Molecular Testing in the Transfusion Medicine Laboratory

Traditionally, transfusion medicine laboratories have relied on serological testing based on hemagglutination principles. Agglutination reactions occur whenever antigens on red blood cells react with antisera or whenever antibodies in plasma react with reagent red cells. 

Although serological testing is present in all transfusion medicine laboratories, it is not without its challenges—technical limitations exist, such as the limited supply of antisera, subjectivity in readings, and the presence of interfering substances. These interferences can include the presence of autoantibodies, treatment with monoclonal antibody drugs, and recent transfusion. 

With these limitations in mind, laboratorians can use molecular testing methods to accurately determine blood type results for patients and blood donors.

"When serological testing produces inconclusive ABO and Rh blood group results for a patient, molecular genotyping can be performed to accurately determine their blood group and allow for the transfusion of group-specific blood."

The genetics of blood group systems

In transfusion medicine, a patient’s blood group is determined based on the different carbohydrate or protein molecules present on the surface of red blood cells.¹ There are currently 43 recognized blood group systems that arise from genetic changes referred to as polymorphisms.^2,3 These are either single-nucleotide polymorphisms, single-nucleotide deletions, gene deletions, sequence duplications, and intergenetic recombinations.³ 

Transfusion medicine laboratories routinely test for several of these blood groups, including the ABO, Rh, Kell, Duffy, and Kidd blood groups. The location of these blood group genes varies within different chromosomes. For example, the ABO blood group gene is located on chromosome 9q34, and the Rh blood group genes are located on chromosome 1p36.^1,4 The Kell blood group gene is located on chromosome 7q33, the Duffy blood group gene is located on chromosome 1q22-223, and the Kidd blood group gene is located on chromosome 18q12-q21.⁵

Applications of molecular testing in transfusion medicine labs

Resolving discrepant results

When serological testing produces inconclusive ABO and Rh blood group results for a patient, molecular genotyping can be performed to accurately determine their blood group and allow for the transfusion of group-specific blood.⁶ In addition, molecular genotyping can accurately reveal variant expression in blood group alleles that hemagglutination methods alone cannot.⁷

Prenatal testing

In transfusion medicine testing, determining whether a pregnant person is Rh(D) positive or negative is critical to effective care. Rh(D)-negative pregnant persons require prophylaxis with Rh(D) immunoglobulin to prevent the production of anti-D antibodies that could result in hemolytic disease of the fetus and newborn, harming them and their child. 

While most people are either Rh(D) positive or negative, other phenotypes exist that put someone at risk of forming anti-D antibodies, such as weak D (excluding type 1, 2, or 3) and partial D, which arise from point mutations in the RHD gene.⁶ Molecular testing of the RHD gene helps identify which patients are at risk and require prophylaxis.⁷ 

RHD typing is also used for testing fetal DNA. In early pregnancy, cell-free fetal DNA can be detected in maternal plasma, allowing for the early determination of Rh(D) status of fetuses.³ If fetal RHD typing is negative, this avoids the unnecessary administration of Rh(D) immunoglobulin to Rh(D) negative patients.¹

"Providing antigen-matched red blood cells through molecular genotyping decreases the risk of alloimmunization and delayed hemolytic transfusion reactions in these patient populations."

Selecting red blood cells for transfusion

Molecular genotyping is very useful in providing antigen-matched red blood cells to patients with special transfusion requirements. Patients with diseases such as sickle cell anemia and thalassemia have chronic transfusion needs, along with high rates of red blood cell alloimmunization, which can make it difficult to find compatible blood. Providing antigen-matched red blood cells through molecular genotyping decreases the risk of alloimmunization and delayed hemolytic transfusion reactions in these patient populations.⁶

Molecular testing platforms in transfusion medicine testing

Polymerase chain reaction (PCR)

One of the first techniques used in ABO genotyping was restriction fragment length polymorphism (RFLP) PCR. This method uses enzymes to distinguish between different alleles within a blood group. Allele-specific primers were later developed and used in sequence-specific primer and multiplex PCR methods.⁴ These methods of detecting DNA amplicons employ gel or capillary electrophoresis. Real-time PCR, or qPCR, can also be used to detect DNA amplicons in real-time using fluorescent probes during PCR reactions. Both PCR and real-time PCR are labor-intensive methods with slow turn-around times.³

Microarray

Other technologies have now been developed to meet the increasing need of molecular testing within transfusion medicine laboratories. Microarrays use glass slides, beads, or microtitre plates with oligonucleotide-specific DNA probes that hybridize amplified DNA targets for easy detection. 

In addition to dry microarrays, fluidic microarray platforms, such as the Luminex testing platform, can also amplify target DNA using biotinylated primers, then after denaturation and neutralization steps, the amplicons are hybridized to probes bound to streptavidin-conjugated fluorescent beads. Fluidic microarrays are highly automated, resulting in a fast turnaround time.⁴

Sequencing

Sequencing methods are a more recent addition to transfusion medicine. Sanger sequencing uses tagged terminating nucleotides in a single direction of PCR. DNA products are then detected via electrophoresis.³ Recently, next-generation sequencing has been used to identify nucleotides by repeatedly sequencing several hundred base pairs of DNA segments over large sections of the genome, allowing for very fast and precise results.¹ Next-generation sequencing can be used to detect unknown or rare blood group gene variants, as well as to screen blood donors for specific complex genotypes, as it is more cost-effective than other genotyping methods.⁸

"Next-generation sequencing can be used to detect unknown or rare blood group gene variants, as well as to screen blood donors for specific complex genotypes."

Challenges of molecular testing in transfusion medicine laboratories

While transfusion medicine laboratories are increasingly using molecular testing, these techniques come with some challenges, such as the cost of implementing these technologies, as well as the limited availability of testing in routine transfusion medicine laboratories. Performing molecular testing also requires specific technical skills. Therefore, most molecular genotyping occurs in reference laboratories. 

Moreover, technical challenges include the risks of false positive or false negative results, which can negatively impact patient care.6 In addition, interpreting genotyping results to infer a person’s phenotype can sometimes be challenging, where variability of blood group genetics, such as large deletions, hybrid alleles, or silenced alleles, can lead to misinterpretation of phenotypes.¹

Further developments

Despite some challenges, the future of molecular diagnostics in transfusion medicine testing looks bright with the increased applications of newer platforms, such as next-generation sequencing. Increased use of this technology will help increase the throughput of genotyping blood donors, allowing more antigen-matched blood to be available for people with rare blood requirements. Further development of this technology may also produce testing platforms customized to test for specific genotypes present within certain ethnic populations. Together, these developments work toward reducing the risk of alloimmunization for patients requiring transfusions, thus improving transfusion care for all.

References:

1. Svensson AM, Delaney M. Considerations of red blood cell molecular testing in transfusion medicine. Expert Rev Mol Diagn. 2015;15(11):1455–1464. doi:10.1586/14737159.2015.1086646.
2. Red Cell Immunogenetics and Blood Group Terminology. International Society for Blood Transfusion. Accessed January 31, 2023. https://www.isbtweb.org/isbt-working-parties/rcibgt.html.
3. Elkins MB et al. Molecular pathology in transfusion medicine: new concepts and applications. Clin Lab Med. 2018;38(2):277–292. doi: 10.1016/j.cll.2018.02.001.
4. Gorakshakar A et al. Evolution of technology for molecular genotyping in blood group systems. Indian J Med Res. 2017;146(3):305–315. doi: 10.4103/ijmr.IJMR_914_16.
5. Dean L. Blood Groups and Red Cell Antigens. Bethesda (MD): National Center for Biotechnology Information (US); 2005. Available from: https://www.ncbi.nlm.nih.gov/books/NBK2261/.
6. Hillyer CD et al. Integrating molecular technologies for red blood cell typing and compatibility testing into blood centers and transfusion services. Transfus Med Rev. 2008;22(2):117–132. doi: 10.1016/j.tmrv.2007.12.002.
7. Castilho L. Molecular typing of blood group genes in diagnostics. Ann Blood. 2021;6:20. doi: 10.21037/aob-20-73.
8. Orzinska A et al. Potential of next-generation sequencing to match blood group antigens for transfusion. Int J Clin Transfus. 2019;7:11–22. doi: 10.2147/IJCTM.S175142.