Flow cytometric evaluation of red blood cells transformed with variable amounts of synthetic A and B glycolipids
Hult, K; Frame, T; Henry, S; Olsson, ML
MetadataShow full metadata
Background: According to national guidelines or directives, monoclonalABO reagents may be required to detect Ax and B weak subgroup red bloodcells (RBCs). Many routine laboratories do not have access to naturallyoccurringABO subgroups that can be used as weak controls for thesereagents. Group O RBCs modified with synthetic analogs of blood group Aand/or B glycolipids (KODE technology) to mimic weak ABO subgroupscould be used for quality control purposes.Aim: Extensive serological testing of KODE RBCs has previously beenperformed. An extended evaluation of KODE RBCs using flow cytometrywas performed to explore the correlation between the concentrations ofsynthetic glycolipids and A/B site density of the resulting RBCs. The aim ofthis study was to examine if KODE RBCs mimic the distinct flow cytometricpatterns of naturally-occurring ABO subgroups.Material and Methods: KODE RBCs were prepared according to a previouslydecribed procedure [Frame et al., Transfusion 2007; 47: 876–82].RBCs were modified with 15 different concentrations of synthetic glycolipids,ranging from 1 mg/mL to 60 ng/mL for KODE-A and 5 mg/mL to0.3 lg/mL for KODE-B. The concentration was decreased by doublingdilution steps. Sensitive and specific flow cytometry [Hult & Olsson.Transfusion 2006; 9S: 32A] was used to characterize and semiquantify thesynthetic A and B antigen levels on RBCs. Relevant control RBCs (A1, A2,Ax, B, Bweak and O) were included in each run. For both KODE-A and KODE-B RBCs, repeat samples were produced for four selected concentrationsand all KODE batches were tested in triplicate.Results: Flow cytometric testing of KODE RBCs modified with highconcentrations of synthetic glycolipids revealed a uniform and evendistribution of antigens in the cell population as shown by a singlenarrow peak in the FACS histograms. When lower concentrations wereused, peaks tended to broaden to a pattern found in Ax and most Bsubgroups indicating a more variable antigen site density on the cells inthe population. The concentrations of synthetic glycolipids that producedKODE cells that resembled the naturally-occurring subgroup control RBCsused in this study are ~2–4 lg/mL for KODE-A and ~10 lg/mL for KODEB.Repeat testing demonstrated good correlation between flow cytometricruns.Discussion and Conclusion: Using very low amounts of syntheticglycolipids, KODE-A and KODE-B RBCs can be made to mimic Ax andBweak subgroup control RBCs, respectively, according to this flowcytometry method. With higher concentrations of synthetic glycolipids, theKODE RBCs demonstrated a more uniform and even distribution of antigensamong the cells. This is in contrast to naturally-occurring subgroupsin which some cells express almost no A or B antigen whilst others haveclose to normal levels. The reason for this is unknown. KODE RBCs obviouslylack A carrying glycoproteins but it is not fully understood to whatextent glycolipid versus glycoprotein epitopes contribute to the phenotypeof weak subgroups.This study indicates that KODE RBCs with weak expression of A and/or Bantigen have characteristics compatible with use as quality controls formonoclonal ABO reagents and could be a valuable addition in theserological laboratory.