Anal Bioanal Chem (2009) 393:1443 1451
DOI 10.1007/s00216-008-2411-3
REVIEW
Recent and future trends in blood group typing
Wim Malomgré & Birgid Neumeister
Received: 2 June 2008 /Revised: 20 August 2008 /Accepted: 12 September 2008 /Published online: 7 October 2008
#
Springer-Verlag 2008
Abstract Blood group typing is the process of testing red It is standard practice to test for A, B, and D (Rh) antigens
blood cells to determine which antigens are present and and to perform tests for other antigens only in selected
which are absent. It is standard practice to test for A, B, and cases; however, regulations may vary from region to region.
D (Rh) antigens and to perform tests for other antigens in When a person does not have a particular RBC antigen, his
selected cases. ABO blood group typing is confirmed by or her serum may contain an antibody directed against this
reverse grouping that detects expected isoagglutinins. Un- antigen. Whether or not the antibody is present in the serum
expected antibodies can be demonstrated by antibody depends on whether the person s immune system has been
screening tests. For transfusion, donor units compatible with previously challenged by, and responded to, this specific
the patient are selected. Prior to transfusion, a crossmatch is antigen or something very similar to it. The human body is
performed as a final check for incompatibility. This article constantly exposed to antigens in pollens, food, bacteria,
describes the recent and future methods of blood group and viruses. Some of these  natural antigens are apparent-
typing and testing of serological compatibility. In addition, ly so similar to human blood group antigens that they
methods for blood bank automation are presented. stimulate almost every susceptible person to produce
antibodies. Thus, certain antibodies are expected in the
. .
Keywords Blood groups Blood group typing Automation serum of anyone whose RBCs lack the reciprocal antigen.
This is especially true for the ABO system. The test to
prove expected antibodies (isoagglutinins) of the ABO
Introduction blood group system is called reverse ABO grouping.
Unexpected antibodies can be demonstrated in the sera of
Many different blood group antigens are found on the a small percentage of people. These people have previously
surface of red blood cells (RBCs) in every individual. been exposed to RBCs which carry antigens different from
Blood group typing is the process of testing RBCs to their own. This exposure to human RBCs may have
determine which antigens are present and which are absent. occurred via transfusion or fetal maternal hemorrhage.
Pretransfusion testing can minimize (but not eliminate)
exposure to nonself blood group antigens, but it is
impractical to test recipients and donors for antigens other
W. Malomgré than those known to be of major significance.
Ortho-Clinical Diagnostics,
In most instances this testing is limited to A, B, and D
Antwerpseweg 19-21,
(Rh). Even though they may have a history of transfusion or
2340 Beerse, Belgium
pregnancy, most people do not have unexpected antibodies
e-mail: [e-mail]
because of the matching that is done prior to transfusion, the
B. Neumeister ( )
administration of Rh immunoglobulin, variability in the
Labor Dr. Gaertner & Partner,
immunogenic potency of different antigens, the number of
Elisabethenstraße 11,
foreign RBCs received, and the variability of the immune
88212 Ravensburg, Germany
e-mail: [e-mail] response in each individual. Antibody screening tests are
1444 W. Malomgré, B. Neumeister
used to detect those people whose sera contain one or more laboratory has occurred in the way the agglutination is
unexpected antibodies. Subsequent to its detection, an captured or visualized.
antibody must be identified so that its significance can be
judged. Some antibodies are harmless in transfusion and
pregnancy, while others are extremely dangerous. Antibody Classical manual blood group typing methods
identification tests define the specificity of the antibody, but
whether an antibody will or will not cause destruction of A whole series of techniques exist which all aim at showing
RBCs bearing the corresponding antigen depends on a the presence of antigen and antibody interaction through the
number of conditions. process of RBC agglutination [2]. This review focuses on the
Donor units compatible with the patient in terms of ABO most commonly used test methods (Table 1), realizing that
and RhD are selected. If an antibody has been detected in the other methods or variations are described in the literature.
recipient, further selection of donor units may be necessary, In the slide test method, a glass or stone tile is used on
depending on the identification and significance of the which a drop of specific antiserum is mixed with blood of the
antibody. Prior to transfusion, a compatibility test called patient or donor. For example, in the case of ABO and RhD
 crossmatch is performed as a final check. In this typing, a drop of anti-A, a drop of anti-B, and a drop of anti-
procedure, the serum of the recipient is tested with the RBCs D are used. The concentration of the RBC suspension may
of each prospective donor/erythrocyte concentrate. The term vary for different reagent manufacturers but usually is more
 crossmatch comes from the original method used to like whole blood. The observed reaction pattern with the
determine if a donor unit and a recipient were suitably different reagents used in the test will determine the ABO
matched. It included the major side of the match (patient and RhD type of the patient or donor. The main advantage of
serum and donor cells) and the minor side of the match the slide test method is that it is a fast and inexpensive test
(patient cells and donor serum). Owing to routine screening that can be executed without the need for specific equipment
for irregular antibodies in the serum of blood donors, the such as a centrifuge. It is, however, not a very sensitive
minor part of the match is no longer necessary. The concept technique and it is only able to detect interaction between
of compatibility encompasses much more than the cross- antigens and antibodies that have direct agglutinating
match alone. It should include all pretransfusion testing of capability (mostly IgM) in a physiological saline environ-
both the recipient and the donor: ABO grouping, Rh typing, ment, so it cannot be used to detect incomplete (IgG)
antibody screening, and crossmatch. antibodies in the serum of the patient. Today, this technique
Another testing approach that has received more and more is used much less and its application is mainly found in a
acceptance is the so-called type and screen approach or quick control of a known blood group.
computer crossmatch whereby the serological test between A very common technique that has been used for many
donor cells and patient serum or plasma is omitted. In this years and that is still used in many places is the tube test.
approach, ABO and RhD types from both the patient and the As the name suggests, reagents and patient material(s) are
donor are confirmed to match by sensitive typing methods transferred into a glass or plastic test tube where they are
and the serum or plasma of the recipient is screened using a allowed to react. The RBCs of the patient are adjusted to a
sensitive antibody screening test for unexpected antibodies. concentration of around 3 5% in serum/plasma or, more
If the antibody screening test is negative, the likelihood for common today, a buffered saline solution. In most cases
the serological crossmatch to detect clinically significant one drop of this suspension is mixed with a drop of anti-A,
antibodies that remained undetected in the screening test is anti-B, or anti-D in a test tube. Then the test tube is
so small that it is acceptable to omit the crossmatch with centrifuged for a short time to bring the RBCs closer to
exception of those categories of patients who are likely to each other and to enhance agglutination. After centrifuga-
have unexpected antibodies. tion, the RBC button in the tube is gently resuspended and
Despite all the increased knowledge, the day-to-day examined for agglutination. In parallel with the antigen
practice in the average blood group serology laboratory has testing, the ABO reverse test is performed by adding two
not changed that much in principle. Karl Landsteiner [1] drops of the patient s serum and one drop of 3 5%
discovered the ABO blood group system in 1900 when he suspended reagent RBCs of groups A1 and B, centrifuged
observed visible clumping when blood of one individual and examined for agglutination. In some countries also A2
was being mixed with blood of another. Today, most of and/or O RBCs are tested. The test tube method can also be
the techniques applied in the blood bank are still based on used to perform antibody screening, identification, and
the principle of interaction between antigen and antibody compatibility testing. For this purpose, 3 5% suspended
and subsequent agglutination of RBCs (positive result). RBCs (reagent or donor erythrocytes) are mixed in the test
The absence of agglutination indicates the lack of inter- tube with the serum of the patient/recipient. In most cases, a
action (negative result). Most of the progress in the potentiator is added to enhance the reaction or shorten the
Recent and future trends in blood group typing 1445
Table 1 Major properties of immunohematological tests
Test Detection of Test principle Technology Reactants Time range Cost range Comments
per assay per assay
(min)
Slide Blood groups Agglutination manual Patient 10 30 ++ Insensitive, fast
erythrocytes
and antiserum
Isoagglutinins Patient serum
and indicator
erythrocytes
Tube Blood groups Agglutination manual Patient 10 30 ++ Sensitive,
erythrocytes time-consuming,
and antiserum centrifugation needed
Isoagglutinins, Patient serum
antibody screening and indicator
(indirect Coombs erythrocytes
test)
Microplate Blood groups Agglutination Manual and Patient 10 30 +++ Sensitive, fast,
automated erythrocytes centrifugation
and antiserum usually not needed
Isoagglutinins, Patient serum
antibody screening and immobilized
(indirect Coombs erythrocyte
test) antigens
Gel Blood groups Agglutination and Manual and Patient 10 45 +++ Sensitive,
centrifugation separation from automated erythrocytes time consuming,
nonagglutinated and antiserum centrifugation needed,
erythrocytes by easy documentation
centrifugation
Isoagglutinins, Patient serum
Antibody screening and indicator
(indirect Coombs erythrocytes
test)
Enzyme Rhesus Agglutination, Manual and Patient serum 30 +++ Highly sensitive,
alloantibodies, can be performed automated and indicator time consuming,
differentiation as tube assay or gel erythrocytes requires special
of alloantibodies centrifugation knowledge, usually
centrifugation needed
Molecular Blood groups Nucleic acid Largely Patient Hours + Highly sensitive, time
difficult to amplification manual leukocytes consuming, no need
determine by techniques for rare antisera, requires
serological (PCR-RFLP, special knowledge, does
methods PCR-SSP, not exclude genes that
PCR-SSO) are not expressed from
detection
Future trends Blood groups Microarray, SNP Still Patient Hours ? Promising but still
difficult to detection, quartz experimental leukocytes confined to
determine crystal microbalance, experimental
by serological surface Plasmon laboratories
methods resonance
For detailed information, see the text.
RFLP restriction fragment length polymorphism, SSP sequence-specific primers, SSO sequence-specific oligonucleotide hybridization, SNP
single-nucleotide polymorphism
1446 W. Malomgré, B. Neumeister
incubation time of the test. There are a variety of After the incubation stage during which the incomplete
potentiators that work according to different principles. antibodies bind to the antigens on the surface of the RBCs in
Since most of the unexpected antibodies that have clinical the test tube, a wash step with saline is performed to remove
significance react best at 37°C, an incubation phase is serum and unbound immunoglobulin from the mixture,
included in the test to allow antibodies to sensitize the leaving only sensitized RBCs in the suspension. Then, an
RBCs. antiglobulin serum is added to the tube, after which it is
Commonly used potentiators are as follows: mixed, centrifuged, and examined for agglutination. This test
is also called the  indirect antiglobulin test or IAT.
& Bovine albumin can be added to reduce the repulsive
The advantage of the tube test over the slide test is that it
energy between the RBCs in the mixture so that they
is a more sensitive method and that it also allows the
can come together and incomplete IgG antibodies can
detection of clinically significant unexpected and incom-
agglutinate the RBCs.
plete antibodies in the serum of the recipient or the donor. It
& Low ionic strength solution accelerates the uptake of
is also a relatively fast test method, but it does require a lot
antibodies onto the RBCs by lowering the ionic strength
of different manual steps to be performed by the laboratory
of the mixture.
technician. A major disadvantage of the tube test with
& Poly(ethylene glycol) is a water-soluble polymer and
individual test tubes is that it is very difficult, if not impossible,
has also been shown to be an effective potentiator of
to automate.
antigen antibody reactions.
Microplate methods were adapted to blood bank use in the
& Cationic polymers such as Polybrene cause aggregation of
late 1960s. Advantages of microplate techniques include
normal RBCs that can be dispersed with sodium citrate;
enhanced sensitivity of reactions, savings in reagents and
however, sodium citrate does not disperse Polybrene-
supplies, and the ability for the dispensing steps to be
induced aggregation of antibody-coated RBCs. (This
automated by pipetting devices. Microplate techniques can
procedure permits detection of IgG antibodies without
be used to test for antigens on RBCs and for antibodies in
use of the indirect antiglobulin technique) [3].
serum or plasma. A microplate can be considered as a matrix
& Proteolytic enzymes such as ficin, papain, or bromelin
of 96  short test tubes. After the reagents and patient
can be used to enhance the reaction strength of certain
materials (RBC suspensions and serum) have been dis-
categories of antibodies (e.g., Rh), but also have clear
pensed, the plate is incubated and centrifuged, after which
limitations since they will destroy other RBC antigens on
the RBC buttons in the wells are resuspended by agitation
the test cells, thus making detection of certain specific-
and examined for agglutination. The agglutination strength
ities of clinically significant antibodies (e.g., Duffy,
can either be estimated through visual inspection with or
MNS) impossible. Antibody screening tests potentiated
without the help of a reader mirror or an automated
by means of enzymes will never be used as a single test
microplate reader can be used. Over time, a number of
but always in addition to an anti-human globulin test.
variations have been applied to the microplate technique.
The use of proteolytic enzymes as potentiators is helpful
Microplates have been developed that have the reactive
in antibody identification.
antisera (anti-A, anti-B, etc.) precoated in a dry format in the
If the antibodies in the serum of patients are complete or
microwells so that only patient materials and reagent RBCs
direct agglutinating antibodies (IgM), simple centrifugation
have to be distributed. Some methods also use proteolytic
of the test tube will be sufficient to cause visible
enzymes as part of the test to enhance the reactivity and
agglutination. But if the antibodies are incomplete (IgG)
increase the reaction strength. Another variation uses
and thus cannot agglutinate in a normal physiological saline
hemagglutination in terraced microplates.
environment or with the help of potentiators, an alternative
For the antibody detection test (screening, identification,
method needs to be applied to cause agglutination of RBCs
and compatibility), alternative methods of hemagglutination
that have sensitized with the antibody during the incubation
have been developed using microwells with known anti-
stage of the test.
gens immobilized on a solid matrix such as the inner side of
In 1945, Coombs, Mourant, and Race described the use
a test well. The serum containing suspected antibodies is
of anti-human globulin serum for the detection of non- then applied to the well and with the help of indicator cells
agglutinating antibodies bound to RBCs, a discovery that
the test is read by adherence. Other variants use wells
must rank as almost as important as the discovery of the
coated with protein A that captures sensitized RBCs by
ABO blood groups. Since then, the test has been in regular
adherence after addition of anti-human globulin serum.
use in laboratories throughout the world and is probably
A significant change in the way agglutination was made
still the most useful single tool at the disposal of the blood
visible was introduced by Lapierre et al. [5] in 1990 with
bank serologist [4]. It is also applied in other techniques
the column agglutination technology in the form of the gel
than the tube test that will be described later in this review.
centrifugation assay.
Recent and future trends in blood group typing 1447
In this method, commercialized by DiaMed (Cressier, affinity principle whereby sensitized RBCs bind to an
Switzerland), agglutinates are captured in a microtube con- active phase in the column. This immune reactive phase
taining gel particles that will trap agglutinated RBCs and will contains protein G, anti-IgM, and anti-C3d bound to a gel
allow nonagglutinated cells to pass through the column to the matrix [6] (Cellbind, Sanquin, The Netherlands).
bottom. Six or eight microtubes are fixed on a plastic card. Over the past 10 15 years, many blood bank laboratories
The test can be modified for different types of tests. around the world have adopted column agglutination technol-
For blood grouping tests (antigen typing), the micro- ogies as their routine grouping and antibody detection method.
columns contain reagents such as anti-A, anti-B, and anti-D.
For reverse grouping, a neutral gel is used and a suspension of
reagent RBCs and serum is dispensed in the upper chamber of
Blood bank automation
the microcolumn where antibodies and antigens can react. The
subsequent centrifugation is intended to first bring the RBCs
Clinical laboratories have been steadily introducing automa-
and serum/antiserum together and then to push the RBCs
tion for many years. Fully automated analyzers have become
through the gel. If RBCs are agglutinated, they will be trapped
integral components of nearly every department. Blood bank
either on top or in the gel column depending on the strength of
laboratories have been much slower in adopting automation.
the agglutination. If they are not agglutinated (in the case of a
Historically, blood banking technology has predominantly
negative reaction), the RBCs will pass through the gel and
used manual testing methods. Blood bank laboratories have
form a pellet at the bottom of the tube.
resisted change, in part because there has been an abundant
For the antibody detection test (screening, identification,
supply of well-trained technologists and inexpensive
and compatibility), the contact between antibodies and
reagents. But more important, blood bank testing methods
antigens takes place in the upper part of the microcolumns
are difficult to automate. With or without automation, there is
where a mixture of reagent RBCs, suspended in low ionic
no acceptable margin of error; each test result or blood type
strength solution, and patient serum or plasma is dispensed.
generated must be correct. More stringent regulations and
After a suitable incubation period of about 10 15 min, usually
 zero tolerance for medical errors have rendered labor-
at 37 °C, the microcolumns are centrifuged and the RBCs
intensive methods inefficient and have increased the risk for
come in contact with the gel that contains an antiglobulin
liability of transfusion services. In the laboratory, the most
reagent to agglutinate RBCs potentially sensitized by incom-
reliable step to reduce error is to reduce opportunities for
plete IgG antibodies. The RBCs used in this test can also be
making mistakes. The laboratory error rate is a function of
mixed with an enzyme solution such as bromelain to enhance
the number of steps in the testing process and the number of
the reactivity of certain types of antibodies, in particular of Rh
human interactions with the system. Laboratory automation
alloantibodies. In this case, a neutral gel is used. This
is one of the most important steps the transfusion service can
technology has a number of advantages over the use of classic
take to ensure safety for the patients.
tubes. It is, in general, easy to use, also for less-skilled
Today, a range of automation systems are available on the
laboratory technicians. There is no more need to  shake
market, ranging from semiautomation to full automation. In
tubes, which removes an important factor of variability among
the early phase of automation in the blood bank laboratory,
different laboratory technicians performing the same test. The
mainly semiautomated solutions were available which focused
washing steps required in the IAT tube test to remove serum
on automating dispensing, washing, and reading steps but still
and unbound proteins after incubation and before addition of
required a lot of manual hands-on work. Today, as many steps
the IAT reagent are no longer needed, which is a significant
as possible should be automated to maximize safety and
saving in time and manipulation. The reactions are stable for a
security and to increase the efficiency of the laboratory.
longer period of time and can be shared with fellow laboratory
Several commercially available full automation plat-
technicians. Another major advantage is that this technology
forms are available on the European market:
can be fully automated. In addition, the sample volumes
required for this test are significantly reduced compared with & The Classic Plus ID-GelStation utilizes the gel test for
those required for the tube test. blood group typing and the antibody detection test and
Variations on the gel technology have been developed the Techno TwinStation employs a combination of full
whereby the gel matrix is replaced by a matrix of glass beads automation for ID-Gel cards and semiautomation for
that will trap the agglutination (Ortho BioVue system). The microplates (both from DiaMed, Cressier, Switzerland).
advantage here is that a faster centrifugation speed can be & The Galileo and the Galileo Echo system (Immucor
applied to the microcolumn, which shortens the time Gamma, Norcross, USA) uses a combination of classic
required for centrifugation, resulting in a faster test result. microplate technology for blood group typing and solid-
Other variations do not make use of agglutination to phase technology for the antibody detection test (Capture-
show sensitization of RBCs with antibodies but use an R). The Galileo system is intended to be the larger
1448 W. Malomgré, B. Neumeister
throughput system, whereas the more recently introduced of the laboratory. If this is not done, the desired
Galileo Echo is suitable for small to medium-sized increase of efficiency may not be achieved. Tradition-
laboratories. ally, when automation is selected, parameters such as
& The Tango Optimo (Biotest, Dreiech, Germany) uses the throughput of the analyzer are used to decide which
ready-for-use microplates coated with dried monoclonal instrument will be the best for the laboratory and
antisera for blood grouping. Antibody detection tests usually the machine that claims the highest throughput
are performed in wells coated with protein A that per hour is chosen. However, if the analyzer is placed
captures sensitized RBCs by adherence after addition of in a broken process or if it does not support the
anti-human globulin (solid screen). laboratory s workflow, it will not meet the expectations.
& The Qwalys system (Diagast, Loos Cedex, France) is a Thus, it is important to look beyond the analyzer and to
system that uses E.M.® technology (erythrocytes observe the total process from the arrival of the sample in
magnetized technology), based on the magnetization the laboratory until the release of the result to the
of RBCs. This eliminates the need for centrifugation. physician or patient. Many analyzers only achieve
This method is used for both blood grouping and the optimal throughput if they process samples in larger
antibody detection test. batches. When samples are processed in smaller numb-
& The Ortho AutoVue Innova/ultra system (Ortho Clinical ers or as individual samples, the analyzers tend to lose a
Diagnotics, Raritan, NJ, USA) that uses the aforemen- lot of their efficiency. In most of the blood bank
tioned BioVue system technology for both blood group laboratories, however, samples do not arrive in large
typing and antibody detection. The BioVue technology batches, so to make the instrumentation run efficiently,
resembles a column agglutination method that uses a samples will have to sit and wait on the bench until a
matrix of glass beads instead of gel beads, which allows a sufficiently large number of samples are available to
shorter centrifugation time. justify a run. This waiting time can be considered pure
waste as it adds to the total time for the availability of the
There are many reasons why blood banks introduce
test result and reduces significantly the value of the high-
automation in their laboratories:
throughput analyzer. Thus, the ability to run both
1. Blood bank laboratories want to increase the safety and
individual samples as well as a larger number of
security of their analytical process. False-positive or false- specimens without significantly affecting the process
negative results may be obtained if inadequate attention is
time is an important feature of a blood group analyzer if
given to proper collection and handling of specimens,
a maximum improvement in efficiency is desired.
serum-to-RBC ratios, RBC concentrations, suspending
3. Financial aspects can also influence the decision to
media, incubation times and temperatures, speed and time
introduce automation into a blood bank laboratory.
of centrifugation, and the manipulation of tubes and their
Laboratories constantly face pressure of limited or
contents while examining for agglutination. Controls
declining budgets and as such the investment in
designed to detect failures of the test system and/or of
automation may look like an expensive operation.
the test operator are essential. Automation can help to
However, there are many areas where automation can
achieve this goal by removing the variability that is
deliver significant savings. Reduction of the costly
typical for manual test methods performed by different
hands-on time skilled laboratory technicians spend with
people. By standardizing all steps of the test process in a
repetitive routine activities such as dispensing samples
consistent way, the chance for error will decline. In
and reagents or a more optimized test process with less
addition, the reduction of manual hands-on activities by
variation (variation being a potential cause of repeat
the laboratory technicians to an absolute minimum will
tests) and waste are just a few examples. In addition,
help to decrease the chance for error because, though we
the value of a safer and more secure test process should
do not like to admit it  to err is human [7].
not be underestimated (what is the value of one
2. A second important reason to introduce automation is
transfusion accident less?) but is not often taken into
to improve the efficiency of the test process. Finding
consideration as it is difficult to quantify.
appropriately qualified staff is becoming increasingly
During the decision-making process for automation, the
difficult, so if automation can take over the routine
costs of purchase rather than the costs of operation are
tasks of the test process such as dispensing reagents and
traditionally considered. Eventually it will be the amount of
samples, incubation, centrifugation, and reading, the
money spent over the total life time of the automation
qualified staff can spend more time on other activities
solution that will define the cost-effectiveness of the solution
that add more value to the process. Here it is very
and not only what is paid to purchase it. So it is important to
important to understand how an analyzer and the
understand how the analyzer operates in the test process and
characteristics of its operation will fit in the test process
which factors affect the cost of operation. Running single or
Recent and future trends in blood group typing 1449
a small number of samples versus a large number of Future developments in analyzer software will not
specimens may have a significant impact on the waste of necessarily focus only on developing new test methods but
reagents or controls. Small differences in hands-on time will also have an increasingly large element of compliance
between the instruments may appear insignificant but if they and evidence to make sure that regulatory requirements are
are repeated several times a day, week, month, etc. they do met.
become significant [8].
4. A last reason why automation should be introduced is
that owing to the optimization of the overall test
Molecular blood group typing
process, the laboratory is able to deliver a faster and
better service to its customers: the physicians and
All of the methods described in the previous sections are
patients. The degree of service improvement is depen- based on antigen antibody interactions and are all directed
dent on the reliability of the instruments and the
to detect directly or indirectly what is expressed on the
customer service. The level of service provided is
surface of RBCs. Molecular blood group typing takes a
becoming increasingly more important as many labo- completely different approach as it investigates the genetic
ratories are presenting themselves as commercial
information in the DNA of the donor or the patient that
organizations in a competitive environment where
encodes the antigen structures on the surface of the RBC.
delivering on customer needs and high-quality services
Molecular methods for blood group genotyping became
is of great value. At the end of the line we should not
available more than 10 years ago as one major aspect of
forget about the patient for whom this potential life- immunogenetics. Since then, the clinical applications have
saving treatment is of extreme importance.
been expanded and refined. Their implementation varies
considerably among different health-care systems, notably
between North America and Europe [9]. In an overview of
molecular methods in immunohematology [10], Marion
Significance of reliable software
Reid writes that the classic method of testing for blood
group antigens and antibodies with hemagglutination is
Software plays an increasingly important role in the overall
simple, inexpensive, and when carried out correctly has a
automated test process: in the laboratory information
specificity and a sensitivity that are appropriate for the
system where a test request is booked, at the interface
clinical care of the vast majority of patients. However, the
between laboratory information system and the analyzer, as
technique has certain limitations:
well as in the analyzer itself. Many of the tests that
& It does not reliably predict a fetus at risk of hemolytic
laboratories performed manually and which they felt they
disease of the newborn.
had a high level of control over are now taken over by
& It is difficult to type RBCs of patients who recently
instruments and computers that sometimes may look like a
received transfusion or of patients suffering from
black box. To ensure consistent and reliable performance it
autoimmune hemolytic anemia whose RBCs are coated
is important that software is properly tested and evaluated
with IgG.
both by the manufacturer as well as within the laboratory
& It does not precisely indicate RhD zygosity in D + people.
process where it will be used as there may be specific
& A relatively small number of donors can be typed for a
characteristics of this process or interactions with other
relatively small number of antigens, thereby limiting
software packages that may not be available to the
antigen-negative inventories.
manufacturer to test. It is critical that sufficient time is
& It requires the availability of specific reliable antisera.
spent on validating that changes in the test configuration
& It is labor-intensive.
will not adversely impact the performance of the test
& The source material is expensive and diminishing.
process. Regulatory bodies are more and more demanding
& The cost of commercial reagents is escalating.
this kind of validation and asking for proof of the
& Many antibodies are not FDA-approved.
operational qualification of the system. Software also needs
& Some antibodies are limited in volume, weakly reactive,
to have the appropriate controls in place that can track and
or not available.
trace activities and detect deviations or errors when they
occur. These can be controls before the start of the test The association of the majority of RBC antigens with
which are used to check whether the system is properly single-nucleotide polymorphisms (SNPs) provides the basis
working (e.g., temperature of the incubator) or in-run to determine blood group antigen expression at the DNA
controls that monitor the system while it is working, report level. Molecular immunohematology not only reduces the
any potential anomaly, and stop the user from proceeding need for increasingly rare serological reagents, but also
unless it is safe to do so. permits the reliable determination of a genotype and
1450 W. Malomgré, B. Neumeister
thereby of the antigen phenotype in situations that are by hemagglutination of predicted antigen negativity is
difficult to resolve by serological methods, especially when recommended using a reagent antibody, if available, or by
the available antibody reagents are only weakly reactive. crossmatching using a method optimal for the detection of
These applications of DNA analysis include the determina- the antibody or antigen in question [10].
tion of weakly or altered expressed antigens, the analysis of Not all blood group polymorphisms can be easily
patients in a variety of conditions, including chemotherapy, analyzed, for example, if a large number of alleles encode
autoimmune hemolytic anemia, and transfusion therapy, one phenotype (e.g., ABO, Rh, and null phenotypes in many
and the identification of fetuses at risk for hemolytic disease blood group systems); or alleles with large deletion (e.g.,
of the newborn [11]. Ge ) or alleles encoded by a hybrid gene (e.g., in the Rh and
The use of molecular typing can be applied for testing both MNS systems); or when the molecular basis is not yet known
patients and donors. Potential clinical applications are [10]: (e.g., Vel, Lan, Jra). In addition, there is a high probability
that not all alleles in all ethnic populations are known.
1. Antigen typing in a patient
Interested readers who want to find out more details about
(a). Identification of fetuses at risk for hemolytic
molecular antigen typing, its application, and the implications
disease of the newborn.
it has on the future blood bank laboratory are encouraged to
(b). Weak or not available antibodies, e.g., anti-Doa,
read more in volume 47 of Transfusion, published July 2007
anti-Dob, anti-Jsa, anti-V/VS.
as a supplement.
(c). Identification of recently transfused patients.
(d). Blood group determination in patients suffering
from autoimmune hemolytic anemia (+DAT).
Future trends in blood group typing
(e). Discrimination of alloantibody and autoantibody.
(f). Detection of weakly expressed antigens (e.g., Fyb
One of the most interesting but still largely experimental new
with the FyX phenotype); where the patient is
approaches in blood group typing is the development of
unlikely to make antibodies in response to
diagnostic microarrays. DNA as well as protein and carbohy-
transfused antigen-positive RBCs.
drate microarrays are under investigation. The biggest impact
(g). Identification of the molecular basis of unusual
could come from combining the various types of conventional
serological results, especially Rh variants.
assays that are currently performed, including blood group
(h). Determination of zygosity, particularly RhD.
typing and pathogen detection in blood donations. Micro-
2. Antigen typing in donors
arrays have the potential to change these testing algorithms to
simultaneous testing all required markers on a single platform,
(a). Screening for antigen-negative donors.
cutting the cost of multiple instruments with dedicated
(b). Weak or not available antibodies, e.g., anti-Doa,
operators and specific reagents, and significantly simplify
anti-Dob, anti-Jsa, anti-Jsb, anti-V/VS.
the data-processing side of operations [12]
(c). Mass screening to increase antigen-negative inven-
As already mentioned, most blood group antigens are
tories and to find donors whose RBCs lack a highly
biallelic and are the result of a SNP. For DNA microarrays,
prevalent antigen.
gene fragments containing SNPs are amplified by multiplex
(d). Resolution of blood group A, B, D, C, and e
PCR after DNA isolation. The PCR products are then
discrepancies.
hybridized to an array containing short allele-specific
(e). Detection of genes that encode weak antigens.
oligonucleotides. For each blood group, different oligonu-
(f). Identification of donors for reagent RBCs (anti-
cleotides are spotted (sense and antisense). The entire array
body screening and identification panels).
is then visualized using an automated microscope and
With this wide range of possibilities it may be presumed image analysis system, the array information is decodified,
that the days of serological testing are over; however, this and the genotypes scored. The first results show that
would probably be too easy. Like all methods, molecular microarrays will provide a reliable and fast procedure,
antigen typing has its advantages but also its limitations, which can be further improved to obtain the necessary
owing to technical, medical, and genetic pitfalls which throughput [13].
cause the phenotype (what is expressed on the surface of For determination of blood groups, protein microarrays
the RBC) to be different from the genotype (what is were developed. They are printed with multiple spotted
encoded in the genes). The majority of DNA-based assays replicates of blood type antigen specific antibodies. This
do not exclude genes that are not expressed from detection method can also be used in microarrays for direct antiglobulin
and this can lead to a donor being falsely identified as testing where monoclonal anti-IgG and anti-C3d are spotted
antigen-positive. This would mean that a valuable antigen- onto a microarray surface and fluorescein isothiocyanate
negative donor would be lost to the inventory. Confirmation labeled, sensitized RBCs are allowed to react.
Recent and future trends in blood group typing 1451
Carbohydrate microarrays are a new technology being neous multiparameter identification of blood groups (ABO,
developed by a number of groups for high-throughput C, c, D, E, e, Cw, and Kell) on the basis of lateral flow
evaluation of carbohydrate macromolecule interactions. assay was developed. The test requires 100 źl of RBC
Carbohydrate microarrays contain many different gly- suspension and 300 źl of diluent. Results are available after
cans immobilized on a solid support in a miniaturized 2 5 min. The speed of performance and the ease of use
format. Binding of proteins, viruses, or cells to every make this card ideal for emergency cases.
component on the array can be simultaneously evaluated
under identical conditions. Using carbohydrate microarrays
containing different glycans and glycoproteins, one can Conclusion
detect antibodies to blood groups A, B, H, and Lewis.
An exciting new feature is the development of a micro- Serological blood group typing by manual methods or
fluidic platform for mass-sensitive analysis of diluted whole automation is well established, sure, and reliable but cost-
blood. The platform consists of a biologically functionalized intensive and time-consuming. Molecular typing and its
quartz crystal microbalance, an insertable microfluidic chip, a application will expand further as more knowledge becomes
microfluidic mixer, and a peristaltic micropump. Blood- available. New technologies such as microarrays, quartz
group-specific antibodies are immobilized on the upper gold crystal microbalance, and lateral flow assays will continue to
electrode of the quartz resonator via protein A. Injection of be developed, but they will not replace serological testing in
whole blood leads to a specific binding of antigen-positive the near future.
RBCs to the sensor surface and thereby to a significant decrease
in the resonance frequency that is detected by a frequency
counter. This platform allows fast and reliable typing of blood
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