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G. Gessoni
Minor Blood Group Systems
• Diego system. Antigens: The Diego blood group system
consists of two pairs of independent antithetic antigens,
called Dia/Dib Wra/Wrb. The system also contains many
low frequency antigens. The antigens of the Diego system
are encoded by alleles located on chromosome 17 and are
expressed on the AE-1 protein which appears to have the
role of anion exchanger across the erythrocyte membrane.
The Dia antigen is present almost exclusively in populations of Asian origin and in North and South American
natives. Antibodies: These are usually immune antibodies
(with the exception of anti-Wra), which can be detected
even in the absence of exposure to nonself red blood cells)
and can be clinically signicant. The anti-Dia antibody
has been associated with PTH and HDFN.The anti-Dib
antibody is rare, but it too can generate HDFN.The antiWra antibody is quite common and, although rarely, can
be associated with PTH and HDFN.
• Cartwright system. Antigens: The Yt (Cartwright) system
consists of two antigens, Yta and Ytb, which are encoded by
a pair of gene alleles located on chromosome 7. The Yt antigens are found on the acetylcholinesterase of red blood cells.
Antibodies: These are immune allo- antibodies of modest
clinical signicance, as they have never been implicated in
cases of PTH or HDFN, although rare cases have been
reported in which an anti-Yta has generated an accelerated
destruction of Yta positive red blood cells transfused.
• Xg system. Xga Antigen: Discovered in 1962, it is an anti-
gen encoded by an on the X chromosome. Anti-Xga
Antibody: It is a rare immune antibody, which reacts only
to the antiglobulin test and is not considered clinically
signicant.
• Scianna system. Antigens: This erythrocyte blood group
is composed of ve antigens, named Sc1, Sc2, Sc3, Rd,
and STAR, expressed on the cell adhesion protein
ERMAP3. Sc1 is a high frequency antigen, while Sc2
occurs very rarely. The gene that encodes the Scianna
antigens is located on chromosome 1. Antibodies: These
are antibodies of rare nding and of modest clinical signicance, not being associated with PTH and HDFN.
• Dombrock system. The Antigens: Initially, this system
consisted of the Doa and Dob antigens. Currently three
other named high-frequency antigens (Gya, Hy, and Joa)
have been associated with this erythrocyte blood group
system. Antibodies: Anti-Doa and anti-Dob are rare antibodies, sometimes identied in sera containing antibody
mixtures. They are not associated with HDFN, but PTH
sustained by these alloantibodies are described.
• Chido/Rodgers system. Antigens: The system is com-
posed of two high frequency antigens called Chido (Ch)
and Rodgers (Rg), which are not part of red cell membrane but are present on the C4 fraction of the comple-
ment and are therefore only subsequently adsorbed by the
erythrocyte membrane. The gene is located on chromosome 6, in the region of the major histocompatibility
complex (class III molecules). Antibodies: These are antibodies substantially devoid of clinical signicance.
• Gerbich system. The Antigens: The Gerbich system
includes eight antigens, three of which are high frequency
(Ge2, Ge3 and Ge4) and ve are low frequency (Wb, Lsa,
Ana, Dha, and GEIS). These antigens are expressed on
Glycophorin C and Glycophorin D. Antibodies: Only
anti-Ge2 antibodies may have any clinical signicance,
having been associated with rare cases of HDFN.
• Colton system. The Antigens: The Colton system consists
of a high frequency antigen (Coa) and a low frequency
antigen (Cob), as well as an antigen considered to be the
product of the Coa gene and the Cob gene, called Co3.
The genes encoding the antigens of the Colton erythrocyte blood group system are located on chromosome 7
and expressed on an aquaporin. Antibodies: The anti-Coa
antibody is considered clinically signicant, since it can
generate PTH and HDFN.
• Cromer system. Antigens: The Cromer system appears to
consist of 10 high frequency antigens and three low frequency antigens. The antigens of this blood-group system
are associated with the complement regulatory protein
called DAF, (decay-accelerating factor), encoded by the
DAF gene on chromosome 1. Antibodies: These are
extremely rare immune antibodies of uncertain clinical
signicance.
• Knops system. Antigens: The antigens belonging to the
Knops erythrocyte blood group system (Kna, Knb, McCa,
McCb, Sla, Yka, Vil, and Sl3) are located on the receptor
(CR1) of the C3b / C4b factors and encoded by a gene
located on chromosome 1. Antibodies: Knops antibodies
usually exhibit weak reactivity and varying intensity in
serum antiglobulin tests. They are not considered to be
clinically signicant.
• Indian system. The antigens of the Indian system include
a high-frequency antigen (Ina) and a low-frequency antigen (Inb). They are associated with a widely spread adhesion molecule (CD44).
• System Ok. The Ok system consists of a single high frequency antigen, called Oka. Ok (a–) subjects are extremely
rare and are exclusively Japanese. It is possible to generate an anti-Oka antibody, which is able to decrease the
survival of transfused red blood cells.
• Raph system. The Raph system consists of a single antigen, MER2, present in 90% of subjects.
• John Milton Hagen system. The John Milton Hagen
(JMH) antigen is located on the CD108 glycoprotein.
• Gill system. In this system, a single high frequency antigen is represented, called GIL, localized on aquaporin 3.
The antibody is not considered clinically signicant.
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• Red blood cell collections. In addition to the erythrocyte
blood group systems already described previously, families or collections (dened collections) of antigens have
been identied which, although having shared characteristics, do not meet the ISBT standards in order to be catalogued in an erythrocyte blood group system. These
antigens were grouped in the Cost, Er and Vel collections.
The Cost collection consists of two named antigens, Csa
present in 98% of the population and Csb which has a
frequency of about 34%. The Er collection consists of two
antigens. It was present in 99% of the population and Erb
which has a frequency of less than 1%. The VEL
Collection includes two high frequency antigens called
VEL and ABTI.Anti-VEL antibodies are frequently IgM,
can x complement, have been associated with PTH
reactions.
• “Public” antigens and “private” antigens. With the term
“public” antigens they are dened high frequency antigens (present in over 99.9% of the population), while with
the term “private” antigens they are dened low frequency
antigens (present in less than 0.5% of the population).
Among the public antigens those named Lan, Jra, AnWj,
Ata, and Sda should be mentioned, while among the private antigens those named: By, Lia, Bi, Milne, Bxa, Ola,
Chra, Pta, HJK, RASM, etc.
Platelet andGranulocyte Antigens
andAntibodies
On the surface of granulocytes and platelets, erythrocyte
antigens, antigens of the HLA system and antigens of platelets and granulocytes can be expressed. This short dissertation will focus on this third type of antigens.
Platelet immunohematology From a clinical point of view,
platelet antigens are important as they are implicated in neonatal alloimmune thrombocytopenia (FNAIT) and posttransfusion purpura (PTP). Almost always these clinical
manifestations are related to the production of an alloantibody directed against the HPA-1a antigen. Forms of autoimmune thrombocytopenia are also described, in which
autoantibodies can be directed against platelet-specic
antigens.
Although numerous platelet-specic antigens have been
dened, for some of them the platelet-specic denomination is not fully correct, being evidenced in many other
cells, especially of endothelial derivation. Of the numerous glycoproteins expressed on the platelet membrane,
only some (GP Ia, GP Ibα, GP Ibβ, GP IIb, GP IIIa, and
CD109) show polymorphisms that make them
immunogenic.
Granulocyte immunohematology
Granulocyte antigens
and related allo-antibodies can be implicated in several clinical syndromes, the two most relevant being neonatal
immune neutropenia (FNAIN) and transfusion-related acute
lung injury (TRALI). Antibodies directed to leukocyte antigens may also be the cause of the most common complication of blood component transfusion, namely the
nonhemolytic posttransfusion febrile reaction. As reported
in Table16.2, seven proper granulocyte antigens have been
described. The HNA-1a antigen and its antithetic HNA-1b
antigen are expressed on CD16 (FcγRIIIb), both have been
associated with TRALI and NAIN.The HNA-2a antigen is
found on CD177, and is also associated with TRALI and
NAIN. Further antigens have also been described (5b or
HNA-3a, MARTa or HNA-4a and ONDa or HNA-5a)
which, although present on granulocytes, are also identiable on other cells. They are therefore not granulocyte specic. In neonatal autoimmune neutropenia, auto antibodies
are directed towards HNA-1a or HNA-1b antigens in more
than half of cases (Tables 16.3 and 16.4).
HLA System
The HLA system includes a complex family of genes and
their protein products that contribute to the recognition of
self and nonself, to the immune responses to antigenic stimuli, and to the coordination of cellular and humoral immunity. HLA major histocompatibility consist in a series of
35–40 closely linked genes physically grouped into three
regions (class I, class II, and class III regions) located on the
short arm of chromosome 6. Class I region encodes genes for
the classic transplantation molecules HLA-A, HLA-B, and
HLA-C. Class II region encodes genes for the molecules
HLA-DR, HLA-DP, and HLA-DQ.Class III region encodes
structurally and functionally diverse molecules, including
complement factors and tumour necrosis factor.
The most important characteristic of HLA genes is that
they are highly polymorphic, and several alleles exist at each
locus. Each person has two alleles for each locus. Both
alleles of a locus are expressed co-dominantly.
Class I molecules are found on the surface of platelets and
of all nucleated cells of the body. Mature red cells surface
usually lack HLA antigens, but nucleate immature erythroid
cells express them. MHC Class II antigens are restricted to a
few immunocompetent cell types such as B lymphocytes,
macrophages, and dendritic cells.
Expression of different HLA antigens has signicant
repercussions in human pathology. It has been determined
that HLA antigens are associated with disease susceptibility:
i.e., narcolepsy is associate with DR2 allele and B27 allele is
associate with ankylosis spondylitis. The HLA system is of
great importance, second only to the ABO, in inuencing the
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G. Gessoni
Table 16.2
Test results with the ve principal Rh blood typing reagents.
Anti-D Anti-C Anti-E Anti-c Anti-e Antigens present
POS POS NEG POS POS D, C, c, e CcDee R1r
POS POS NEG NEG POS D, C, e CCDee R1R1
POS POS POS POS POS D,C,c,E,e CcDEe R1R2
POS NEG NEG POS POS D, c, e ccDee RoRo / Ror
POS NEG POS POS POS D, c, E, e ccDEe R2r
POS NEG POS POS NEG D, c, E ccDEE R2R2
POS POS POS NEG POS D,C, E, e CCDEe R1Rz
POS POS POS POS NEG D,C, c, E CcDEE R2Rz
POS POS POS NEG NEG D, C, E CCDDEE RzRz
NEG NEG NEG POS POS c, e Ccdee rr
NEG POS NEG POS POS C, c, e Ccdee r’r
NEG NEG POS POS POS c, E, e ccdEe r”r
NEG POS POS POS POS C, c, E, e CcdEe r’r”
Commercial anti-D, anti-C, anti-E, anti-c and anti-e antisera are used for the determination of the Rh phenotype. The set of antigens detected on a
subject’s red blood cells therefore constitutes its Rh phenotype. In this table columns from one to ve report the pattern of reactivity, column six
report phenotype, column seven the common denomination and column eight the most probable genothype. D negative subjects lack the RHD gene
that encodes the D antigen, or have a nonfunctional RHD gene. Most of the negative D subjects are homozygous for the RHce allele, which represents the gene encoding the antigens c and e. Less frequently they may have RHCe or RHcE alleles, which code for C and e or for c and E,
respectively. The RHCE gene, which produces the C and E antigens, is very rare in D negative individuals. The D genotype of D positive persons
cannot be determined with serological tests, as molecular biology techniques must be used
Table 16.3
System Antigen Frequency Gene Localization Associated diseases
HPA-01 HPA-1a, HPA-1b
HPA-02 HPA-2a, HPA-2b
HPA-03 HPA-3a, HPA-3b
HPA-04 HPA-4a, HPA-4b
HPA-05 HPA-5a, HPA-5b
HPA-06 HPA-6bw <1% ITGB3 GP IIIa NAIT,
HPA-07 HPA-7bw <1% ITGB3 GP IIa NAIT,
HPA-08 HPA-8bw <1% ITGB3 GP IIIa NAIT,
HPA-09 HPA-9bw <1% ITGA2B GP IIb NAIT,
HPA-10 HPA-10bw <1% ITGB3 GP IIIa NAIT,
HPA-11 HPA-11bw <1% ITGB3 GP III a NAIT,
HPA-12 HPA-12bw <1% GOIBB GP Ib NAIT,
HPA-13 HPA-13bw <1% ITGA2 GP Ia NAIT,
HPA-14 HPA-14bw <1% ITGB3 GP IIIa NAIT,
HPA-15 HPA-15a, HPA-15b
HPA-16 HPA-16bw <1% ITGB3 GP IIIa NAIT,
This table indicates the name of the blood group system in the rst column while the second column shows the name of the individual antigens,
and their frequency is indicated in the third column. The name of the gene alleles is reported in the fourth column, while their chromosomal location is reported in the fth column. The possible associations with the pathology are indicated in the last column., PTP: posttransfusion purpura,
NAIT: neonatal immune thrombocytopenia
Principal RH gene and antigens encoded
Platelet antigens
HPA-1 (a+b−) 72%, (a+b+) 28%, (a−b+) <1%
HPA-2 (a+b−) 85%, (a+b+) 15%, (a−b+) <1%
HPA-3 (a+b−) 37%, (a+b+) 48%, (a−b+) 15%
HPA-4 (a+b−) 99%, (a+b+) <1%, (a−b+) <1%
HPA-5 (a+b−) 80%, (a+b+) 19%, (a−b+) 1%
HPA-15 (a+b−) 35%, (a+b+) 42%, (a−b+) 23%
Conventional
name Probable phenotype
ITGB3 GPIIIa NAIT, PTP
GPIBA GP Ib NAIT,
ITGA2B GP IIb NAIT, PTP
ITGB3 GP IIIa NAIT, PTP
ITGA2 GP Ia NAIT, PTP
CD109 CD109 NAIT, PTP
Table 16.4
System Antigen Frequency Gene Localization Associated diseases
NA NA1
SH HNA-1c 5% caucasici, 25% neri SH CD16
NB HNA-2a 97% NB1 CD177
5 HNA-3a <1% 5b GP 95kD
MART HNA-4a 99% MART CD11a
OND HNA-5a 99% OND CD11b
This table indicates the name of the blood group system in the rst column while the second column shows the name of the individual antigens,
and their frequency is indicated in the third column. The name of the gene alleles is reported in the fourth column, while their chromosomal location is reported in the fth column. The possible associations with the pathology are indicated in the last column. TRALI transfusion relatedLung
Injury, NAIN neonatal immune neutropenia
Granulocytes antigens
NA2
HNA-1a 45%
HNA-1b 85%
NA1
NA2
CD16 TRALI, NAIN
TRALI, NAIN
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long-term survival of transplanted solid organs (kidney, liver,
lug, heart) and is of paramount signicance in hematopoietic
progenitor cell (HPC) transplantation. HLA antigens and
antibodies are also important in such complications of transfusion therapy as platelet transfusion refractoriness,
transfusion- related acute lung injury (TRALI), neonatal alloimmune neutropenia NAIN), neonatal alloimmune thrombocytopenia (NAIT), and posttransplant and post transfusion
graft-vs-host disease (GVHD).
Analytical Aspects ofErythrocyte
Immunohematology
It is strongly recommended that an immunohematology laboratory can have access to different methods to face complex
clinical cases overcoming the intrinsic limitations of a single
method. For example, in our laboratory, which is in a provincial reference hospital, we routinely have three methods for
the serological study of antigens and antibodies: tube tests,
gel tests, and solid-phase tests. We also have a molecular
biology laboratory using a micro-arrays-based assay for the
genotyping of blood group antigens.
As previously reported, demonstration of red cell antigenantibody reactions are key to immunohematology. The combination of antibody with antigen may produce a variety of
observable results: agglutination, hemolysis, and precipitation. Agglutination is the antibody-mediated clumping of
particles that express antigen on their surface, and it is the
endpoint for most tests involving red cells and blood group
antibodies. Agglutination is a reversible chemical reaction
that occurs in two stages: (1) sensitization, the attachment of
antibody to antigen on the red cell membrane; (2) formation
of bridges between the sensitized red cells to form the agglutinate. Agglutination is also affected by physical and chemical conditions such as temperature, pH, ionic strength,
relative antigen-to-antibody concentrations, time of incubation. Usually in routine immune hematology laboratory were
in use some enhancement medium for antibody detection
such as albumin, Polyethylene Glycol (PEG), low-ionicstrength solution (LISS). Proteolytic enzymes reduce the red
cell negative surface charge cleaving sialic acid molecules
from polysaccharide chains. Any mechanism that reduces
the net charge should enhance red cell agglutination.
Moreover, while enhancing agglutination by some antibodies, enzymes destroy certain red cell antigens, notably M, N,
S, Fya, and Fyb.The proteolytic enzymes used most often in
immunohematology laboratories are bromelin, cin, papain,
and trypsin. Figure16.4 shows solid-phase tests.
Determination oftheABO Group
In the direct determination of the ABO group, the patient’s
RBCs are tested against known antisera. The presence of the
antigens (agglutinogens) present on the surface of the red
cells is detected by the agglutination that occurs after contact
with the antisera. A positive reaction (agglutination) indicates
Fig. 16.4 Solid phase tests. In this gure are reported, on the left, the picture of a 96 wells microplate and, on the right, the results interpretation.
A “button” in the bottom of the wells represent a positive reaction while a diffuse pattern represents a negative result
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that the corresponding antigen is present on the surface of
the red cells under examination. In the indirect determination
of the ABO group, the patient’s serum (or plasma) is tested
against red blood cells of a known group. The patient’s serum
contains natural antibodies (agglutinins IgM) against antigens that are not present on the surface of their own red cells.
A positive reaction (agglutination) indicates that antibodies
are present in the serum, and therefore the relative antigen is
missing on the surface of the red cells. Figure16.5 shows
direct and indirect ABO group analysis with RHD type
determination.
In rare cases, subjects who do not react with anti-A, anti B and anti-A, B sera strongly agglutinate the red cells of
group O.If this agglutination is not due to the presence of
cold agglutinins (in this case it disappears completely after
5minutes at 37°C), it could be the so-called Bombay phenotype (hh), an exceptional nding.
Weak variants of A and B are known. The best known A2
reacts well (4+) with anti-A, B sera and weaker (2+) with
anti-A sera, does not react with anti-A lectin. A1 but reacts
well with anti-H lectin. Natural anti-B antibodies are always
present in the serum, and in 2–3% of cases anti-A antibodies
may be present which react with group A1 red cells but not
with group A2 ones. The variants called A3 and B3 have the
characteristic of reacting very weakly with the anti-A and
anti-B sera (respectively), while they react well with the anti A, B serum. The expected natural antibodies are present in
the serum of these subjects.
From these observations it follows that the ABO group
must be determined using always and, on all samples, complete anti-A, anti-B and anti-A, B sera. The search for “natural” antibodies in the serum must be carried out using at least
group A1 and B red blood cells.
There may be discrepancies between direct and indirect
determination of the ABO group, mostly attributable to technical causes or anomalies present in the sample.
Technical causes: False negatives may be attributable to
failure to add a reagent, errors in interpretation or registration
(hemolysis) of the results, incorrect relationship between red
blood cells and antisera, too low working temperature. False
positives can be due to excessive centrifugation, use of contaminated reagents, misinterpretation or recording of results.
Sample anomalies: Interferences with the direct test can
be observed in patients recently transfused with nonhomo
group red blood cells (i.e., red blood cells O to a recipient A)
or with weak subgroups of A or B, in case of poly agglutination of the red blood cells, presence of Wharton’s gelatine in
umbilical cord) or high concentrations of abnormal proteins
(myeloma), cold agglutinins. Interference with the indirect
test can be observed in specimens with clots, presence of
allo antibodies to erythrocytes, auto-antibodies to erythrocytes, immunosuppressed patients, children under 6 months.
Transplanted with ABO-incompatible allogenic marrow,
recent transfusions with nonhomo group plasma. Figure16.6
shows direct ABO determination with evidence of a double
red cells population.
Search and titration of immune or natural anti-A and anti B antibodies is done primarily to support ABO incompatible
solid organ and hematopoietic stem cells transplant programs. The titration of anti-A and anti-B is carried out by
diluting the serum to the doubling in saline and evaluating
the reaction after immediate centrifugation. The last dilution
of the serum in which hemolysis and / or agglutination is
highlighted determines the titer of the hemolysins or anti-A
and anti-B agglutinins. Subjects with high titer (>1/128) of
anti-A and anti-B have antibodies of an immune nature
alongside the natural ones. Search for anti-A and anti-B
immune antibodies can be carried out with the same method
after denaturation of the IgM with 2-mercaptoethanol.
Alternatively, two doubling dilutions of the serum can be
prepared, one incubated at 37°C and the other at 4°C.The
difference in the titer (always greater in that incubated at
37°C) will be attributable to the presence of IgG.Group O
subjects naturally have IgG class anti-AB.
Determination ofType D andRh Phenotype
In direct determination of Rh type and Rh phenotype, the
patient’s red cells are tested against known antisera. The
presence of the antigens (agglutinogens) on the surface of
the red cells is subtended by the agglutination that occurs
after contact with the antisera. A positive reaction (agglutination) indicates that the antigen corresponding to the
surface of the red cells under examination is present. As
there are no natural antibodies, the indirect test is not used.
Search for weak D Pre-prepared columnsare used for the
execution of the Coombs test in Liss. A 1% suspension of the
patient’s red blood cells is prepared, and a drop of the suspension is placed in the incubation chamber of a microtube
of the coupon. Then 25 μL of an IgG monoclonal anti-D
serum or a serum blend in which an IgG component is present is added. It is incubated for 10–15minutes at 37°C, centrifuged for 10minutes at 900rpm on a dedicated centrifuge
and visually assessed the presence of agglutination against a
light source. In the latter circumstance, the patient will be
classied as Du (Fig.16.7).
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Fig. 16.5 Determination of the direct and ndirect ABO Group, with
RHD type determination; some examples. The rst sample reacts with
the anti-A and anti-AB sera, does not react with the anti-B serum, it
reacts with the anti-D sera (both Dvi− and Dvi+), plasma reacts with
group B but not group A red blood cells, therefore the subject is A D
positive. The second sample does not react with the anti-A serum, but
reacts with the anti-B and anti-A sera, B, reacts with the anti-D sera
(both Dvi− and Dvi+), plasma reacts with group A but not group B red
blood cells. It is, therefore, a B D positive. The third sample reacts with
the anti-A, anti-B and anti-A,B sera, does not reacts with the anti-D sera
(both Dvi− and Dvi+), plasma does not react with group A and group B
red blood cells. It is therefore a subject of AB D negative group. The
fourth sample does not react with the anti-A, anti-B, anti-A,B sera, such
as with the anti-D sera (both Dvi− and Dvi+), plasma reacts with group
A and group B red blood cells. It is, therefore, a subject of group O D
negative
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Study oftheAntigens Belonging toOther
Erythrocyte Blood Group Systems
The search for the antigenic specicities of the different
erythrocyte blood group systems is carried out using specic
commercial antiserum. The methodology varies according to
the technology used in the individual laboratories. In any
case, the presence of agglutination indicates a positive reaction and therefore underlies the presence of the antigen.
Figure 16.8 describes research for MN, Ss, Kidd, and
Lutheran blood groups antigens.
The Search forAnti-erythrocyte Antibodies
Fig. 16.6 Direct ABO determination with evidence of a double red
cells population. This is a sample from a B group patient that in emergency room received two units of O group packed red cells. In the anti B column are present two RBC population: the patient’s B RBC (top)
and the transfused O RBC (bottom) of the tube
Irregular anti erythrocyte alloantibodies are dened as antibodies directed against erythrocyte antigens, except for natural anti-A and anti-B antibodies. Immunization against
erythrocyte blood group antigens can usually be traced back
Fig. 16.7 Sample with a variant D.The image above shows the determination of the direct and indirect ABO group D type. The red blood
cells of the sample under examination do not react with the anti-A, anti B, and anti-AB sera, while the plasma of the test sample reacts strongly
with red blood cell tests of groups A and B.The patient is therefore of
group O.As regards the determination of D type, negativity with the
rst antiserum (DVI−) and a reactivity (+++−) with the second antiserum (DVI+), suggest the presence of a D variant. Further analysis using
genotyping study are highly recommended
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Fig. 16.8 Research for MN, Ss, Kidd and Lutheran blood groups
antingens. This sample is positive for M (rst column), negative for N
(second column), positive for S and s (third and fourth column), posi-
Table 16.5
Antibody Ig class
Anti-H IgM Ye s Yes – No
Anti-A IgM
Anti-B IgM
Anti-A,B IgG
Anti-A1 IgG Yes Rare – No
Anti-D IgG Few Ye s Ye s Enhance No No Few Severe
Anti-c IgG Few Yes Yes Enhance No No Few Severe
Anti-C IgG Few Ye s Ye s Enhance No No Few Severe
Anti-E IgG
Anti-e IgG Few Yes Yes Enhance No No No Mild to
Anti-Cw IgG
This Table reports the main characteristics of antibodies directed against erythrocyte blood groups antigen of ABO and RHD antigens. The rst
column report antibody specicity, the second the Ig class, the third the presence of reactivity in NaCl at +22°C, the fourth the presence of reactivity al +37°C, the fth the presence of reactivity by using a Coombs antiserum, the sixth the sensitivity to enzymes, the seventh the ability in
complement activation with invitro hemolysis (eight column), the ninth and the eleventh columns report data about antibody’s signicance: posttransfusion hemolysis and newborn hemolytic disease
Serological characteristics of allo-antibodies directed towards erythrocyte blood groups antigens of ABO and RHD systems
Reactivity in NaCl
at 22°C
Yes Ye s – No
(IgG*)
Yes Ye s – No
(IgG*)
Yes Ye s – No
(IgM*)
Few Yes Ye s Enhance No No No Mild to
(IgM*)
Few Yes Ye s Enhance
(IgM*)
Reactivity al
37°C
Reactivity in
AHG Enzymes
tive for Jka and Jkb (fth and sixth column), negative for Lua (seventh
column), and positive for Lub (eighth column). Phenotype of this subjects was M+N−, S+s+, Jka+Jkab+, Lua-Lua+
Complement
binding
Yes Yes Yes Mild
change
Yes Yes Yes Mild
change
Yes Yes Yes Mild
change
Yes Yes Yes Mild to
change
Rare Rare No No
change
No No No Mild to
In Vitro
hemolysis PTH NHD
moderate
moderate
moderate
moderate
to exposure to nonself red blood cells, such as pregnancy or
previous transfusions. In some rare cases, however, no exposure to nonself red blood cells is identiable. If an antierythrocyte alloantibody is discovered, it is important to
determine its specicity, titer, and to evaluate its clinical signicance. An anti-erythrocyte alloantibody is dened clini-
cally signicant when it can generate a PTH (or is able to
signicantly reduce the survival of transfused red cells), or
when it is implicated in cases of hemolytic diseaseof the foetus and neonate. Usually, the evaluation of the clinical signicance of an anti-erythrocyte antibody is carried out on the
basis of literature data, as reported in Tables 16.5 and 16.6.
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Table 16.6 Serological characteristics of allo-antibodies directed towards erythrocyte blood groups antigens other than ABO and RHD
Antibody Ig class
Anti-K IgG, (IgM*) Some Some Most No
Anti-k IgG Few Few Most No
Anti-Kpa IgG Some Some Most No
Anti- Kpb IgG Few Few Most No
Anti-Jsa IgG Few Few Most No
Anti-Jsb IgG No No Most No
Anti-M IgM, (IgG*) Most Few Few Destroy No No Few Mild to Severe
Anti-N IgM Most Few Few Destroy No No Rare Moderate
Anti-S IgG Few Some Most Variable Some No Ye s Mild
Anti-s IgG Few Few Most Variable Rare No Ye s Mild to severe
Anti-U IgG Rare Some Most No
Anti-Jka IgG Few Few Most Enhance Yes Some Yes Mild
Anti-Jkb IgG Few Few Most Enhance Yes Some Yes Mild
Anti-Fya IgG Rare Rare Most Destroy Rare No Ye s Mild to severe
Anti-Fyb IgG Rare Rare Most Destroy Rare No Yes Mild
Anti-Lua IgM, (IgG*) Most Few Few Variable Some No No Mild
Anti-Lub
Anti-Lea IgM Most Few Few No
Anti-Leb IgM Most Few Few No
Anti-I IgM Most Few Few Enhance Most Few Rare No
Anti-i IgM Most Few Few Enhance Most Few No Mild
Anti-P1 IgM Most Some Rare Enhance Rare Rare No No
This Table reports the main characteristics of antibodies directed against erythrocyte blood groups antigen other than ABO and RHD antigens. The
rst column report antibody specicity, the second the Ig class, the third the presence of reactivity in NaCl at +22°C, the fourth the presence of
reactivity al +37°C, the fth the presence of reactivity by using a Coombs antiserum, the sixth the sensitivity to enzymes, the seventh the ability
in complement activation with invitro hemolysis (eight column), the nineth and the eleventh columns report data about antibody’s signicance:
posttransfusion hemolysis and newborn hemolytic disease
IgG Few Few Most Variable Some No No Mild
Reactivity in
NaCl at 22°C
Reactivity al
37°C
Reactivity in
AHG Enzymes
change
change
change
change
change
change
change
change
change
Complement
binding
Rare No Ye s Mild to severe
No No Yes Mild
No No Yes Mild
No No Yes Moderate
No No Yes Moderate
No No Yes Mild
No No Yes Mild to severe
Most Few No No
Most Few No No
In vitro
hemolysis PTH HDFN
G. Gessoni
Indirect Antiglobulin Test (Indirect Coombs
Test)
The indirect antiglobulin test (IAT) is used to detect the presence of serum antibodies directed to erythrocyte blood group
antigens, there must be between 100 and 200 IgG or C3 molecules on the cell to obtain a positive reaction.
The presence of these antibodies is identied by reacting
(in vitro) the test serum with suitably selected red blood
cells. The reaction is revealed by using a Coombs serum. If
the indirect Coombs’ is positive, titration of the antibody
and differentiation between IgG or IgM antibodies must
always be carried out. It is also necessary to identify the
erythrocyte blood group antigen towards which the antibody is directed. For the titration of the antibody a scalar
dilution to doubling the serum in physiological is prepared.
A IATis then prepared from each dilution, using a polyspecic antiglobulin serum. The titer of the antibody is indicated by the highest dilution at which agglutination of the
red cells that have given a positive reaction to the IATstill
occurs.
To proceed with the identication of the specicity of an
antibody, special RBC panels are used. If maternal-foetal
incompatibility due to ABO incompatibility is studied, a
panel consisting of 4 red blood cells of groups A1, A2, B and
O is used. a panel consisting of group O polyantigenic red
cells characterized by different phenotypes is used.
Figure16.9 shows screening for Indirect Antiglobulin Test
using a three cells panel.
This screening is positive for cells 1 and 2. An antibody
identication should be performed using an extended panel
of fteen group O polyantigenic red cells.
16 Immunohematology
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Fig. 16.9 Screening for Indirect antiglobulin test using a three cells
panel. The gure above shows a screening search for anti-erythrocyte
antibodies carried out using a panel of red blood cells test polyantigenic
group O.This screening is positive for cells 1 and 2. Antibody specicityidenticationperformed using a more extensivepanelisreported in
Fig.16.10
Figure 16.10 describes characterization of an alloantibody mixing using an extended identication panel
untreated and treated with enzymes and Fig.16.11 antibody
titration.
After reporting the reactivity pattern on the appropriate
sheet, it is compared with the results reported on the accompanying sheet of the panel (identigram) (Fig.16.12), excluding the antigens based on the negative results. It is good
practice, after presumptively identifying an alloantibody
directed towards a specic erythrocyte antigen, to demonstrate its absence on the patient’s red cells.
Interpreting Results
Antibody screening results are interpreted as positive or negative based on the presence or absence of reactivity.
Interpretation of panel results can be a more complex process because panel results generally will include a mix of
both positive and negative results at different phases of testing, with different reactivity score. Moreover, patient’s red
cell phenotype also plays roles in the nal interpretation and
in differentiation between auto and allo antibodies.
Both positive and negative reactions are important in
antibody identication: positive reactions (considering
phase and strength of reactivity) can suggest certain specicities. Positive reactions also can be compared to the anti-
215
gen patterns expressed by the panel cells to help assign
specicity. Single alloantibodies usually show denite positive and negative reactions that create a clear-cut pattern
with antigen- positive and -negative reagent red cell samples.
Negative reactions are important in antibody identication
because they allow tentative exclusion of antibodies to antigens expressed on the nonreactive cells. Exclusion (crossing-out) of antibodies is a widely used rst approach to the
interpretation of panel results. Once results have been
recorded on the worksheet, the antigen prole of the rst
nonreactive cell is examined. If an antigen is present on the
cell and the serum did not react with the cell, the presence
of the corresponding antibody may be, at least tentatively
ruled out. After all antigens present on that cell have been
crossed off, interpretation proceeds with the other nonreactive cells and additional specicities are excluded. In most
cases, this process will leave a group of antibodies that still
have not been excluded. Next, the cells reactive with the
serum are evaluated. The pattern of reactivity for each non
excluded specicity is compared to the pattern of reactivity
obtained with the test serum. If there is a pattern that matches
exactly, that is most likely the specicity of the antibody in
the serum. However, if there are remaining specicities that
have not been excluded, additional testing may be needed to
eliminate remaining possibilities and to conrm the specicity identied.
For example, patient reported in Figs.16.9 and 16.10 is
group O and has the CCDee Kk, control negative, IAT positive. Because negativity od autologous control, we can
exclude the presence of antibodies present on the red blood
cells in the patient: D, C, e, K, and k.
Considering the negative cells in the panel not treated
with enzymes: cells 6, 11 and 12; we can provisionally
exclude the antigens c, Cw, Kpa, Fya, Fyb, Jka, Jkb, N, S, s,
Lua, Lub, Lea, P1, N, S, s, and Xg. On the basis of the analysis of this panel it is not possible to exclude antibodies
directed to the antigens E, Jsa, Lea, M, lua, and Coa. It was
therefore considered, also considering the differentiated
reactivity scores, to evaluate a second extended panel of
polyantigenic red blood cells of group O treated with
enzymes. The treatment with enzymes eliminates the reactivity for cells 1, 2, 3, 7, 8, 10, 14, and 15 while the reactivity
for cells 4, 5, and 13 remain. This differentiated reactivity
allows us to hypothesize the presence of an antibody directed
towards an antigen sensitive to the treatment with enzymes
that according to the reactivity panel we can identify with an
anti-M associated with an antibody directed towards an antigen resistant to the enzymatic treatment that we can identify
with an anti-E.
The presence of these allo antibodies is in agreement with
the patient’s phenotyping.
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