**4. Molecular testing: current technologies**

Polymerase chain reaction (PCR) and further advancements in molecular methods such as DNA sequencing and recombinant DNA technology allowed elucidation of the molecular basis of most of the blood group antigens and subsequent use of molecular techniques employed for blood group genotyping initially as in-house assays or laboratory developed tests (LDT) and later as commercially available assays.

While molecular basis for many of the blood group systems is relatively straightforward, with two antithetical alleles differing by a single nucleotide polymorphism (SNP) and responsible for one of the two possible phenotypes, some blood group systems are very complex. RH blood group system has over 50 antigens described


**209**

making it possible to predict the phenotype.

*Other techniques developed for blood group genotyping.*

*Accuracy of Blood Group Typing in the Management and Prevention of Alloimmunization*

**Name Principle Number of polymorphisms/**

Denaturation of PCR product followed by fragment analysis in polyacrylamide gel

by enzyme digestion (PCR-RFLP)

followed by gel electrophoresis

Amplification of multiple targets using both specific and universal-FAM-labeled primers detected by electrophoresis

Amplification followed by enrichment with a known sample and DHPLC

MLPA probes hybridized to DNA target sequence followed by fragment amplification and detection

Multiplex PCR followed by singlenucleotide extension and hybridization to complementary oligonucleotides

PCR with gene-specific primers and probes followed by melting curve analysis

Multiplex PCR followed by SNaPshot reaction and fragment analysis

to probes coupled to fluorescent

fragment using dye terminators

Sequencing of entire genome or specific regions. Millions of small fragments of DNA are simultaneously sequenced

Chimeric primers (specific-universal tag) are used along with fluorescent-label universal primers followed by products hybridization to a DNA array

allele-specific primers

**antigens identified**

Singleplex or multiplex

1 blood group

exons of *RHD*

Entire *RHD* gene

18 blood groups

Various—customized

Various—customized

Various—customized

Various—customized

6 blood groups

systems

antigens

Varies

9 polymorphisms associated with

Polymorphisms associated with 6

10 exons of both *RHD* and *RHCE*

48 alleles encoding blood group antigens and 112 variant alleles of

12 polymorphisms associated with 6 blood groups and platelet

8 polymorphisms associated with

Gene fragments of 19 different RBC and platelets antigen

to date, more than 500 alleles, and 2 highly homologous genes that, in addition to SNPs, insertions, and deletions, may have hybrid configurations. Other systems with more than 50 alleles currently identified include MNS, Diego, and Kell. Genotyping strategies are often based on a SNP altering a restriction site or punctuating otherwise perfectly matched sequence for primers or probe annealing site. These approaches may be suboptimal for designing genotyping assays of complex systems and fail to reveal rare variants. Next generation sequencing (NGS) is the only method that can reliably detect the exact genetic composition of the site

*DOI: http://dx.doi.org/10.5772/intechopen.90095*

PCR-RFLP [68–74] Amplification of target sequence followed

PCR-SSP [75–77] Amplification of single alleles using

Multiplex-PCR [78, 79] Amplification with multiple primers

analysis

Microsphere-based array [87] Multiplex PCR followed by hybridization

Sanger sequencing [89] Classical DNA sequencing of single

microspheres

Multiplex PCR-single-strand conformation polymorphism (SSCP) analysis [67]

Quantitative multiplex PCR of short fluorescent fragments (QMPSF) [80]

Denaturing high-performance liquid chromatography (DHPLC) analysis [81]

Multiplex ligation-dependent probe amplification (MLPA)

High-resolution melting analysis [84, 85]

Single nucleotide primer extension minisequencing assay (SNaPshot) [86]

Multiplex PCR and DNA microarray hybridization [88]

Next generation sequencing

(NGS) [90]

**Table 3.**

[82]

GenomeLab SNPStream [83]

#### **Table 2.**

*Commercial solutions available for blood group genotyping.*

*Accuracy of Blood Group Typing in the Management and Prevention of Alloimmunization DOI: http://dx.doi.org/10.5772/intechopen.90095*


#### **Table 3.**

*Human Blood Group Systems and Haemoglobinopathies*

**4. Molecular testing: current technologies**

Immucor PreciseType HEA Molecular BeadChip

BioArray RHCE and RHD

 [57] BioArray Solutions

[58]

 [59] Progenika Biopharma

[62]

[64]

Test#,\$ [56] BioArray Solutions

BeadChip\$

ID CORE XT#,\$

ID RHD XT\$

[60, 61] Agena Bioscience

eXtend\$ [63] inno-train diagnostik

GMBH

GMBH

assays

*FDA approved.*

*CE mark certification.*

HIFI Blood 96\$

RBC-ReadyGene\$

inno-train diagnostik

TaqMan genotyping on OpenArray [65, 66] BioTrove

Pre-designed TaqMan

Thermo Fisher Scientific

AXO Science

Progenika Biopharma

Hemo ID DQS Panel\$

RBC-FluoGene vERYfy

Polymerase chain reaction (PCR) and further advancements in molecular methods such as DNA sequencing and recombinant DNA technology allowed elucidation of the molecular basis of most of the blood group antigens and subsequent use of molecular techniques employed for blood group genotyping initially as in-house assays or laboratory developed tests (LDT) and later as commercially available assays. While molecular basis for many of the blood group systems is relatively straightforward, with two antithetical alleles differing by a single nucleotide polymorphism (SNP) and responsible for one of the two possible phenotypes, some blood group systems are very complex. RH blood group system has over 50 antigens described

**Name/manufacturer Principle Number of polymorphisms/antigens** 

Multiplex PCR followed by hybridization of amplified DNA to probes attached to spectrally distinguishable beads and

Multiplex PCR followed by hybridization of amplified DNA to probes attached to spectrally distinguishable beads and

Multiplex biotinylating PCR followed by hybridization to probes on color-coded

Multiplex biotinylating PCR followed by hybridization to probes coupled to color-coded

Multiplex end-point PCR and single base primer extension

Automated multiplex PCR and microarray-based assay

Nanofluidic TaqMan assays performed in OpenArray plates

Real-time allelic discrimination

PCR

*Commercial solutions available for blood group genotyping.*

elongation

elongation

microspheres

microspheres

**identified**

Hemoglobin S

24 polymorphisms associated with 38 antigens plus phenotypic variants and

35+ *RHCE* variants; 80+ *RHD* variants

29 polymorphisms associated with 37 antigens and phenotypic variants

6 *RHD* variants and HPA-1

101 antigens associated with 16 systems, and 23 platelet and neutrophil antigens, in modules

associated with 12 systems

and variant phenotypes within 13

Polymorphisms associated with 8

16, 32 or 64 assays (custom designed)

antigens

systems

systems

Automated TaqMan-based assay 70 antigens and phenotypic variants

PCR-SSP-based assay 16 modules encompassing antigens

15 polymorphisms associated with 24

**208**

**Table 2.**

*#*

*\$*

*Other techniques developed for blood group genotyping.*

to date, more than 500 alleles, and 2 highly homologous genes that, in addition to SNPs, insertions, and deletions, may have hybrid configurations. Other systems with more than 50 alleles currently identified include MNS, Diego, and Kell. Genotyping strategies are often based on a SNP altering a restriction site or punctuating otherwise perfectly matched sequence for primers or probe annealing site. These approaches may be suboptimal for designing genotyping assays of complex systems and fail to reveal rare variants. Next generation sequencing (NGS) is the only method that can reliably detect the exact genetic composition of the site making it possible to predict the phenotype.

Genotyping methodologies vary widely and include labor-intensive techniques that are best suited to test individual samples for limited number of polymorphisms (i.e. PCR-RFLP and PCR-SSP), high-throughput commercial kits that are relatively easy to use (i.e. real-time PCR and arrays), and methods that require specialized equipment to differentiate between alleles in multiple blood group systems at once (i.e. NGS). Most of assays described to date rely on enzyme-mediated DNA amplification at some point in their workflow and on sequence-specific primers or probes (**Tables 2** and **3**).

At the time of this writing, several platforms have been commercialized, but only two commercial assays have been approved by the US FDA. Immucor PreciseType by BioArray Solutions was approved in May 2014 and ID CORE XT manufactured by Progenika Biopharma was approved in October 2018. Due to less stringent requirements to obtain the European Conformity (CE mark), this certification has been granted to most of the commercial devices described here (see **Table 2**). Commercial assays not approved by the FDA can be labeled and utilized for research or investigational use only. Some commercial platforms (such as OpenArray or GenomeLab SNPStream) are used to run LDT assays to increase the throughput (**Table 3**).
