59
0.035
sS


60
0.034
sS


61
0.034
sS


62
0.034
sS


63
0.033
sS


64
0.033
sS


65
0.032
sS


66
0.032
sS


67
0.032
sS


68
0.032
sS


69
0.032
sS


70
0.031
sS


71
0.030
sS


72
0.030
sS


73
0.029
sS


74
0.016
sS


75
0.009
SS
S/S < 0.009


76
0.008
SS


77
0.007
SS


78
0.007
SS


79
0.006
SS


80
0.006
SS


81
0.005
SS


82
0.005
SS


83
0.004
SS


84
0.004
SS


85
0.004
SS


86
0.004
SS


87
0.003
SS


88
0.003
SS


89
0.002
SS


90
0.002
SS


91
0.002
SS


92
0.002
SS


93
0.002
SS


94
0.001
SS


95
0.001
SS


96
0.001
SS


97
0.000
SS



















TABLE 10






Allelic ratio




Nr.
b/(a + b)
Genotype


















1
1.000
Lub/Lub
Lub/Lub < 0.880


2
1.000
Lub/Lub


3
1.000
Lub/Lub


4
1.000
Lub/Lub


5
1.000
Lub/Lub


6
1.000
Lub/Lub


7
1.000
Lub/Lub


8
0.998
Lub/Lub


9
0.986
Lub/Lub


10
0.985
Lub/Lub


11
0.980
Lub/Lub


12
0.970
Lub/Lub


13
0.969
Lub/Lub


14
0.958
Lub/Lub


15
0.957
Lub/Lub


16
0.956
Lub/Lub


17
0.956
Lub/Lub


18
0.950
Lub/Lub


19
0.949
Lub/Lub


20
0.947
Lub/Lub


21
0.947
Lub/Lub


22
0.947
Lub/Lub


23
0.947
Lub/Lub


24
0.946
Lub/Lub


25
0.945
Lub/Lub


26
0.944
Lub/Lub


27
0.942
Lub/Lub


28
0.941
Lub/Lub


29
0.940
Lub/Lub


30
0.940
Lub/Lub


31
0.939
Lub/Lub


32
0.938
Lub/Lub


33
0.936
Lub/Lub


34
0.933
Lub/Lub


35
0.932
Lub/Lub


36
0.930
Lub/Lub


37
0.928
Lub/Lub


38
0.927
Lub/Lub


39
0.925
Lub/Lub


40
0.922
Lub/Lub


41
0.912
Lub/Lub


42
0.910
Lub/Lub


43
0.907
Lub/Lub


44
0.907
Lub/Lub


45
0.901
Lub/Lub


46
0.901
Lub/Lub


47
0.899
Lub/Lub


48
0.898
Lub/Lub


49
0.892
Lub/Lub


50
0.872
Lub/Lub


51
0.651
Lua/Lub
0.540 < Lua/Lub < 0.695


52
0.627
Lua/Lub


53
0.575
Lua/Lub


54
0.301
Lua/Lua
Lua/Lua < 0.307


55
0.291
Lua/Lua


56
0.289
Lua/Lua

















TABLE 11





System
Allelic ratio for the determination of genotype*


















MNS
s/s > 0.860
0.059 > S/s > 0.014
S/S < 0.009


Duffy
Fyb/Fyb > 0.931
0.902 > Fya/Fyb > 0.752
Fya/Fya > 0.050


Kell
Kpb/Kpb > 0.867
0.364 > KPa/KPb > 0.342
Kpa/Kpa < 0.031



Jsb/Jsb > 0.831
0.562 > Jsa/Jsb > 0.509
Jsa/Jsa < ND**



k/k > 0.647
0.550 > K/k > 0.502
K/K < 0.036


Lu
Lub/Lub > 0.880
0.695 > Lua/Lub > 0.540
Lua/Lua < 0.307


Co
Coa/Coa > 0.941
0.754 > Coa/Cob >
Cob/Cob <




0.652
0.116


Jk
Jka/Jka > 0.870
0.260 > Jka/Jkb > 0.175
Jkb/Jkb < 0.016





*reference ranges obtained from the average of the allelic ratios of the single samples plus and/or minus two standard deviations


**no available Jsa/Jsa samples






BIBLIOGRAPHY


    1) Castilho L. et al. Transfusion 2002; 42(2):232-240
    2) Montalvo L. et al. Transfusion 2004; 44(5):694-702
    3) Reid M E. Vox Sanguinis 2002; 83(1): 91-93
    4) Ferri G. et al. Journal of Forensic Sciences 2006; 51:357-360
    5) Denomme G. et al. Transfusion 2005; 45: 660-666
    6) Petrik J. Vox Sanguinis 2001; 80: 1-11
    7) Beiboer S. et al. Transfusion 2005; 45:667-679
    8) Kellar K L. et al., J. Immunol. Methods 2003; 279(1-2): 277-285
    9) Kettman J R et al. Cytometry 1998; 33(2): 234-243
    10) Fulton R F et al. Clinical Chemistry 1997; 43(9): 1749-1756
    11) Earley M C et al. Cytometry 2002; 50(5): 239-242
    12) Colinas R F et al. Clinical Chemistry 2000; 46 (7): 996-998
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    14) Dunbar S A et al. 2005; Methods Mol Med 114: 147-71
    15) Dunbar S A et al. 2006; Clinica Chimica Acta (363) 71-82
    16) Lee et al. 1997; Vox Sanguinis 73 (1): 1-11
    17) Hashmi et al. 2005; Transfusion 45: 680-688
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    19) Irshaid et al. 1998; British Journal of Haematology 102: 1010-1014
    20) Deregt D. et al. 2006; Journal of Virological Methods 136:7-23
    21) Schmitt M. et al. 2006; J. Clin Microbiol (44) 2: 504-512
    22) Diaz M. JCM August 2005; (43) 3662-3672

## Claims

1. A method for the identification and typing of at least one single nuclear polymorphism (SNP) of the erythrocyte system X for discriminating between heterozygote and homozygote individuals which comprises:
a) contacting said single nuclear polymorphism (SNP) with at least one set or more than one set of oligonucleotide probes amino-modified at the 5′-end, wherein said oligonucleotide probes have a sequence length ranging from 18 to 20 nucleotides containing at or near the center of the probe sequence, the specific single nuclear polymorphism (SNP) for each target alleles belonging to the erythrocyte system X that are selected from the group consisting of Kpa/Kpb, and Fya/Fyb, said probes being capable of specifically hybridizing to each of said target alleles; wherein said probes are coupled to a microsphere labeled with at least one fluorescent substance and consist of at least one probe/probe set of oligonucleotide sequences selected from the group consisting of
Probe/probe set 1: Kpb/ATCACTTCACggCTgTTCCA (SEQ ID NO: 20) and Kpa ATCACTTCATggCTgTTCCAg (SEQ ID NO:21); and probe/probe set 7: Fya/GAgACTATggTgCCAACCTg (SEQ ID NO: 35) and Fyb TggAgACTATgATgCCAACC (SEQ ID NO: 36);
b) hybridizing said at least one set or more than one set of oligonucleotide probes to target alleles containing the one single nuclear polymorphism (SNP) at the following hybridization temperature ranges: probe set 1: 45-50° C.; and probe set 7: 52-56° C.; for the identification and typing of at least one single nuclear polymorphism (SNP) of an allelic pair X selected from the group consisting of Kpa/Kpb and Fya/Fyb using non-specific negative probes as control probes and
c) detecting the presence of fluorescence with a flow cytometer-based instrument to identify and type said at least one single nuclear polymorphism (SNP) in order to discriminate between heterozygote and homozygote individuals wherein a negative control is used with probe set 1 consisting of AACTCTACggggCTCTTCgA (SEQ ID NO: 22); and a negative control is used with probe set 7 consisting of AggCTATCCTgACAAgCTT (SEQ ID NO: 37).

2. A method for the identification and typing of at least one single nucleotide polymorphism (SNP) of the erythrocyte system X in heterozygote and homozygote individuals for discriminating between homozygous and heterozygous samples, comprising the following phases:
a) extracting DNA from a biological sample;
b) amplifying by PCR, the genomic locus comprising the SNP of the erythrocyte system of interest, by means of at least one specific pair of primers for a target allele selected from the group consisting of: pair 1: Kpa/Kpb/Fw: TgAggCCAggAgAAAAgCA (SEQ ID NO:3) and Rw: TgACCATCTggAAgAgCTTgC (SEQ ID NO:4); and pair 2:Fya/Fyb/Fw: CTTCCggTgTAACTCTgATgg (SEQ ID NO:13) and Rw:ATCCAgCAGGTTACAggAgT (SEQ ID NO:14) wherein at least one primer is biotinylated at the 5′-end with biotin to obtain biotinylated PCR products;
c) hybridizing the biotinylated PCR products obtained in step b) with one set or more than one set of oligonucleotide probes and adding streptavidin-phycoerythrin at a temperature for each probe/probe set at the following hybridization temperature ranges: probe set 1: 45-50° C.; and probe set 7: 52-56° C.;
Probe/probe set 1: Kpb ATCACTTCACggCTgTTCCA (SEQ ID NO: 20) and Kpa ATCACTTCATggCTgTTCCAg (SEQ ID NO:21); and probe/probe set 7: Fya GAgACTATggTgCCAACCTg (SEQ ID NO: 35) and Fyb TggAgACTATgATgCCAACC (SEQ ID NO: 36);
d) detecting any fluorescence with a flow cytometer-based instrument to identify and type at least one single nucleotide polymorphism (SNP) of the erythrocyte system X in heterozygote and homozygote individuals in order to discriminate between heterozygote and homozygote individuals wherein a negative control used with probe set 1 consisting of is AACTCTACggggCTCTTCgA (SEQ ID NO: 22); and a negative control is used with probe set 7 consisting of AggCTATCCTgACAAgCTT (SEQ ID NO: 37.

3. The method of claim 2 wherein the target allele is Kpa/Kpb.

4. The method of claim 2 wherein the target alleles are Fya/Fyb.

5. The method of claim 1 wherein the target allele is Fya/Fyb.