CLS 306 - ABO Blood Group System

ABO Blood Group System

Overview

• The ABO blood group system was the first human blood group identified.

• It is unique because it has naturally occurring antibodies (Abs) without requiring a foreign red blood cell antigen (Ag) to stimulate their production.

• ABO Abs, also known as isohemagglutinins, have specificity opposite to the corresponding antigens on the red blood cells (RBCs).

• These antibodies are non-red cell stimulated, likely resulting from environmental exposure and are referred to as "EXPECTED" antibodies.

• The ABO blood group is highly significant in transfusion practice.

Importance in Transfusion Medicine

• Common isohemagglutinins include Anti-A and Anti-B, which are IgMs that readily activate the normal complement cascade (C').

• Transfusion of incompatible RBCs can lead to intravascular hemolysis, potential renal failure, and even death.

• Detection of ABO incompatibility is a primary foundation for safe pretransfusion testing; The Joint Commission (TJC) and College of American Pathologists (CAP) proficiency testing (PT) require 100% accuracy in ABO typing tests at all times.

• ABO compatibility is also critical in transplant patients.

• ABO-incompatible organs may cause acute humoral rejection, that can result in graft rejection/failure [aka, Graft-vs-Host Disease (GVHD)], potentially leading to the recipient's death, depending on the transplanted organ.

USA ABO Blood Groups Approximate Population Frequencies (%)

Inheritance

• The ABO system has 3 common alleles: A, B, and H (O).

• Inheritance follows straightforward Mendelian genetics.

• Individuals inherit one ABO autosomal gene from each parent.

• A, B, and O genes are expressed as codominant; the "O" gene is an amorph, producing no transferase enzyme and resulting in no detectable O antigen.

• Rare, mutant alleles exist within the human population.

• The ABO locus is on chromosome 9.

Possible Blood group Genotypes

Inheritance Question

• If both parents are type A, can they produce a type O baby?

• Answer: Yes. Mom (type A) AA or AO, Dad (type A) AA or AO can have:

  • Child 1 (type A): AA or AO

  • Child 2 (type O): OO

ABO Phenotype / Genotype

*When determining possible genotypes, use the heterozygous form of the genes.

ABO Blood Group

• ABO Phenotype, Antigen on the RBC, Circulating Antibody in the Plasma

  • Group O: H (not an O ag) (No A or B ags), Anti-A, Anti-B, Anti-A,B* a single, IgG2 subclass cross-reacting antibody

  • Group A: A, Anti-B

  • Group B: B, Anti-A

  • Group AB: A and B, None

ABO GENETICS

ABH Antigens (ags)

• ABH = ABO; "O" blood type contains H ag, i.e., there is no “O” ag.

• Antigens are carbohydrate structures.

• Specific glycosyl-transferases add individual sugars sequentially to sites on sugar chains on the common precursor substance.

• A common precursor substance produces H, A, and B antigens.

• Interaction of ABO, Hh, and Sese genes affects antigen expression.

ABH antigens (ags)

• H precursor substance is a 4-sugar chain: Glucose (Glu); Galactose (Gal); N-acetylglucosamine (GlcNAc); Galactose (Gal).

• It is primarily of Type 2 chains with beta 1 → 4 linkage on the RBC surface antigen.

Effects of the H Gene

• H gene (FUT 1 gene)

• 2 allele system (H & h): HH, Hh, or hh

• h is an amorph; h is very rare

• Inherited independently of ABO genes

• The H gene is required for the production of A or B antigens

• H gene produces the α-2-L-fucosyltransferase that adds L-fucose to the precursor substance.

• The H antigen is the result of the attached L-fucose (Fuc).

Effects of the A or B Gene

• A and B alleles / genes encode for glycosyl-transferases which add sugars to the H antigen.

• The A gene produces the α-3-N-acetylgalactosaminyltransferase that adds the GalNAc (N-acetyl-D-galactosamine) sugar to the H antigen.

• The B gene produces the α-3-D-galactosyltransferase that adds the Gal (D-galactose) sugar to the H antigen.

• When both A and B genes are present to produce the AB phenotype, the B enzyme seems to compete more efficiently for the H antigen than the A enzyme. As a result of this competition, the number of A antigens on the RBC are less than the number of B antigens.

Genetic Pathway of the ABO System

• Genes Hh or HH, hh

• antigens (rbc) AO or AA, BO or BB, AB, 00

• A & H B & H A, B, & H H none detecteced

• any combination P.S. P.S.

Effects of the H Gene

• Over 99.99% of the human population has the H gene.

• ABO Types A and B can exist as a "subgroup" which is inherited. Subgroups usually have a lower number of A or B antigens on the RBCs.

• The more A and / or B antigens produced, the more H antigen is converted. Thus, the density of H antigen on cells is (from greatest to least amounts): O > A2 > B > A2B > A1 > A1B.

Effect of H Allele

Interaction of 3 Genes for ABO Expression

• H (FUT 1) chromosome 19

• Se (FUT 2) chromosome 19

• ABO chromosome 9

Interaction of 3 Genes for ABO Expression

1. H

   • H substance on RBCs

   • h Silent / Amorph

2. Se

   • Secretor; H substance found in secretions

   • se Non-secretor; Silent / Amorph

3. A, B

   • A, B, & H antigens on RBCs, when H is present

   • A and B in secretions, when Se is present

   • "O" Silent / Amorph H antigen is present

Secretor Gene

• Se (secretor) locus FUT 2

• 2 allele system – SeSe, Sese, or sese

• Individual (of appropriate ABO group) who has A, B, antigens in gastrointestinal, genitourinary, and respiratory tract secretions, as well as in milk, sweat, tears, & amniotic fluid (NOT in CSF).

• Secreted antigens are mostly glycoproteins (mucins), but also can be "free oligosaccharides" in milk & urine.

Secretor Gene

• Se has expression over the se amorph allele

  • SeSe / Sese ~ 80% secretors of A, B, and / or H

  • sese ~ 20% nonsecretors

ABH antigens & Se

• Glycolipids - imbedded in cell membranes

• Glycoproteins - secreted into exocrine fluids

• Fucosly-transferase adds L-fucose to make H antigen primarily on Type-1 chains

Distinction of ABH antigens & ABH Soluble Substances

Effect of secretor (FUT 2) Allele

Effects of the h Amorph Gene

• The hh genotype is called "Bombay" (designated as "Oh")

• No L-fucosyl-transferase produced

• No H substance / no H antigen

• No A or B antigens present on RBCs

• Only precursor substance found on RBCs

Bombay (Oh) Phenotype

• Cannot produce L-fucosyl-transferase; no H antigen produced - results in RBCs with no H, A, or B antigen.

• Bombay RBCs will NOT agglutinate with reagent Anti-A, reagent Anti-B, reagent Anti-A,B or reagent Anti-H (no antigens present); the patient types as an "O".

• Bombay people have Anti-A, Anti-B, Anti-A,B, and a potent Anti-H as “naturally occurring” Abs in their plasma.

• Agglutinating ALL ABO blood groups. What blood ABO blood group would you use to transfuse this patient??

ABO Subgroups

A Subgroups

• A1 & A2 both encode for the enzyme α-3-N-acetyl-galactos-aminyl-transferase; puts an immunodominant sugar N-acetyl-D-galactose on H antigen: A1 gene is very potent and produces much more enzyme than the A2 gene.

• A1 allele is prevalent over A2

  • A1A2 genotype → A1 phenotype

  • A2A2 or A2O genotype → A2 phenotype

• Most group A infants "appear to be" A2 at birth - ABH antigens not yet fully developed.

A Subgroups

• A2 cells do not always react well with Anti-A reagent.

• Quantitative differences:

  • Type # antigens / cell

  • A1 800,000 - 1,200,000

  • A2 ≈ 250,000

• Qualitative differences:

  • 1 - 8% of A2 people have Anti-A1 in sera

  • 22 -35% of A2B people have Anti-A1 in sera

  • A1 enzyme transferase is 5-10x more active which converts most of the H antigen to the A1 antigen.

A Subgroups

• Phenotypic reactions Reactions with:

  • Anti-A Anti-A1 (lectin)

  • A1 + +

  • A2 + -

• Frequency of A subgroups

  • Type % of A types % of all A types (A, AB)

  • A1 80 33

  • A2 20 8

Weak A Subgroups

• <1 % of all A types, e.g., A3, Ax, ABantu, Ael

• Most are a result of mutant A allele inheritance

• To detect:

  1. Anti-A,B sera obtained / manufactured from O donors

  2. Anti-H

  3. Adsorption-elution tests with Anti-A

  4. Saliva studies for A and H antigens

  5. Monoclonal Ab reagents are commonly used & because of their specificity, it makes weak subgroup reactions nearly irrelevant

• Also look for presence of Anti-A1 in the patient sera of A2 & A2B phenotypes.

B Subgroups

• Subgroups & weak subgroups of B are very rare.

• Classified & detected similarly to A subgroups, i.e., absorption- elution technique.

• Unlike an A Subgroup, the presence of an "anti-B1" does NOT exist.

"CIS AB"

• A and B genes are "next to each other"

• Antigens appear to be inherited together: AB parent + O parent = AB child Normally, what would be the offspring type?

• Gene mutations identified resulting in single transferase capable of transferring both GalNAc and Gal.

ABO Blood Group Testing

Laboratory Specimens

• Patient samples used for Blood Bank testing can be collected in any of these specimen tube types:

  • Purple top tube (contains RBCs & plasma)

  • Red top tube (contains serum and a clot)

  • Pink top tube (contains RBCs & plasma) - considered the official BB specimen

NOTE: To separate serum / plasma from the RBCs, spin specimen tube.

Blood Bank Patient Sample(s)

• Purple top tube (Hematology specimen)

  • Contains EDTA (Ethylenediaminetetraacetic acid) in either of 2 forms:

    • K2 EDTA - dried form coated along sides of plastic specimen tube

    • K3 EDTA - liquid form found in glass specimen tubes

  • EDTA binds Ca^{+2} to prevent clotting

  • Both types of EDTA are used in Hematology for CBCs, differential smears, platelet counts, sed rates, etc.

Blood Bank Patient Sample(s)

• Red top tube (Chemistry specimen)

  • Contains no anticoagulant or any additives; just a plain glass or plastic tube; blood will clot in this tube

  • Prior to performing BB tests, spin specimen tube for approx. 5 minutes

  • Use serum for Ab detection & identification

  • Use the sloughed- / sluffed-off RBCs found at the bottom of this specimen tube for blood typing

Blood Bank Patient Sample(s)

• Pink top tube (BB specimen)

  • Contains K2 EDTA - dried form coated along sides of a plastic specimen tube

  • Does NOT contain the liquid K3 EDTA anticoagulant, commonly used in Hematology analyzers.

    • Liquid K3, causes a slight dilution in the patient's plasma which may result in the undetection of a low-titer Ab. The Hematology analyzers account for the dilution effect from K3 EDTA tubes when analyzing the CBC.

  • This EDTA anticoagulant binds Ca^{+2} in the blood and prevents the blood from clotting

  • Preferred BB sample for all BB testing

  • Use both plasma & RBCs; must spin down sample

  • If you need to use a purple top tube from Hematology, ensure you use the K2 EDTA plastic tube…NOT the liquid K3 glass tube.

Blood Bank Reagents

• Blood banking reagents are categorized into 5 basic groups:

  1. Antisera - possess antibodies (Abs) of known specificity

  2. Red Cell Reagent - RBCs that possess antigens of known specificity

  3. Antiglobulin Reagents - detects IgG and / or C' on RBCs

  4. Potentiators / Enhancement Media - reagent use to increase Ab uptake to the corresponding antigen OR enhance RBC agglutination

  5. Lectins - plant extracts that react to specific human RBC antigens

Reagent - Common Lectin Reagents

Blood Bank Reagents

• When using any BB reagents, you must strictly follow these guidelines:

  • Carefully review the package inserts

  • Adhere to the package insert instruction for use

  • Adhere to the manufacturer’s restrictions & precautions - know your reagent's limitations!

Blood Bank Reagents - Antisera

• IgM - Usually contained in a "saline" media

• IgG - Usually contained in a "potentiator" media to enhance reactivity to RBCs OR in a "saline" media to react at the AHG phase of testing (antiglobulin reagent)

• IgA - There is no known IgA reagent, at this time

Blood Bank Reagents - Antisera (Polyclonal vs Monoclonal)

• Polyclonal

  • Usually an immune response

  • Produced by multiple B-cell lines

  • Broader specificity due to mixture of antibodies to multiple epitopes.

• Monoclonal

  • Produced by a single B-cell line

  • Specificity directed at single epitope (best type of reagent)

  • Identical antibody molecules

Blood Bank Reagents - Antisera Monoclonal Ab Advantages:

• Unlimited Production

• No human source - animal source Ab usually obtained through hybridoma technology

• IgM Ab, so direct agglutination occurs in shorter time

• No contaminating Ab(s)

• Standardized reagents

• Very little or no variation between batches

Blood Bank Reagents - Red Cell Reagents

• Used with patient sera to identify patient Abs that are present

• Used as "positive" & "negative" controls, when using a specific antisera reagent

ABO Blood Group System

• For typing a patient's ABO blood type, testing BOTH the patient's RBC & the plasma / serum is required.

• The testing scheme terminology using different reagents is known as:

  • For RBC testing - known as the "Forward" type / group

  • For the plasma / serum testing - known as the "Reverse" type / group

ABO Testing: Forward Type / Group

• Performed to detect A or B antigens on RBC membrane:

  • Always performed:

    • Unk. RBCs + Anti-A = aggl. ?

    • Unk. RBCs + Anti-B = aggl. ?

  • Optionally performed:

    • Unk. RBCs + Anti-A,B = aggl. ? (subgroups)

    • Unk. RBCs + Anti-H = aggl. ? (Bombay)

    • Unk. RBCs + Anti-A1 = aggl. ? (A subgroup)

    • Lectins : Anti-A1 (Dolichos biflores); Anti-H (Ulex europeous)

• Anti-A,B reagent is known as "group O" typing sera; contains anti-A,B of IgG2 subclass

• Forward types typically give 4+ reactions, unless subgroup or mixed field

ABO Forward Type / Group

*Anti-A,B use is optional

ABO Testing: Reverse Type / Group

• Performed to detect circulating Abs to A, B, AB, or H

• Uses known RBCs (reagent red cells).

  • Always performed:

    • Unk serum + A1 cells = aggl. ?

    • Unk serum + B cells = aggl. ?

  • Optionally performed:

    • Unk serum + O cells = aggl. ? (Bombay or unexpected alloantibodies)

• Should not be performed on children < 6 mos

• All forward / reverse discrepancies must be resolved!

Serologic Testing

• Please note the "order" of adding the reagents:

  • Add 1 drop of reagent antisera + 1 drop of patient cells; mix; spin; & read

  • Add 2 drop of patient serum + 1 drop of red cell reagent; mix; spin; & read

• Always add the "clear" substance(s) FIRST…this ensures that you've added the reagent (or serum) to the test system.

Serologic Testing

• Always add the "cells" LAST…this accomplishes 3 things:

  1. The reagent / serum was added to the test.

  2. The test is properly setup for serological testing.

  3. A "false" negative test result due to the lack of adding reagent or serum can be avoided.

ABO Reverse Type / Group

ABO Blood Typing Summary

ABO Antibodies (Abs)

• Aka, isohemagglutinins

• Mostly are IgM, i.e., primarily IgMs are found in groups A & B individuals; both IgM & IgG2 subclass found in group O individuals.

• Readily binds C'; aka, hemolysins

• Usually gives strong (4+) agglutination at IS / RT…Cold to thermo tolerant Abs

• IgM does not cross the placenta barrier

• “Naturally occurring” - all normal, healthy people have Abs to ABH antigens which they lack

ABO Abs

• Titers vary, e.g., type O people have higher titers of Anti-A than do B people

• May cause serious / fatal incompatible transfusion reactions

• May cause severe Hemolytic Disease of the Fetus & Newborn (HDFN); usually seen in group O mothers giving birth to group A neonates

ABO Abs

• Specificity

  • Anti-A

  • Anti-B

• Special attributes for group O individuals

  • Anti-A & anti-B IgMs both present

  • Anti-A,B (an IgG2 subclass Ab) is also present

  • Anti-A,B is not a "combination" of each anti-A & anti-B, but rather a single "cross-reacting" IgG Ab unique to Group O types

ABO Abs Age

• Before 3 to 6 mos -

  • Low titers of ABO Abs due to immaturity / undeveloped immune system

  • Do NOT perform reverse typing on a baby less than 6 mos old

  • In clinical practice, some hospitals will defer reverse typing until age 1 yr.

• By age 5 to 10 - adult levels of Abs are generally reached

• > 65 yrs - may have decreased ABO titers due to aging & weakened immune system

ABO Red Cell Compatibility Table

| Donor | O- | O+ | A- | A+ | B- | B+ | AB- | AB+ |
| :------ | :- | :- | :- | :- | :- | :- | :-- | :-- |
| Recipient |
| O- | ✓ | × | × | × | × | × | × | × |
| O+ | ✓ | ✓ | × | × | × | × | × | × |
| A- | ✓ | × | ✓ | × | × | × | × | × |
| A+ | ✓ | ✓ | ✓ | ✓ | × | × | × | × |
| B- | ✓ | × | × | × | ✓ | × | × | × |
| B+ | ✓ | ✓ | × | × | ✓ | ✓ | × | × |
| AB- | ✓ | × | ✓ | × | ✓ | × | ✓ | × |
| AB+ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ |

ABO Typing Discrepancies

• Technical / Clerical errors

• RBC reactivity with antisera:

  • Weak or mixed field

  • Unexpected reaction

• Plasma / serum reactivity with reagent RBCs:

  • Unexpected reaction

  • Missing or weak plasma reaction

Common ABO Discrepancy Causes

Common ABO Discrepancy Causes

ABO Discrepancy 1 - Test Results

What is the ABO Type?

ABO Discrepancy 1

ABO Discrepancy 1

Adding the Reverse Type What is the "possible" ABO Type?

ABO Discrepancy 1

What could be causing the Reverse Grouping problem?

ABO Discrepancies

• For A subgroup chart - see p 131, Table 6-14

• For B subgroup chart - see p 133, Table 6-15