Isotype-specific detection of ABO blood group antibodies using a novel flow cytometric method

Isotype-specific detection of ABO blood group antibodies usinga novel flow cytometric method

Naturally occurring antibodies (Ab) such as anti-A/B Ab play

an important role in transfusion and transplantation medicine.

The analysis of ABO antigens (Ag) and anti-A/B Ab prior to

blood product transfusion and organ transplantation is part of

the daily routine in blood bank laboratories. Ignoring the

ABO-blood group match between donor and recipient may

lead to life-threatening complications, such as haemolytic

transfusion reactions and (hyper) acute vascular graft rejec-

tion. ABO-specific Ab belong to the group of naturally

occurring Ab first described by Landsteiner (1901) and later

studied extensively by other groups (Avrameas, 1991; Cou-

tinho et al, 1995). Anti-A/B Ab formation requires the

presence of accessible Ag, which is provided by microorga-

nisms in the gastrointestinal tract (Springer & Horton, 1969).

Although ABO Ag have only subtle chemical differences, anti-

A/B are, in general, highly specific for their respective blood

group Ag (Rydberg, 2001; Watkins, 2001). Nevertheless,

despite this high specificity shown for anti A/B natural Ab, a

variety of mainly IgM Ab, including monoclonal anti-D, show

polyspecificity and polyreactivity to unrelated exo- and

autoantigens (Thompson et al, 1992; Thorpe & Bailey, 1993).

Anti-A/B Ab differ from most naturally occurring Ab in so far

as they are expressed exclusively in individuals lacking the

corresponding Ag. Hence, they are not auto-reactive. However,

human plasma contains naturally occurring autoantibodies to

several red blood cell (RBC) proteins including the anion

transport protein, band 3 (Lutz, 2004). ABO typing on whole

RBC populations with anti-human IgG does not interfere with

binding of anti-band 3 autoantibodies in situ, because anti-

band 3 autoantibodies bind to oxidatively stressed and

senescent RBC (Lutz et al, 1988).

Blood group typing depends on the detection of ABO Ag on

the surface of RBC by means of haemagglutinating antibodies

against the A or B Ag (Brecher, 2002). The quantification of

anti-A/B Ab is carried out most frequently by haemagglutina-

tion and haemolysis techniques. These techniques are based on

G. Stussi,1 K. Huggel,1 H. U. Lutz,2 U.

Schanz,3 R. Rieben4 and J. D. Seebach1

1Laboratory for Transplantation Immunology,

Department of Internal Medicine, University

Hospital Zurich, Zurich, 2Institute of

Biochemistry, ETH Hoenggerberg, Zurich,3Division of Haematology, Department of Internal

Medicine, University Hospital Zurich,

Switzerland, and 4Department of Clinical

Research, University of Bern, Bern, Switzerland

Received 10 March 2005; accepted for

publication 22 June 2005

Correspondence: PD Dr med. Jorg D. Seebach,

University Hospital Zurich, Department of

Internal Medicine, Laboratory for

Transplantation Immunology, Ramistrasse 100,

C HOER 31, CH-8091 Zurich, Switzerland.

E-mail: [email protected]

Summary

Several methods to detect anti-A/B antibodies based on haemagglutination

and haemolysis have been described. These methods measure predominantly

anti-A/B immunoglobulin (Ig)M, whereas anti-A/B IgG and IgG subclasses

are less well examined. We established a flow cytometry method (ABO-

fluorescence-activated cell sorting; ABO-FACS) to quantify binding of anti-

A/B IgM, IgG and IgG subclasses to human A or B red blood cells. Anti-A/B

IgM were present in the majority of 120 blood donors, as expected from

blood group typing. The sensitivity and specificity of anti-A/B IgM to predict

the blood group was 93% and 96% respectively. Anti-A/B IgG was found in

34/38 blood group O samples (89%). Anti-B IgG in blood group A or anti-A

IgG in blood group B was present in 4/28 (14%) and 1/28 (4%) samples,

respectively, and absent in 26 AB sera. IgG2 was the predominant IgG

subclass. The correlation of anti-A/B IgM and IgG in the ABO-FACS with

haemagglutination titres was 0Æ870 and 0Æ783, respectively (n ¼ 240;

P < 0Æ001) whereas the comparison of ABO-FACS with ABO-enzyme-

linked immunosorbent assay was less significant. In conclusion, ABO-FACS

is a valid method to quantify anti-A/B IgM, IgG and IgG subclasses. It opens

the possibility of isotype-specific monitoring of anti-A/B antibodies levels

after ABO-incompatible solid organ and stem cell transplantation.

Keywords: ABO blood group, antibodies, flow cytometry, enzyme-linked

immunosorbent assay, immunoglobulin G subclasses.

research paper

doi:10.1111/j.1365-2141.2005.05705.x ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 130, 954–963

crosslinking of standardised RBC by haemagglutinins in the

serum of interest. Yet, haemagglutinating Ab are predomin-

antly of the IgM class and the determination of anti-A/B IgG in

clinical practice is more challenging. A variety of assays have

been used for this purpose, making it difficult to compare the

results of different studies (Hershko et al, 1980; Sniecinski

et al, 1988; Bar et al, 1995; Nussbaumer et al, 1995). More-

over, none of these techniques allows the determination of IgG

subclasses. Therefore, several years ago an enzyme-linked

immunosorbent assay (ELISA)-based method, to measure the

binding of anti-A/B Ab to synthetic or animal-derived A and B

substances, was developed, enabling the discrimination

between IgM, IgG and IgG subclasses (Buchs & Nydegger,

1989; Rieben et al, 1991). However, this assay depends on the

binding to synthetic or animal-derived Ag, rather than the

actual human ABO Ag on RBC. We therefore developed a new,

semi-quantitative assay based on flow cytometry to determine

the binding of IgM, IgG and IgG subclasses to ABO Ag

expressed on RBC (ABO-fluorescence-activated cell sorting,

ABO-FACS). Using this method we measured the total amount

of anti-A/B Ab in 120 healthy blood donors and compared the

results with haemagglutination and the ABO-ELISA.

Materials and methods

Sera and red blood cells

The amount of anti-A/B Ab in the sera of healthy donors was

evaluated by measuring binding to human RBC. For this

purpose, 120 sera were obtained from the local transfusion

service and stored at )20�C (28 A, 28 B, 38 O, and 26 AB).

Blood group typing was performed by standard techniques and

all samples were negative for human immunodeficiency virus,

hepatitis B virus and hepatitis C virus. The mean age of the

blood donors was 50 years, ranging from 21 to 70 years. Forty-

six per cent were male and 76% were rhesus positive. Eighty-

eight per cent of blood group A or AB donors had the A1 or

A1B subgroup and the remainder had the A2 or A2B subgroup.

Statistical analyses of age, rhesus blood group and gender did

not reveal significant differences among the ABO blood groups

(data not shown). Rhesus negative human RBC of type A1, B,

and O were obtained from the local transfusion service.

Porcine RBC and serum were used as controls and were kindly

provided by the local slaughterhouse. The RBC were isolated

from heparinised venous blood and washed three times in

phosphate-buffered saline (PBS). To prevent haemagglutina-

tion, the cells were fixed according to a previously described

method (Pins et al, 1997). Briefly, 100 ll of highly concentra-

ted RBC (haematocrit 80%) were incubated for 20 min at 4�Cin 4 ml Karnovsky buffer, which contains formaldehyde and

glutaraldehyde. Thereafter, the RBC were washed once in PBS

containing 6% bovine serum albumin (BSA; Fluka Biochem-

icals, Buchs, Switzerland) and twice in PBS containing 0Æ6%BSA. After fixation a 1% solution of RBC was prepared

resulting in a concentration of 1–5 · 108 RBC/ml. RBC were

stored at 4�C and used for up to 6 weeks without changes in

Ab binding levels.

For antibody absorption, serum was incubated with washed,

non-fixed A, B, or O RBC at different ratios (106–108/ml) for

30 min at RT under rotation using a disc rotator (Fischer

Bioblock Scientific, Illkirch, France: 33 rpm). Subsequently the

samples were centrifuged for 4 min at 1700 g and the

supernatant was harvested. This procedure was performed

consecutively three times and the absorbed sera were tested by

ABO-FACS. For degradation of IgM molecules, serum was

incubated for 45 min with 20 mmol/l dithiotreitol (DTT; Sigma

Chemical Co., St Louis, MO, USA) and tested by ABO-FACS.

ABO-FACS

Undiluted serum (35 ll) was incubated with 25 ll of a 1%

solution of fixed RBC for 30 min at 4�C and subsequently

washed once in 3 ml of FACS buffer (0Æ1% BSA in Hank’s

buffered salt solution). Binding of anti-A/B Ab was measured

by indirect flow cytometry using secondary phcoerythrin-

labelled Ab (goat anti-human IgG; Zymed Laboratories, Inc.,

San Francisco, CA, USA; goat anti-human IgM; Sigma

Chemical Co.). A minimum of 10 000 events was acquired

on the flow cytometer. Human AB-serum from a single-donor

was further depleted of potentially autoreactive anti-A/B Ab by

incubation with highly concentrated A and B RBC and used as

negative control. To exclude binding of irregular RBC Ab, a

commercially available panel of O RBC expressing different

Rh, MNS, P, Lewis, Lutheran, Kell, Duffy, Kidd and sex-linked

RBC Ag was pooled and included into all assays (Screening test

for irregular RBC Ab; Reagent RBCs for antibody identifica-

tion, Gamma Biologicals, Inc., Houston, TX, USA). As positive

control human serum of a healthy donor of blood group O

with high anti-A/B Ab levels was included in all assays. Using

serially diluted sera, antibody binding, as measured by ABO-

FACS, was clearly dose dependent (data not shown). In

addition, each serum was incubated with fixed porcine RBC

because the serum of the majority of healthy individuals

contains xenoreactive natural Ab. To compare the levels of

anti-A/B Ab binding the geometric mean fluorescence intensity

ratios (MFIR) were calculated by dividing the mean fluores-

cence intensity of the sera of interest with the mean fluores-

cence intensity of the negative control. To validate the results,

all measurements were performed in two independent experi-

ments and displayed as mean values. Comparison of three

series of experiments yielded a good interassay correlation

(Spearman rs ¼ 0Æ945 between series 1 and 2; and rs ¼ 0Æ969between series 2 and 3 experiments, both P < 0Æ001).In 15 samples with high total IgG, IgG-subclass analysis was

performed using specific biotinylated secondary Ab for IgG1,

2, 3, and 4 (Zymed Laboratories, Inc.) and a streptavidin-

fluorescein isothiocyanate (FITC) conjugate (BD Biosciences,

Pharmingen, San Diego, CA, USA). The sera were incubated

with RBC for 30 min at 4�C and washed once with Hank’s

buffered saline. Biotinylated secondary Ab were added for

Detection of ABO Antibodies by Flow Cytometry

ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 130, 954–963 955

30 min at 4�C and washed twice thereafter. In a third step,

streptavidin-FITC was incubated for 30 min at 4�C and

washed twice. The samples were then measured by flow

cytometry and the results displayed as MFIR.

Haemagglutination

Anti-A/B and anti-porcine haemagglutination titres were

measured with a microplate haemagglutination assay (Craw-

ford et al, 1970). Serially diluted serum was incubated with a

1% suspension of RBC in 0Æ9% NaCl for 30 min at room

temperature in 96-well U-bottom plates (Nunclon Surface;

NUNC, Roskilde, Denmark). Thereafter, the plates were

centrifuged at 300 g for 2 min and placed in an upright

position for 10 min. As discussed by Crawford et al (1970),

haemagglutination was judged positive when the RBC

remained as a clot and did not run down the well of the

96-well plate. Titres were expressed as the highest dilution that

caused haemagglutination. All experiments were performed

twice; in cases of more than one titre difference, a third

experiment was performed, and the median value was taken

for further analysis.

ABO-ELISA

Enzyme-linked immunosorbent assays were used to detect

anti-A/B Ab as described previously (Rieben et al, 1991). In

brief, polystyrene microtitre plates (NUNC maxisorp, NUNC

A/S) were coated overnight at 4�C with synthetic A or B

trisaccharides coupled to polyacrylamide (Syntesome, Mos-

cow, Russia). Subsequently, the plates were washed thrice in

PBS containing 0Æ05% Tween 20 (Sigma Chemicals Co.).

Serum samples were diluted 1:40 in PBS containing 1% BSA

(Fluka Biochemicals) and 5% Tween 20 and incubated for

90 min at 37�C on a shaker. Binding of human anti-A/B IgM

and IgG to the coat antigen was detected by incubation with

mouse monoclonal anti-human IgG or IgM (clones HB43 and

HB57 from ATCC), followed by biotinylated goat anti-mouse

IgG1 (Southern Biotechnology Associates, Birmingham, AL,

USA) for 1 h at 37�C on a shaker and streptavidin-alkaline

phosphatase conjugate (Amersham, Bucks, UK) for 30 min at

37�C. As a chromogenic substrate 4-nitrophenyl phosphate

was used (Fluka Biochemicals), and optical density (OD)

values were read in an ELISA reader at 405 nm. A value of 0Æ35OD was chosen as cut-off. As internal standard, a serum with

high anti-A/B titres was included in the assay. All assays were

run in duplicates and the blank was subtracted from the mean

values of the duplicates.

Statistics

All statistical analyses were performed with the Statistical

Package for the Social Sciences (SPSS) software (SPSS for

Windows, version 11.0). The groups were compared by

Student’s t-test or nonparametric tests as appropriate. Stand-

ard deviation (SD) or range was used as an estimate of

variance. Spearman’s rank correlation test was performed for

comparison between haemagglutination, ABO-ELISA and

ABO-FACS assays. Sensitivity and specificity were analysed

by receiver operating characteristic curves (ROC). All reported

P-values are two-sided, and P < 0Æ05 was assumed to be

statistically significant.

Results

IgM and IgG anti-A/B antibodies in healthy blood donors

The ABO-FACS was established and validated with 120 sera of

healthy blood donors. Fig 1A demonstrates the cell size

visualised by flow cytometry on a linear scale. Porcine RBC

showed a second population in the range of 15–25% after

fixation, most probably representing microagglutinates. A

second population indicating microagglutinates was not seen

in human RBC. The somewhat larger range of the human RBC

population was attributed to cellular debris. Characteristic

results of the ABO-FACS for one donor of each blood group

are demonstrated in Fig 1B. As expected, anti-A IgM was

detected in the sera of blood group B and O, but not in the sera

of blood group A and AB. Conversely, anti-B IgM was detected

in the sera of blood group A and O, but not in the sera of

blood group B and AB. The sera shown were all positive for

anti-porcine IgM and negative for irregular RBC Ab. As

described below, IgM and IgG were considered to be positive if

the MFIR was greater than 3Æ7 and 2Æ0 respectively. Table I

shows the numbers and percentages of positive samples in the

four blood groups. Anti-porcine IgM were present in 112 of

120 samples (93%) and anti-porcine IgG in 103 of 120 samples

(86%), irrespective of the ABO-blood group of the donor. The

screening test for irregular RBC IgM was positive in 13/120

samples (11%) and negative for irregular RBC IgG in all

samples. If the screening test for irregular RBC Ab was positive,

unexpected binding to RBC expressing the self-ABO Ag was

considered to be false positive and these samples were excluded

from the specificity calculations below. In the majority of

samples the presence of anti-A/B IgG was restricted to blood

group O: anti-A and/or anti-B IgG were found in 34/38

samples (89%). In contrast, only 4/28 samples (14%) with

blood group A had anti-B IgG, 1/28 samples (4%) with blood

group B had anti-A IgG, and none of the samples with blood

group AB had anti-A or anti-B IgG. There was a considerable

variability in the level of both anti-A/B IgM and IgG in all

blood groups. The median anti-A/B MFIR levels for 28 A, 28 B,

38 O, and 26 AB sera are displayed in Fig 2. Blood group O

samples contained higher levels of anti-A IgG than anti-B IgG

(mean MFIR 9Æ2 vs. 3Æ8), corresponding to the higher number

of anti-A IgG positive samples when compared with the anti-B

IgG positive samples shown in Table I (76% vs. 66%). In 15

blood group O samples with high anti-A/B or anti-porcine IgG

levels, IgG subclass analysis was performed. As shown in Fig 3,

the predominant IgG subclass was IgG2. Low levels of IgG1

G. Stussi et al

956 ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 130, 954–963

and IgG3 were found in seven and four donors respectively.

IgG4 was negative in all samples measured.

Reverse blood group typing

To determine the optimal diagnostic cut-off level of the ABO-

FACS, ROC were calculated. This statistical procedure pro-

vides the sensitivity and 1-specificity for continuous variables,

such as the MFIR. The levels of anti-A/B IgM were compared

with conventional blood group typing and produced an

optimal sensitivity (93%) and specificity (96%) at an MFIR

cut-off level of 3Æ7 (Fig 4). The area under the curve (AUC) of

the ROC analysis was 0Æ972 [n ¼ 214; 95% confidence interval

(CI), 0Æ951–0Æ993; P < 0Æ001], indicating a high accuracy of

anti-A/B IgM to predict the correct blood group. In contrast,

anti-A/B IgG FACS values were not useful for reverse blood

group typing. The mean levels of anti-A and anti-B IgG MFIR

in blood group B and A sera were 1Æ5 and 1Æ2 respectively.

Meaningful calculations of the sensitivity could only be

obtained at an MFIR cut-off level of 2Æ0. Using this cut-off

level, the presence of anti A/B IgG was highly specific for blood

group O samples (data not shown).

Comparison of ABO-FACS with haemagglutination

The MFIR levels of anti-A/B Ab as determined by ABO-FACS

were compared with the anti-A/B Ab titres obtained by

microplate haemagglutination assays in 120 sera. As shown in

Fig 5, an overall correlation coefficient of 0Æ870 for IgM and

0Æ783 for IgG was obtained by Spearman’s rank testing. The

correlation between anti-A IgM MFIR and anti-A haemagglu-

tination titre was 0Æ862 and between anti-B IgM MFIR and

anti-B haemagglutination titre 0Æ880. Eight samples with

negative haemagglutination titres were positive for anti-A/B

IgM in the ABO-FACS, but five of these eight samples were

also positive in the screening test for non-ABO anti-RBC Ab.

Thus, overall, only 3/82 samples (4%) of blood group A, B, or

AB were positive in the ABO-FACS and negative in the

haemagglutination assay, indicating the occurrence of anti-A/B

Ab that bind to RBC but do not induce haemagglutination. On

the contrary, 6/94 samples (6%) of blood group A, B, or O

expressed low haemagglutination titres (range: 1:2–1:32), while

the anti-A/B IgM MFIR was below the cut-off level. These

ABO-FACS results were therefore considered to be false

negative. This finding indicated that in some cases low

A B O AB

Cel

l nu

mb

er

Mean fluorescence intensity(logarithmic scale)

Anti-Scr

101 102 103 101 102 103 101 102 103 101 102 103

1·0 1·3 1·3

14·5 26·2 15·4

Anti-Pig

6·2

Anti-A

Anti-B

1·1

25·7

18·7

1·3

14·2

9·8

1·1

1·5

1·2

Donor blood group

Mean fluorescence intensity(linear scale)

A

B

Po RBC

SS

C

FSC

Human RBC

SS

C

FSC

21%

Fig 1. (A) Density plots of porcine (Po) and human RBC incubated

with human serum derived from a blood group O donor. The cell size

was visualised by FACS on a linear scale, the X-axis represents the size

of the cells by forward scatter (FSC) measurement, the Y-axis repre-

sents the granularity of the cells by sideward scatter (SSC) measure-

ment. (B) Measurement of anti-A/B IgM by ABO-FACS: the anti-A/B

IgM MFIR levels were determined by ABO-FACS in sera of blood

donors of blood group A, B, O, and AB, respectively. The X-axis

represents the mean fluorescence intensity (logarithmic scale), the

Y-axis the cell number. Open histograms represent the staining with

absorbed human AB serum for human RBC and porcine serum for

porcine RBC (negative control). The filled histograms represent

binding of IgM Ab present in the tested serum to blood group A (anti-

A), B (anti-B), porcine (anti-Pig), or screening RBC for irregular RBC

Ab (anti-Scr). Anti-porcine IgM (positive control) were present in all

four sera, whereas the screening test for irregular RBC Ag (anti-Scr)

was negative in all four sera. Numbers indicate mean fluorescence

intensity ratios (MFIR), for calculation see Materials and Methods.

Table I. Frequency of anti-A/B and anti-porcine antibodies.

Blood group

A B O AB Total

Number 28 28 38 26 120

IgM antibodies [n (%)]

Anti-A IgM* 1 (4) 26 (93) 36 (95) 1 (4) 64 (53)

Anti-B IgM* 26 (93) 0 36 (95) 1 (4) 63 (52)

Anti-Po IgM 27 (96) 25 (89) 35 (92) 25 (96) 112 (93)

Screening IgM 4 (14) 3 (11) 4 (11) 2 (8) 13 (11)

IgG antibodies [n (%)]

Anti-A IgG 0 4 (14) 29 (76) 0 33 (28)

Anti-B IgG 1 (4) 0 25 (66) 0 26 (22)

Anti-Po IgG 27 (96) 22 (79) 38 (100) 16 (61) 103 (86)

Screening IgG 0 0 0 0 0

*If the screening test for irregular RBC Ab was positive, sera were not

included into the analysis.



concentrations of anti-A/B Ab are able to induce haemagglu-

tination, whereas Ab-independent haemagglutination is rather

unlikely.

ABO-ELISA in comparison with haemagglutination andABO-FACS

Another method to measure anti-A/B Ig isotypes is the ABO-

ELISA using synthetic A and B trisaccharides coupled to

polyacrylamide as coating substance. The anti-A/B Ab MFIR

levels obtained by ABO-FACS were compared with the OD

levels of the ABO-ELISA. For this purpose, 29 serum samples

of blood group A (9), B (6), O (12), and AB (2) were randomly

chosen. Spearman’s rank correlation of the ABO-ELISA with

the ABO-FACS assay was 0Æ654 for anti-A/B IgM and 0Æ684 foranti-A/B IgG. Correlation between ABO-ELISA and haemag-

glutination yielded an overall correlation of 0Æ550 for anti-A/B

IgM and 0Æ513 for anti-A/B IgG. Furthermore, a ROC curve

IgM

IgG

A (n = 28) B (n = 28) O (n = 38) AB (n = 26)

Donor blood group

Anti-A/B/pig antibodies

A B Pig A B Pig A B Pig A B Pig

0

50

100

150

0

10

20

30

40MF

IR

Fig 2. Levels of anti-A/B antibodies in the sera of 120 healthy blood donors: Anti-A/B IgM and IgG levels were measured in the sera of 120 healthy

blood donors, 28 A, 28 B, 38 O, and 26 AB respectively. The box plots show the median MFIR values and the 25th and 75th percentiles, the whiskers

represent the 95% confidence intervals. Open box plots represent anti-A Ab, light grey box plots anti-B and dark grey box plots anti-porcine (Pig)

antibodies. The upper panel shows the results of IgM measurements, the lower panel IgG.

0

5

10

15

IgG1 IgG2 IgG3 IgG4

P < 0·001

MF

IR

Fig 3. Anti-A/B IgG subclass analysis in sera of 15 blood group O

donors: IgG subclasses were analysed by ABO-FACS in 15 blood group

O donors with high anti-A/B IgG levels. The bars represent mean

values (+SD). All six 15 donors were positive for the IgG2 subclass,

whereas seven donors were positive for IgG1 and four donors for IgG3.

IgG4 was negative in all samples. The differences among the four

subclasses were statistically significant using a Kruskal-Wallis test as

indicated by the P-value. The detection limit (MFIR ¼ 1) is indicated

with a horizontal line.

1-specificity

Sen

siti

vity

10·750·500·25

1

0·75

0·50

0·25

AUC 0·972, P < 0·001

Fig 4. ROC curve analysis to determine sensitivity and specificity of

the ABO-FACS: ROC curve analysis was used to determine the optimal

sensitivity and specificity of the continuous MFIR data obtained by

ABO-FACS. The area under the curve (AUC) indicates the accuracy of

the assay. Comparison between anti-A/B IgM MFIR levels and the

effective ABO blood group as determined by conventional forward

blood group typing yielded a 96% specificity and 93% sensitivity at a

cut-off level of 3Æ7. The AUC was 0Æ972 (95% confidence interval

0Æ951–0Æ993, P < 0Æ001).

G. Stussi et al


analysis was performed to determine the cut-off level for

optimal sensitivity and specificity of the ABO-ELISA to detect

the correct blood group. At a value of 0Æ35 OD the sensitivity

and specificity were 62% and 63%, respectively (AUC 0Æ675,95% CI 0Æ5270–0Æ823; P ¼ 0Æ032). Anti-A/B IgM against self-

ABO Ag were detected in as many as 7/17 samples (41%) with

blood group A, B, or AB. The haemagglutination titre was

negative in all cases, whereas one of these seven samples

yielded a positive ABO-FACS result. However, this sample

could be considered a false positive result, because the

screening test for non-ABO RBC Ab was also positive. Three

of 17 samples (18%) were positive for anti-A/B IgG against

self-ABO Ag in the ABO ELISA, whereas autoreactive anti-A/B

IgG were not detectable in blood group A, B and AB samples

by ABO-FACS (Table I). According to the low sensitivity of

the ABO-ELISA obtained by ROC analysis a high number of

false negative results were observed. Anti-A/B IgM was below

the cut-off level of the ABO-ELISA in 12/27 samples (44%) of

blood group A, B, or O, all of which had positive haemag-

glutination titres. In one sample anti-A IgM was negative in

both ABO-ELISA and ABO-FACS, but showed low-level

haemagglutination (1:2).

Comparison of ABO-FACS with standard methods todetermine anti-A/B IgG

In clinical routine, anti-A/B IgG is determined by addition of

DTT to the serum followed by a crosslinking anti-IgG Ab

(indirect Coombs assay). Therefore, the effect of DTT on the

MFIR levels of IgM and IgG as determined by ABO-FACS was

analysed by the incubation of four sera with DTT. At a

concentration of 20 mmol/l, DTT predominantly degraded

IgM by reduction of intermolecular disulphide bonds, whereas

the intramolecular disulphide bonds of IgG were only affected

at higher concentrations. As shown in Fig 6A, DTT completely

abrogated the binding of IgM to the RBC, whereas IgG binding

was only slightly reduced by DTT, suggesting a minimal

destruction of IgG molecules. Another technique often applied

to discriminate IgG from IgM is the use of differential binding

qualities of IgM and IgG Ab at 4�C and 37�C. As displayed in

Fig 6B, overall binding of anti-A/B IgM as well as IgG was

higher at 4�C than at room temperature or 37�C. These data

seemed to confirm the usefulness of DTT to determine the

presence of IgG antibodies in a functional assay, whereas the

temperature method did not seem to be appropriate.

Cross-reactive anti-A/B antibodies

In the final series of experiments we studied whether Ab cross-

reacted to A and B Ag. For this purpose, seven blood group O

sera were absorbed with either O, A, or B RBC prior to ABO-

FACS analysis. After absorption, there was a 8%, 100% and

17% mean reduction of anti-A IgM, respectively, and a 7%,

33% and 100% mean reduction of anti-B IgM respectively

(Fig 7, upper panel). Similarly, there was a 3%, 100% and

18%, mean reduction of anti-A IgG, and a 4%, 18% and 100%

mean reduction of anti-B IgG MFIR levels after absorption

(Fig 7, lower panel). As expected, anti-A and anti-B Ab were

completely removed from blood group O sera by absorption

with A or B RBC, respectively. In contrast, absorption with A

RBC also reduced anti-B MFIR levels by a mean of 17% (0–

39%) and incubation with B RBC reduced anti-A MFIR levels

by a mean 33% (0–89%). This suggested the presence of cross-

reactive IgM in some of the O donors. This finding was specific

for blood group O sera, as absorption of blood group A sera

with A RBC did not change the level of anti-B IgM. The anti-B

IgM present in blood group A sera was completely removed by

incubation with B RBC. Conversely, incubation of blood group

B sera with A RBC almost completely reduced the anti-A IgM

levels whereas incubation with B RBC did not alter anti-A IgM

levels (data not shown).

Discussion

This study has shown that the ABO-FACS is a valid tool to

quantify the binding of anti-A/B Ab to human RBC. As

compared with classical functional assays, such as tube

haemagglutination and indirect antiglobulin testing, and a

previously described ABO-ELISA, the ABO-FACS offers several

advantages: First, it measures the presence of all anti-A/B Ab

binding to the respective ABO Ag expressed on RBC, not only

haemagglutinating or haemolysing Ab. Secondly, binding of

anti-A/B Ab to natural ABO Ag on RBC was clearly more

sensitive and more specific than binding to synthetic ABO Ag

immobilised on ABO-ELISA plates. Thirdly, the ABO-FACS

directly quantifies the binding of different anti-A/B Ab isotypes

and IgG subclasses using specific secondary Ab. For these

reasons the ABO-FACS offers a novel high throughput and

Agglutination (1/x)

MF

IR (

log

arit

hm

ic s

cale

)

0·1

1

10

100

1000

0 1 2 4 8 16 32 64 128 256 512 1024

MFIR 3·7

r = 0·870, P < 0·001

Fig 5. Correlation between ABO-FACS and haemagglutination: MFIR

values obtained by ABO-FACS (Y-axis) and haemagglutination titres

(X-axis) in all 120 tested sera are shown using logarithmic scales. The

overall correlation as calculated by Spearman’s rank testing yielded a

coefficient of 0Æ870 (P < 0Æ001). The cut-off level of the IgM ABO-

FACS (MFIR ¼ 3Æ7) is indicated with a horizontal line.



observer-independent method that can be used to monitor

anti-A/B Ab levels in large studies and to validate other

techniques.

Technically, the ABO-FACS analysis could be hampered by

RBC aggregates in the presence of haemagglutinating Ab.

Fixation of RBC with formaldehyde and glutaraldehyde

(Karnovsky buffer) largely prevented haemagglutination. A

subpopulation of microagglutinates consisting of two RBC was

observed using fixed porcine RBC, but not with human RBC of

blood group A or B. However, the flow cytometric analysis

using a logarithmic scale was not hampered by these micro-

agglutinates. Other flow cytometry studies to measure anti-A/B

Ab applied vacuum filtration or centrifugation over a filter

membrane to disperse RBC in an attempt to obtain single-cell

suspensions (Roback et al, 2003, 2004).

Although the exact mechanism leading to inhibition of

agglutination by fixation is unknown, cross-linking of proteins

stabilises RBC membranes and thereby impairs lateral move-

ments of ABO Ag, which is required for efficient haemagglu-

tination. Theoretically, fixation with Karnovsky buffer might

also result in structural alterations of RBC epitopes, including

ABO Ag, and thereby might change the anti-A/B Ab binding

pattern. In the current analysis we did not have any evidence

that the specificity or density of ABO Ag was transformed,

changes in the expression of other RBC Ag however cannot be

ruled out. Although Berneman et al (1991) reported that

formaldehyde fixation reduces the total number of ABO Ag on

RBC, we did not detect major differences of anti-A/B Ab levels

using RBC of several donors and after serial fixations with

Karnovsky buffer. Thus, the ABO Ag density was relatively

constant after fixation with Karnovsky buffer (data not shown).

Nonetheless, to minimise potential differences, we stored large

batches of single donor RBC in liquid nitrogen following shock

freezing, which can be used for ABO-FACS up to 6 weeks after

thawing and fixation without deterioration of the Ab binding.

Cel

l nu

mb

er

Negative control

30 min, 4°C

30 min, 37°C

Temperature

IgM IgG

MF

IR

IgMA BIgG

0

10

20

30

40

50

+0

5

10

15

20

25

30

DTT treatment

Negative control

Native serum

DTT treatment

+

IgM/G MFI (logarithmic scale) IgM/G MFI (logarithmic scale)

MF

IRC

ell n

um

ber

0

10

20

30

40

50

4°C RT 37°C0

5

10

15

20

25

4°C RT 37°C

Fig 6. Effect of dithiotreitol (DTT) and temperature on anti-A/B Ab binding: (A) Measurements of anti-A/B IgM (left panel) and IgG (right panel)

by ABO-FACS following incubation of the serum with DTT. The upper panel shows a representative binding of anti-A/B Ab with (light grey

histograms) or without DTT treatment (dark grey histograms), and staining with absorbed human AB serum as negative control (open histograms).

The lower panel summarises the effect of DTT on anti-A/B IgM MFIR values in four sera (left panel) and anti-A/B IgG MFIR values in three sera

(right panel) respectively. The figure represents one of two independent experiments. (B) Measurements of anti-A/B IgM (left panel) and IgG (right

panel) by ABO-FACS performed at 4�C, room temperature and 37�C respectively. The filled histograms represent binding of anti-A/B Ab at 37�C(light grey) or at 4�C (dark grey). The lower panel summarises the temperature effect on anti-A/B IgM MFIR values (left panel) and anti-A/B IgG

MFIR values (right panel) in seven sera respectively. The figure represents one of five independent experiments.

0

20

40

60

80

100

Anti-A

0

20

40

60

80

100

0

20

40

60

80

100

Anti-B

0

20

40

60

80

100

Red blood cells used for absorption

% M

FIR

(u

nab

sorb

ed c

on

tro

l ser

um

)

IgM

IgG

O A B O A B

O A B O A B

Fig 7. Evidence for the presence of cross-reactive anti-A/B Ab in blood

group O sera: blood group O sera were incubated with RBC and

analysed for the presence of anti-A/B IgM (upper panel) and anti-A/B

IgG (lower panel) by ABO-FACS. The bars represent relative mean

MFIR ± SD of anti-A (left panel) and anti-B (right panel) Ab binding

after absorption with blood group O (open), A (grey), or B RBC

(black) relative to the anti-A/B Ab MFIR of unabsorbed serum. The

data shown are pooled from four independent experiments repre-

senting seven blood group O sera.

G. Stussi et al


Controversial results have been reported on the measure-

ment of anti-A/B IgG and IgG subclasses by gel tests, ELISA,

immunofluorescence and flow cytometry (Owen, 1954; Raw-

son & Abelson, 1960; Kay & Locke, 1986; Brouwers et al, 1987;

Rieben et al, 1991; Lynen et al, 1995; Fabijanska-Mitek et al,

1997). Our data with FACS analysis on IgG subclasses in 15

samples with high anti-A/B IgG Ab revealed a predominance of

IgG2 Ab. IgG1 and IgG3 were found only in seven and four

donors, respectively, and IgG4 was not detectable. These

findings are in line with the results of some earlier studies, but

contrast with others (Kay & Locke, 1986; Brouwers et al, 1987;

Fabijanska-Mitek et al, 1997), thereby reflecting methodologi-

cal problems, such as the choice of the antigenic source, the

specificity of secondary Ab and the sensitivity of the detection

system. Although we cannot draw a final conclusion on the

distribution of IgG subclasses from analysing 15 samples, the

ABO-FACS avoids many of the technical problems that

hampered previously used methods. In addition, it parallels

the situation of other bacterial polysaccharides predominantly

evoking IgG2 subclass responses. Although it is still contro-

versial whether IgG2 subclass deficiency is associated with a

higher incidence of bacterial infections, it has been shown in

numerous studies that IgG2 are highly active against bacterial

polysaccharides (Siber et al, 1980; Maguire et al, 2002).

Moreover, differentiation between IgM and IgG Ab is difficult

to achieve by using standard techniques and depends on

surrogate procedures, such as performance at different tem-

peratures or after dissociation of IgM molecules by reducing

agents. We could confirm that IgM generally binds more avidly

at 4�C, however, more importantly, that binding of anti-A/B

IgG was also reduced at room temperature and 37�C in several

samples. Thus, mere differentiation of IgM and IgG by

temperature seems inappropriate. In contrast, the DTT

treatment seemed suitable for the determination of anti-A/B

IgG.

Cross-reactive anti-A/B Ab binding to both A and B Ag,

have been demonstrated by several groups, especially after

immunisation with A and B Ag (Owen, 1954; Holburn, 1976;

Contreras et al, 1984). In the present study, we provide

indirect evidence for the occurrence of cross-reactive anti-A/B

Ab in the sera of some, but not all O donors. In contrast, cross-

reactive Ab were not detected in the sera of blood group A or

B, presumably because subjects with blood group O lack the

selective pressure to eliminate B-lymphocytes producing cross-

reactive anti-A/B Ab.

Comparison of ABO-FACS MFIR values and haemaggluti-

nation titres showed good correlation between the two assays

and the measurement of anti-A/B IgM by ABO-FACS enabled

the correct prediction of the blood group (reverse typing) in

the majority of the samples. These findings indicate that the

majority of anti-A/B IgM are haemagglutinating Ab. On the

contrary, the correlation between ABO-ELISA and ABO-FACS

was weak, as was the correlation between ABO-ELISA and

haemagglutination, even lower than originally reported (Rie-

ben et al, 1991). In the current study, a high number of

samples containing potentially autoreactive Ab was found by

ABO-ELISA (41% IgM, 18% IgG), but not by ABO-FACS (4%

IgM, 0% IgG). These findings might be explained by the use of

synthetic ABO Ag in the ABO-ELISA, which exhibit an

incomplete antigenic structure and are bound to a carrier

substance, such as polyacrylamide or BSA. Binding of

low-affinity and low-specificity Ab may have potentially

overestimated the levels of anti-A/B Ab and the presence of

auto-reactive Ab (Rieben et al, 1991). In addition, the

screening test for non-ABO RBC Ab can identify false positive

samples in the ABO-FACS and increases thereby the accuracy

of the ABO-FACS assay. Thus, using the ABO-FACS, the

incidence of autoreactive anti-A/B Ab is lower than expected

from the results obtained by the ABO-ELISA.

In contrast to anti-A/B IgM, ‘immune’ anti-A/B IgG are

predominantly found in blood group O sera or after immu-

nisation with the respective ABO Ag (Rawson & Abelson, 1960;

Contreras et al, 1984). In fact, the present study revealed anti-

A/B IgG by ABO-FACS in 89% of the blood group O samples,

but only in 6% of non-O sera, a value similar to that found

earlier (Rawson & Abelson, 1960; Kay & Locke, 1986). To date,

there is no explanation for the latter finding, but low anti-A in

B and low anti-B in A may provide an advantage to women

with these blood groups during ABO incompatible pregnan-

cies, because only IgG are transferred through the placenta,

exposing the child to the risk of haemolytic disease. This view

is supported by the clinical finding, that only newborns of

blood group O mothers develop haemolytic disease after ABO-

incompatible pregnancies (Brouwers et al, 1988). The apparent

lack of antibody class switch in blood group A and B

individuals may represent an interesting in vivo model to

study the development of the natural Ab repertoire.

Beside the ABO-FACS reported here, another elegant

method quantifying anti-A/B IgM and IgG by surface plasmon

resonance was recently reported (Kimura et al, 2005). This

method measures changes of the physical properties of Ab/

ABO Ag complexes of which the latter are immobilised to a

sensor chip. The classical tube haemagglutination assay

remains the gold standard in clinical routine. However, novel

methods to determine anti-A/B Ab levels are being developed

to closely monitor these Ab in different clinical settings, such

as ABO-incompatible transplantations, providing an excellent

model to study humoral immune responses in vivo. Accidental

ABO incompatibility in solid organ transplantation usually

results in catastrophic hyperacute rejection because of the

expression of ABO Ag on graft endothelial cells (Cooper,

1990). Nevertheless, sophisticated protocols have allowed

ABO-incompatible living donor kidney and liver transplanta-

tions (Cacciarelli et al, 1995; Takahashi et al, 2004; Winters

et al, 2004; Tyden et al, 2005). The importance of anti-A/B Ab

measurements in these settings is stressed by the fact that high

pretransplant anti-A/B IgG haemagglutination titres were

associated with poor graft survival, irrespective of pretrans-

plant procedures to lower these Ab titres (Shimmura et al,

2000). Interestingly, ABO-mismatched haematopoietic stem



cell transplantation does not influence the patient’s outcome

(Hershko et al, 1980; Bensinger et al, 1982; Kalaycioglu et al,

1995; Seebach et al, 2005), presumably because of regulatory

processes on the level of Ab production reported both after

incompatible solid organ and haematopoietic stem cell trans-

plantations (Mohiuddin et al, 2003; Takahashi, 2004). Alter-

natively, incompatible Ab may completely disappear after

transplantation, as found in both ABO-incompatible HSCT

(Stussi et al, 2005) and neonatal ABO-incompatible heart

transplantations (West et al, 2001; Fan et al, 2004).

In conclusion, we developed a sensitive and accurate

method to quantify the concentrations of anti-A/B Ab as

well as IgG subclasses by means of flow cytometry. While

this work has focused on anti-A/B Ab levels in healthy blood

donors, it is suitable to study anti-A/B Ab in clinical

situations, such as ABO-incompatible HSCT, solid organ

transplantation and haemolytic disease of the newborn. In

ABO-incompatible solid organ and stem cell transplantation,

accurate measurements of ABO antibodies will give insights

into the immunological mechanisms leading to the graft

acceptance including antibody class switch, accommodation

and tolerance induction. Moreover, analysing anti-A/B Ab

levels prior ABO-incompatible kidney transplantation can

identify patients suitable for ABO-incompatible kidney

transplantations and post-transplant monitoring can identify

those patients at risk for humoral rejection. In mothers with

ABO-incompatible pregnancies, the assay can be used to

detect stimulation of the maternal immune system due to

the ABO-incompatible fetal Ag during pregnancy or at

delivery. Finally, this current method can be applied for the

monitoring of removal of A and B Ag from RBC by enzyme

degradation.

Acknowledgements

This work was supported by grants from the Swiss National

Science Foundation (4046-058668/1) and the Julius-Muller

Stiftung to J.D.S. and a grant from the Heuberg Stiftung to

G.S. The authors thank Katja Matozan for performing the

ABO-ELISA experiments and the local transfusion service for

providing the serum and RBC samples. Dr F. Salomon is

acknowledged for critical reading of the manuscript and

helpful discussions.

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Isotype-specific detection of ABO blood group antibodies using a novel flow cytometric method

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