MLS 323 LEC – Immunohematology

Block Exclusive Notes

MAJOR BLOOD GROUP: ABO Blood Group System

ABO Blood Group System (001)

• Most important of all blood groups in transfusion practice
• ABO Genotypes and Phenotypes
o ABO genes are inherited in a codominant manner following simple Mendelian genetics laws
• Several other genetic loci interact with the ABO locus: ___________________________
o The gene that codes for A, B, and O is at the long arm of chromosome _____
• Single genetic locus having 3 possible alleles: A, B or O
o A and B genes are codominant; O represents the lack of either A or B
o ABO Allele Theories
3 allele theory by Bernstein 4 allele theory by Thompson
Phenotype Possible Genotypes Phenotype Possible Genotypes
A A1
B A2
AB A1B
O A2B
B
O
o Frequency (%) of ABO Phenotypes
Phenotype Whites Blacks Native Americans Asians
A 42 27 16
B 10 20 4
AB 4 4 <1
O 44 49 79

• Landsteiner’s Laws
1. The antigen on the RBC determines the blood group
2. The corresponding antibody is never found in the individual’s serum
3. The opposite antibody is always present in the individual’s serum

ABO Antigens and Antibodies

1. ABO Antigen
o ABO group inheritance is controlled by the A, B, and H genes
• These genes code not for the antigens themselves but for the production of glycosyltransferases, which are
involved in the formation of their respective antigenic determinants
• A and B antigens develop from precursor H substance
o Demonstrated as early as the second month of fetal life
• Developed in utero at 5-6 weeks of gestation ® full expression occurs between 2-4 years of age
• Persist throughout life unaltered
o Found on RBCs, lymphocytes, platelets, tissue cells, bone marrow and organs
• May be found in saliva, pancreatic secretions and gastric secretions
• May be found in bacteria and other species
o Precursor Oligosaccharide Chains
Type 1 Type 2
Linkage
Origin
Controlling genes
o Formation of ABO Antigens
Gene Glucosyltransferase Immunodominant Sugar Acceptor Antigen
H α-2-L-fucosyltransferase L-fucose
A α-3-N-acetylgalactosaminyltransferase N-acetyl-D-galactosamine
B α-3-D-galactosyltransferase D-galactose
α-3-N-acetylgalactosaminyltransferase N-acetyl-D-galactosamine
AB
α-3-D-galactosyltransferase D-galactose
O --- --- ---

Page 1 of 6
DISCLAIMER: This Block Exclusive Lecture Notes is intended ONLY for the use of MLS 323 Class Codes 8426 and 8433 of Academic Year 2022-2023.
This handout shall NOT, by any means, replace the reference textbooks as the principal source of learning Immunohematology.
 MLS 323 LEC – Immunohematology
Block Exclusive Notes

o Comparison of ABH Antigens on RBCs and in Secretion

ABH Antigens on Red Cells A, B and H Soluble Substances
RBC antigens can be glycolipids, glycoproteins, or Secreted substances are _______________
glycosphingolipids
RBC antigens are only synthesized on type ___ Secreted substances are primarily synthesized on
precursor chains type ___ precursor chain
Enzyme: α-2-L-fucosyltransferase Enzyme: α-2-L-fucosyltransferase

o ABH Substances in Saliva

RBC Blood Group Secretor Status ABH Substances found in Saliva
A Secretor
B Secretor
AB Secretor
O Secretor
A, B, AB, O Non-secretor
• Fluids in which A, B, and H substances can be detected in secretors:
o Saliva, tears, urine, digestive juices, bile, milk, amniotic fluid
o Pathologic fluids: pleural, peritoneal, pericardial, ovarian cyst

2. ABO Antibodies
o Production initiated at birth
• Detectable titers: 3-6 months ® peaks at 5-10 years
o Naturally occurring in the serum
• Predominantly IgM; activates the complement
• Optimum temperature: 21°C or colder
o Antibodies:
Blood Group Antibody Produced Characteristics
A IgM
o Naturally occurring antibodies
B

AB --- ---
O Mostly IgM (some IgG)

ABO Typing Technique

1. Forward Grouping
o Using known sources of reagent antisera (anti-A, anti-B) to detect antigens on an individual’s red cells
Reaction with
Blood Group Antigen Interpretation
Anti-A Anti-B Anti-AB
A
B
AB
O
o Characteristics of Routine Reagents Used for ABO Forward Grouping:
Anti-A Reagent Anti-B Reagent
• Monoclonal antibody • Monoclonal antibody
• Highly specific • Highly specific
• IgM • IgM
• Clear blue colored reagent • Clear yellow colored reagent
o Determination of Secretor Status:
• Principle: _________________________
Page 2 of 6
DISCLAIMER: This Block Exclusive Lecture Notes is intended ONLY for the use of MLS 323 Class Codes 8426 and 8433 of Academic Year 2022-2023.
This handout shall NOT, by any means, replace the reference textbooks as the principal source of learning Immunohematology.
 MLS 323 LEC – Immunohematology
Block Exclusive Notes

2. Reverse Grouping
o Using reagent cells with known A1 and B antigens and testing the serum of the patient for ABO group antibodies
Reaction with
Blood Group Antibody Interpretation
A1 Cells B Cells
A
B
AB
O
o Characteristics of Routine Reagents Used for ABO Testing: Reverse Grouping
Reagent A1 and B Cells
• Human source • 4-5% red cell suspension
• Lectins
o Plant extracts that bind to carbohydrate portions of certain red cell antigens and agglutinate the red cells
o Lectins Useful in Blood Group Serology:
Lectin Antigen Reactivity Serologic Specificity
For ABO antigens
Ulex europaeus L-fucose
N-acetyl-D-glucosamine
Lotus tetragonolobus L-fucose
Dolichos biflorus α-N-acetyl-D-galactosamine
Helix pomatia α- or β-N-acetyl-D-galactosamine
Griffonia simplicifolia or α-D-galactose (BSI)
Bandeiraea simplicifolia N-acetyl-D-glucosamine (BSII)
For other blood group antigens
Iberis amara
Vicia graminea β-D-galactose
For antigens causing polyagglutination
Arachis hypogaea β-D-galactose
Glycine soja N-acetyl-D-galactosamine
Salvia horminum α-N-acetyl-D-galactosamine
Salvia sclarea α-N-acetyl-D-galactosamine

Other ABO Phenotypes

• ABO Subgroups
o Subgroups of A
Reaction with
Phenotype Antigens Present Population Frequency
Anti-A (from B sera) Anti-A1 lectin
A1
A2
o Other Subgroups of A
Phenotypes Description
A3 o Mixed field agglutination with anti-A and/or anti-A,B
Ax o Weak agglutination with anti-A,B only
Aend o <10% red cells show very weak mf agglutination
o No agglutination with anti-A and anti-A,B
Am
o Secretors demonstrate quantities of A substance in saliva
o No agglutination with anti-A and anti-A,B
Ay
o Secretors contain small amount of A substance in saliva
o No agglutination with anti-A and anti-A,B
Ael
o Secretors contain only H substance and no A substance in saliva
o Subgroups of B
Phenotypes Description
B3 o Mixed field agglutination with anti-B and/or anti-A,B
Bx o Agglutination with anti-A,B (wk/0 with anti-B)
o No agglutination with anti-B and anti-A,B
Bm
o Secretors demonstrate quantities of B substance in saliva
o No agglutination with anti-B and anti-A,B
Bel
o Secretors contain only H substance and no B substance in saliva
Page 3 of 6
DISCLAIMER: This Block Exclusive Lecture Notes is intended ONLY for the use of MLS 323 Class Codes 8426 and 8433 of Academic Year 2022-2023.
This handout shall NOT, by any means, replace the reference textbooks as the principal source of learning Immunohematology.
 MLS 323 LEC – Immunohematology
Block Exclusive Notes

• Bombay Phenotype
o hh genotype ® no H antigens formed; therefore, no A nor B antigens formed
• Anti-A, anti-B, anti-A,B and anti-H present in the serum
o General Characteristics:
• Absence of H, A, and B, antigens; no agglutination with anti-A, anti-B, or anti-H lectin
• Presence of anti-A, anti-B, anti-A,B and a potent wide thermal range of anti-H in the serum
• A, B, H nonsecretor (no A, B, or H substances present in saliva)
• Absence of α-2-L-fucosyltransferase (H-enzyme) in serum and H antigen on red cells
• Presence of A or B enzymes in serum (depending on ABO genotype)
• A recessive mode of inheritance (identical phenotypes in children but not in parents)
• RBCs of the Bombay phenotype will not react with the anti-H lectin (Ulex europaeus)
• RBCs of the Bombay phenotype are compatible only with the serum from another Bombay individual
o Phenotyping of Type O and Oh
Forward Typing Reverse Typing
Phenotype
Anti-A Anti-B Anti-H A cells B cells O cells
O
Oh
o Characteristics Reported or Postulated for Categories of H-Deficient Phenotypes
Classification Category 1 Category 2 Category 3
Description o Bombay Phenotype o RBC H-Partially o Para-Bombay
Deficient, Nonsecretor phenotype

Proposed Genes Inherited o hh sese o hh se o hh Se

(weak variant present
at the Hh locus)
o A and/or B (dependent
on ABO genotype)
Glycosyltransferase o None o A and/or B (dependent o A and/or B (dependent
o A and/or B (dependent on ABO genotype) in on ABO genotype) in
on ABO genotype) in serum or RBC stroma serum/RBCs, H in
serum or RBC stroma serum (weak)
Red Cell Antigens: o None detectable o Weak A/B (dependent o Weak A/B (dependent
A, B and H Detected on ABO genotype) on ABO genotype) and
o Residual H when A or B H
immunodominant
sugar is removed with
appropriate enzymes
A, B and H Soluble o None detectable o None detectable o H substance (normal
Substances in Secretions amounts)
o A/B dependent on ABO
genotype (all normal
amounts)
Antibodies in Serum o Anti-A, anti-B, anti-H o Anti-H, Anti-A/anti-B o Weak Anti-A/anti-B
(dependent on ABO (dependent on ABO
genotype) genotype)

ABO Discrepancies
• Common Sources of Technical Errors Resulting in ABO Discrepancies
o Incorrect or inadequate identification of blood specimens, test tubes, or slides
o Cell suspension is either too heavy or too light
o Clerical errors or incorrect recording of results
o A mix-up in samples
o Missed observation of hemolysis
o Failure to add reagents
o Failure to add sample
o Failure to follow manufacturer’s instructions
o Uncalibrated centrifuge
o Overcentrifugation or undercentrifugation
o Contaminated reagents
o Warming during centrifugation
Page 4 of 6
DISCLAIMER: This Block Exclusive Lecture Notes is intended ONLY for the use of MLS 323 Class Codes 8426 and 8433 of Academic Year 2022-2023.
This handout shall NOT, by any means, replace the reference textbooks as the principal source of learning Immunohematology.
 MLS 323 LEC – Immunohematology
Block Exclusive Notes

• Group Discrepancies
1. Group I Discrepancies: Weakly Reacting or Missing Antibodies
o Newborns
o Elderly patients
o Patients with leukemia demonstrating hypogammaglobulinemia
o Patients with lymphomas demonstrating hypogammaglobulinemia
o Patients using immunosuppressive drugs that yield hypogammaglobulinemia
o Patients with congenital agammaglobulinemia
o Patients with immunodeficiency diseases
o Patients with bone marrow transplantations
o Patients whose existing ABO antibodies may have been diluted by plasma transfusion or exchange
o ABO subgroups

2. Group II Discrepancies: Weakly Reacting or Missing Antigens

o Subgroups of A and/or B
o Leukemia
o Hodgkin’s disease
o Excess amounts of blood group-specific soluble substances (BGSS) present in the plasma in association with
certain diseases
o Acquired B phenomenon
• Group A with acquired B antigen: group A1 individual with disease of the lower gastrointestinal tract,
cancer of the colon and rectum, intestinal obstruction, or gram-negative septicemia
o Antibodies to low incidence antigens

3. Group III Discrepancies: Protein or Plasma Abnormalities Resulting to Rouleaux Formation

o Elevated levels of globin from certain disease states
o Elevated levels of fibrinogen
o Plasma expanders
o Wharton’s jelly

4. Group IV Discrepancies: Miscellaneous

o Polyagglutination
• Refers to the agglutination of altered red blood cells (RBCs) by a large proportion of ABO-compatible
adult human sera
• Categories:
o Acquired
• Microbially-associated
T o Caused by bacteria, such as pneumococci, Clostridium perfringens,
and Vibrio cholerae, and viruses such as the influenza virus
Th o Caused by several microbial organisms including Clostridia,
Bacteroides, Escherichia coli, and Proteus
Tk o Caused by Bacteroides fragilis, Serratia marcescens, Aspergillus niger,
and Candida albicans
Tx o Caused by pneumococcal infections
Acquired B o Caused by enzymes produced by certain strains of Escherichia coli,
Clostridium tertium, and probably by certain strains of Proteus vulgaris
VA o H antigens of VA polyagglutinable RBCs are significantly depressed
Passive Adsorption of o Adsorption of bacterial products onto RBCs
Bacterial Products

• Non-microbially-associated
Tn o Caused by a mutation in the hematopoietic tissue (gives rise to a clone
of cells that lack β-3-D-galactosyltransferase)

o Inherited
Cad o An inherited autosomal dominant condition that gives rise to a
permanent polyagglutinable state
NOR o Inherited dominant form of polyagglutination
Hemoglobin M-Hyde Park o RBCs showed incomplete biosynthesis with exposure of terminal GluNAc
Hereditary Erythroblastic o Congenital dyserythropoietic anemia type II (CDA II)
Multinuclearity with o An autosomal recessive condition that is characterized by abnormal RBC
Positive Acidified membranes, multinucleated erythroblasts in the bone marrow, and
Serum (HEMPAS) RBCs that have a second membrane internal and parallel to the external
membrane
Page 5 of 6
DISCLAIMER: This Block Exclusive Lecture Notes is intended ONLY for the use of MLS 323 Class Codes 8426 and 8433 of Academic Year 2022-2023.
This handout shall NOT, by any means, replace the reference textbooks as the principal source of learning Immunohematology.
 MLS 323 LEC – Immunohematology
Block Exclusive Notes

• Panel of Lectins for Detection of Polyagglutination:

Tn Cad T Tk
Arachis hypogaea
Salvia sclarea
Salvia horminum
Glycine soja
o Cold reactive antibodies
o Unexpected ABO isoagglutinins
o Antibodies other than anti-A and anti-B may react to form antigen-antibody complexes that may then
adsorb into the patient’s red cells
o RBCs with the cis “AB phenotype”
• Cis-AB refers to the inheritance of both AB genes from one parent carried on one chromosome and an O
gene inherited from the other parent

• Overview of ABO Discrepancies

Problems with Category Causes
Red Cell Testing Weak/missing red cell ABO subgroup
reactivity Pathologic etiology – leukemia/malignancy
Transfusion
Intrauterine fetal transfusion
Transplantation
Excessive soluble blood group substance
Extra red cell reactivity Autoagglutinins/excess protein coating red cells
Unwashed red cells: plasma proteins
Unwashed red cells: antibody in patient’s serum to
reagent constituent
Transplantation – hematopoietic progenitor cell
transplants
Acquired B antigen
B(A) phenomenon: group B individual who acquires
reactivity with anti-A reagents in ABO red cell testing
Out-of-group transfusion
Polyagglutination

Mixed-field red cell Recent transfusion

Serum/Plasma Testing
reactivity Transfusion of group O to group A, B, or AB
Transplantation – hematopoietic progenitor cell
transplants
Fetomaternal hemorrhage (FMH)
Twin or dispermic (tetragametic) chimerism
A3 phenotype
Weak/missing serum Age-related (<4-6 months old, elderly)
reactivity ABO subgroup
Pathologic etiology – hypogammaglobulinemia
Immunosuppressive therapy for transplantation
Extra serum reactivity Cold autoantibody
Cold alloantibody
Serum antibody to reagent constituent
Excess serum protein
Transfusion of plasma components
Transplantation
Infusion of intravenous immune globulin (IVIG)

Page 6 of 6
DISCLAIMER: This Block Exclusive Lecture Notes is intended ONLY for the use of MLS 323 Class Codes 8426 and 8433 of Academic Year 2022-2023.
This handout shall NOT, by any means, replace the reference textbooks as the principal source of learning Immunohematology.