AABB News: The Evolution of Blood Group Genotyping

This article originally appeared in AABB News, a benefit of AABB membership.

Genotyping of blood groups is becoming an increasingly common alternative to more traditional serological antibody-based methodology to help transfusion medicine professionals determine the optimally matched unit of blood for patients in need of a transfusion.

Blood group genotyping allows medical professionals to identify blood products that are matched to recipients for a wide variety of antigens. Experts say blood group genotyping can lead to improved matching and, therefore, reduce the risk of potential transfusion-related adverse events. As this area of transfusion medicine continues to evolve, the field may see even more comprehensive testing and matching options – all of which can help provide the best possible unit of blood for every patient in need.

Blood group genotyping has been used to some degree in various European countries for more than 2 decades. But it was not until the last decade that this methodology started coming into common usage in the United States. There are scientific and technological reasons for this.

Advancing Blood Group Genotyping

Margaret Keller, PhD, executive director of National Laboratories and senior director of the American Rare Donor Program at the American Red Cross in Philadelphia, explained that many blood group antigens are determined by single nucleotide variants (SNVs) and that there have been methods to test for SNVs for more than a quarter of a century. “As far as use of molecular methods to interrogate SNVs to predict blood group antigen phenotypes, a small number of blood centers, mostly in Europe, have been doing this since the 1990s,” she said. “In the U.S., this testing began in earnest in the early 2000s.”

Gregory R. Halverson, MT(ASCP)SBB, DLM, manager of LifeSouth Community Blood Center’s Immunohematology Reference Laboratory, reflected back on the early 1980s — the HIV era — to provide a relevant example of what it took to overcome the AIDS pandemic. “We needed methods to detect and monitor infection,” he said. “It was the discovery of a heat-resistant bacteria that utilized stable deoxyribonucleic acid [DNA] polymerase with a temperature optimum of 80° C purified from the extreme thermophile Thermus aquaticus cultured from the thermal pools in Yellowstone Park that enabled the polymerase chain reaction [PCR] allowing for the study of human genes on the molecular level.”

This proved important to reducing the risk of transfusion-transmitted HIV and allowed for development of hundreds of molecular testing modalities for genetic diseases such as cystic fibrosis, while at the same time offering a means of predicting the red blood cell surface phenotype, an important parameter that can be used to optimize treatment in transfusion medicine.

“The key advances required for this testing to become a reality were first, the invention of PCR that facilitates amplification of a specific region of the genome that can then be used in a genotyping assay,” said Keller. “Second, the discovery of the gene associated with a specific blood group system and third, the knowledge of the genetic variation that is responsible for gain or loss of blood group antigens. This work is actually still ongoing.”

Once the means of testing was available, the question became one of the efficiency and cost of the tests. “One of the major reasons why blood group genotyping is becoming more widely used is the development by several companies of platforms and methods for genotyping in a single-run multiple blood group systems or many positions for the more complex systems,” said Aline Floch, MD, PhD, Etablissement Français du Sang – Ile de France, Site de Créteil – Hôpital Henri Mondor. “This lowers the time and cost per sample than was previously spent on performing multiple assays. Together with the manufacturer’s automated interpretation of the results, this also lowers the expertise required to perform genotyping.”

However, as Floch warned, these methods and platforms may create a false sense of simplicity, and there is also the risk of misinterpretation. “Expert genotyping labs remain very important in this dynamic and should be called upon for any unexpected findings or ambiguity,” she said. “A second important limitation is that the phenotype can only be deduced — not determined — from genotyping the few targets included by manufacturers; rare or silent alleles may not be detected.”

Genotyping versus Phenotyping

Currently, common methods of genotyping analyze relatively small samples of the molecular make-up of blood group antigens. “Genotyping in its current form mostly probes for the presence of single nucleotide polymorphisms [SNPs], but they do not search for changes all through the  gene,” said Halverson. “This is changing with the advent of next generation sequencing [NGS].”

“Both phenotyping and genotyping are strategies that enable the matching process, each with limitations,” explained Floch. “While phenotyping many will not detect very low levels of antigen expression, or the lack of certain epitopes [partial antigens], current genotyping strategies only target the main polymorphisms and will miss null alleles. Both strategies can miss rare and novel variants. Perhaps if comprehensive methods such as NGS become standard of care, the balance will lean toward genotyping, however the costs remain too high at present.”

Genotyping can provide key information about the makeup of blood products.

“RBC genotyping can provide a comprehensive antigen phenotype that cannot be obtained serologically because antisera are not available for all antigens, and for others, the antisera that are available are unreliable,” said Keller. “So, an RBC profile obtained using molecular methods can be used to then select blood products to match.” She reiterated that this is often how blood products are matched in the case of patients with warm autoantibodies and in patients with prophylactic matching protocols, such as those with sickle cell disease.

As this area of medicine continues to evolve, there is much room for additional advancements. Limitations remain, but blood group genotyping has many benefits that make it preferable to serological testing, at least in some situations.

Keller noted that genotyping is not impacted by either recent transfusion or weak antigen expression. In addition, genotyping can identify variant antigens encoding partial phenotypes and predict weak antigen expression that can be missed by serology. Finally, genotyping isn’t limited to serologic reagent availability.

“Genotyping is a great tool for many reasons, including understanding gene variants and searching for matched donors,” said Halverson. “There is, however, another significant area where we need to learn why certain people are prone to make antibodies and what those genes are. If we can understand who is going to respond to transfused allogeneic blood, we can look to find ways to minimize or mitigate the immune response.”

“If we could determine the genetic profile of ‘responders,’ the patients at highest risk of making alloantibodies to red cell antigens transfused red cells, we could put those patients on the more stringent matching protocols and perhaps lessen the stringency for patients who are not predicted to be ‘responders,’” added Keller.

Immunotherapy Drugs

Genotyping can be used in some situations in which serology would not produce accurate results, particularly in patients being treated with immunotherapy drugs. The use of immunotherapy drugs can hamper the ability to perform an antibody screen.

Anti-CD38 is a biologic agent that binds to CD38, which is overexpressed on some malignancies such as multiple myeloma, said Keller. “The problem is that CD38 is also expressed on red blood cells. So the RBCs of patients on this drug will be coated with the drug and this makes antibody identification problematic. RBC phenotyping before the drug is started can be useful, but we cannot test for all antigens,” she said. RBC genotyping, on the other hand, can be done at any point, not only before the drug is started, she added.

“The development of antibodies to CD38 caused quite a problem at first because of the gap between the evolving treatment regimens and the lack of communication to the blood transfusion facilities,” said Halverson. “When anti-CD38 first arrived for treatment of relapsing multiple myeloma, it was thought that the CD38 protein was not expressed on human red blood cells. We now know that it is, and the learning curve for transfusion facilities slowly caught up with methods to test for antibodies in the presence of the drug to exclude most common antibodies. The exception is those that are sensitive to treatment with DTT, such as those in the KEL blood group system.”

As the use of immunotherapy drugs expands, blood bankers need to be aware of their potential complications on serologic results. “New therapies are on the horizon and we need to be alert for new drugs and treatment regimens that could interfere with serology and compatibility testing,” added Halverson. “Reporting these new drugs in abstracts to blood bank meetings helps to get  the word out about the drugs and how to work around their interference.”

Keller said that the blood banking community learned lessons when it began identifying the links between daratumamab treatment and inaccurate serologic results. “One important lesson we learned with anti-CD38 is the importance of communicating and educating blood bankers  regarding potential interference of the therapy on standard laboratory tests and provide guidance on how to manage that interference,” said Keller. “AABB provided that to the community at that time with AABB Association Bulletin #16-02, and that was very helpful. As far as future immunotherapies and the potential for interference with antigen typing and/or antibody  identification, it’s important for blood bankers to know that the use of genotyping to predict the red cell antigen phenotype can be a useful means to assess risk of alloimmunization and that it can be performed on a blood specimen before or after immunotherapy has begun.”

The Future of Blood Group Genotyping

Blood group genotyping is already the standard of care in the U.S. at many institutions for  specific populations. “I think it is already standard procedure to phenotype match donors to patients with sickle cell disease and warm autoimmune hemolytic anemia,” said Halverson.

Keller also noted its increasingly common usage. “It is the standard of care at many institutions for obtaining red cell phenotype in patients being treated with immunotherapies, including anti-CD38, and at many institutions for the resolution of serologic weak D phenotype in women of child-bearing age; but more education is needed,” she said.

Keller added that she expects this trend to continue. “As more and better tests become available and as clinicians learn more, I do believe this testing will grow,” she said. “I believe the strength of molecular methods related to red cell phenotyping lies in the fact that it only needs to be done once. Unless you have a hematopoietic stem cell transplant (HSCT), your red cell phenotype remains the same. Therefore, the cost of red cell genotyping is a worthwhile investment, as it can be used to provide the best matched blood products and determine candidacy for Rh immune globulin in the case of women of child-bearing age. The challenge in the U.S. is integrating this type of clinical genomic data into the electronic health record of the patient and sharing it across institutions while protecting patient privacy. I think this is where the most change will be seen in the next 20 years.”

Experts say blood group genotyping remains in its early stages and much of its potential may yet still be realized. “The use of genotyping will continue to expand, especially with automation,” predicted Floch. “Many countries are only beginning to use genotyping. Genotyping has the potential of being easier to scale up, especially because it does not require rare reagents, and multiple targets can be interrogated in a single assay.”

Beyond blood group genotyping, the most important use of gene therapy may be in potential applications of such technologies as CRISPR to treat certain inherited diseases, according to Halverson. “The study of immunology and mechanisms of gene therapy to resolve inherited disease is likely to be where the biggest and most beneficial changes will be made,” he said. “The last time a great discovery in the treatment and prevention of disease and death among newborns was made was the development of Rh immune globulin to prevent alloimmunization to RhD. That was over 50 years ago. We still have inherited diseases such as SCD, thalassemia and diabetes that have yet to be solved by science.”