Background: According to national guidelines or directives, monoclonal
ABO reagents may be required to detect Ax and B weak subgroup red blood
cells (RBCs). Many routine laboratories do not have access to naturallyoccurring
ABO subgroups that can be used as weak controls for these
reagents. Group O RBCs modified with synthetic analogs of blood group A
and/or B glycolipids (KODE technology) to mimic weak ABO subgroups
could be used for quality control purposes.
Aim: Extensive serological testing of KODE RBCs has previously been
performed. An extended evaluation of KODE RBCs using flow cytometry
was performed to explore the correlation between the concentrations of
synthetic glycolipids and A/B site density of the resulting RBCs. The aim of
this study was to examine if KODE RBCs mimic the distinct flow cytometric
patterns of naturally-occurring ABO subgroups.
Material and Methods: KODE RBCs were prepared according to a previously
decribed 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 to
0.3 lg/mL for KODE-B. The concentration was decreased by doubling
dilution steps. Sensitive and specific flow cytometry [Hult & Olsson.
Transfusion 2006; 9S: 32A] was used to characterize and semiquantify the
synthetic 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 concentrations
and all KODE batches were tested in triplicate.
Results: Flow cytometric testing of KODE RBCs modified with high
concentrations of synthetic glycolipids revealed a uniform and even
distribution of antigens in the cell population as shown by a single
narrow peak in the FACS histograms. When lower concentrations were
used, peaks tended to broaden to a pattern found in Ax and most B
subgroups indicating a more variable antigen site density on the cells in
the population. The concentrations of synthetic glycolipids that produced
KODE cells that resembled the naturally-occurring subgroup control RBCs
used in this study are ~2–4 lg/mL for KODE-A and ~10 lg/mL for KODEB.
Repeat testing demonstrated good correlation between flow cytometric
runs.
Discussion and Conclusion: Using very low amounts of synthetic
glycolipids, KODE-A and KODE-B RBCs can be made to mimic Ax and
Bweak subgroup control RBCs, respectively, according to this flow
cytometry method. With higher concentrations of synthetic glycolipids, the
KODE RBCs demonstrated a more uniform and even distribution of antigens
among the cells. This is in contrast to naturally-occurring subgroups
in which some cells express almost no A or B antigen whilst others have
close to normal levels. The reason for this is unknown. KODE RBCs obviously
lack A carrying glycoproteins but it is not fully understood to what
extent glycolipid versus glycoprotein epitopes contribute to the phenotype
of weak subgroups.
This study indicates that KODE RBCs with weak expression of A and/or B
antigen have characteristics compatible with use as quality controls for
monoclonal ABO reagents and could be a valuable addition in the
serological laboratory