(pt 2) exam #1 - immunohematology (cls 544)

history of the ABO blood group system

• 1901: Discovered by Dr. Karl Landsteiner

  • Noted three different patterns agglutination named A, B, C

ABO blood group system

most important of all blood groups in transfusion medicine

• blood group system in which people have antibodies in their blood to antigens that are absent from their RBCs without any prior exposure (transfusion or pregnancy)

• Due to these antibodies, transfusion of incompatible ABO type may result in immediate lysis of donor RBCs

  • Anti-A/B antibodies are IgM

• Transfusion of ABO-incompatible RBC units remains the leading cause of immediate hemolytic transfusion reaction (HTR) and HTR-related deaths reported by the FDA

  • After TRALI and TACO

forward/front typing

test individual's RBCs with commercial antisera

• looks for RBC antigens

reverse/back typing

tests patient plasma/serum with commercial RBCs

• looks for antibodies against RBCs

relationship between forward and reverse typing?

inverse reciprocal relationship

• the results of the two tests act as a built-in cross-check for accuracy

• ex: someone with A antigen will have anti-B antibody

reagents for ABO testing

• Anti-A, Anti-B, Anti-D, & Rh control for forward typing

• A1 and B cells for reverse typing

• A2 cells are also available (not routinely used)

T/F: reverse typing should be done on infants

false ; only forward typing should be performed

• ABO antibody production is too low at birth

  • Most antibodies in cord blood are maternal

• Reciprocal ABO antibodies are naturally occurring antibodies

  • Fully developed at 3 to 6 months after birth

ABO antibodies

• Predominantly IgM (pentamer)

• There are small quantities of IgG present

  • O blood type produce Anti-A, Anti-B and Anti-A,B

  • Anti-A,B is IgG and is a separate "cross-reacting" antibody

• Activate complement, cause intravascular hemolysis

• React best at room temperature (22 C) or colder

• Produce strong direct agglutination reactions

patient with type A blood has what type of antibodies? who can they donate/receive blood from?

• antibodies: Anti-B

• can donate blood to: type AB & A

• can receive blood from: type A & O

patient with type B blood has what type of antibodies? who can they donate/receive blood from?

• antibodies: Anti-A

• can donate blood to: type AB & B

• can receive blood from: type B & O

patient with type AB blood has what type of antibodies? who can they donate/receive blood from?

• antibodies: none

• can donate blood to: type AB

• can receive blood from: type A, B, AB, O

patient with type O blood has what type of antibodies? who can they donate/receive blood from?

• antibodies: Anti-A & Anti-B

• can donate blood to: type A, B, AB, O

• can receive blood from: type O

what blood type is known as the universal donor for RBC donation? universal recipient?

• universal donor: type O (specifically O neg)

• universal recipient type AB (specifically AB+)

**remember that it is the reverse for plasma !!!

inheritance of ABO blood groups

•  based on Mendelian genetics

  • Codominant expression (A, B)

  • O gene is considered an amorph

    • No detectable antigen produced by the inheritance of this gene, only H precursor substance (H antigen)

  • O phenotype can only be produced by two O genes (OO), autosomal recessive

what chromosome is the ABO blood groups on?

• chromosome 9

  • Three alleles: A, B, or O

  • A and B are co-dominant, A and B are dominant over O

H gene (FUT1)

inherited independent of the ABO genes, must be present to form ABO antigens on the RBCs

• Located on chromosome 19

• Codes for L-fucosyltransferase

  • L-fucose = H antigen, which the A and/or B antigens are attached

  • HH genotype very common (99.99%)

  • Hh rare

  • hh extremely rare (Bombay)

• H antigen is precursor substance for ABO

ABO immunodominant sugars

sugar that makes one antigen different from another antigen

• ABO antigens reside on sugar molecules attached to outside of RBC membrane

immunodominant sugar for H antigen + enzyme that adds the sugar

• sugar: L-fucose / fucose

• enzyme: α-1,2-L-fucosyltransferase

immunodominant sugar for A antigen + enzyme that adds the sugar

• sugar: N-acetyl-D-galactosamine

• enzyme: α-1,3-N-acetyl-galactosaminyltransferase

immunodominant sugar for B antigen + enzyme that adds the sugar

• sugar: D-galactose

• enzyme: α-1,3-D-galactosyltransferase

if H and A genes are present, what are the corresponding enzymes made? what are the antigens on the RBCs?

• enzymes: α-1,2-L-fucosyltransferase + α-1,3-N-acetyl-galactosamyltransferase

• antigens: H and A ; blood type would be A

if H and B genes are present, what are the corresponding enzymes made? what are the antigens on the RBCs?

• enzymes: α-1,2-L-fucosyltransferase + α-1,3-D-galactosyltransferase

• antigens: H and B antigens ; blood type would be B

if H and O genes are present, what are the corresponding enzymes made? what are the antigens on the RBCs?

• enzymes: α-1,2-L-fucosyltransferase

• antigens: H antigen ; blood type would be O

if H, A and B genes are present, what are the corresponding enzymes made? what are the antigens on the RBCs?

• enzymes: α-1,2-L-fucosyltransferase + α-1,3-N-acetyl-galactosaminyltransferase + α-1,3-D-galactosyltransferase

• antigens: H, A, and B ; blood type would be AB

ABO antigens

• ABO antigens are carbohydrate structures

  • H antigens serves as the precursor substance for both A and B antigens

• ABO antigen frequency and subgroups

  • The most common ABO blood group is O

  • Subgroups = genetic variation in ABO gene

    • e.g. A subgroup and B subgroup

formation of A, B, & H antigens

results from the interaction of genes at three separate loci: ABO, Hh (FUT1), and Se (FUT2)

• These genes code for specific glycosyltransferases that add sugars to a basic precursor substance

• H antigen is the precursor structure on which the A and B antigens are found

• ABH antigens are integral parts of the RBC membrane, endothelial cells, platelets, lymphocytes, and epithelial cells

at what age are ABO antigens fully expressed in humans? what age are an individuals own ABO antibodies present?

• ABO antigens are fully expressed at 2-4 years of age

• ABO antibodies not present until 3 to 6 months

**RBCs of newborns only carry 25-50% of antigens found on adult RBCs—forward typing will be weaker

glycoproteins

RBC carbohydrate/sugar molecules that carry A, B, H antigens attached to protein

• ABO and H blood group system carbohydrate chains are composed of hexoses

type I precursor substances

• found in gut lining, plasma, and secretions

• precursor substances are built off of the type I precursor chain (aka the β-1,3 connection in red—see image)

type 2 precursor substances

• found in A, B, H antigens ON RBC MEMBRANE

• build off type 2 precursor chains (aka the β-1,4 connection in red—see image)

molecular genetics of ABH antigens

• Investigation of ABO alleles, epitope structures, and exons

• ABO gene located on chromosome 9 and consists of seven exons

• Cis-AB

  • Very rare genotype

  • Inheritance of both A and B genes from one parent on same (cis) chromosome 9

  • O gene inherited from the other parent

  • Offspring inherit 3 ABO genes instead of two

formation of ABH soluble antigens

• H (FUT1) and Se (FUT2) genes are closely linked and located on chromosome 19

• Se gene must be inherited to form the ABH antigens in all body secretions

• ABH-soluble antigens found in all body secretions

• 80% of the random US population are known secretors

  • SeSe, Sese

secretors

• People who have ABH antigens in their body fluids

• Inheritance of Se (FUT2) gene codes for production of transferase that modifies the type 1 precursor substance in secretions = forms H substance

• H substance can be modified to express A and B substance in secretions (e.g. saliva)

  • Group A = secrete glycoproteins carrying both A and H antigens

non-secretors

• People who are sese do not produce any A, B, or H antigen in their secretions

• Make up ~20% of random US population

  • presence of Lewis antigens can help determine whether an individual is a secretor or non-secretor

  • Presence in secretions is dependent on ABO and secretor genes inherited

ABH substances in secretions vs ABH antigens on RBCs

• A, B, and/or H soluble substances in secretions are glycoproteins

• A, B, and/or H antigens on RBCs are glycolipids, glycoproteins, or glycosphingolipids

ABO subgroups

phenotypes that show weaker and variable serological reactivity with the commonly used human polyclonal Anti-A, Anti-B, and Anti-A,B reagents

• Common subgroups of A

  • A1 and A2 antigens

  • Other subgroups: A₃, A_x, A_el, and A_m → ID by molecular analysis

• Subgroups of B extremely rare

  • B₃, B_x, B_el and B_m

• Monoclonal typing reagents used routinely in ABO grouping tests currently

• Donors with very weak A antigen expression may be mistyped as group O

type A subgroups

• First described in 1911 by von Dungen

  • A1: react with Anti-A, and anti-A1

  • A2: react with Anti-A only

• Differences between A1 and A2 are both quantitative and qualitative

  • 80% of all A (or AB) are A1 (or A1B)

  • 20% of all A (or AB) are A2 (or A2B)

  • 1-8% of A2 individuals

• A subgroups are generally more common than B subgroups

how can you differentiate between A1 and A2 subgroups?

use anti-A1 lectin reagent

• Anti-A1 lectin agglutinates A1 or A1B cells but does not agglutinate A2 or A2B cells

characteristics of A₁ allele

• A1 transferase has proline at amino acid 156

• Elicits higher concentration of transferase

• Transferase is more efficient

• Converts almost all of H to A₁

• 810,000 to 1,170,000 antigen sites/RBC

characteristics of A₂ allele

• A₂ transferase has leucine at amino acid 156 and single base deletion

• Lower concentration of transferase

• Less efficient transferase

• 240,000-290,000 antigen sites/RBC

(lectin reagents) Dolichos biflorus

• aka anti-A₁ lectin

• agglutinates A₁ or A₁B

(lectin reagents) Bandeiraea simplicifolia

• aka anti-B lectin

• agglutinates B cells

(lectin reagents) Ulex europaeus

• aka anti-H lectin

• agglutinates O cells (H specificity) and other ABO blood groups depending on the amount of H antigen available on RBC surface

  • The amount of H antigen on RBCs of different blood types:

    • O  >  A₂ > B > A₂B > A₁ > A₁B

weak A subgroups

include: A₃, A_x, A_end, A_m, A_y, and A_el

• Most often discovered via ABO discrepancy

  • Unexpected reactions in the forward and/or reverse typing

• Varying expression of four characteristics

  • Decreased number of A antigen sites per RBC

  • Degree of agglutination by anti-A,B

  • Increased detectability of H antigen

  • Presence of absence of anti-A₁ in serum

weak B subgroups

• Very rare and less frequent than A subgroups

• Usually recognized by variations in the strength of the reaction using anti-B and anti-A,B antisera

• Result of alternative alleles at the B locus

• Serological techniques characterize B subgroups in the following categories

  • B₃, B_x, B_m, B_el

criteria for differentiation of weak B phenotypes

• Reactivity with anti-B, anti-A,B and anti-H

• Presence or absence of ABO antibodies

• Absorption-elution with anti-B

• B substance in saliva

• Molecular testing

bombay phenotype

• First reported by Bhende in 1951 in Bombay, India

• Inheritance of a double dose of the h gene, producing the genotype hh

• No H antigen made

  • ABO genes cannot be expressed

  • ABH antigens cannot be formed

  • Anti-A, Anti-B, Anti-A,B, and anti-H present

if an individual had the genotype hh and sese, would they have RBC antigens and ABH substances in their secretions?

nope

• lacks H antigen to attach immunodominant sugars for a blood type

• lacks Se gene for ABH to be present in secretions

para-bombay phenotype

• hh and SeSe/Sese genotypes

• H, A, and/or B antigens in secretions and plasma

bombay phenotype & testing

• Fail to react with anti-A, anti-B, anti-A,B and anti-H antisera

• In blood group testing, the Bombay would phenotype as an O blood group

• Unlike the anti-H found occasionally in the serum of A₁ and A₁B individuals, the Bombay anti-H can often be potent and reacts strongly at 37 C

• IgM antibody → can bind complement → cause intravascular hemolysis

• Can only be transfused with another Bombay (O_h)

list the blood types in order of most to least H antigen present on the RBCs

O > A₂ > B > A₂B > A₁ > A₁B

• Greatest amount of H antigen found on O cells

• Least amount of H found on A₁B cells

  • two transferase are present to attach sugar onto the H antigen, meaning that almost all H antigen is converted into A₁/B antigen

anti-H antibody

• Anti-H is occasionally found in the A₁ and A₁B serum

  • H antigen well hidden by N-acetyl-D-galactosamine

    • Reminder = conversion of H antigen into A antigen

• Anti-H naturally occurring IgM cold agglutinin that reacts best below RT

  • Can cause intravascular hemolysis

• Possible problems in antibody screening procedures

• Use of anti-H antisera (anti-H lectin)

bombay phenotype transfusion considerations

• Only blood from another Bombay individual will be compatible

• Underlying molecular defect of the Bombay phenotype

ABH antigens & antibodies in disease

• Hypogammaglobulinemia

  • Leukemias, e.g. CLL (absence of isoagglutinins)

• Depress antigen strength

  • Other leukemias with chromosome 9 translocations

  • Any hemolytic disease inducing stress hematopoiesis (e.g. thalassemia)

• "Acquired B" phenomenon in group A1 individuals

  • Group A1 RBCs absorb bacterial-like polysaccharide = reacts with anti-B reagent

ABO hemolytic disease of the fetus and newborn (HDFN)

• Maternal IgG antibodies cross placenta causing hemolysis of fetal RBCs with corresponding antigen

• Mainly occurs when mother is blood group O (anti-A,B = IgG) and fetus is blood type A or B

• May occur with the first pregnancy

• More common than Rh HDFN but not as severe

• Common cause of jaundice in newborn

  • Spherocytes observed on smear