ABO BLOOD GROUP SYSTEM  These antibodies are naturally occurring because they are produced without any

exposure to RBCs.
 Marked the beginning of the concept of “individual uniqueness” defined by RBC  IgM activates complement and react at RT or colder.
antigens (Ag) in the RBC membrane.
 The most important of all blood group system in which individuals predictably have
antibodies in their serum to antigens that are absent on the RBC membrane (occurs Blood Group RBC Antigen Serum Antibody
without any exposure to RBCs by transfusion or pregnancy) A A Anti – B
 Only blood group that has naturally occurring antibodies B B Anti – A
 Transfusion of an incompatible ABO type can result in the almost immediate lysis of
AB A, B none
donor RBCs (due to presence of antibodies)
 Simplest but the most crucial test Anti – A, Anti – B,
O none
Anti A, B
HISTORY
 Discovered by Dr. Karl Landsteiner (University of Vienna) in 1901, A. Decastrello Production of Antibodies
and A. Sturli identified the 4th blood group (AB) in 1902.  Initiated at birth but titers are generally low for detection until the individual is 3-6
 Bernstein postulated the experience of 3 allelic genes (A, B and C) in 1924. months of age.
 Thompson proposed a 4-allele theory inheritance, encompassing the 4 allelic genes o Only forward grouping is done. Reverse grouping cannot be done due to
(A1, A2, B and O). low titers.
o Result of serum from 3 – 6 months is invalid
o ABO FORWARD and REVERSE grouping tests are required to be o Most antibodies found in cord blood serum are of maternal origin.
performed on ALL donors and patients. o Do not use cord blood for reverse grouping because blood is still
o An inverse reciprocal relationship exists between forward and reverse maternal in origin
testing thus, one serves to check the other. o Antibody production peaks between 5 - 10 years of age and declines
later in life.
FORWARD GROUPING  Can cause rapid INTRAVASCULAR HEMOLYSIS if the wrong ABO group is transfused
→ Using known sources of commercially prepared sera (anti-A, anti-B) to detect and can result to death.
antigens on an individual’s red blood cells. o Anti-A (from group B individuals) and Anti-B (from group A individuals) is
→ Specimen: Needleprick/ EDTA whole blood predominantly of IgM class and smaller quantities of IgG.
→ Sera contains antibodies o Predominant immunoglobulin class of antibodies in O individual’s serum
is IgG.
REVERSE GROUPING  Serum from O individuals contains not only anti-A and anti-B,
→ Detection of ABO antibodies in the patient’s serum by using known red blood cells but also anti-A,B which reacts with A and B cells.
(A1 and B cells).  Anti-A, B antibody activity (originally thought to be just a
→ Reagents: contains Ag mixture of anti-A and anti-B) cannot be separated into a pure
specificity when adsorbed with either A or B cells.
IMPORTANCE of ABO  Cross reacting so if tested with A and B, it causes
 Almost all normal healthy individuals above 3-6 months of age have naturally agglutination0
occurring antibodies to the ABO antigens that they lack.  Not part of routine blood typing but important for
 These naturally occurring antibodies are mostly ______ class. They are antibodies diagnosis of HDN
capable of agglutinating in saline or low protein suspended RBCs without  If O serum is adsorbed with either A or B cells, the antibody
enhancement and may activate complement cascade at 37 0C and react at room eluted will react both to A and B cells. Therefore, Anti-A,B
temperature (20-240C) or colder. possess serologic activity NOT found in mixtures of anti-A plus
 Bacteria, pollen particles and other substances present in nature anti-B.
→ Chemically similar to A & B antigen o Knowledge of the amount of IgG anti-A, anti-B or anti-A,B in a woman’s
serum sometimes allows prediction or diagnosis of HDN caused by ABO
ABO ANTIBODIES incompatibility.
 Individuals normally produce antibodies directed against the A and/or B antigen(s) o Testing RBCs with anti-A,B reagent is NOT REQUIRED as a ROUTINE part
absent from their RBCs. of RBC testing.
 ABO Groups of the Offspring from various Possible ABO Matings:
Reagent Anti-A,B can be prepared using: ABO Frequencies
→ Blended monoclonal anti-A and anti B Phenotype White (%) Black (%) Oriental (%)
→ Polyclonal human anti-A,B or A 41 27 27
→ Blend of monoclonal anti-A, anti-B and anti-A,B B 9 19 25
AB 4 4 5
Both immunoglobulin classes (IgM, IgG) of ABO antibodies react preferentially at room O 45 49 40
temperature (20-24oC) or colder and efficiently activate complement at 37 oC.
FORMATION of A, B and H RBC Antigens
ABO Grouping is REQUIRED for all of the following:  Formation of ABH antigens result from interaction of genes at 3 separate loci (ABO,
 Blood donors Hh, and Se).
 Transfusion recipients  H gene codes for the precursor substance on which A and B gene products act.
 Transplant candidates and donors o These genes do NOT code for the production of antigens but rather produce
 Prenatal patients specific glycosyltransferases that add sugars to a basic precursor substance.
 Newborns o The product of A and B genes are enzymes that act as specific
 Paternity testing TRANSFERASES.
 H gene product is an enzyme that produces H substance.
INHERITANCE of the ABO BLOOD GROUPS  A and B gene products are enzymes that convert H substance to A & B
Antigen.
 First discovered by Bernstein in 1924
 An individual inherits 1 ABO gene from each parent and that these 2 genes
determine which ABO antigens are present on RBC membrane.
 Follows simple Mendelian Genetics
 ABO is codominant in expression (like most other blood group systems).
 1 position or locus, on each chromosome 9 is occupied by an A, B, or O antigen.

Phenotypes (blood types) – outward or physical manifestation

A, B, AB, O

Genotypes – blueprints, code or genetic make – up

AA, BO, OO
ABH Antigen
o In case of an O individual, both phenotypes and genotype are the same because the  Present NOT only in red cells but on many other cells and in most body fluids.
individual is homozygous for gene.  Presence of A, B or O antigens on red cells depend upon the inheritance of allelic
 Gene is Amorph, no detectable antigen is produced in response to genes A, B, and O.
inheritance of this gene.  A, B, and H antigens are formed from the same basic precursor material
o Individual with phenotype A (or B) can have genotype AA or AO (or BB or BO). (Paragloboside or Lacto-N-neotetraosylceramide) to which sugars are attached in
o Phenotype and genotype are the same in AB individual because of inheritance of response to specific enzyme transferases elicited by an inherited gene.
both A and B gene.  Precursor substance on RBC is referred to as Type 2.
o The terminal galactose on precursor substance is attached to N-
acetylglucosamine in a β 1-4 linkage.
Mating Offspring Possible Mating Offspring Possible  Type 1 -> a β 1-3 linkage between galactose and N-acetylglucosamine.
Phenotypes Phenotypes (Genotypes) Phenotypes Phenotypes (Genotypes)  ABH antigens on the RBC are constructed on oligosaccharide chains of Type 2
precursor.
A+A A(AA,AO),O(OO) A+O A(AO),O(OO)
B+B B(BB,BO),O(OO) A+AB AB(AB),A(AA,AO),B(BO) Development of ABH Antigens:
AB+AB AB(AB),A(AA),B(BB) B+O B(BO),O(OO) → Develops as early as 37th day of fetal life
O+O O(OO) B+AB AB(AB),B(BB,BO),A(AO) o Newborn RBCs carry about 25-50% antigenic sites found on adult RBC.
A+B AB(AB),A(AO),B(BO),O(OO) AB+O A(AO),B(BO)
 o Reactions of newborn RBCs with ABO reagent antisera are frequently
weaker than with adult cells.
o A and B antigens expression fully develop by 2-4 years of age and
remains constant for life.
o ABH antigens phenotypic expression may vary with race, genetic
interaction, and disease states.

Group “O” individuals inherit at least:

 1 H gene (genotype HH or Hh)
 2 O genes

H gene
→ elicits the production of an enzyme α-2-L-fucosyltransferase
o Transfers the sugar L-fucose to an oligosaccharide chain on the
terminal galactose of Type 2 chains.

IMMUNODOMINANT SUGARS
→ Sugar responsible for blood group specificity
→ L-FUCOSE
o Sugar responsible for H specificity
o The O gene at the ABO locus (AMORPH) does NOT elicit the production of In the formation of blood group “B”:
catalytically active polypeptide.  B gene (BB,BO) codes for α-3-D-galactosyltransferase
o Therefore, H substance remains unmodified. o Attaches D-galactose (Gal) sugar to H substances
o “O” blood group has the highest concentration of H antigen.

 H substance (L-fucose) must be formed for the other sugars to be attached in

response to an inherited A and/ or B gene.
o Allele of H, is quite rare
o Genotype “hh” is extremely rare
 hh genotype
o Does NOT elicit production of α-2-L-fucosyltransferase
o L-fucose is NOT added to Type 2 chain
o H substance is NOT expressed on RBC

BOMBAY
 Refers to the phenotype that lacks normal expression of ABH antigens due to
inheritance of “hh” genotype.

BOMBAY “hh” INDIVIDUALS

 may inherit ABO genes but normal expression (formation of A, B or H antigens)
does NOT occur.

In the formation of blood group “A”:

 A gene (AA, AO) codes for production of α-3-N-acetylgalactosaminyltransferase
o Transfers an N-acetyl-D-galactosamine (GalNAc) sugar to H substance.
 Responsible for A specificity (“A”)
  610,000 – 830,000 B antigen sites exist on adult RBC in response to conversion of H A Subgroups
antigen by α-3-D-galactosyltransferase produced by B gene.
When both A and B genes are inherited:
 B enzyme (α-3-D-galactosyltransferase) compete more efficiently for H substances
than A enzyme (α-3-N-acetylgalactosaminyltransferase).
o Average number of A antigens on AB adult cell is approximately 600,000
sites
o B antigens average of 720,000 sites

Formation of A, B and H Soluble Antigens:

 Found in ALL body secretions
 Their presence is dependent on the ABO genes inherited as well as the inheritance
of another set of genes (secretor “Se” genes) that regulate their formation.
 80% of random U.S. population is known as secretors.
o They have inherited a Secretor gene (SeSe or Sese).
 Inheritance of an Se gene codes for the production of a transferase (α-2-L-
fucosyltransferase) that results in the modification of the type 1 precursor
substance in secretions to express H substance.
o H substance can then be modified to express A and B substance (if the
corresponding gene is present) in the secretions (ex. saliva).
o Se gene does NOT affect the formation of A, B, or H antigens on RBCs.
o The presence of the Se-gene-specified α-2-Lfucosyltransferase that
determines
o People who inherit the sese gene are termed nonsecretors.

Comparison of ABH Antigens on RBCs and in Secretions:  In 1911, von Dungern described 2 different A antigens based on reactions between
 ABH Antigens on RBCs group A RBCs and anti-A and anti-A1.
A1
1. RBC antigens can be glycolipids, glycoproteins, or glycosphingolipids.
2. RBC antigens are only synthesized on Type 2 precursor chains.
3. The enzyme produced by the H gene (α-2-L-fucosyltransferase) acts primarily on
type 2 chains, which are prevalent on the RBC membranes.

 A, B & H Soluble Substances

1. Secreted substances are glycoproteins.
2. Secreted substances are primarily synthesized on Type 1 precursor chain.
3. The enzyme produced by Se gene (α-2-L-fucosyltransferase) preferentially acts on
type 1 chains in secretory tissues.

Type 1 Type 2
Linkage β1-3 β1–4
Synthesized on erythrocyte
Origin Plasma
precursor
Controlling gene H, A, B, Se & Lewis H, A, B genes
ABH SUBSTANCES IN THE SALIVA of SECRETORS (SeSe or Sese)
ABO Subgroups
 → are A RBCs that reacts with both Anti-A and Anti-A1.  Group A1 individuals will not possess a great deal of H antigen
o In the presence of A1 gene, almost all of the H antigen is converted to A 1
A2 antigen by placing the large N-acetyl-D- galactosamine sugar on H
→ are A RBCs that reacts with anti-A (not to anti-A1) substance.
→ 1-8% of “A2” produce anti-A1 in their serum o May not be available to react anti-H sera
→ 22-35% of “A2B” produce anti-A1 in their serum  Weak subgroups of A antigen will often have a RECIPROCAL relationship between
the amount of H antigen on the RBCs and the amount of A antigens formed.
 RBCs from A1 and A2 individuals react equally strong with reagent anti-A in ABO
forward typing tests. Anti-H
 Approximately 80% of all group A (or AB) individuals are A1 (or A1B)  Naturally occurring IgM cold agglutinin that reacts best below room temperature
o 20% are A2 (or A2B) or weaker subgroups  Reacts most strongly with cells of group O individuals (which have the greatest
 Production of both types of antigens is a result of an inherited gene at the ABO amount of H substance on their RBCs)
locus.  Insignificant antibody in terms of transfusion purposes, it has no reactivity at body
o Inheritance of A1 gene elicits production of high concentrations of α-3-N- temperature.
acetylgalactosaminyltransferase o However, high-tittered anti-H may also present a problem in AB screening
 Converting almost all of the H precursor structure to A 1 procedures because reagent screening cells are group O.
antigens on RBCs  Anti-H LECTIN
 Antigen sites of A1 : 810,000 to 1,170,000 o From Ulex europaeus extract closely parallels the reactions of human
 Antigen sites of A2 : 240,000 to 290,000
o Immunodominant sugar for both A1 and A2 RBCs: N-acetyl-D-
galactosamine
 There must be some difference between antigenic structure of
A1 and A2 because, even though the same sugar is attached by
the same transferase, A2 and A2B individuals CANNOT recognize
the A1 antigen as being part of their own RBC make up and are anti-H.
immunologically stimulated to produce a specific A 1 antibody
that does not cross react with A2 RBCs. WEAK A SUBGROUPS
 Occur infrequently and most often recognized through ABO discrepancy.
Anti-A1  Only 1% of group A encountered in the laboratory.
→ Naturally occurring IgM cold antibody o Mainly of academic interest
o Unlikely to cause a transfusion reaction because it usually reacts better or
only at temperatures well below 37oC. Characteristics of weak ABO subgroups:
o Considered clinically significant if it is reactive at 37 oC. 1. Decreased number of A antigen sites per RBC.
2. Varying degrees of agglutination by human anti-A,B
 Serum from group B individuals contains anti-A and anti-A 1, therefore, this Ab 3. Increased variability in the detectability of H antigen resulting in strong reactions
mixture reacts with both A1 and A2 RBCs because both cells have the A antigen. with anti-H.
4. Presence or absence of anti-A1 in the serum.
 If serum from type B (anti-A an anti-A 1) individuals is adsorbed with A2 cells (only A
antigen), anti-A will bind to RBC.  Secretor studies and Adsorption-Elution tests can be utilized to subdivide A
o The serum left after the cells and attached anti-A are removed by individuals into A3, Ax, Aend, etc.
centrifugation is referred to as “Adsorbed Serum” and contains anti- A 1.
o This serum will react only with A1 antigen sites. Weak A phenotypes can be serologically differentiated using the following techniques:
 Forward grouping of A and H antigens with anti-A, anti-A,B and anti-H
 The seed of the plant Dolichos biflorus (another source of anti-A1) is known as anti-  Reverse grouping of ABO isoagglutinins and the presence of anti-A1
A1 lectin.  Adsorption-elution tests with anti-A
o Agglutinates A1 (or A1B) cells but does NOT agglutinate A2 (or A2B) cells.  Saliva studies to detect the presence of A anf H substances
 Additional special procedures such as Glycosyltransferase studies for detection of A
o LECTIN – seed extracts that agglutinate human cells with some degree of
enzyme
specificity.
A3 → Adsorption and elution of anti-A confirms the presence of A antigens
→ Mixed-field pattern of agglutination with anti-A and most anti-A,B reagents → Trace amounts of A glycosyltransferase is detectable in serum
→ A antigen sites is approximately 35,000 per RBC → Saliva secretor studies demonstrate A and H substance, with A substance present in
→ Weak α - 3-N-acetylgalactosaminyl – transferase activity in serum below-normal quantities
→ Divided into 3 groups: → Usually do not produce anti-A1 in their sera
o GROUP 1 – A3 phenotypes with an enzyme that had an optimal pH of → Recessive mode of inheritance
approximately 7.0 and very low activity
o GROUP 2 – A3 individuals with no detectable A transferase Ael
o GROUP 3 – A3 phenotype demonstrating a serum A enzyme with an → Unagglutinated by anti-A or anti-A,B
activity equivalent to 1/3 of that normally found in group A 1 serum and → Adsorption and elution can be used to demonstrate the presence of A antigen
optimal pH of 6.0 → No detectable A enzyme activity can be demonstrated
 Capable of converting O RBCs into A RBCs that did not show the → Usually produce an anti-A1 that is reactive with A1 cells and sometimes produce
mixed-field agglutination pattern characteristics of A3 anti-A which agglutinates A2 RBCs
→ Anti-A1 may be present in serum of A3 individuals → Secretor studies demonstrate the presence of only H substance in the saliva.
→ A substance detected in saliva of A3 secretors
B Subgroups
Ax → Are very rare and less frequent that A subgroups
→ Not agglutinated by anti-A reagent but do agglutinate with most examples of anti- → No B subgroups reported that are equivalent to Aend of Ay.
A, B → Usually recognized by variations in the strength of the reaction using anti-B and
→ A antigen sites: approx. 4000/RBC anti-A, B.
→ Anti-A can be adsorbed and the eluted from Ax cells without difficulty → Inheritance of B subgroups is similar to that of majority of A subgroups, is
→ Transferase is not usually detectable in serum considered to be a result of alternate alleles at the B locus.
→ Almost always produce anti-A1 in their serum
→ Secretor studies detect the presence of only H substance in Ax secretors WEAK B SUBGROUPS
→ Ax secretors contain A substance detectable only by agglutination/inhibition studies Criteria used for differentiation of weak B phenotypes:
using Ax RBCs as indicators 1. Strength/type of agglutination with anti-B, anti-A,B and anti-H.
Aend 2. Presence or absence of ABO agglutinins in the serum
→ Mixed-field agglutination with antiA and anti-A,B (only a very small percentage 3. Adsorption-elution studies with anti-B
≤10% of RBCs agglutinate) 4. Presence of B substance in saliva
→ A antigen sited : approx. 3500/RBC
→ No detectable A antigens are demonstrated on RBCs that do not agglutinate B3
→ No A glycosyltransferase is detectable in serum or RBC membranes → Mixed-field pattern of agglutination with anti-B and anti-A,B
→ Secretor studies detect presence of only H substance in saliva of Aend secretors → B glycosyltransferase is present in serum but not in RBC membrane
→ Anti-A1 is found in some Aend sera → Anti-B is absent in serum but B substance is present in normal amounts in saliva
→ Variants of Aend subgroup: secretors
o Afinn → The most frequent weak B phenotype
o Abantu
Am Bx
→ Not agglutinated or agglutinated only weakly by anti-A or anti-A,B → Weak agglutination with anti-B and anti-A,B antisera
→ Strongly positive adsorption/elution of anti-A confirms the presence of A antigen → B glycosyltransferase has not been detected in serum or RBC membrane
sites. → Weakly reactive anti-B is usually produced
→ A antigen sites: 200-1900/RBC → Readily adsorb and elute anti-B
→ A enzyme of either A1 or A2 type is detectable in the serum → Secretor studies demonstrate large amounts of H substance as well as some B
→ Usually do not produce anti-A1 in their sera substance that can often be detected only by inhibition of agglutination of Bx cells
→ Normal quantities of A and H substance are found in the saliva of Am secretors. with anti-B.
Bm
Ay → Unagglutinated by anti-B or anti-A,B
→ Not agglutinated by anti-A or anti-A,B → Easily adsorb and elute anti-B
 → B glycosyltransferase is present in serum but usually lower in activity, only very Parabombay
small amounts of B transferase activity in RBC membrane → Rare
→ Anti-B is not characteristically present in serum → RBCs are completely devoid of H gene
→ Normal quantities of H and B substance found in saliva of secretors → Small amount of H antigen is present
Bel
→ Unagglutinated by anti-B or anti-A,B H gene (HH or Hh genotypes)
→ Extremely rare phenotype determined by adsorption and elution of anti-B → Necessary for the formation of A and B antigens
→ No B glycosyltransferase.
→ Weak anti-B may be present in serum H-DEFICIENT PHENOTYPES:
→ Only H substance is demonstrated in saliva of secretors → Those rare phenotypes in which RBCs are completely devoid of H antigens or have
small amounts of H antigen present
Anti-B LECTIN → 3 Categories:
→ From Griffonia (Bandeiraea) simplicifolia (Modified BS-1 lectin) 1. RBC H-Deficient, Nonsecretor ; Bombay Phenotype (hh sese)
→ Prepared for differentiating group B variants  Inherits the “hh” genotype, therefore, lacks normal expression
o Ability to differentiate true B antigens from “Acquired B-like antigens” of ABH antigens
(does not agglutinate modified BS-1 lectin) 2. RBC H-Partially Deficient, Nonscretor (Ah, Bh or Abh)
 RBCs of these individuals express weak forms of A and B (which
Acquired B Phenomenon are primarily detected by adsorption and elution studies).
 In group A individuals converted to B  If a person is genetically A or B, the respective enzymes can be
 Due to passage in the gastrointestinal tract of bacterial polysaccharides (ex. E.coli detected, but NO H enzyme is detectable, even though it has
serotype O86, Proteus vulgaris infection) been shown that there is LIMITED production of H antigen on
o Modifies the immunodominant sugar that highly resembles the D - RBCs.
galactose  Notations Ah, Bh, and ABh have been used to describe these
 A red cells absorbs B-like polysaccharides which reacts with anti-B (reaction is individuals.
always weaker than the true A antigen).  Ah, Bh, and ABh have been reported mainly in individuals of
European origin.
BOMBAY PHENOTYPES (Oh)  No H, A or B antigen is present in the saliva, and anti-H is
 First reported by Bhende in 1952 in Bombay, India. present in the serum.
 Represents the inheritance of a DOUBLE dose of “h” gene (hh genotype)  Ah individuals contains anti-B and no anti-A (although anti-A1 is
 ABO genes CANNOT be expressed → ABH antigens CANNOT be formed. usually present).
 (RBC) fails to react with anti-A, anti-B, anti-A,B and anti-H  Bh individuals contains anti-A and anti-B may be detected.
 In RBC testing, using anti-A and anti-G, the Bombay would phenotype as an O blood  Theory:
group. o Homozygous inheritance of a mutant H gene (FUT 1)
o However, RBCs of the Bombay phenotype (Oh) do NOT react with codes for the production of LOW levels of H
the anti-H lectin unlike those of the normal group O individual transferase activity.
 Bombay serum contains anti-A, anti-B, anti-A,B and anti-H. o The small amount of H substance on the RBC is
 Bombay anti-H can often be potent and reacts strongly at 37 oC (unlike those anti-H completely used by the A and/or B antigen present on
in A1 and A1B individuals). the RBC with NO detectable H antigen.
o IgM antibody that can bind to complement and cause RBC lysis o Anti-H present is weaker in reactivity than the anti-H
 Transfusing normal group O blood to a Bombay recipient would in Bombay phenotype, although may be active at
cause immediate cell lysis. 37oC.
 Only blood from another Bombay individual will be compatible 3. RBC H-Deficient, Secretor (Parabombay)
and can be transfused.  RBCs have little or no A, B and H antigens
 ABH substance is absent in saliva.  RBCs of Oh secretors are not agglutinated by most examples of
 When family studies demonstrate which ABO genes are inherited in the Bombay anti-H but may be agglutinated by strong anti-H reagents.
phenotype, the genes are written as superscripts (OhA, OhB, OhAB).  A weak H-like antibody (anti - IH) that is reactive at low
 Can falsely be typed as O temperature is almost always present in the serum
 o This antibody is non - reactive with cord cells and is Anti - A Anti - B Anti – A,B Interpretation (Blood group)
not inhibited by secretor saliva 0 0 0 O
 Due to their secretor status (Se), normal levels of H substances + 0 + A
are present in saliva. 0 + + B
o A and B substances are present in the secretions + + + AB
when A and B genes are present. 0 0 + Weak A subgroup

BLOOD TYPING Procedure Notes:

 Do not rely on the color of dyes to identify reagent antisera. All tubes must be
FORWARD TYPING properly labeled.
 Do not perform tests at temperature higher that room temperature.
Principle:  Perform observations of agglutination with a well-lighted background, not a warm
The ABO blood groups (A, B, AB, O) represents the ANTIGENS on the erythrocytes view box.
of each group. When an antibody and its corresponding antigens are combined in vitro, it  Record results immediately after observation.
results in clumping/agglutination of RBCs expressing the antigen.  Remember that contaminated specimens, reagents or supplies may interfere with
the test results.
Specimen:
 No special preparation is required specimen collection. REVERSE BLOOD TYPING
 The patient must be properly and positively identified when the specimen is
collected. Principle:
 The specimen must be labeled at bedside (patient’s full name, age, date and time The reverse (serum) grouping procedure to confirm ABO blood grouping is based
of collection, patient’s hospital ID number, phlebotomist’s initials). on the presence or ansence of the ANTIBODIES (anti-A, anti-B) in serum. If these antibodies
 Blood should be drawn by an aseptic technique. are present in serum, agglutination should be demonstrated when the serum is combined
 Specimens should be tested as soon as possible. If delayed, blood should be with the reagent erythrocytes expressing either A or B antigens.
refrigerated.
 Plain or EDTA evacuated tubes could be used. Quality Control:
Reagent erythrocytes should be tested daily with known antisera.
Quality Control:
Reagent antisera should be tested daily with erythrocytes of known antigenicity. Procedure:
1. Label 2 test tubes: 1 with letter A, the other with letter B. (Underline the letters A
Procedure: and B to denote reverse grouping)
1. Check the patient’s name and ID numbers on the blood specimen and requisition. 2. To each of the 2 test tubes, add 2 drops of the serum or plasma to be tested.
2. Prepare a2-5% suspension of the patient’s red cells in normal saline. 3. To test tube labeled A, add 1 drop of thoroughly mixed A1 reagent erythrocytes.
3. Label 3 test tubes: 1 with the letter A, another 1 with letter B and the 3rd tube with 4. To test tube labeled B, add 1 drop of thoroughly mixed B reagent erythrocytes.
AB). 5. Mix well and centrifuge both tubes for 15 minutes at 3400 rpm.
4. To test tube labeled A, add 1 drop of anti-A antiserum. 6. Resuspend and examine macroscopically for agglutination.
5. To test tube labeled A,B, add 1 drop of anti-A,B antiserum.
6. Using a disposable pipette, add 1 drop of cell suspension to each of the test tubes. Reporting:
7. Mix well and centrifuge the test tubes for 15 seconds at 3400 rpm.  AGGLUTINATION indicates that an antibody specific for either A or B antigens is
8. Resuspend the cells with gentle agitation and examine microscopically for present in the serum or plasma being tested.
agglutination.  NO agglutination indicates that an antibody specific for either A or B antigen is
absent in the serum or plasma being tested.
Reporting:
 AGGLUTINATION of RBCs with specific antiserum is interpreted as a POSITIVE (+)
result and indicates the presence of the corresponding antigen.
 NO agglutination of RBCs produces a NEGATIVE (0) result and indicated that the
corresponding antigen is not present. ABO DISCREPANCIES
Reactions of Patient’s Red Cells to Known Antigens
 → Occur when unexpected reactions are observed in the forward and reverse → Some causes:
grouping. o Subgroups A (or B) may be present
o Leukemias
Common Sources of Technical Errors Resulting in ABO Discrepancies: o Hodgkin’s disease
 Inadequate identification of blood specimens, test tube or slides o “Acquired B” phenomenon (associated with diseases of the digestive
 Cell suspension either too heavy or too light tract)
 Clerical errors Resolution:
 Mix-up in samples  Incubate the test mixture at RT for up to 30 minutes.
 Missed observation of hemolysis  If NEGATIVE, incubate the test mixture at 4oC for 15-30 minutes.
 Failure to add reagents  RBCs may also be pretreated with enzymes and retested with reagent antisera.
 Failure to follow manufacturer’s instructions  Include group “O” and autologous cell as controls.
 Uncalibrated centrifuge
 Contaminated reagents GROUP III Discrepancies
 Warming during centrifugation → Between forward and reverse grouping caused by protein or plasma abnormalities
resulting in rouleaux formation.
 It is essential to acquire information regarding the patient’s age, diagnosis, → Attributed to:
transfusion history, medications and history of pregnancy. o Elevated globulin levels
 When a discrepancy is encountered, results must be recorded, but interpretation of o Elevated fibrinogen levels
the ABO type must be delayed until the discrepancy is resolved. o Plasma expanders
o Wharton’s jelly
GROUP I Discrepancies  Mucopolysaccharide present on cord red blood cell
→ Associated with unexpected reactions in reverse grouping due to weakly reacting Resolution:
or missing antibodies  Washing the patient’s RBCs several times with saline or adding 1-2 drops
→ Reason: Patient has depressed antibody production or cannot produce the ABO saline to test tubes will free cells from rouleaux formation.
antibodies  Saline dilution or saline replacement technique will FREE cells in rouleaux
→ Common populations with discrepancies of Group 1: formation of reverse typing.
o Newborns  Washing the cord cells 6 – 8 times with saline should alleviate rouleaux
o Elderly patients formation due to Wharton’s jelly.
o Leukemia or Lymphoma patients o Patients using immunosuppressive o Alternative: adding 1 – 2 drops of saline to test tubes
drugs
o Patients with congenital aggamaglobulinemia or immunodeficiency GROUP IV Discrepancies
diseases → Between forward and reverse grouping due to miscellaneous problems with the
o Bone marrow transplantation patients o Patients whose ABO antibodies following causes:
may have been diluted by plasma transfusion or exchange o Cold reactive autoantibodies
o ABO subgroups o Patient has circulating RBCs of more than 1 ABO group due to RBC
o Rare cause: Chimerism transfusion or marrow transplant
 presence of 2 cell population in a single individual o Unexpected ABO isoagglutinins
Resolution: o Unexpected non-ABO alloantibodies
 Enhance the weak or missing reaction in serum by incubating the patient’s serum Resolution:
with reagent A1 and B cells at RT for 15-30 minutes.  Patient’s RBCs could be incubated at 37 oC for a short period, then washed with
 If there is still NO reaction after centrifugation, the serum-cell mixtures can be saline at 37oC, 3 times and retype.
incubated at 4oC for 15-30 minutes. o If NOT successful, patient’s RBCs can be treated with 0.01M ditheotreitol
 AUTOCONTROL and “O” cell control must always be tested concurrently with (DTT) to disperse IgM-related agglutination.
REVERSE typing.  Patient’s serum: Reagent RBCs and patient serum are warmed to 37 oC, then mixed,
tested and read at 37oC.
GROUP II Discrepancies o Test can be converted to AHG phase if necessary.
→ Associated with unexpected reactions in forward grouping due to weakly reacting
or missing antigens
 o If result if NEGATIVE (and a POSITIVE result was expected), an
AUTOABSORPTION could be performed to remove theautoantibodies
from the serum.

 Unexpected ABO isoagglutinins in patient’s serum:

o Serum grouping can be repeated using at least 3 examples of A1, A2, B
cells, O cells and an autologous control.
o Specificity of Ab can be determined by examining the pattern of
reactivity.
o Patient’s RBCs can be tested with Dolichos biflorus.

 Unexpected alloantibodies in the patient’s serum (other than ABO isoagglutinins

like anti-M) may cause discrepancy in reverse grouping.
o A PANEL can be performed with the patient’s serum.
o Once the unexpected alloantibody(ies) is(are) identified, A1 and B cells
negative for the corresponding antigen can be used in reverse typing, or;
o Once again, the reverse typing can be repeated at 37oC (if the ABO
isoagglutinins react at this temperature and there is no interference from
the unexpected alloantibody.