Discriminating the hemolytic risk of blood type A plasmas using the complement hemolysis using human erythrocytes (CHUHE) assay

Abstract

BACKGROUND The agglutination‐based cross‐matching method is sensitive for antibody binding to red blood cells but is only partially predictive of complement‐mediated hemolysis, which is important in many acute hemolytic transfusion reactions. Here, we describe complement hemolysis using human erythrocytes (CHUHE) assays that directly evaluate complement‐mediated hemolysis between individual serum‐plasma and red blood cell combinations. The CHUHE assay is used to evaluate correlations between agglutination titers and complement‐mediated hemolysis as well as the hemolytic potential of plasma from type A blood donors. STUDY DESIGN AND METHODS Plasma or serum from each type A blood donor was incubated with AB or B red blood cells in the CHUHE assay and measured for free hemoglobin release. RESULTS CHUHE assays for serum or plasma demonstrate a wide, dynamic range and high sensitivity for complement‐mediated hemolysis for individual serum/plasma and red blood cell combinations. CHUHE results suggest that agglutination assays alone are only moderately predictive of complement‐mediated hemolysis. CHUHE results also suggest that plasma from particular type A blood donors produce minimal complement‐mediated hemolysis, whereas plasma from other type A blood donors produce moderate to high‐level complement‐mediated hemolysis, depending on the red blood cell donor. CONCLUSION The current results indicate that the CHUHE assay can be used to assess complement‐mediated hemolysis for plasma or serum from a type A blood donor, providing additional risk discrimination over agglutination titers alone.

Challenges in the treatment and prevention of delayed hemolytic transfusion reactions with hyperhemolysis in sickle cell disease patients

“…Consistent with this, prior observations suggest that many incompatible RBC transfusions fail to result in a hemolytic transfusion reaction, highlighting that the outcome of incompatible RBC transfusion even when the target antigen is known can be quite varied . Recent tools have been developed, including the monocyte monolayer assay, to aid in the prediction of adverse events following RBC transfusion . Unfortunately, many of these tests do not lend themselves to rapid turnaround time and/or do not accurately predict in vivo hemolytic potential, reducing their utility in the management of patients at risk for or acutely experiencing hemolytic events.…”

“…43,44 Recent tools have been developed, including the monocyte monolayer assay, to aid in the prediction of adverse events following RBC transfusion. [45][46][47] Unfortunately, many of these tests do not lend themselves to rapid turnaround time and/or do not accurately predict in vivo hemolytic potential, reducing their utility in the management of patients at risk for or acutely experiencing hemolytic events. Ultimately, the varied nature of RBC antigens coupled with incompletely understood recipient factors that likely influence the outcome of DHTRs make it difficult to predict what approach, if any, may be optimal in a given patient.…”

Who's afraid of incompatible plasma? A balanced approach to the safe transfusion of blood products containing ABO‐incompatible plasma

“…The monocyte monolayer assay shows some promise in distinguishing between clinically significant and insignificant alloantibodies of IgG type . Other in vitro assays, such as the recently described complement hemolysis using human erythrocytes (CHUHE) test, might also be useful in predicting the hemolytic potential of group A plasma . However, until more clinical correlations between the results of these assays and actual recipient outcomes can be made, antibody titration is the most accepted method available for predicting the risk of an HTR from ABO‐incompatible plasma transfusions.…”

“…37 Other in vitro assays, such as the recently described complement hemolysis using human erythrocytes (CHUHE) test, might also be useful in predicting the hemolytic potential of group A plasma. 38,39 However, until more clinical correlations between the results of these assays and actual recipient outcomes can be made, antibody titration is the most 41 Gel 64, 256 Cooling et al 6 Gel NT, 128 Quillen et al 42 Gel 250, NT Karafin et al 19 Gel 512 Pittsburgh, PA 16 Tube 100, NT UK national guidance 43 Automated Tube 34 Tube 50, NT accepted method available for predicting the risk of an HTR from ABO-incompatible plasma transfusions. Apart from the titer technique, selection of a titer threshold requires some cognizance of the potential impact that the threshold will have on the available donor pool; the lower the selected titer threshold, the fewer donors that are likely to be suitable.…”

Complement activation by human red blood cell antibodies: hemolytic potential of antibodies and efficacy of complement inhibitors assessed by a sensitive flow cytometric assay

“…Because of the potential fatality of hemolytic reactions, we designed an in vitro assay that allows the evaluation of complement activation by antibodies using a model for intravascular hemolysis . Normally, a hemolysis assay using human RBCs and homologous complement lacks sensitivity and is suitable only for heavily complement‐activating antibodies such as certain isoagglutinins . The resistance of human RBCs to hemolysis is due to the presence of membrane‐bound complement regulators on the RBC membrane, especially CD59, which restricts the lytic complement reaction.…”