**5. Molecular testing: standards for RBC antigens**

The use of molecular typing for characterization of blood group genes has been steadily increasing in the last few decades due to the transfusion benefits for the patients, technological advances in molecular techniques, and expanding availability of mass-scale genotyping. While blood group genotyping is becoming increasingly used, typing errors have been documented indicating the need for quality control.

International Workshops on Molecular Blood Group Genotyping have reported discrepancies between laboratories studying the same samples indicating that there is room for improvement [91, 92]. As a genotyping test may only be done once in a person's lifetime, errors can have serious consequences. A major challenge in performing routine RBC genotyping is controlling for process variability of molecular assays that arises due to scarcity of reference materials. This limitation has been circumvented by using clinical specimen leftovers from previous testing, which lack proper characterization. Reference materials are critical for the development and manufacture of testing kits, for test calibration and for monitoring of assay performance.

Recommendations on which targets and recommended controls to use for prediction of RBC antigens have been published by international societies offering proficiency test programs such as INSTAND [93], The Consortium for Blood Group Genes (CBGG) [94], College of American Pathologists (CAP), and International Society of Blood Transfusion (ISBT) [91, 92] and also by The American Association of Blood Banks (AABB) [95] whose focus is on creating guidelines for AABB accreditation aiming to certify laboratories that perform molecular testing for red cell, platelet, and neutrophil antigens.

Molecular testing laboratories participate in accreditation programs to validate their activities. In the US, the requirements for AABB accreditation are described in the 4th edition of standards for molecular testing for red cell, platelet, and neutrophil antigens (updated in October 2018) [95]. According to the document, "laboratories shall use appropriate reference DNA to validate and control the reported test" and further, "the reference DNA needs to contain the target polymorphisms reported by the laboratory". The same publication lists the blood group

**211**

donor [97].

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

alleles within 17 blood group systems that should be included in the reference DNA materials by the lab seeking to meet the minimum requirements for accreditation. Various preparations have been used as controls to monitor performance of blood group genotyping assays, including synthetic DNA (such as PCR products, plasmid-cloned PCR products) and genetic material from human samples col¬lected and characterized for that purpose following standard guidelines [96]. PCR products and plasmid-cloned PCR products are simple and easy to produce and use as controls for SNPs, but they lack the genomic complexity of human sample and do not represent the clinical analyte (genomic DNA); moreover, the synthetic material can be a source of contamination for the laboratory if not carefully handled and well diluted. Although well-characterized human specimens are the best representation of clinical samples, they are of limited source and the replenishment may not come from the same donor. The alternative to overcome source limitation is to transform human cells into immortalized cell lines and use those for characterization and formulation of reference reagents. This approach has been successfully used to produce reference DNA using B-lymphoblastoid cell lines (B-LCLs), which are an appropriate representation of the genetic material of the

B-LCLs can be generated by Epstein Barr Virus (EBV) infection of peripheral mononuclear cells from genetically characterized donors carrying specific blood group antigens and variants. The infection leads to proliferation and subsequent cell immortalization, providing an unlimited source of donor's genomic DNA. Once the B-LCLs are established, master and working cell banks are maintained in liquid nitrogen to ensure long-term survival of the cell line and continuous supply of the DNA. To produce the DNA reagent, B-LCLs from the working cell banks are expanded for bulk DNA extraction and subsequent DNA lyophilization. Tests on the lyophilized reagents are performed for assessment of their stability under accelerated degradation conditions and under normal conditions over longer periods of time as long-term stability. The material must be validated via collaborative studies

with laboratories that routinely perform molecular blood typing.

DI, YT, SC, CO, LW, CR, KN, IN, and OK) (**Table 5**) [89].

Participants in the international collaborative study to validate the CBER panel used traditional molecular techniques and additional genotyping techniques not available at the time of the first WHO IRR production. The most common methods

The first DNA reference reagents for blood group genotyping developed using B-LCLs were produced in 2013 by the National Institute for Biological Standards and Control (NIBSC) and serve as World Health Organization (WHO) International Reference Reagents (IRR) for common blood group alleles found in ancestral Caucasians and Black African populations. The panel includes four DNA samples covering the most clinically important homozygous and heterozygous genotypes within RH, FY, KEL, JK, DO, and MNS blood group systems (**Table 4**). The material was validated in an international collaborative study by PCR-ASP or PCR-SSP, PCR-RFLP, Multiplex SSP assays, real-time PCR, Immucor Beadchip array, Progenika BLOODchip array, Luminex array, and 5′ Nuclease assay [98]. Additional cell lines were produced by CBER-FDA for use as source of genomic DNA for development of reference reagents to expand the number of red cell blood group polymorphisms represented in the first WHO IRR from 2013 for blood group genotyping. The CBER reference panel consists of 18 members, covering genotypes associated with 40 polymorphisms within 17 blood group systems, including alleles present in the existing WHO IRR-2013 except for *RHD\*ψ* (**Table 4**). The CBER panel was also characterized and validated for additional genotypes belonging to systems already included in the WHO IRR-2013 (RH, KEL, FY, JK, DO, and MNS) (**Table 4**), and for additional systems only represented in CBER panel (ABO, LU,

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

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

alleles within 17 blood group systems that should be included in the reference DNA materials by the lab seeking to meet the minimum requirements for accreditation.

Various preparations have been used as controls to monitor performance of blood group genotyping assays, including synthetic DNA (such as PCR products, plasmid-cloned PCR products) and genetic material from human samples col¬lected and characterized for that purpose following standard guidelines [96]. PCR products and plasmid-cloned PCR products are simple and easy to produce and use as controls for SNPs, but they lack the genomic complexity of human sample and do not represent the clinical analyte (genomic DNA); moreover, the synthetic material can be a source of contamination for the laboratory if not carefully handled and well diluted. Although well-characterized human specimens are the best representation of clinical samples, they are of limited source and the replenishment may not come from the same donor. The alternative to overcome source limitation is to transform human cells into immortalized cell lines and use those for characterization and formulation of reference reagents. This approach has been successfully used to produce reference DNA using B-lymphoblastoid cell lines (B-LCLs), which are an appropriate representation of the genetic material of the donor [97].

B-LCLs can be generated by Epstein Barr Virus (EBV) infection of peripheral mononuclear cells from genetically characterized donors carrying specific blood group antigens and variants. The infection leads to proliferation and subsequent cell immortalization, providing an unlimited source of donor's genomic DNA. Once the B-LCLs are established, master and working cell banks are maintained in liquid nitrogen to ensure long-term survival of the cell line and continuous supply of the DNA. To produce the DNA reagent, B-LCLs from the working cell banks are expanded for bulk DNA extraction and subsequent DNA lyophilization. Tests on the lyophilized reagents are performed for assessment of their stability under accelerated degradation conditions and under normal conditions over longer periods of time as long-term stability. The material must be validated via collaborative studies with laboratories that routinely perform molecular blood typing.

The first DNA reference reagents for blood group genotyping developed using B-LCLs were produced in 2013 by the National Institute for Biological Standards and Control (NIBSC) and serve as World Health Organization (WHO) International Reference Reagents (IRR) for common blood group alleles found in ancestral Caucasians and Black African populations. The panel includes four DNA samples covering the most clinically important homozygous and heterozygous genotypes within RH, FY, KEL, JK, DO, and MNS blood group systems (**Table 4**). The material was validated in an international collaborative study by PCR-ASP or PCR-SSP, PCR-RFLP, Multiplex SSP assays, real-time PCR, Immucor Beadchip array, Progenika BLOODchip array, Luminex array, and 5′ Nuclease assay [98].

Additional cell lines were produced by CBER-FDA for use as source of genomic DNA for development of reference reagents to expand the number of red cell blood group polymorphisms represented in the first WHO IRR from 2013 for blood group genotyping. The CBER reference panel consists of 18 members, covering genotypes associated with 40 polymorphisms within 17 blood group systems, including alleles present in the existing WHO IRR-2013 except for *RHD\*ψ* (**Table 4**). The CBER panel was also characterized and validated for additional genotypes belonging to systems already included in the WHO IRR-2013 (RH, KEL, FY, JK, DO, and MNS) (**Table 4**), and for additional systems only represented in CBER panel (ABO, LU, DI, YT, SC, CO, LW, CR, KN, IN, and OK) (**Table 5**) [89].

Participants in the international collaborative study to validate the CBER panel used traditional molecular techniques and additional genotyping techniques not available at the time of the first WHO IRR production. The most common methods

*Human Blood Group Systems and Haemoglobinopathies*

(**Tables 2** and **3**).

the throughput (**Table 3**).

control.

performance.

cell, platelet, and neutrophil antigens.

**5. Molecular testing: standards for RBC antigens**

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

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 use of molecular typing for characterization of blood group genes has been steadily increasing in the last few decades due to the transfusion benefits for the patients, technological advances in molecular techniques, and expanding availability of mass-scale genotyping. While blood group genotyping is becoming increasingly used, typing errors have been documented indicating the need for quality

International Workshops on Molecular Blood Group Genotyping have reported discrepancies between laboratories studying the same samples indicating that there is room for improvement [91, 92]. As a genotyping test may only be done once in a person's lifetime, errors can have serious consequences. A major challenge in performing routine RBC genotyping is controlling for process variability of molecular assays that arises due to scarcity of reference materials. This limitation has been circumvented by using clinical specimen leftovers from previous testing, which lack proper characterization. Reference materials are critical for the development and manufacture of testing kits, for test calibration and for monitoring of assay

Recommendations on which targets and recommended controls to use for prediction of RBC antigens have been published by international societies offering proficiency test programs such as INSTAND [93], The Consortium for Blood Group Genes (CBGG) [94], College of American Pathologists (CAP), and International Society of Blood Transfusion (ISBT) [91, 92] and also by The American Association of Blood Banks (AABB) [95] whose focus is on creating guidelines for AABB accreditation aiming to certify laboratories that perform molecular testing for red

Molecular testing laboratories participate in accreditation programs to validate their activities. In the US, the requirements for AABB accreditation are described in the 4th edition of standards for molecular testing for red cell, platelet, and neutrophil antigens (updated in October 2018) [95]. According to the document, "laboratories shall use appropriate reference DNA to validate and control the reported test" and further, "the reference DNA needs to contain the target polymorphisms reported by the laboratory". The same publication lists the blood group

**210**


**213**

**Table 5.**

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

MNS *GYPA\*M/GYPA\*M GYPA\*M/GYPA\*M* (59C/C)

*GYPA\*M/GYPA\*N GYPA\*M/GYPA\*N* (59C/T) *GYPA\*N/GYPA\*N GYPA\*N/GYPA\*N* (59 T/T)

*GYPB\*S/GYPB\*s GYPB\*S/GYPB\**s (143C/T) *GYPB\*s/GYPB\*s GYPB\*s/GYPB\*s* (143C/C)

> c.526C>G C/C; G/C c.703G>A G/G; G/A c.796C>A C/C; C/A c.803G>C G/G; G/C

c.4768A>G A/A; A/G c.4801A>G A/A; A/G; G/G

c.261delG G/G; G/delG, delG/delG

*Notes: Additional polymorphisms and genotypes included in the CBER panel are in bold; in parentheses are genotypes represented in the CBER panel members; descriptions of the polymorphism positions/genotypes are not available for* 

*Alleles or genotypes included in the WHO IRR and CBER RR for the overlapping blood group systems.*

**Blood group system Gene Polymorphism Genotypes included**

ABO *ABO* c.1061delC CC; C/delC

Lutheran *BCAM* c.230G>A A/A; G/A; G/G Diego *SLC4A1* c.2561C>T C/T; C/C Cartwright *ACHE* c.1057C>A C/C; C/A; A/A Scianna *ERMAP* c.169G>A G/G; G/A Colton *AQP1* c.134C>T C/C; C/T; T/T Landsteiner-Wiener *ICAM4* c.299A>G A/A; A/G Cromer *CD55* c.679G>C G/G; G/C Knops *CR1* c.4681G>A G/G; G/A

Indian *CD44* c.137G>C G/G OK *BSG* c.274G>A G/G

*Genotypes from additional blood group systems covered by the CBER panel members.*

*For additional details, see reference [89].*

*GYPB\*S/GYPB\*S* (143 T/T)

**Additional polymorphisms:** *GYPB* **c. 230C>T (genotype C/C)** *GYPB* **c. 270+5G>T (genotype G/G)**

**Blood group system WHO IRR [98] CBER RR [89]**

used by the collaborators were PCR-SSP, either single- or multiplex, the HEA (human erythrocyte antigen), RHD, and RHCE BeadChip arrays from Immucor, Sanger sequencing, PCR-RFLP, and real-time PCR based assays. Less common methods included ID-CORE XT and BLOODchip REFERENCE by Progenika, MALDI-TOFbased assays such as Hemo ID from Agena Bioscience, NGS, RBC-Ready Gene and

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

*WHO panel. For additional details see references [89, 98].*

**Table 4.**

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


*Notes: Additional polymorphisms and genotypes included in the CBER panel are in bold; in parentheses are genotypes represented in the CBER panel members; descriptions of the polymorphism positions/genotypes are not available for WHO panel. For additional details see references [89, 98].*

#### **Table 4.**

*Human Blood Group Systems and Haemoglobinopathies*

**Blood group system WHO IRR [98] CBER RR [89]**

*RHD\*01 N.01/RHD\*ψ*

Rh *RHD* positive (zygosity not determined) *RHD\*01/RHD\*01* (*RHD* homozygous)

*RHD\*01 N.01/RHD\*01 N.01 RHD\*01 N.01/RHD\*01N.01* (homozygous

*RHCE\*C/RHCE\*c RHCE\*C/RHCE\*c* (307T/C; 109 bp intron

*RHCE\*C/RHCE\*C RHCE\*C/RHCE\*C* (307T/T; 109 bp intron

*RHCE\*c/RHCE\*c RHCE\*c/RHCE\*c* (307C/C; 109 bp intron

*RHCE\*E/RHCE\*e RHCE\*E/RHCE\*e* (676C/G) *RHCE\*e/RHCE\*e RHCE\*e/RHCE\*e* (676G/G)

*KEL\*01/KEL\*01 KEL\*01/KEL\*01* (578T/T) *KEL\*02/KEL\*02 KEL\*02/KEL\*02* (578C/C)

*FY\*02/FY\*02 FY\*02/FY\*02* (125A/A)

*JK\*01/JK\*01 JK \*01/JK\*01* (838A/A) *JK\*02/JK\*02 JK \*02/JK\*02* (838G/G)

*DO\*02/DO\*02 DO\*02/DO\*02* (793G/G)

*FY\*02N.01/FY\*02N.01 FY\*02N.01/FY\*02N.01* (─67T/T)

Kell *KEL\*01/KEL\*02 KEL\*01/KEL\*02* (578T/C)

Duffy *FY\*01/FY\*02 FY\*01/FY\*02* (125G/A)

Kidd *JK\*01/JK\*02 JK\*01/JK\*02* (838G/A)

Dombrock *DO\*01/DO\*02* (793A/G)

*RHD* deletion)

2 ins present)

2 ins present)

2 ins absent)

**C/C)**

**T/T)**

**C/C)**

**Additional polymorphisms:**

*RHCE* **c. 122A>G (genotypes A/A; A/G)** *RHCE* **c. 106G>A (genotype G/G)** *RHCE* **c. 733C>G (genotype G/C; G/G;** 

*RHCE* **c. 1006G>T (genotype G/G)**

**Additional polymorphisms:**

*FY\*01/FY\*01*(125G/G)

*FY\*01/FY\*02N.01* (─67T/C) **Additional polymorphisms:** *FY* **c. 265C>T (genotypes C/C; C/T)**

*DO\*01/DO\*01* (793A/A)

**T/T)**

**Additional polymorphisms:**

*DO* **c. 323G>T (genotypes G/G; G/T)** *DO* **c. 350C>T (genotype C/C; C/T;** 

*KEL* **c. 841C>T (genotypes C/C; C/T;** 

*KEL* **c. 1790T>C (genotypes T/T; C/T;** 

*RHD\*01/RHD\*01N.01* (*RHD* hemizygous)

**212**

*Alleles or genotypes included in the WHO IRR and CBER RR for the overlapping blood group systems.*


#### **Table 5.**

*Genotypes from additional blood group systems covered by the CBER panel members.*

used by the collaborators were PCR-SSP, either single- or multiplex, the HEA (human erythrocyte antigen), RHD, and RHCE BeadChip arrays from Immucor, Sanger sequencing, PCR-RFLP, and real-time PCR based assays. Less common methods included ID-CORE XT and BLOODchip REFERENCE by Progenika, MALDI-TOFbased assays such as Hemo ID from Agena Bioscience, NGS, RBC-Ready Gene and

RBC-FluoGene by Inno-train Diagnostik GmbH, droplet digital PCR, HI-FI Blood by AXO Science, SNaPshot, and high-resolution melting analysis (HRMA) [89].
