I M M U N O H E M A T O L O G Y

Flow cytometry evaluation of red blood cells mimicking naturallyoccurring ABO subgroups after modification with variable

amounts of function-spacer-lipid A and B constructs_3268 247..251

Annika K. Hult, Tom Frame, Scott Chesla, Stephen Henry, and Martin L. Olsson

BACKGROUND: Kodecytes bearing synthetic bloodgroup A and B antigens are increasingly being used intransfusion laboratories as serologic mimics of redblood cell (RBC) Aweak and Bweak subtypes. The aim ofthis study was to compare the flow cytometry profile ofkodecytes with native ABO subgroups.STUDY DESIGN AND METHODS: A series of A/Bkodecytes, each with decreasing A or B antigen expres-sion, were prepared from group O RBCs that weremodified with dilutions of function-spacer-lipid KODEtechnology (FSL) constructs representing a wide sero-logic range. Using an established flow cytometrymethod designed for the detection of low levels of A/Bantigens, kodecyte profiles were compared with thoseof native subgroup cells.RESULTS: Kodecytes with positive tube serology from4+ to 1+ were created with 15 to 2 mg/mL FSL-A or 78to 10 mg/mL FSL-B transformation solutions. The kode-cytes created with higher concentrations of FSL con-structs revealed a uniform and/or even distribution ofantigens as seen by a single flow cytometry peak morenarrow than the broader peaks produced with lowerFSL concentrations similar to those found in native Ax

and most Bweak subgroups.CONCLUSIONS: Although kodecytes are created artifi-cially, they can be designed to mimic the serologic andflow cytometric profiles of native ABO subgroup RBCs.

According to UK national1 guidelines or Euro-pean directives,2 monoclonal ABO reagents arerequired to detect Ax and Bweak subgroup redblood cells (RBCs). However, many routine

laboratories do not have access to naturally occurringABO subgroups, and those who do seldom characterizethem genetically to confirm their subgroup status. GroupO RBCs can be controllably modified with synthetic bloodgroup A and/or B function-spacer-lipid (FSL) constructs,3

and thus have the potential to be ABO subgroups mimics.These so-called kodecytes4 (KODE construct modifiedcells) have been used in quality control (QC) systems4 andmore recently to determine in vivo RBC survival, mimicincompatible transfusion reactions, and neutralize anti-Ain an animal model.5,6 The aim of this study was toexamine if kodecytes can be created to mimic the distinctflow cytometric patterns recently established for nativeABO subgroups7 and the optimal concentration of FSLsrequired to achieve this.

MATERIALS AND METHODS

FSL constructsTwo FSL constructs were obtained from KODEBiotech Materials Ltd (Auckland, New Zealand;

ABBREVIATION: FSL = function-spacer-lipid KODE Technology

construct.

From the Division of Hematology and Transfusion Medicine,

Department of Laboratory Medicine, Lund University, and

Department of Clinical Immunology and Transfusion Medicine,

University and Regional Laboratories Region Skåne, Lund,

Sweden; ImmucorGamma, Norcross, Georgia; and the Biotech-

nology Research Institute, AUT University, Auckland, New

Zealand.

Address correspondence to: Steve Henry, Biotechnology

Research Institute, AUT University, Private Bag 92006, Auckland

1142, New Zealand; e-mail kiwi@aut.ac.nz.

Received for publication March 17, 2011; revision received

May 19, 2011, and accepted June 6, 2011.

doi: 10.1111/j.1537-2995.2011.03268.x

TRANSFUSION 2012;52:247-251.

Volume 52, February 2012 TRANSFUSION 247

http://www.kodebiotech.com): FSL-A with a bloodgroup A trisaccharide epitope GalNAca3(Fuca2)Galb(FSL-A(GALNa3[Fa2]GALb)-SA1-L1, Cat. No. 421604) andFSL-B with a blood group B trisaccharide epitopeGala3(Fuca2)Galb (FSL-B(GALa3[Fa2]GALb)-SA1-L1, Cat.No. 199283). These constructs are also available fromSigma-Aldrich (St Louis, MO).

KodecytesKodecytes were prepared in two independent batches atdifferent times according to a previously described proce-dure.3 Group O RBCs were modified (transformed) withdifferent concentrations of FSLs. The first batch containedconcentrations of FSLs down to 2 and 9 mg/mL for FSL-Aand FSL-B, respectively, whereas the second batch con-tained extended dilution steps (Table 1), ranging from 15to 0 mg/mL for FSL-A and 78 to 0 mg/mL for FSL-B (asserial twofold dilutions). Native RBCs of different ABOphenotypes were used as controls. Manual tube sero-logy was performed by the immediate-spin method(15 sec, 900 ¥ g) with 3% to 4% kodecyte suspensions inphosphate-buffered saline (PBS) against monoclonalanti-A (AM201-1) and anti-B (BM154-1; ImmucorGamma,Inc., Atlanta, GA). Kodecytes are defined as cells that canbe shown to have acquired the FSL construct5,6 and can bedescribed by the concentration and type of constructinserted, for example, 4 mg/mL A kodecytes.

Flow cytometryKodecytes and control RBCs were washed three times inPBS and 25 mL of RBCs were suspended in 900 mL of PBS.Flow cytometry was as previously described using a highlysensitive method developed to detect A and B antigens inABO subgroup phenotypes using ABO reagents; anti-A,ES-15 (Serologicals Limited, West Lothian, UK); anti-B,9621A8 (Diagast, Loos, France) and phycoerythrin-labeledrat anti-mouse Ig kappa light chain (Becton Dickinson,San Jose, CA) as secondary antibody.7 Each batch wastested three times during a 3-week period. Additional ABO

reagents used were anti-A, AM201-1, anti-B, BM154-1,anti-AB, and ABM201-1 (ImmucorGamma, Inc.). Bothbatches of kodecytes were tested with the additional ABOreagents, the first batch tested once and the second testedtwice, 3 weeks apart. Relevant control RBCs A1, A2, Ax (Ax-1/O1 genotype), B, Bweak (Bw-3/O1 genotype), and group Owere included in each run, but for simplicity the histo-grams reported show only the most relevant, selectedcontrols.

RESULTS

A range of A and B kodecytes with decreasing antigenexpression was prepared twice from the same group ORBC, starting from the FSL transformation solution con-centration that gave a maximum 4+ serologic result by thestandard tube method. The group O RBC used for thepreparation of the A and B kodecytes were also tested byserology and flow cytometry against anti-A, -B, and -H andbehaved like a normal group O control (results notshown).

Tube serologyFSL-A transformation solutions over a four-tube dilutionrange (15-2 mg/mL) created A kodecytes that gave positivetube serology from 4+ to 1+ (Table 1). The 4 and 2 mg/mL Akodecytes gave serologic reactions in the range expectedof Aweak subgroups. Similarly FSL-B was also able to createpositive tube serology from 4+ to 1 + over a four-tube dilu-tion, but in the range of 78 to 10 mg/mL. The 20 and10 mg/mL B kodecytes gave serologic reactions in therange expected of Bweak subgroups.

Flow cytometryFlow cytometric testing of kodecytes prepared withhigher FSL construct solutions revealed a uniform, repro-ducible, and even distribution of antigens in the cellpopulation as seen by a single, quite narrow peak in theflow cytometry histograms (Fig. 1). When kodecytes were

TABLE 1. Aweak, Bweak kodecytes created by modification of O cells with different concentrations of FSLtransformation solutions

FSL-A–modified O cells FSL-B–modified O cells

Transformation solution Tube serology Flow cytometry* Transformation solution Tube serology Flow cytometry*

FSL-A (mg/mL) AM201-1 AM201-1 FSL-B (mg/mL) BM154-1 BM154-1

15 4+ Positive 78 4+ Positive8 4+ Positive 39 3+ Positive4 2+ Positive 20 2+ Positive2 1+ Positive 10 1+ Positive1 – Positive 5 – Positive0.5 – – 3 – –0.2 – – 1 – –

* Positive MFI in relation to the group O control cell (for details see Figs. 1 and 2).

HULT ET AL.

248 TRANSFUSION Volume 52, February 2012

prepared with lower FSL concentrations, peaks tended tobroaden to resemble a pattern found in Ax and most Bweak

subgroups,7 indicating a more variable antigen sitedensity on the cells in the population, although this ispartially explained by the logarithmic scale used todisplay the results. The concentrations of FSLs that pro-duced kodecytes that resembled the naturally occurringsubgroup control RBCs used in this study are approxi-mately 2 to 4 mg/mL for FSL-A and approximately10 mg/mL for FSL-B. Repeat testing demonstrated goodcorrelation between flow cytometric runs and kodecytebatches (data not shown).

DISCUSSION

Kodecytes are becoming more evident in some transfu-sion laboratories where they are being routinely used asQC cells expressing low levels of A and B blood groupantigens.4 However, as A/B kodecytes are created byinserting simple glycolipid-like FSL constructs into groupO RBCs the question of whether they are representative ofnative ABO subgroups,8 which bear a range of simple andcomplex ABO antigens on both glycolipids9 and glycopro-teins, requires addressing. We used the same methodologyin our recent report characterizing low levels of A/B

15 mg/mL

O

O

A2AxB

BBw

84

2 1

0.5

78 mg/mL39

20

10

5

3

Anti-A-derived fluorescence

Cel

l co

un

tC

ell c

ou

nt

Anti-B-derived fluorescence

FSL-A

FSL-B

Fig. 1. Representative histograms of Aweak kodecytes (top histograms, blue fill), Bweak kodecytes (bottom histograms, yellow fill), and

natural RBCs against monoclonal reagents (anti-A clone ES-15 and anti-B clone 9621A8). Kodecytes (red lines) of decreasing

antigen expression are indicated by a number that represents the mg/mL FSL construct concentration in the transformation solu-

tion used to create them (Table 1). Control RBCs analyzed at the same time as the kodecytes are included in each histogram (and

labeled in the first histogram) and are as follows: group O (black line), group B (blue line), and group A2 (green line) plus natural

subgroups Ax with the Ax-1/O1 genotype and Bw with the Bw-3/O1 genotype are shown as orange lines.

FLOW CYTOMETRY OF ABO KODECYTES

Volume 52, February 2012 TRANSFUSION 249

antigen on a large variety of ABO subgroups,7 to investi-gate A and B kodecytes. A similar approach has also beenapplied as QC of group A, B, and AB RBCs converted bybacterially derived exoglycosidases to type as group Owith CE-marked and FDA-approved monoclonal anti-Aand -B reagents.10 Using appropriate concentrationsof FSL-A and -B constructs, kodecytes could be easilycreated that gave manual tube serology and flow cytom-etry profiles identical to those observed for ABO sub-groups expressing low levels of A and B antigens. It wasinteresting to note that flow cytometry was only slightlymore sensitive than manual tube serology when the same

antibody was used (Table 1), and although different anti-bodies did show differences in mean fluorescence withkodecytes (Fig. 2), the endpoints were essentially thesame. However, in this article the monoclonal antibodiesused were specifically selected for their high sensitivitywith some shown to detect native ABO subgroups7 andremaining A and B antigens after exoglycosidase treat-ment.10 It would be expected that different clones wouldshow different sensitivity profiles, as has been shown byserology.3 Recently, we have also found a few qualitymonoclonal ABO reagents that do not react with FSL-A/Bconstructs and others which react with FSL-A/B con-

100

200

0 0

0

00

100

200

400

300

600

100

200

400

300

Anti-A (ES-15)

Anti-B (9621A8)

Anti-AB (ABM201-1)

Anti-A (AM201-1)

Anti-B (BM154-1)

300

200

100

15

mg/mL FSL-A kodecytes

mg/mL FSL-B kodecytesmg/mL FSL-B kodecytes

mg/mL FSL-A kodecytes or mg/mL FSL-B kodecytes

mg/mL FSL-A kodecytes

78

8

39

4

20

2

10

1

5

0.5

3

O

O

Ax

Bw

15 8 4 2 1 0.5 O Ax

78 39 20 10 5 3 O Bw

15/78 Bw8/39 4/20 2/10 1/5 0.5/3 O Ax

Fig. 2. Mean fluorescence intensity (MFI) comparison of a representative flow cytometric run of Aweak and Bweak kodecytes against

different monoclonal reagents. Natural O, Ax, and Bweak samples (gray bars) are included for comparison. Black bars compare Aweak

kodecytes labeled with two monoclonal anti-A reagents (ES-15 and AM201-1). White bars show Bweak kodecytes labeled with mono-

clonal anti-B (9621A8 and BM154-1). The lower bar graph shows the reactivity with the same kodecytes as above but tested against

monoclonal anti-A,B (ABM201-1), with the A kodecytes in black and B kodecytes in white. Note: the scale range for the y-axis is

different between images.

HULT ET AL.

250 TRANSFUSION Volume 52, February 2012

structs but do not react with natural glycolipid antigens ofthe same specificity (unpublished). It is clear that furtherknowledge of the specificity of synthetic antibodies(monoclonal reagents) and synthetic antigens (FSL con-structs) is still necessary.

Again3 the requirement for more blood group B thanblood group A antigen was evident, with five times moreFSL-B transformation required to be serologically equiva-lent to FSL-A transformation. This phenomenon is con-sistent when either monoclonal or human polyclonalantibodies are used and against different variants of bothnatural and synthetic B antigen (unpublished). It is nowbelieved to be due to the structure of the B antigen, whichhas an inherently lower affinity for specific antibody, incontrast to its antithetical partner the A antigen, whichhas the N-acetyl protrusion on its terminal sugar. Thisconcept is also supported by clinical observations thatanti-B less frequently causes hemolytic disease of thefetus or newborn.11

In agreement with earlier reports3,4 on the agglu-tination serology of kodecytes, by simply altering theconcentration of FSL construct in the transformationsolution “antigen dilutions” were introduced to the kode-cytes. By flow cytometry and with higher concentrationsof FSLs, the kodecytes created demonstrated a relativelyuniform and even distribution of antigens among thecells. Using very low amounts of FSL-A and FSL-B, kode-cytes could be made which mimicked the broad antigendensity flow cytometry profiles of some natural Ax andBweak subgroup RBCs. However, not all naturally occur-ring ABO subgroups are equal and some may have eithera narrower or wider range of antigen distribution pro-files7 than are seen with kodecytes. The reason for thisinteresting phenomenon is unknown. It was somewhatsurprising that the lower concentrations of FSL solutionsproduced broader antigen distributions on kodecytes.Even if unlikely, it cannot be excluded that this may bean artifact of preparation, potentially due to transfor-mation equilibration or contact times or possibly a pre-ference of FSLs for some RBCs (perhaps due to lipiddifferences as a consequence of age). Interestingly,para-Bombay samples, whose weak A phenotype isdependent on acquiring A glycolipids from plasma (andtherefore the natural analog to kodecytes), showed amore uniform and even antigen distribution.7 The samenarrow histogram peaks have also been shown to occurafter human in vivo plasma incubation, where group ORBCs transfused to group A individuals with the secretorphenotype, will acquire A glycolipids from the plasma ofthe recipient.12,13

This study indicates that kodecytes with low expres-sion of A and/or B antigen have characteristics compatiblewith use as QCs for monoclonal ABO reagents and couldbe a valuable addition in the serologic laboratory.

CONFLICT OF INTEREST

TF and SC are employees of ImmucorGamma, a licensee of KODE

technology. SH is the CEO/CSO and a stockholder of KODE

Biotech Ltd, the patent owner of KODE cell surface engineering

technology. AH and MLO have no conflicts of interest regarding

this study.

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2. Francesco D. The New European Directive on in vitro diag-

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4. Henry S. Modification of red blood cells for laboratory

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