Ficin-Treated Red Cells Help Identify Clinically Significant Alloantibodies Masked as Reactions of Undetermined Specificity in Gel Microtubes

Ben C. Hill, MD; Courtney A. Hanna, MD; Jill Adamski, MD, PhD; Huy P. Pham, MD, MPH; Marisa B. Marques, MD; Lance A. Williams III, MD


Lab Med. 2017;48(1):24-28. 

In This Article

Abstract and Introduction


Non-specific antibodies or antibodies of undetermined significance (AUS) often pose problems for a blood bank technologist and physician. It is well known that antibodies can weaken and evanesce over time, thus eluding detection by routine blood bank techniques. Special enhancement techniques exist (eg, ficin treatment); however, they are often underutilized due to concerns over expense. Ficin is known to enhance reactivity caused by antibodies in the ABO, Rh, Kidd, Lewis, I, and P blood group systems, while destroying reactivity of antibodies in the Duffy, and MNS blood group systems. Herein, we discuss our protocol for using ficin treatment to determine the specificity of antibodies that would otherwise be classified as AUS. Of the 97 AUS specimens that were treated with ficin, we were able to identify 25 new alloantibodies that would have otherwise been missed without ficin treatment. Thus, we believe our protocol enhances transfusion safety, while minimizing additional workload and cost.


Although the risk of viral transmission by transfusion has decreased substantially in the past 20 years, other residual risks remain.[1] According to the United States Food and Drug Administration (FDA), the presence of non-ABO alloantibodies ranked as the third-highest cause of transfusion-related mortality nationwide between 2009 and 2014.[2] Typically, alloimmunization follows exposure to blood via pregnancy or transfusion; donor and recipient factors influence its occurrence.[3] However, much remains unknown regarding prevention of alloimmunization and antibody persistence to improve transfusion safety.

Although large transfusion services identify alloantibodies multiple times per day, the greatest concern regards antibodies that may not have been detected. Some clinically significant alloantibodies are difficult to detect because of their evanescent nature or due to the low sensitivity of the testing method. Tormey and Stack[4] demonstrated that nearly 67% of alloantibodies identified at least once in 304 military veterans disappeared within 5 years of formation. Therefore, a negative antibody screening result does not guarantee that a patient will not develop hemolysis after an allegedly in vitro compatible red blood cell (RBC) transfusion. For this reason, the transfusion service must maintain transfusion records indefinitely and check for the history of alloantibodies every time a patient returns to the same institution for care.[5]

Another confounding factor in pretransfusion testing is interpretation of unexplained agglutination reactions, or antibody of undetermined specificity (AUS), as recently explored by Liu and Grossman.[6] According to the experience of those authors using gel microtubes, AUS was the most common antibody reported, representing 18% of all alloantibodies. Although ficin-treated reagent RBCs can be used to explain antibody panel reactions, many institutions do not routinely use them because of their cost and time consuming nature. However, we believe that if used in a limited number of scenarios, ficin treatment can be useful.

For example, in December of 2011, we encountered a 41-year-old African American man with a history of multiple transfusions; assay of his specimen in a gel microtube yielded a positive antibody screening result. Review of our records from approximately 18 months earlier revealed an AUS via the same method. Subsequent RBC units transfused had proved to be crossmatch compatible at the antiglobulin phase using gel methodology; the patient did not have any signs of hemolysis.

In the most recent sample from this patient, AUS was still present, and all clinically significant alloantibodies were, again, ruled out with homozygous cells. However, we elected to test his plasma with ficin-treated reagent cells in gel microtubes, recognizing that this proteolytic enzyme increases the sensitivity of the assay, helping it to identify antibodies to the ABO, Rh, Kidd, Lewis, P, and I antigens while destroying Duffy and MNS systems antigens. To our surprise, the pattern of reactivity revealed 2 clinically significant antibodies: anti-C and anti-e, which are antigens that the patient phenotypically lacked. After this index case, we instituted a laboratory policy whereby all AUS identified in gel microtubes is followed by testing of the specimen in question using ficin-treated cells. This report details the findings of our experience with that policy and the practicality of our approach.