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RED CELL REACTIONS WITH ANTI-A &/OR ANTI-A,B
WEAKLY AGGLUTINATED
MIXED-FIELD AGGLUTINATION WITH
(1+-3+) ANTI-A,B ONLY
A3 Ax
FIGURE 9-7 Investigation of A subgroups.
with anti-A,B to confirm that they are not actually
weak subgroups of A.
The reason for this special caution is that all group
O people have in their serum anti-A,B, which is the
most potent antibody capable of reacting with the
weaker subgroups of A. Failure to detect a weak sub-
group of A or B in a patient population usually causes
few if any problems. If the people fail to demonstrate
a cell–serum group discrepancy, they would be trans-
fused with RBCs as if to disregard the presence of the
weak A or B antigen. The weaker examples of the A or
B antigen also are unlikely to lead to significant
hemolysis if transfused with incompatible plasma
because the plasma will be diluted with existing pa-
tient blood volume (e.g., a weak subgroup of A trans-
fused with group O plasma).
RBCs of the Aint, A3, Ax, Am, or Ael subgroups are
rarely seen in transfusion practice. Classification of the
subgroups of A depends on several different testing
procedures. Correct classification of the subgroups of A
depends on patterns of reactivity with anti-A, A1 lectin,
anti-A,B, and H lectin, as well as the presence or absence
of anti-A1 in the subject’s serum and the presence of A or
H antigens in the saliva of secretors. Box 9-3 lists the
BOX 9-3
Methods of Classification of the Subgroups
of A
• Agglutination pattern with anti-A and anti-A1
• Agglutination pattern with anti-A,B
• Agglutination pattern with Ulex europaeus
(H lectin)
• Presence or absence of anti-A1 in the patient’s
serum, resulting in ABO discrepancy
• Presence or absence of A and H substances in the
saliva of secretors
CHAPTER 9 The ABO Blood Group System 129
NO AGGLUTINATION
ABSORBS AND ELUTES ANTI-A
SECRETOR SALIVA SECRETOR SALIVA
CONTAINS A AND H CONTAINS H
SUBSTANCES SUBSTANCE ONLY
Am Ael
procedures used in the classification of the subgroups of
A, and Figure 9-7 demonstrates a flow chart that may
be used systematically to classify these subgroups.
Table 9-7 gives the reactions expected with suggested
testing.
SUBGROUPS OF B
The subgroups of B are even more infrequent than the
weaker subgroups of A. They are initially identified
by variability of reaction with anti-B and anti-A,B. The
subgroups B3, Bx, Bm, and Bel are classified similarly to
their counterparts in the classification of the A sub-
groups. See Table 9-7 for representative reactions of
these weak subgroups of B.
DISCREPANCIES IN ABO GROUPING
The importance of ABO blood grouping is under-
scored by the fact that all ABO blood grouping, with
the exception of newborns, consists of both a cell
grouping and a serum grouping. In most cases, the in-
terpretation of these two tests supports a common
conclusion, and the ABO group is confirmed. In some
instances, cell grouping and serum grouping tests re-
sult in different interpretations of the ABO type of the
patient. In these cases, a cell–serum group discrep-
ancy exists. All discrepancies between cell and serum
grouping must be resolved before a definitive type
can be assigned to the patient. Discrepancies can be
grouped according to their probable causes to facili-
tate resolution of the discrepancy. Common causes of
discrepancy are discussed in the following sections.
Technical Errors
Technical errors leading to ABO discrepancy are
common in student laboratories and may occur more
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130 UNIT 4 Red Blood Cell Groups and HLA

TABLE 9-7 Results of Testing that May Be Used for Subgrouping of A and B

Reaction of Subject Red Blood Cells with NRSC Antibodies

Antigen in Presence of A/B
Type Anti-A Anti-B Anti-A,B Anti-H Common Other Secretions Transferases

A1 4⫹ 0 4⫹ 0 Anti-B A, H A

A2 3 to 4⫹ 0 3 to 4⫹ 2 to 3⫹ Anti-B Anti-A1 A, H A

A3 2 ⫹ mf 0 2 ⫹ mf 3⫹ Anti-B Anti-A1 A, H Weak A

Ax Weak/0 0 1 to 2⫹ 4⫹ Anti-B Anti-A1 H Very weak A

Aend Weak mf 0 Weak mf 4⫹ Anti-B Anti-A1 H No A

Am 0 0 0 4⫹ Anti-B A, H Weak A

Ae1 0 0 0 4⫹ Anti-B Possible H No A

anti-A1

B 0 4⫹ 4⫹ 2⫹ Anti-A B, H B

B3 0 2 ⫹ mf 2 ⫹ mf 3⫹ Anti-A B, H Weak B

Bx 0 Weak/0 Weak/1⫹ 3⫹ Anti-A Weak H No B

Anti-B

Bm 0 0 0 3 to 4⫹ Anti-A B, H Weak B

Bel 0 0 0 3 to 4⫹ Anti-A May be H No B

weak anti-B
NRCS, non–red cell stimulated.

frequently than they should in the clinical laboratory.

The best defense against technical errors is a well-
written and meticulously followed testing protocol
and attention to detail. Common errors that may lead
to ABO discrepancies are listed in Box 9-4. The best
way to prevent these errors is to follow the testing
procedure exactly, and the first step in resolving a dis-
crepancy is to repeat the testing procedure to ensure it
was followed.
Weak or Missing Antibodies
The most frequent ABO discrepancy is due to weak
or missing antibodies. ABH antibodies have been
traditionally reported as being in highest titer during
adolescence and gradually weakening with age. It
has been demonstrated repeatedly that reduction in
titer of ABH antibodies does not come necessarily
with age but with poor health and nutritional status.
The very young and the very sick or debilitated may
demonstrate such a low titer of ABH antibodies that
routine testing may not detect them (Box 9-5). Other
patients may show a depression of the normal levels
BOX 9-4
Errors that May Cause ABO Discrepancies
Clerical errors
• Improper identification of patient sample/testing
• Improper recording of reactions
Technical errors
• Failure to follow manufacturer’s directions
• Contaminated or expired reagents
• Improper concentration of subject red blood cells
• Failure to add reagents/sample or improper
amounts
• Improper centrifugation
• Warming of test
of ABH antibodies due to disease or iatrogenic
causes.
This discrepancy may best be resolved by opti-
mizing the reverse grouping reaction. Although
routine serum grouping is performed at room
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BOX 9-5
Patients Who May Express ABO Discrepancy
Due to Weak or Missing Isoagglutinins
• Neonatal patients
• Elderly patients
• Patients with hypogammaglobulinemia
• Immunosuppressed patients (drug/disease)
• Bone marrow transplant patients
temperature, the antibodies that are detected in this
procedure react best at reduced temperatures.
Therefore, enhancement will occur if the testing is
performed at 18°C or 4°C. However, care must be
taken that other cold-reacting antibodies are not
mistaken for ABH antibodies. When incubating the
reverse grouping at reduced temperature, an auto-
control and group O screening cells should be in-
cluded to rule out autoantibodies or alloantibodies,
causing the expected pattern of agglutination. If the
autocontrol and the screening cells are negative and
the expected reaction occurs after incubation for 15
to 60 minutes, the discrepancy is resolved and may
be attributed to weakened antibodies. An appropri-
ate note should be included in the person’s record.
Table 9-8 shows an example of a discrepancy due to
weak or missing isoagglutinins and the proper res-
olution of the discrepancy. Most discrepancies in
this category may be resolved with testing under
enhanced conditions. However, rare people may
lack detectable ABH antibodies in their serum for a
variety of reasons.
Weak or Absent Antigens
Causes of discrepancies under this classification may
include the presence of subgroups of A or B and weak-
ening of antigenic strength in leukemia, as previously
mentioned. If apparent weakening of the antigen is
TABLE 9-8 Example of ABO Discrepancy Due to Weak or Missing Isoagglutinins
Forward Grouping Reaction of
Subject RBCs with
Anti-A Anti-B Anti-A,B
RT testing 4+ 0 NT
a
18/4 C testing 4+ 0 NT
a
Repeat testing of subject sample at reduced temperature will enhance reaction. O cells and autotesting must be included and show no agglutination.
RBCs, red blood cells; RT, room temperature; NT, not tested.
CHAPTER 9 The ABO Blood Group System 131
suspected, attempts to prove the presence of the anti-
gen with adsorption and elution may be worthwhile.
Refer to the Procedural Appendices at the end of this
chapter for instructions on the demonstration of weak
antigens by adsorption and elution. The depression is
rarely complete, and it should be possible properly to
group the patient.
Unexpected Cold-reactive Autoantibodies
Panagglutination is the ability of a particular serum to
agglutinate all or almost all cells in a particular popula-
tion. A good example of a panagglutinin is a person
whose serum contains a strong auto anti-I. This anti-
body agglutinates all but approximately 0.01% of the
adult population because the I antigen is strongly ex-
pressed in almost all adults. Patients with cold-reactive
autoantibodies such as autoanti-I may demonstrate
ABO discrepancies. Patients with high titers (⬎126) of
cold agglutinins may have cells that autoagglutinate at
room temperature. The cooler the testing temperature,
the greater the likelihood that a patient with cold au-
toagglutinins will have an ABO discrepancy. High-
titered cold autoagglutinins also may interfere with
reverse grouping if the reagent cells have the appro-
priate antigens on their surface. Refer to Table 9-9 for
an example of ABO discrepancy due to cold-reactive
autoantibodies.
The resolution of discrepancies due to cold-reactive
autoantibodies may be difficult without first obtaining
a new specimen. Two problems exist. As the person’s
sample is allowed to cool after drawing, the cells
become sensitized or coated with IgM antibody. This
antibody may cause autoagglutination and not only
interfere with blood bank testing, but may cause the
RBCs to clump together, interfering with the counting
and sizing necessary for automated hematologic test-
ing. Prewarming the specimen tube and keeping it at
37°C prevents the autoantibody from attaching to
the person’s RBCs in vitro. The patient’s cells should
be washed with 37°C saline three or more times
before being used for testing to remove any previously
Reverse Grouping of Subject Serum
with Reagent RBCs
A1 Cells A2 Cells B Cells O Cells Auto
0 NT 0 0 0
0 NT 2+ 0 0
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132 UNIT 4 Red Blood Cell Groups and HLA

TABLE 9-9 Example of ABO Discrepancy Due to Unexpected Cold-reactive Autoantibody

Forward Grouping Reaction Reverse Grouping Reaction

of Subject Red Blood Cells with of Subject Serum with
Anti-A Anti-B Anti-A,B A1 Cells A2 Cells B Cells O Cells Auto

RT testing 4+ 2+ NT 2+ 2+ 4+ 2+ 2+

After adsorptiona 4+ 0 NT 0 0 2 to 4+ 0 0
a
Serum either autoadsorbed or adsorbed with rabbit red blood cell stroma before retesting. Subject’s sample redrawn and kept at 37⬚C. Cells washed five
times with warm saline prior to being tested.
RT, room temperature; NT, not tested.

attached warm-reactive antibodies. Reverse grouping
of patients with strong cold autoantibodies may be dif-
ficult to perform. The sample may be allowed to clot
and then stored at refrigeration temperatures to allow
autoagglutination to occur.
Alternatively, the person’s serum may be autoad-
sorbed or adsorbed with commercially prepared rabbit
RBC stroma to remove all cold-reactive autoantibody be-
fore reverse grouping is performed. Ensuring that all
reagents have come to room temperature after removal
from the refrigerator can sometimes prevent this problem.
Unexpected Cold-reactive Antibodies
Unexpected cold-reactive antibodies may lead to ABO
cell–serum discrepancies by causing unexpected reac-
tions in the reverse grouping of subjects. These antibod-
ies may be related to the ABO BGS, such as anti-A1 in the
serum of a group A2 individual, or they may be com-
pletely unrelated to the ABO BGS. It must always be re-
membered that the reagent A1 and B cells used for
reverse grouping have not only A1 and B antigen, but
many other antigens. Antibodies that react at reduced
temperatures (usually the IgM class) may sometimes be
present in the subject’s serum and may react with the
appropriate antigen on the reagent RBCs. The antibody
screen performed using O reagent cells usually allows
proper identification of these antibodies as being “atypi-
cal” as opposed to the typical antibody (ABO isoagglu-
tinins) found in most subjects. Chapter 10 discusses the
detection and identification of atypical antibodies. Once
the specificity of the atypical antibody has been deter-
mined, examples of A1 and B cells that lack the antigen to
this atypical antibody may be selected and the discrep-
ancy resolved. Table 9-10 gives an example of ABO dis-
crepancy due to unexpected cold-reactive antibodies.
Rouleaux
Abnormal levels of proteins, plasma expanders such as
Dextran, and Wharton jelly (coating cord tissue of the
fetus) can cause RBCs to stick together in a manner that
may resemble agglutination. This false agglutination, or
rouleaux, may result in an ABO discrepancy. The most
common cause of this type of discrepancy is due to ele-
vated protein levels, as might be seen in multiple
myeloma, Waldenstrom macroglobulinemia, and other
plasma cell dyscrasias. Increased levels of protein may

TABLE 9-10 Example of ABO Discrepancy Due to Unexpected Cold-reactive Antibody

Forward Grouping Reaction Reverse Grouping Reaction

of Subject Red Blood Cells with of Subject Serum with
Anti-A Anti-B Anti-A,B A1 Cells A2 Cells B Cells O Cells Auto

Example 1:
RT testinga 4+ 0 NT 2+ 2+ 4+ 2+ 0

Example 2:
RT testingb 4+ 0 NT 2+ 0 4+ 0 0
a
Subject shows evidence of atypical antibody. Antibody identification should be performed.
b
Subject has probable anti-A1. Subject’s cells should be tested with A1 lectin, and subject’s serum should be tested against a panel of at least three different
A1 and A2 cells. Subject cells should be negative with lectin and should react only with the A1 cells on the panel.
RT, room temperature; NT, not tested.
82043_ch09.qxd 11/11/09 6:10 PM Page 133
TABLE 9-11 Example of ABO Discrepancy Due to Rouleaux
Forward Grouping Reaction
of Subject Red Blood Cells with
Anti-A Anti-B Anti-A,B
RT testing
saline tube 4+ 2+ NT
Replacement
testinga 4+ 0 NT
a
Subject demonstrates loose 2⫹ reactions that when examined under the microscope appear as “stacks of coins.” Saline tube replacement can be used to
disperse the rouleaux and yield results indicated. In this procedure, the testing is performed as usual, but before resuspending the cell button, all sera are
removed with a piper and gently replaced with two drops of saline. The test is then examined for agglutination.
NT, not tested; RT, room temperature.
interfere in cell grouping, serum grouping, or both. In-
terference in cell grouping tests may be overcome by
multiple washing of the cells to be tested so that all
nonattached protein is removed. Interference with serum
grouping is more difficult to deal with and may be over-
come by the saline tube replacement technique. See
Table 9-11 for an example of this type of discrepancy.
Miscellaneous
Miscellaneous causes of ABO discrepancy include
interferences caused by alterations in normal subject
blood samples such as increased BGSS, acquired B
phenomenon, antibodies to low-incidence antigens
present in reagent antisera, and polyagglutination.
Problems related to increases in BGSS due to carcinoma
of the stomach and pancreas were noted previously.
Adequate washing of a person’s RBCs before testing
alleviates this cause of ABO discrepancy.
Acquired B phenomenon may result from intestinal
obstruction, carcinoma of the colon or rectum, or any
disorder of the gastrointestinal tract that may lead to ob-
struction or slowing of intestinal movement, sufficient
to allow passage of intestinal bacteria through the in-
testinal wall and into the bloodstream. Acquired B may
result from alteration of A antigen in group A people by
bacterial enzymes or by adsorption of a B-like antigen
from bacteria, such as may occur in group A or group O
patients. In the latter case, bacterial polysaccharide from
Proteus vulgaris and Escherichia coli O86 may be adsorbed
onto the cell surface and result in alteration of group A
to apparent group AB and group O to group B. The re-
sult is a person who carries the antibody to an apparent
antigen he or she carries on the RBCs but fails to agglu-
tinate his or her own cells. Proof of the nature of the ac-
quired antigen may lie in the testing of secretors for the
presence of the antigen in secretions.
Occasionally, a human or animal source reagent
serum, although exhaustively tested by the manufac-
CHAPTER 9 The ABO Blood Group System 133
Reverse Grouping Reaction
of Subject Serum with
A1 Cells A2 Cells B Cells O Cells Auto
2+ 2+ 4+ 2+ 2+
0 0 4+ 0 0
turer, will demonstrate an antibody to a private anti-
gen (one carried by the RBCs of only a few people).
When used to group the RBCs of those people carry-
ing the private antigen, the serum reacts positive. This
may lead the technologist to believe that he or she is
witnessing the reaction of the antibody named in the
reagent (e.g., anti-A) with the A antigen on the surface
of the cells, rather than the actual interaction of a pri-
vate antigen. This discrepancy may be resolved by
testing the patient with more than one manufacturer’s
reagent antiserum. The likelihood of two manufactur-
ers’ sera containing the same antibody to a private
antigen is extremely remote.
Polyagglutination is the spontaneous agglutina-
tion of RBCs by all or almost all normal human sera.
There are several causes of polyagglutination. The
first type to be discovered was due to T activation.
Bird showed that a plant lectin made from the peanut,
Arachis hypogaea, was able to detect an antigen on the
surface of the RBCs of rare people who seemed to de-
velop an antigen labeled T on the surface of their
RBCs.5 Further work led to the discovery that a num-
ber of lectins could be used to differentiate polyagglu-
tinable cells (Table 9-12). People may sometimes suffer
from T activation without pathologic causes, whereas
in others the change seems to indicate some poten-
tially serious latent disease. T activation occurs when
a portion of the normal RBC membrane is cleaved en-
zymatically to expose a previously unexposed anti-
gen. This may occur in vivo or in vitro. Once it is
exposed, the T antigen is free to react with the IgM
anti-T that is normally present in the serum of most
normal adults. People who have in vivo T activation
do not usually demonstrate anti-T in their serum and
thus have a negative autocontrol. T activation in vivo
is a transient condition caused by exposure to bacte-
rial enzymes. Once the cause of the exposure is
eliminated, the cell will no longer be activated. Organ-
isms noted as a cause of T activation include E. coli
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134 UNIT 4 Red Blood Cell Groups and HLA

TABLE 9-12 Reactions of Polyagglutinable Cells with Various Lectins

T Tn Tk Cad

ABO-compatible adult human serum ⫹ ⫹ ⫹ ⫹

Arachis hypogaea ⫹ 0 ⫹ 0

Salvia sclarea 0 ⫹ 0 0

Salvia horminum 0 ⫹ 0 ⫹

Glycine soja ⫹ ⫹ 0 ⫹

Dolichos biflorus 0 ⫹ 0 ⫹

and Vibrio cholerae as well as other bacteria and
viruses.
Testing with cord serum may allow proper
typing of the subject because newborns have not
formed the anti-T antibody, anti-A, or anti-B in the
cord serum (in appropriate samples) therefore may
detect the A or B antigen on the surface of T-activated
RBCs without anti-T interfering. In addition, testing
the person with a panel of lectins useful in identifica-
tion of suspected polyagglutination may be helpful.
A similar situation results in the exposure of a dif-
ferent antigen, Tk. Tk activation results from similar
causes and reacts in a similar manner to T activation.
Also, like T activation, Tk activation is transient.
Another type of polyagglutination is a result of
unknown causes and may occur much less frequently.
This polyagglutination is permanent and results in Tn
activation. It has not been simulated in vitro. How-
ever, Tn is destroyed by enzyme, and thus enzyme-
treated cells may be used for further testing. Tn
activation results in cells that react in a mixed-field
pattern and fail to react with the lectin from A. hy-
pogaea. Bird and Wingham showed that when prop-
erly diluted, a lectin from the plant Salvia sclarea had
specificity for Tn.6
Finally, there are several forms of inherited polyag-
glutination. The first is that resulting from the presence
of the antigen Cad. The Cad antigen (Sda) is present in
variable amounts in most people. Some people express
an extremely large amount of Cad antigen and react
with the sera of most normal people, which contain a
small amount of Cad autoantibody.
Hereditary erythroblastic multinuclearity with a
positive acidified serum test is a form of chronic
dyserythropoietic anemia. It results in increased sus-
ceptibility to agglutination and complement destruc-
tion by the small amount of cold agglutinins found in
most normal sera.
SUMMARY
Although the ABO BGS may seem complex, the level
of knowledge required for routine testing is funda-
mental. The student of immunohematology should be
aware of the complex interaction of genes and rare
causes of discrepancies that may lead to problems in
grouping patients. However, the fundamental princi-
ples of ABO grouping are simple and straightforward.
Once committed to memory, these simple principles,
along with following proper protocols for testing, will
suffice in most instances. If the world of immunohe-
matology were a book, then the ABO system would be
only the first chapter. Although the study of many
other BGSs follows, no other system is more impor-
tant to the routine practice of blood banking.

Review Questions
1. The H antigen is found in highest concentration on 2. The lectin used for detection of the H antigen is
what type of red blood cell? called:
a. group A a. Arachis hypogaea
b. group B b. Dolichos biflorus
c. group AB c. Ulex europaeus
d. group O d. Salvia sclarea

(continued)
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CHAPTER 9 The ABO Blood Group System 135

REVIEW QUESTIONS (continued)

3. Common sources of ABO discrepancies due to techni- 8. Approximately what percentage of A2 individuals have
cal error include all the following except: evidence of anti-A1 in the sera?
a. clerical mix-ups a. none
b. contaminated reagents b. 1% to 8%
c. warming of the test c. 13% to 18%
d. patients with agammaglobulinemia d. 50% or more
4. An individual must inherit which of the following to be 9. The antibody normally found in the serum of group B
classified as a group AB? individuals is:
a. the A gene a. anti-A
b. the B gene b. anti-B
c. the H gene c. anti-H
d. answers a and b d. anti-A,B
e. answers a, b, and c. 10. Approximately what percentage of adult levels of A and
5. The immunodominant sugar associated with the B antigen are present at birth?
A antigen is: a. 10%
a. L-fucose b. 25%
b. N-acetylgalactosamine c. 50%
c. D-galactose d. 100%
d. D-glucose 11. Fetal development of ABO antigens begins in the:
6. The gene that controls the presence or absence of the a. first week of fetal life
H substance in body secretions is the: b. second week of fetal life
a. H gene c. sixth week of fetal life
b. Se gene d. second trimester
c. h gene 12. ABO grouping discrepancies may occur due to which
d. B gene of the following causes?
7. Group A2 constitutes approximately what percentage a. rouleaux
of group A individuals? b. clerical error
a. 2% c. atypical antibody
b. 20% d. all of the above
c. 40%
d. 60%
e. 80%

REFERENCES 4. Yamamoto F, McNeill PD, Kominato Y, et al. Molecular

genetic analysis of the ABO blood group system: 2, cis-
AB alleles. Vox Sang. 1993; 64: 120–123.
1. Issitt PD. Applied Blood Group Serology. 3rd ed. Miami:
5. Bird GWG. Anti-T in peanuts. Vox Sang. 1964; 9: 748.
Montgomery Scientific; 1985.
6. Bird GWG, Wingham J. Haemagglutinins from Salvia.
2. Mollison PL, Englefreit CP, Contreras M. Blood Transfu-
Vox Sang. 1974; 26: 163.
sion in Clinical Medicine. 8th ed. Oxford: Blackwell Scien-
tific Publications; 1988.
3. Yamamoto F, Clausen H, White T, et al. Molecular
genetic basis of the histo-blood group ABO system.
Nature. 1990; 345: 229–233.
82043_ch09.qxd 11/11/09 6:10 PM Page 136
PROCEDURAL APPENDIX I
ROUTINE ABO GROUPING
Slide Testing
Principle
Anti-A, anti-B, and anti-A,B are prepared from the
sera of appropriate people who lack other atypical
antibodies. These reagents are used to test for the
presence of the A and B antigens on the surface of ery-
throcytes. The test is routinely performed at room
temperature and must not be heated. The manufac-
turer’s directions always must be meticulously fol-
lowed. Some manufacturers specify the use of whole
blood, whereas others specify differing concentrations
of RBCs in saline or in autologous serum or plasma.
1. Place one drop of each reagent to be tested
(anti-A, anti-B, and, when appropriate, anti-
A,B) on a clean glass slide or tile allowing
approximately a 20 ⫻ 40 mm area for each test.
A wax pencil or other method may be used to
ensure that the reagents do not become inap-
propriately mixed together.
2. To each drop of reagent from step 1, add one
drop of a well-mixed suspension of RBCs (in
saline, autologous serum, or plasma and in a
concentration recommended by the reagent
manufacturer).
3. Mix the reagents and RBC suspensions thor-
oughly using a clean wooden applicator stick
while spreading the mixture over an area ap-
proximately 20 ⫻ 40 mm.
4. Gently tilt the slide back and forth for 2 minutes
while observing for agglutination. Read and
record the results.
Interpretation
Agglutination in the presence of specific reagents
indicates the presence of the appropriate antigen on
the RBC surface. Lack of agglutination indicates a lack
of the appropriate antigen and thus a negative result.
Tube Testing
Tube testing may be used for forward and reverse
grouping of RBCs. The manufacturer’s directions must
be followed at all times.
136
Forward Grouping
1. Place one drop of the appropriate reagent
(anti-A, anti-B, or anti-A,B) in a clean and appro-
priately labeled 10 ⫻ 75-mm or 12 ⫻ 75-mm
glass test tube.
2. Add one drop of a 2% to 5% suspension of
RBCs to be tested (in saline, serum, or plasma)
to each tube. Alternatively, an equivalent num-
ber of RBCs may be transferred from a whole-
blood sample with a wooden applicator stick.
3. Mix the reagent and RBCs, and centrifuge at
100g for 15 to 20 seconds.
4. Gently resuspend the RBC buttons while
observing for agglutination and record results.
Interpretation
Agglutination with appropriate reagents indicates the
presence of that antigen on the surface of the RBCs
tested.
Reverse Grouping
Reverse grouping is performed to confirm the presence
of expected naturally occurring antibodies in the serum
or plasma. Reagent RBCs demonstrating the strongest
antigenic makeup for A and B are used (A1 and B cells).
1. Place two drops of the serum or plasma to be
tested in each of two appropriately labeled
tubes (A1 cells and B cells).
2. Place one drop of reagent A1 cells in the tube
marked “A1 cells” and one drop of reagent B cells
in the tube marked “B cells.”
3. Mix thoroughly and centrifuge at 1000g for
15 to 20 seconds.
4. Gently resuspend the RBCs while observing
for agglutination, and record results.
Interpretation
Agglutination with the appropriate reagent RBC
suspension demonstrates the presence of the appro-
priate antibody in the serum or plasma tested.
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PROCEDURAL APPENDIX II
SECRETOR STUDY
ABH Antigens in Secretion
Principle
Approximately 78% of people (those who inherit at least
one Se gene) are capable of secreting H substance in
their body secretions. If they also inherit A or B genes,
the transferases in their secretions will result in the con-
version of this H substance to A or B antigen. The pres-
ence of H, A, or B antigens in secretions may be
demonstrated by testing the saliva of an individual with
agglutination inhibition testing. The saliva is collected,
heat inactivated, and used to attempt to neutralize weak
reacting antiserum. Neutralization of this antiserum by
soluble ABH substances results in complete inhibition
of agglutination, resulting from the mixing of the anti-
sera with appropriate RBCs.
Collection of Saliva
1. Collect approximately 10 mL of saliva and
place it in an appropriately labeled test tube. To
encourage salivation, the subject may be given
a small amount of paraffin to chew.
2. Place the test tube in a boiling water bath for
10 minutes to inactivate innate salivary en-
zyme activity, which may interfere with sub-
sequent testing.
3. Centrifuge the heat-inactivated saliva for
10 minutes at high speed, and remove the clear
supernatant. Save the supernatant for further
testing.
4. If the test is not to be completed immediately,
refrigerate until testing is completed later that
day. Alternatively, the sample may be stored
frozen until needed.
Dilution of Antisera to be Tested
1. Prepare doubling dilutions of the antisera to be
tested.
2. Combine one drop of the diluted antiserum with
one drop of a 2% to 5% suspension of appropri-
ate RBCs (A cells when testing for A substance,
B cells when testing for B substance, and O cells
when testing for H substance).
3. Centrifuge at 1,000g for 15 to 20 seconds.
4. Gently resuspend and observe for agglutina-
tion. Select for further testing the dilution that
gives a 2⫹ agglutination.
Testing of Neutralized Saliva
1. Add one drop of the appropriately diluted an-
tiserum (see step 4) to an appropriately labeled
tube. Repeat for each soluble antigen that you
wish to test. A saline control tube also should
be used.
2. Add one drop of saliva to each tube from step 1.
Allow the tubes to incubate at room tempera-
ture for at least 10 minutes.
3. Add one drop of a 2% to 5% saline suspension of
appropriate indicator cells to each tube, mix, and
incubate for 60 minutes at room temperature.
4. Centrifuge at 1,000g for 15 to 20 seconds.
5. Gently resuspend the cells, observe for aggluti-
nation, and record the results.
Interpretation
See Table 9-8 for aid in interpretation of saliva testing.
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PROCEDURAL APPENDIX III

ABSORPTION AND ELUTION TESTS 9. Centrifuge the eluate at 1,000g for 10 minutes
FOR WEAK ANTIGENS to pack the RBCs and separate the saline or
6% albumin containing any eluted antibody.
Remove the supernatant, and discard the RBCs
Principle
and RBC stroma. The supernatant (eluate) will
Erythrocytes having weak A or B antigens may not be be cherry red because of the amount of RBC
directly agglutinated by anti-A, anti-B, or anti-A,B in hemolysis that has occurred.
routine forward grouping tests. However, the pres- 10. Test the eluate and final wash (control) from step
ence of A or B antigens on the erythrocyte surface can 5 with at least three examples of group O, group
be proven by the adsorption and subsequent elution A, and group B RBCs by placing two drops of
of one or more of these antibodies. the eluate or control in an appropriately labeled
tube, along with one drop of a 2% to 5% saline
1. Wash 1 mL of the RBCs to be tested at least three
suspension of the RBCs to be tested.
times with a large volume of saline. Remove the
11. Test the eluate and control at 4⬚C, 37⬚C, and in
supernatant from the last wash, and save for a
the AHG phase.
control.
2. Add an equal volume (1 mL) of appropriate
reagent (anti-A, anti-B, or anti-A,B). Interpretation
3. Mix the RBCs and antiserum, and incubate for
The final wash (control) sample should show no agglu-
1 hour at 4⬚C.
tination at any phase of testing, indicating that no anti-
4. Centrifuge the mixture for 10 minutes at 1,000g
body remained in the supernatant of the final wash.
to ensure that the RBCs and antiserum are well
Agglutination of group A cells would indicate that
separated.
there is a weakened form of the A antigen on the sur-
5. Remove the antiserum and discard. Wash the
face of the RBCs tested. Agglutination of group B cells
RBCs at least five times with large volumes of
would indicate that there is a weakened form of the B
saline (at least 10 ⫻ RBC volume). Save an
antigen on the surface of the RBCs tested. It is impor-
aliquot of the final wash, and label as a control.
tant that no reactivity be demonstrated against group
6. Add an equal volume of saline or 6% albumin
O cells. Reactivity with O cells would indicate the pres-
to the washed, packed RBCs and mix well.
ence of an atypical antibody that would not support
7. Elute the adsorbed antibody (if any) by placing
the determination of a weak subgroup of A or B.
the tube in a 56⬚C water bath for 10 minutes.
8. Mix the sample several times during the elu-
tion procedure.

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