The Importance of Extended Antigen Typing in Transfusion Medicine

Matt: Hi my name is Matt Dawson and welcome to ORTHO Science BYTES! Today I am joined by Tony Casina, thank you for joining us today, Tony can you tell everyone a little bit about yourself?

Tony: Thank you Matt.  Hi everyone, for those of you who do not know me, I have been involved with Transfusion Medicine as a blood banker and immunohematologist for a little over 40 years. There have been many advances and innovations in the immunohematology world that have occurred over those years. Many of those will be discussed during these podcasts.

Matt: To open this podcast edition, let’s start with the following question Tony, what role does extended antigen typing or phenotyping play in transfusion medicine?

Tony: Well, let’s start with defining what blood group antigen phenotyping is. Most who have worked in blood banking have done some form of phenotyping.  Presently, tests for a basic ABO blood group and Rh(D) type is a simple, yet most important form of phenotyping. Phenotype is the physical expression of a characteristic that is defined by genes. The concept of extended antigen typing or extended antigen phenotyping has application across a variety of situations and rationales. Beyond transfusion, pregnancy, relationship/paternity and disease state are the common ones where phenotyping is utilized. For those of us in transfusion medicine, we think mainly about the application in transfusion and pregnancy.

Matt: So, Tony can you tell us how has phenotyping for red cell antigens evolved on the history of transfusion medicine?

Tony: Yes Matt, that is a very good question. I should start with a little bit of history on how phenotyping came to be and where it is today. It all revolves around the discovery of new blood groups when antibody development in humans through transfusion or pregnancy occurred. These antibodies were tested against populations or in family studies to show frequency and prevalence along with genetic inheritance. Of course, the use of the antibody then became a way to find compatible blood for transfusion. As a fun fact they were named based on who developed the antibody, who discovered the antibody or based on the blood group system they ultimately belonged to in the end. Eventually, then came the concept of having a high-quality source to assure that the “reagent” really was a high-quality reagent capable of accurate antigen typing.  Commercial sources became the norm with reagents manufactured from human, animal and plant (lectin) based sources and regulatory agency approved.  Then in the early 80’s with monoclonal based technology, a new source for reagent production became available.  Most of the early reagents were used on slides but as tube-based tests became available extended antigen typing transitioned to tube based approaches. Further transitions into new technologies have occurred using microwell/microplates and column-based tests.

Matt: You mentioned prevalence of blood group antigens, can you talk a bit more about that?

Tony:  Well, as blood group antigens were discovered and further defined population studies were done, these studies focused on defined ethnic groups and regions of the world demonstrating that differences exist in frequency of specific genes and therefore antigens expressed. So Matt, there are many examples of blood group antigens that differ in the population; in the African American population the Fy(a-b-) is very common but rare in the Caucasian population, Di(a+) individuals are of higher prevalence in South America, Central America and Japan, Jk(a-b-) in Polynesians and  in Japan finding an Fy(a-) individual in the Japanese population is considered rare as greater than 99% of Japanese are Fy(a+). I could probably go on with even more examples but these all relate back to an ancestral lineage. As an example of a commonly tested antigen, Jka prevalence breaks down as follows: Caucasian 77%, Blacks 92% and 72% in Asians.

For those who are passionate about this data and polymorphisms, there are two text books I recommend: one from Mourant and colleagues, is the “The Distribution of Human Blood Groups and other Polymorphisms”, and more recent information including differences at the molecular level between blood group antigens can be found in “the Blood Group Antigens Facts Book” by Reid, Francis-Lomas and Olsson.

Matt: So, when we talk about extended antigen typing, how do hospitals and donor centers use it?

Tony: Well Matt, red cell phenotyping in patients is mainly associated with antibody identification. Let me explain this a bit further; when a patient develops a blood group antibody the confirmation of the identity of that antibody is done by demonstrating that the patient lacks the antigen therefore is capable of developing that antibody. Phenotyping of a patient can play an even bigger role  in 2 ways:  first when a patient has what appears to be multiple antibodies that are difficult to sort through, identifying the phenotype of the patient can help further identify the possible antibodies that can be formed by the patient. This then allows us the ability to appropriately select cells to sort through the possibilities of antibodies that may be present and finding that a phenotype matched cells are compatible.  Of course, if they are incompatible then there may be more to the antibody identification story.

Secondly phenotyping becomes a valuable tool to help prevent the development of antibodies when you know which red cell antigen(s) the patient lacks.

Matt:  Can you please expand on this concept Tony?

Tony: Absolutely! what I mean is this approach helps identify what antigen(s) to avoid transfusing to the patient and is critical to apply to those patients who are frequently transfused.  This plays a significant role in transfusion of sickle cell, thalassemia and myelodysplastic syndrome patients as well as other chronically transfused patients. Preventing the development of antibodies avoids the challenges that come later with complex antibody problems and helps finding compatible blood for the patient.

Matt:  Then, you have the other side of phenotyping donors, correct?

Tony: Exactly! Searching through donors for antigen negative blood can be quite laborious.  In hospitals, finding donor units in their blood bank refrigerator requires phenotyping with the antiserum to locate antigen negative donors for their patient. Knowing the prevalence of the antigen allows to know how many units need to be tested. As an example, a patient with anti-Jka needs to be transfused with 2 units. The search of their blood inventory will require that about 10 units of blood be tested with reagent anti-Jka to find the 2 units. That is because we know that about 75% of the population is Jk(a+). In blood centers, testing of donors, particularly new donors that become repeat donors is necessary to keep a ready supply of antigen negative units at the ready for patients in hospitals.  Donor centers pursue multiple antigen negatives to be ready to supply hospitals by testing with a variety of antisera.   In some cases, some centers use DNA based genotyping to identify multiple antigen negative units, followed by serological phenotyping. Once a donor is antigen typed based on typing of two different donations, that historical record can be used to label the units in the future.

Matt: You mentioned antigen typing can be laborious? Why is that the case?

Tony: Well Matt just consider if you need to find antigen negative blood for a patient by testing a bunch of donors. Then consider the need to do this manually, recording donor numbers, labelling tests, selecting the right antisera,  dispensing antisera, selecting red cell controls, preparing  red cell suspensions, dispensing the red cell suspensions, incubating ( if necessary), test processing ( centrifugation, washing etc) and finally reading  and recording results.  Let’s take the same concept to phenotyping a patient for multiple antigens and applying all of the same manipulations just discussed but now you are testing with various antisera. The key here is that whenever testing is done manually there are lots of opportunity for errors. There is lots of record keeping required for extended antigen typing.

Matt: So can we make phenotyping for red cell antigens less arduous?

Tony: Absolutely! through automating the test we can eliminate many of the manual interactions that are prone to errors in performing the test and at the same time minimize the record keeping activities that take place.  So, with full traceability of process steps delivered by automation along with reaction grading, interpretation and image capture, full process control is attained. Keep in mind that the instrument captures all the data and that can be transferred through interfacing to the laboratory information system.   The bottom line here is that the benefits that automation delivers on routine testing can be achieved for extend antigen typing improving the outcomes and bringing confidence to the transfusion for that patient.

Matt Dawson: Well, that brings us to the end of this podcast. As you can see during pre-transfusion testing, labs routinely encounter patient samples that require extended antigen typing for blood group system antigens beyond those most commonly tested (A,B,D). These patients have developed atypical antibodies (ie K, Lea, Leb), which require additional testing to find compatible blood. Tony, thank you again for taking the time to speak with us and share your expertise.

I hope you enjoyed this podcast edition about the importance of extended antigen typing in Transfusion Medicine. Make sure to review the section within the podcast description for Reading materials suggested in the reference list.

Now, based on our Podcast Today, I leave you with the Ortho pop quiz of the day: So what is the prevalence of Fy(a+) in the Japanese population?

Thank you for listening today.

Please subscribe to Ortho Science BYTES, our monthly podcast, where we will be discussing more complex questions, we face every day in our Labs.  Brought to you by Ortho Clinical Diagnostics, pioneering advances in diagnostics for 75 years, because every test is a life.

Take care and stay safe.