Blocking phenomenon occurs with murine monoclonal antibodies (anti-Fy(a)) in a neonate with a positive direct antiglobulin test due to maternal anti-Fy(a).

Abstract

Gaining an accurate type for patients’ red blood cells that have a positive direct antiglobulin test (DAT) is problematic using serological methods. Attempting to type red cells heavily coated with immunoglobulin, complement or both by an indirect antiglobulin technique will be falsely positive because of the in vivo coating. Currently, only limited direct saline agglutinating reagents (often IgM monoclonals) are available. Alternatively, chemical treatment can be applied to remove IgG antibodies (allo-antibodies or auto-antibodies) from the DAT-positive red blood cells. Chloroquine diphosphate1 is a chemical used in blood transfusion laboratories to dissociate IgG from red blood cells without compromising the integrity of the red cell membrane antigens by neutralisation of the charged groups on amino acids that govern the tertiary structure of antibody molecules (i.e. R groups that are involved with intermolecular bonds). However, chloroquine diphosphate may not totally remove the coating of antibody from red cells and it is not able to remove complement component 3 (C3) coating. ZZAP2 is a combination of an enzyme/reducing agent composed of dithiothreitol and papain; it removes antibody coating by disruption of membrane-bound IgG through the combined action of a protease and a thiol reagent. EDTA/citric acid3 removes antibodies from red blood cells by lowering the pH of antigen and antibody proteins such that they both become protonated. The antigens and antibodies lose their ability to attract one another through electrostatic bonding and may be forced apart by repulsion of like charges. The tertiary structure of proteins may be affected, hydrogen (H+) ions attracted to hydroxyl (OH−) groups on aspartic acid and glutamic acid resulting in molecular unfolding with consequential loss of structural complementarity between antibody and antibody. Several other methods are also available for removing antibody to allow serological testing; however, the red blood cells are not suitable for typing after these treatments because there is damage to the cells as a result of loss or denaturing of some red blood cell antigens. This leads to invalid typing results with regards to some MNS, Duffy, Kell and Yt system antigens2–9. Murine monoclonal antibodies are known not to cross species specificity. These antibodies have been successfully used to type DAT-positive patients’ red blood cells9,10. A number of murine antibodies from the New York Blood Center (New York, USA) were used in those studies, including MIMA-8 (anti-Jsb), MIMA-19 (anti-Fya), MIMA-22 and −23 (anti-K), MIMA-21 and −27 (anti-Kpa), and MIMA-174 and −180 (anti-s) (unpublished data). The antigen blocking phenomenon is not uncommon in examples of human anti-D and -K11,12. MIMA-19 (anti-Fya), a very specific antibody which can be used in the testing of human DAT-positive red cells, was shown by Lee et al. to be blocked by a high level of anti-Fya. Failure to recognise this false negative result obtained using the MIMA-19 would have led to a false negative type being reported, even though the murine monoclonal antibodies were used as described by the Authors previously. We report a case of a false negative Fya type when using a murine monoclonal anti-Fya (MIMA-19) due to blocking by a maternal anti-Fya.