ORIGINAL ARTICLE

A comparison of two automated methods for the detection

and identification of red blood cell alloantibodies

Giovanni Garozzo, Vincenzo Licitra, Rosario Criscione, Nunzio Comitini, Chiara Noto,
Rosario Lomagno, Daniela Ruta, Giovanni Spadola, Valeria Zago, Pietro Bonomo

Servizio di Medicina Trasfusionale ed Immunoematologia, Azienda Ospedaliera “Civile Maria Paternò Arezzo”,
Ragusa, Italy

Background. The aim of this study was to compare the routine use of two automated systems
(OrthoAutoVue Innova, microcolumn, and Immucor Galileo, solid phase) for the screening and identification
of irregular red blood cell alloantibodies in samples, analysed in our Transfusion Service during 6 months
of normal activity. The study focused particularly on an evaluation of the repeatability of the screening
tests, the identification of antibody specificities and the identification of antibodies in samples showing
discordant results.
Materials and methods. Overall 2,229 samples from potential blood donors (A), multiply transfused
patients with blood disorders (DH), potential transfusion recipients (TS), and external cases (E) were
studied.The protocols were carried out according to the manufacturers’ recommendations.
Results. The screening tests detected 78 samples that were positive with both systems, while 18
were positive only with Immucor and 11 only with Ortho (thus, overall, Immucor detected 96 positive
samples and Ortho 89 positive samples). The use of the respective identification panels enabled us to
identify the antibodies in 65 samples with Immucor and in 61 samples with the Ortho system; 74
antibodies were identified with Immucor (55 with a single specificity and 19 with mixed specificities) and
68 antibodies with Ortho (51 and 17, respectively). In the remaining cases (31 samples for Immucor and
28 for Ortho), the antibody specificity was not identified. The two systems were found to be essentially
similar. The Immucor system revealed a greater number of antibodies, mainly because of its greater
sensitivity at detecting anti-D antibodies.
Conclusions. Both systems showed a repeatability of over 85%, demonstrating that automation of
immunohaematological tests is advantageous. The specificity of the antibody was identified in 68% of the
samples. Furthermore, using the two systems led to the identification of ten new antibodies (6 anti-D, 2
anti-E, 1 anti Lea, and 1 anti-Vel), which would not have been detected had only one of the two methods
been used.

Key words: red blood cell antibodies, antibody screening and identification, automated methods.

Introduction In order to evaluate the attachment of the antibody to

There are two phases of the immunological reaction
that leads to red cell agglutination: in the first phase the
antibody attaches to the red cell antigen, while in the
subsequent phase, a lattice of red blood cells, united by
antibody bridges, is formed and manifested as visible
agglutination.
the antigen in the first phase of the immunological reaction
more directly and to improve the reading of the agglutination
further, already by the end of the 1980s, techniques based
on agglutination on columns and reactions in solid phase
had been introduced into immunohaematology
laboratories1. Alongside this evolution in techniques,

Blood Transfus 2007; 5: 33-40 DOI 10.2450/2007.0022-06 33

© SIMTI Servizi Srl

automated systems for the preparation and performance of
red cell immunohaematology tests became used routinely
in order to obtain a complete guarantee of the tests’
performance and standardisation of the procedures2.
The purpose of this study was to compare, as in other
published studies3-8, the routine use of two automated
systems for the screening and identification of irregular
antibodies in a large number of serum samples, analysed in
our Service of Immunohaematology and Transfusion
Medicine (SIMT) in 6 months of normal activity. There
were numerous parts of this study (agreed with the
manufacturers of the automated systems). In detail, we
evaluated the repeatability of the screening tests, the results
achieved in different categories of subjects, the number
and the validity of the panels of test red blood cells used to
identify the antibody specificity, the identification of
discordant specificities in samples that were positive
according to both methods and the antibody specificity
when only one of the systems gave a positive result.
Materials and methods
Between 22 August 2005 and 28 February 2006, we
analysed 2,299 samples from the following categories of
subjects: 560 potential blood donors (A), 260 patients with
blood disorders or haemoglobinopathies, followed in the
Haematology and Thalassaemia Units of the SIMT of
Ragusa (DH), 1,181 inpatients in surgery wards in our
hospital, who were candidates for transfusion therapy (TS)
and 298 external subjects undergoing indirect antiglobulin
tests for various reasons, including Rh-negative pregnant
women, being monitored for anti-D prophylaxis (E).
All the samples were tested, in a completely automated
manner, with both the systems used in our Service, that is,
with the Immucor Galileo (Immucor Italia srl, Opera, MI,
Italy), which uses solid phase technology, and Ortho
AutoVue Innova (Ortho-Clinical Diagnostics, Milan, Italy)
which uses microcolumn technology. The aim was to
compare the performance of the two systems used in a
routine context.
It should be highlighted that the operative protocol
(reported in diagram 1) was agreed upon with both the
companies, which provided the reagents for carrying out
the double testing. All the tests were carried out following
their instructions scrupulously.
The samples were taken at the blood collection centre
of the SIMT of Ragusa or in the wards of the Maria Paternò
Arezzo Civil Hospital of Ragusa and analysed according to
the agreed protocol.
The screening tests were carried out with red blood cell
34
Garozzo G et al.
panels from the Immucor company (four-cell Capture-R
Ready Screen, for the Immucor Galileo analyser) and from
Ortho (three-cell Surgiscreen at 0.8±0.2%, column
agglutination, for the Ortho AutoVue Innova analyser). The
specificities of the antibodies were investigated using,
respectively, the appropriate panels from Immucor (first
panel: 13-cell Capture-R Ready-Id; second panel: 14-cell
Extend I, D-positive; third panel: 14-cell Capture-R Ready-
Id Extend II, D negative panel) and those from Ortho (first
panel: Resolve Panel C Untreated; second panel: Resolve
Panel B; third panel: Resolve Panel C Ficin Treated, all with
11 cells at 0.8±0.2%).
The search for irregular antibodies and the reading of
the results were conducted using only these two automated
methods; the data were interpreted by at least two different
people.
The indirect antiglobulin test and the "type and screen"
were carried out on the same day with the two automated
systems, according to routine methodology in the SIMT.
Tests that gave positive or doubtful results were
repeated the same day with both methods and the results
reported on the registration forms. The samples, that were
confirmed to be positive or that gave doubtful results in
the screening tests, were stored at 4 °C for identification of
the antibody specificity in the following days. A doubtful
result was one, whose result in the automated screening
was given as "NTD " by Immucor and as "?" by the Ortho
system.
Results
Table I reports the total number of samples that were
positive according to both screening tests, those positive
only with the Immucor system and those positive only
with the Ortho system.
Table I - Number of samples found to be positive by the
screening tests
Samples positive with both systems 78
Samples positive only with Immucor 18
Samples positive only with Ortho 11
Total n. of samples positive with Immucor 96
Total n. of samples positive with Ortho 89
Repeatability of the tests
The repeatability (or not) of the tests, expressed in
absolute values and as percentages, is reported in table II.
As can be seen, the repeatability was almost identical for
the two systems.
Blood Transfus 2007; 5: 33-40 DOI 10.2450/2007.0022-06
Antibody detection: comparison between 2 automated methods

FLOW CHART
SCREENING TEST FOR THE DETECTION
OF IRREGULAR RED CELL ANTIBODIES
NEGATIVE 3-CELL ORTHO AND STOP
4-CELL IMMUCOR
RESULT

NEGATIVE
STOP RESULT

DOUBTFUL REPEAT
RESULT TEST
POSITIVE DUBBIO
RESULT
POSITIVE OR
FIRST PHASE ON
THE SAME DAY

DOUBTFUL RESULT
REPEAT
TEST

POSITIVE OR NEGATIVE
DOUBTFUL RESULT RESULT

IDENTIFICATION
PANELS
SECOND PHASE

11-CELL ORTHO “C” 13-CELL IMMUCOR

PANEL NOT TREATED
PANEL
WITH FICIN

ANTIBODY
IDENTIFIED ?

STOP YES YES STOP

NO NO
“B” AND
“C”(FICIN-TREATED) EXTEND I AND II
ORTHO PANELS IMMUCOR PANELS

EVALUATION

Blood Transfus 2007; 5: 33-40 DOI 10.2450/2007.0022-06 35

 Garozzo G et al.

Table II - Results and repeatability of the screening tests with both systems

Screening panels used Positive, Doubtful, Doubtful, Repeatability

confirmed reactive confirmed reactive not confirmed reactive (a)+(b)
(a) (b) (c) (a)+(b)+(c)

Immucor 96/2,299 3/2,299 15/2,299 86.8%

Capture-R Ready-Screen (4.2%) (0.13%) (0.6%)

Ortho 89/2,299 2/2,299 17/2,299 84.2%

Surgiscreen (3.9%) (0.08%) (0.7%)

Table III - Results of the screening tests divided by category of samples and type of panels

Results A DH TS E Total

Immucor Capture-R Ready Screen

Positive, confirmed reactive 4/560 34/260 22/1,181 36/298 96/2,299

(0.7%) (13.1%) (1.9%) (12%) (4.2%)
Doubtful, confirmed reactive 1/560 2/260 0/1,181 0/298 3/2,299
(0.2%) (0.8%) - - (0.13%)
Doubtful, not confirmed 5/560 1/260 8/1,181 1/298 15/2,299
(0.9%) (0.4%) (0.7%) (0.3%) (0.6%)

Ortho Surgiscreen

Positive, confirmed reactive 2/560 34/260 18/1,181 35/298 89/2,999

(0.3%) (13.1%) (1.5%) (11.7%) (3.9%)
Doubtful, confirmed reactive 1/560 1/260 0/1,181 0/298 2/2,299
(0.2%) (0.4%) - - (0.08%)
Doubtful, not confirmed 2/560 5/260 10/1,181 0/298 17/2,299
(0.3%) (1.9%) (0.8%) - (0.7%)

Table IV - Samples divided according to category, system used and number of panels necessary to reach a certain identification of
the antibodies

Number of panels used A DH TS E Total

Immucor panels

1 panel 1/2 10/21 9/15 17/27 37/65 (57%)

3 panels 1/2 11/21 6/15 10/27 28/65 (43%)

Ortho panels

1 panel 1/2 10/20 7/13 13/26 31/61 (51%)

3 panels 1/2 10/20 6/13 13/26 30/61 (49%)

Results of the screening tests divided by system
and category of samples
Table III presents the results obtained with the screening
tests, divided according to the category of the samples
examined and the red cell panels used for the screening
tests.
The 32 samples whose results were doubtful results
and not confirmed, that is 15 samples examined with the
Immucor system and 17 samples examined with the Ortho
system, were considered negative and therefore
identification of the antibody specificity was not
undertaken. Only one of these 32 samples gave doubtful

36 Blood Transfus 2007; 5: 33-40 DOI 10.2450/2007.0022-06

Antibody detection: comparison between 2 automated methods

Table V - Results of the use of panels for identifying the antibodies present in samples that gave positive
results, subdivided by the testing system and outcome (Ab: antibodies, Id: identified)

Panels used Samples with Ab Id Samples with Ab not Id Panagglutinated samples

Immucor Panels 65/96 19/96 12/96

(67.7%) (19.8%) (12.5%)

Ortho Panels 61/89 22/89 6/89

(68.5%) (24.7%) (6.7%)

results not subsequently confirmed according to both the Table VI - Samples in which the antibodies were identified,
divided by the system used and category of sample.
Immucor and Ortho screening tests.
Table IV reports the number of panels used to identify
Panels used A DH TS E Total
the antibodies, with the data divided according to the
category of samples and the system used. Identification Panels
with the first panel was attributed only when the antibody Immucor 2/4 21/34 15/22 27/36 65/96
(50%) (61.8%) (68.2%) (75%) (67.7%)
specificity was certain.
Identification with "1 panel" means that the antibody
specificity was determined using the Capture-R Ready- ID Panels 2/2 20/34 13/18 26/35 61/89
Ortho (100%) (58.8%) (72.2%) (74.3%) (68.5%)
panel (for Immucor) or the Resolve Panel C panel (for Ortho),
whereas identification with "3 panels" means that the
antibody specificity was determined using, for Immucor,
Differences found in the specificities of the
also the Capture-R Ready–ID Extend I and Extend II panels
alloantibodies in samples positive according
or, for Ortho, also the Resolve Panel-C treated with ficin
to both the screening methods
and Resolve Panel B.
As shown in table I, 78 samples were positive according
to both screening tests. Using the identification panels of
Results of the panels
the two systems the same antibody specificity was found
for antibody identification
in 51 out of 55 samples, whereas, in the other four samples,
The use of the antibody identification panels, tested
different specificities were found.
on samples that were positive according to either of the
Table VIII reports the data on the positive samples,
two systems, as defined above, gave the results shown in
divided by category and results (concordant and
table V.
discordant), whereas table IX presents the data on the four
The samples with unidentified antibodies reacted with
samples that gave discordant results, in relation to the type
only some cells of the panels used.
of panel used.
Despite this, it was not possible to reach a certain
identification of the antibodies.
Positive screening results with only one of the
The panagglutinated samples were positive with all the
methods used and identification of the
cells of the panels used, including the screening panels.
antibodies
This made automated identification of the antibodies
impossible.
The comparison of the two systems showed that 18
Table VI reports the number of samples in which
out of 2,299 (0.78%) samples were positive with the
antibodies were identified, divided by category of sample
Immucor screening test and negative with the Ortho
and the system used.
screening test and 11 out of 2,299 (0.47%) samples were
Overall, the use of the pertinent panels enabled the
positive with the Ortho screening test and negative with
identification of 74 antibodies with the Immucor system
the Immucor screening test (see also table I).
and 68 antibodies with the Ortho system. These totals
The panels used to identify the antibodies in these cases
obviously include the samples with mixtures of antibodies
provided the results and specificities shown in table X.
(that is, with multiple specificities, table VII).

Blood Transfus 2007; 5: 33-40 DOI 10.2450/2007.0022-06 37

 Garozzo G et al.

Table VII - Antibodies identified, divided by the system and number of panels (in brackets) used to reach certain identification of the
antibodies

anti -D anti -C w anti –c anti-E anti-V anti-K anti-Kp a anti-Jk b anti-M anti-Le a Total

Immucor panels

28/39 (1); 1/1 (3) 1/1 (1) 4/7 (1) 1/1 (3) 2/2 (1) 1/2 (1) 1/1 (3) 1/1 (3) 55
11/39 (3) 3/7 (3) 1/2 (3)

Multiple antibodies: 1: anti D+C (1), 1: anti D+V (3), 2: anti E+Cw (3); 1: anti Kpa+ 1 not identified (3); 19
1:anti K+ Kpa+ 1 not identified (3); 1: anti Jka+ Kpa (3); 1: anti E+ c (3); 2: anti c+ Jkb (3)

Total 74

Ortho panels

18/34 (1); 1/1 (1) 2/2 (1) 2/5 (1) 1/2 (1) 1/1 (1) 2/2 (3) 1/1 (3) 1/2 (1) 1/1 (1)
16/34 (3) 3/5 (3) 1/2 (3) 1/ 2 (3) 51

Multiple antibodies: 2: anti D+C: 2/2 (1), 1: anti E+K (3), 2: anti E+Cw (1/2: 1; 1/2: 3), 1: anti K+ 1 not identified (3),
1: Jka+Kpa (3), 1: anti E+c (3), 1: anti c+anti Jkb (3) 17

Total 68

Table VIII - Differences found in the specificities of antibodies Discussion

(Ab), divided by category of the samples
The screening tests for the presence of antibodies
found 78 samples that were positive according to both
Category Positive samples Positive samples
of samples with concordant Ab (%) with discordant Ab (%) systems. The Immucor system detected another 18 different
samples that were positive and the Ortho system another
A 2/2 (100%) 0/2 (0%) 11 samples. Overall, therefore, the Immucor system detected
DH 16/19 (84.2%) 3/19 (15.8%) 96 positive samples and the Ortho system 89 positive
TS 11/11 (100%) 0/11 (0%) samples.
E 22/23 (95.6%) 1/23 (4.4%)
The repeatability of the tests with the Immucor Galileo
system was 86.8%, whereas that of the Ortho AutoVue
Total 51/55 (92.7%) 4/55 (7.3%) Innova system was 84.2%. The performance of the two
systems was, therefore, essentially similar. Likewise, the
two systems differed only slightly in the number of positive
Table IX - Antibodies (Ab) detected by the two systems in samples repeatedly reactive (4.2% for Immucor and 3.9%
positive samples showing discordant specificities
for Ortho).
(the category of the samples is shown in brackets)
It should be underscored again that the testing in this
Positive samples Immucor Ortho
study was carried out completely as routine practice in our
with discordant Ab Service. Importantly, all the samples were handled in the
same way and by the same staff, therefore eliminating the
1 (DH) anti E anti E+K
variability inherent in multicentre studies.
2 (DH) anti K+Kpa anti K The considerable percentage of samples found to be
positive in the screening tests is due to the large number of
3 (DH) anti D+Vel anti D
patients in our series with blood disorders (mostly
4 (E) anti c+Jkb anti c thalassaemia), who have received multiple transfusions
(DH): among these patients, the rate of positive results
was 13.1%, and was identical according to both systems.
In conclusion, the use of the Immucor system led to the The rate of positivity was slightly lower in the samples
identification of 81 antibodies, while the Ortho system from external patients (E): 12% according to Immucor and
identified 71 antibodies. 11.7% according to Ortho. It should be noted that these

38 Blood Transfus 2007; 5: 33-40 DOI 10.2450/2007.0022-06

Antibody detection: comparison between 2 automated methods
Table X - Antibodies (Ab) identified in the samples reacting positive in only one of the two screening tests used
Panels used Samples Panagglutinated
with unidentified Ab samples
Immucor panels 6/18 5/18
(33.3%) (27.7%)
Ortho panels 7/11 1/11
(63.6%) (9.1%)
external subjects included pregnant women, whose
positivity was usually due to antenatal administration of
anti-D immunoglobulin for prophylactic purposes. The
incidence of positive samples was clearly lower among the
subjects undergoing surgery (TS), among whom the rate
of positivity was 1.9% according to Immucor and 1.5%
according to Ortho, and even lower among potential blood
donors (A), whose rates of positivity ranged from 0.7%
(Immucor system) to 0.3% (Ortho system).
The attempt to identify the antibodies not only in those
samples positive in the screening test and confirmed as
such in a second test, but also in those with doubtful results
at screening and doubtful results again in the second test,
enabled a thorough comparison of the two systems.
In the samples that were positive or doubtful in the
screening tests, the use of the respective identification
panels determined the antibody specificity in 65 out of 96
samples (67.7%) with the Immucor system and in 61 out of
89 samples (68.5%) with the Ortho system, these
percentages being essentially the same.
On the other hand, in 32.3% of the cases investigated
with the Immucor system and in 31.5% of these examined
by the Ortho system, it was not possible to determine the
specificity of the antibodies detected: the Immucor
identification panels and the Ortho panels were unable to
identify the antibodies in, respectively, 19 of 96 samples
(19.8%) and 22 of 89 (24.7%), while the Immucor system
showed panagglutination in 12 of the 96 samples (12.5%)
and the Ortho system in 6 of 89 cases (6.7%).
The evaluation of the number of panels used to identify
the antibodies showed a slight advantage for Immucor,
which enabled the identification of 57% of the cases using
only one panel, compared to 51% for Ortho. To the best of
our knowledge, a comparison of this parameter has never
previously been published.
As far as concerns the specificity of the antibodies, the
Immucor system detected a higher number of antibodies
than did the Ortho screening test (74 versus 68). The most
frequent specificities were those of the Rh system, in
Blood Transfus 2007; 5: 33-40 DOI 10.2450/2007.0022-06
Samples with Ab specificity
identified Ab
7/18 anti D: anti E:
(38.9%) 5/18 2/18
3/11 anti D: anti Lea: anti Vel:
(27.2%) 1/11 1/11 1/11
particular anti-D, in direct relationship with antenatal
prophylaxis against haemolytic disease of the newborn.
Investigations of the 78 samples that were positive
according to both screening tests failed to identify the
antibody specificity in 23 cases; in the other 55 cases the
specificities were determined (although in four cases the
results were not concordant).
Finally it should be noted that of the 2,999 samples
screened, 18 (0.78%) were positive only according to the
Immucor system and 11 (0.48%) only according to the Ortho
system. Attempts to identify these antibodies were
successful in 38.9% of the cases using Immucor and in
27.2% using the Ortho system.
Conclusions
The performances of the two systems were essentially
similar.
The Immucor system detected a greater number of
antibodies, mainly due to its greater sensitivity for anti-D
antibodies, while maintaining a good specificity.
Testing with both systems was shown to be repeatable
(in over 85% of the cases), bearing witness to the advantage
of using automation in immunohaematological tests.
Both systems were able to identify the antibody, in a
completely automated manner, in about 68% of the positive
samples. The capacity of both systems to identify the
antibodies decreased in direct relation to the complexity of
the patients’ immunological profile.
The use of both technologies identified ten additional
antibodies (specificities: 6 anti-D, 2 anti-E, 1 anti-Lea, 1 anti-
Vel), that would not have been detected and identified, had
only one of the methods been used. Having two
technologies is an undoubted advantage, although
systematic testing of all samples with two systems, as done
in our study, is certainly not feasible.
Although the level of automation achieved was high,
there is room for further improvement of this aspect, both
in the management of the analytic phase and in the
interpretation of the results.
39

Finally, it is to be hoped that the companies ensure
consistency between the antigens present on the red blood
cells used in the screening tests and those present in the
identification panels, in order to avoid the occurrence of
positive screening tests and negative results with the
identification panels, because of the absence of some
antigens from the relevant panels.
Acknowledgements
We thank the Ortho and Immucor companies for their
kind collaboration and for having provided the reagents
necessary for duplicating the tests.
We also thank the laboratory staff of the SIMT of
Ragusa, who made this study possible, by doubling the
number of tests carried out over the whole period of 6
months.
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Received: 7 July 2006 – Revision accepted: 2 October 2006
Correspondence: Dr. Pietro Bonomo,
SIMT Azienda Ospedaliera “Civile-Maria Paternò Arezzo”
Piazza Igea, 1
97100 Ragusa, Italy
e-mail: Bonomo@ospedaleragusa.it
Blood Transfus 2007; 5: 33-40 DOI 10.2450/2007.0022-06