Unit4: Other Major Blood Groups, Antiglobulin, Antibody Identification, & Crossmatch

Lewis Blood Group System

1946: Made & reported by Mourant, 1948: Ab anti-Le^b discovered reacting w/Le(a-) pts
Le^a & Le^b aren’t antithetical antigens by not coming from alternative alleles of a single gene
Comes from genes Le & Se encoding Fucosyltransferases interaction to produce it

Fucosyltransferase (FUT)

Enzyme that transfers L-fucose (FUC) from Guanosine Diphosphate (GDP)-fucose donor substrate to an acceptor substrate

Lewis Antigen Characteristics

Glycosphingolipid antigens made by tissue cells
Passively adsorbed on Type 1 Glycosphingolipid Chains onto the RBC membrane via plasma
Secreted in body fluids as antigens/glycoproteins

Se Gene (Secretor Gene)

Shares Chromosome 19 w/H gene
2 alleles in Secretor Locus: Se & se
se= amorph, no detectable product in secretions
Codes FUT to add FUC to Type 1 Precursor Chains on Body Fluids vs H gene codes FUC to type 2 precursor chains

Secretors

Individuals w/secretor genotype, present water-soluble ABH antigens in their body secretions, make up 80% of random pop
Homozygous: SeSe, Heterozygous: Sese

ABH Secretor Groups

Group O: H antigen in secretions
Group A or B: H w/A or B antigens in secretions
Group AB: H, A and B antigens in secretions

Nonsecretors

Individuals w/ homozygous se gene, fail to produce any ABO Antigens in their secretions, make up 20% of random pop

Lewis Gene (FUT3)

Linked to Se(FUT2) & H(FUT1) on chromosome 19, depends on fucosylate Type 1 interactions to produce Lewis Antigens by Lewis & Se gene
2 Alleles in Lewis Locus: Le & le

Lewis Allele Function

Le codes α1,4-L-FUT transferring FUC to Type 1H chain on glycoproteins/lipids= Le^b, if A/B gene= ALe^b/BLe^b
Small amount of Le^a made prior to secretor enzyme adds Terminal FUC

le= amorph= Le(a-b-) phenotype

Le(a+b-) phenotype

Le/H genes present= Le/H antigens, Se gene absent (sese)= A,B,H antigen Nonsecretor= no Le^b, A &/or B Antigens
Le^a secreted due to Le gene

Le(a-b+) phenotype

Inherited Le, Se & H gene= Le^a, Le^b, A, B, & H antigens
Le/Se present= Le^b made, only one adsorbed unto RBC surface from plasma

Le(a-b-) phenotype

No Lewis gene (lele)= no α1,4-L-FUT= no Le Antigens
Se gene present= A,B,H Antigens in secretions
No Se gene= no antigens in secretions

Genotype 4x more common in African Americans than Caucasians

Le(a+b+) phenotype

Weak Se gene= ineffective competition between Se & Le FUT= weak antigen expression, Insignificant & rare

Lewis Antigen Post Birth Developments & Changes

Undeveloped at birth, require adsorption to appear, Cord blood & newborn RBCs= Le(a-b-), detectable in plasma 10 days post birth
Transforms to true Lewis Phenotype (Le(a-b+) post 6yrs
If Le/sese genotype= no Lewis antigens in cord cells except Le^a in saliva

LeSe Gene Development

Le(a-b-)→ Le(a+b-) post 10d→ Le(a+b+)→ Le(a-b+) post 6 yrs (true phenotype)

Le sese Gene Development

Le(a-b-)→ Le(a+b-) post 10d

lele Gene Development

Le(a-b-) for life

Lewis Antigen Clinical Significance

Not intrinsic to RBC membrane, shed from transfused RBCs a few days post transfusion, transfused plasma neutralize anti-Le^a/Le^b= rarely hemolyzes transfused RBCs

Le^a Antigen Formation

Made from Le FUT acting on Type 1 Precursor, found in secretions, ISBT#: LE1

Le^b Antigen Formation

Made from transferring FUC to 1H chain, ISBT#: LE2

Le^ab

Anti-Le^ab reacts w/Le(a+b-) & Le(a-b+) RBCs from adults & 90% cord RBCs, ISBT#: LE3

Le^bH

Anti-Le^bH reacts w/O Le(b+) & A₂ Le(b+) RBCs, ISBT#: LE4

Ale^b

Anti-Ale^b reacts with group A₁ Le(b+) & A₁B Le(b+) RBCs
ISBT#: LE 5

Ble^b

Anti-Ble^b reacts with group B Le(b+) & A₁B Le(b+) RBCs
ISBT#: LE 6

Pregnancy & Lewis Antigens

Antigen strength declines dramatically, physiologic changes in RBC composition affect Lewis glycolipid distribution between plasma & RBCs, large↑ in plasma lipoprotein:RBC mass ratio

Pregnant females make transient Lewis Abs (Anti-Le^a)

Lewis System Antibodies

Anti-Le^a/Le^b found in Le(a-b-) Secretor serum, naturally occurring Abs
ID Techniques: Ab neutralization w/Prepped Soluble Lewis Antigens or Enzyme-Treated Cells that enhance Ab reactivity

Anti-Le^a Antibody

Most common, reacts w/30% RBCs in reagent red cell panel (3/10 crossmatched units), if Le(a-b+)= no Anti-Le^a Abs
Clinically Insignificant: IgM majority & common in prenatal serum
Reacts best at RT, disappears post 37°C incubation & AHG Phase

Anti-Le^b Antibody

Uncommon, weaker than Anti-Le^a, made by Le(a+b-) pts who receive Le^bpos RBCs (very rare, poor immunogen)
Clinically Insignificant: IgM majority & often reacts RT, poor complement activator

Lewis Antibody Clinical Significance

Generally Insignificant: Neutralized by Lewis Substances in plasma & decreased in quantity, Antigens dissociate from RBC as easily as they bind to it, cannot cross placenta nor cause HDFN

Lewis Antibodies Biological Significance

Extremely significant, assoc.w/Peptic Ulcers, Cardiac Ischemia, Cancer, Kidney Transplant Rejection, Helicobacter pylori

Helicobacter pylori

Pathogen, causes gastric/duodenal ulcers, muscosa-assoc. lymphoid tissue lymphoma, atrophic gastritis & adenocarcinoma
Lewis Antigens have receptors to interact w/it

MNS Blood Group

Discovered by Landstein & Levine recovering MN Abs from rabbits immunized w/human RBCs
Walsh & Montgomery discovered S antigen w/Antiglobulin Test
Molecular Genetics show close linkage between genes controlling M,N, & S/s antigens

M&N Antigens

Found on Glycophorin A (GPA), Antithetical & differ in amino acid residue at position 1&5
M= serine at 1, glycine at 5, N= leucine at 1, glutamic acid at 5
Phenotypes: M+N-, M+N+, M-N+

S/s Antigens

Located on smaller glycophorin B (GPB, similar to GPA), differs by amino acid at position 29- S=Methionine, s=Threonine
Phenotypes: S+s-, S+s+, S-s+, S-s-U-

Anti-M/Anti-N Antibodies

Clinically insignificant, often IgM, common in human sera, cold agglutinin (RT, optimal @4°C), weak/unreactive at 37°C, exhibit dosage w/M+N- cells, pH dependent (6.5 optimal), M antigen destroyed by enzyme treated red cells=Anti-M unreactive

Anti-M/Anti-N Clinical Significance

Insignificant if unreactive at <37°C or isn’t an IgG Anti-M (causes HTRs/HDFN), Anti-N rare, weak, IgM cold agglutinin, can’t activate complement= insignificant, express dosage w/M-N+ cells

Anti-S/Anti-s Antibodies

Uncommon Abs, IgG & immunogenic, react best at 37°C & reacts in AHG Phase= clinically significant, bind complement, assoc. w/fatal HTRs & HDFN, but require exposure to obtain

Anti-U Antibody

Rare Ab, high frequency, IgG & react best at 37°C=clinically significant, almost exclusive in African Americans, assoc. w/HTRs & HDFN
U antigen present in 100% Caucasians & 99% African Americans, consult AABB Rare Donor Registry for Uneg transfusions (<10 in US)

En(a-) Phenotype

Rare homozygous gene deletion at GYPA locus, GYPB unaffected, produces Anti-En^a Abs, transfusion near impossible w/siblings as potential donors

M^k Phenotype

Single near-complete deletion of GYPA/GYPB, null MN system phenotype

MN Antigen Enzyme Treatment

Destroyed by Ficin, Papain, Bromelin, Trypsin, Pronase, ZZAP, DTT+Papain/Ficin, AET (2-aminoethylisothiouronium)

S/s Antigen Enzyme Treatment

Destroyed by Ficin, Papain, Bromelin, Pronase, Chymotrypsin

P & Globoside Blood Group Antigens

Found on RBC & WBCs, synthesized by glycosyltransferases adding sugars to lactosylceramide
Antigens: P, P1, P^k, Luke (LKE)

P Antigen (GLOB 028)

Glob gene in chromosome 3, produces transferases transforming P^k to P antigen, high RBC incidence: 14×10⁶ P antigen vs 5×10⁵ P1 antigen copies

P1 Antigen (P1PK 003)

Most common P Ag in P BGS, req. 7 yrs to mature expression
P1 gene in Chromosome 22, codes transferase to make P1 on glycosylated Type 2 Precursors= Carries ABH Antigens

P1 Antigen Characteristics

black pop P1 expression> white pop P1 expression
Inhibited by rare dom gene ln(lu) type Lu(a-b-) RBCs= P1 neg
Deteriorates fast, high false neg w/Anti-P1 Ab
Found in pts w/parasitic tape worm hydatid cyst infections

P^k Antigen

Rare low freq. antigen, expressed on all RBCs except phenotype p, undetected w/P present, on chromosome 22 codes P^k production
Like P, found as glycosphingolipids in plasma & glycoproteins in hydatid cysts

P Blood Group Phenotypes

P₁, P₂, P, P₁^k, P₂^k

P Blood Group System Antibodies

Separable through adsorption techniques, efficiently bind complement & made of IgM & IgG type components, all produced by p individuals

Ab Types: Anti-P₁, Anti-P, Anti-P^k, & Anti-P+P₁+P^k

Anti-P₁ Antibody

Often naturally occuring IgM= clinically insignificant: Found in P₂ pts, reacts optimally at 4°C & not assoc. w/HDFN
Clinically significant if reacts at 37°C= immediate/delayed HTRs

Anti-P (Donath-Landsteiner) Antibody

Found in P^k pts, clinically significant biphasic hemolysin IgG, must have Pneg RBCs, cause spontaneous abortions in P^k & p females

Biphasic Hemolysin

Ab that attaches to RBCs in the cold & lyse them when warmed

Anti-P^k Antibody

Clinically significant IgG, made by p individuals

Anti-P+P₁+P^k Antibody

All 3 P/GLOB system Abs found in p individuals, clinically significant IgG type (Anti-P/P^k)= severe HTRs & HDFN + effective complement binding

P Antigen Enzyme Treatment

Resistant to treatment w/Ficin+Papain, DTT, Chloroquine, & Glycine-Acid EDTA

I_adult antigen & i Antigen Relationship

Developmental regulated reciprocal relationship, all human RBCs have both antigens but express in different stages of life development, I antigen= I_adult

icord antigenic structure

Linear structure in newborn RBCs w/repeating N-acetyllactosamine units in umbilical cord cells, rich in i antigens but little to no I_adult antigens

I_adult antigen development

Depends on branching N-acetyllactosamine units as individual matures, gradually develops until 2yrs of age
↑I_adult=↓i antigen during maturity, RBCs at ≥2yo express I_adult pos & weak/neg for i antigen

i_adult Characteristics

I antigen never develops, retain I-i+ phenotype & are strong pos for RBC i antigen, can only receive rare i blood due to rare auto anti-i ab & mask presence of other significant abs

Anti-i Antibody Characteristics

Fairly rare autoAb, seen in infectious mononucleosis (EBV), myeloid leukemia, alcoholic cirrhosis & rerticulosis

Auto Anti-I Antibody Characteristics

Nonspecific cold agglutinin autoAb in I pos people, interferes w/in vitro testing at low temp, doesn’t prevent transfusions due to shared I antigen
Blood prewarmed prior to infusion at 37°C, hemolyzes at ≥40°C

Auto Anti-I Serological Properties

Naturally occurring, 1-6°C optimum temp, no react/weak in RT, IgM, enhanced activity w/albumin enzyme media, sensitized to complement at antiglobulin phase

Anti-i Antibody Serological Properties

Rarely causes hemolysis, weak neg reaction w/I adult & autologous cells, Cold Ab w/same serological properties as anti-I, naturally occurring

Auto-anti I & Cold Agglutinin Diseases

Autoanti-I made via pathogen stimulation carrying I-like antigens, assoc.w/Mycoplasma pneumonia= transient acute abrupt hemolysis upon infection resolving

autoanti-i assoc.w/Infectious Mononucleosis (EBV)

Ii Antigen Enzyme reactions

Enhanced reactivity w/ficin & papain
Resistant to DTT & glycine-acid EDTA

Kell (006) Blood Group System

32 antigens encoded by closely linked gene loci made of subloci w/alleles
Major Alleles: K& k- makes K & k antigens
Alternate Alleles/antigens: Kp^a, Kp^b, Js^a, Js^b
Amorph: K_o

K_x Blood Group System (019)

XK gene makes K_x proteins, found in X chromosome, only expressed in males, protein covalently linked to Kell glycoproteins, essential for Kell system antigen expression on RBCs

Kell Antigen General Characteristics

Mature at birth, unaffected by ficin/papain, destroyed by DTT/2-ME, show dosage, artificially prep RBCs lacking Kell Antigens to ID Kell Abs, may impair/destroy reactivity of other antigens

Kell Antigen Types

K- powerful immunogen 2^nd to D antigen, induces alloimmunization, low freq (found in 9% whites, 4% blacks)
k- high freq antigen detectable in 7wks
Kp^b & Js^b- high freq antigen
Kp^a- low freq antigen found primarily in whites
Js^a- low freq antigen found primarily in blacks

K_o (Kell Null) Phenotype

inherited from K_oK_o, lacks all K antigens but express lots of K_x antigens, assoc.w/ structural & functional RBC abnormalities
Makes clinically significant Anti-Ku ab, requires K_o neg blood

AET & ZZAP enzymes destroys all K antigens= artificially induce Kell null RBCs

Common Kell Phenotypes

K/k: K-k+, K+k+, K+k-
Kp: Kp(a+b-), Kp(a+b+), Kp(a-b+)
Js: Js(a+b-), Js(a+b+), Js(a-b+)

Anti-K Antibody

Most clinically significant after ABO/Rh Abs, IgG, detected at 37°C & AHG phase, assoc.w/HTRs & HDFN

Anti-k/Kp^a/Kp^b/Js^a/Js^b Antibody Characteristics

Rare abs, similar serology & clinically significant, assoc.w/HDFN
low freq (anti-Kp^a/Js^a) detected via unexpected incompatible crossmatch
High freq (Anti-k/Kp^b/Js^b) rare due to few people lacking antigen

McLeod Syndrome

Discovered by Allen et al. in 1961, appears Kell null but shows weak k, Kp^b & Js^b expression via adsorption-elution methods
Lacking XK gene= poor Kell antigen expression

Signs/Symptoms of McLeod Syndrome

Shortened RBC survival, acanthocytosis, anisocytosis, ↓H₂O permeability=↑Osmotic Fragility, reticulocytosis, cardiomyopathy & splenomegaly
high risk of developing Chronic Granulomatous Disease (CGD)

Kell/Kx Antigen Enzyme Treatment

Destroyed by trypsin & chymotrypsin w/Thiol reducing agents (DDT, 2-ME, AET, & ZZAP)
Unaffected by Ficin & Papain

Duffy (008) Blood Group System

F_y gene makes transmembrane carrier for Duffy glycoproteins, easily removed/destroyed by enzymes, responsible for malaria susceptibility from Plasmodium vivax
Fy^x gene= no distinct antigen, inherited weak Fy^b reacts w/some but not all Duffy Abs like Anti-Fy^b

Duffy System Antigens

6 Antigens: Fy^a, Fy^b, Fy3, Fy4, Fy5, Fy6
Alleles: Fy^a & Fy^b, expresses Fy^a & Fy^b antigens that are mature at birth & codominant, destroyed by ficin, papain & bromelin

Fy(a-b-) Phenotype

Lack Fy6 receptor, malaria resistant, found in approx.100% African blacks & 68% in American Blacks, rare in whites

Genetic Origins of Fy(a-b-)

Blacks express Fy4 antigen, whites express Fy3 antigen
Blacks make anti-Fy3 Abs receiving blood from whites= HTR from future donors, require Fy3 neg RBCs

Anti-Fy^a/Anti-Fy^b Antibodies

Most common Fy Abs, clinically significant IgG, react best in IAT phase, exhibit dosage= homozygous Fy(a+b-) ID Anti-Fy^a, Fy(a-b+) ID Anti-Fy^b
Weak duffy Abs may not react w/Fy(a+b+) RBCs

Duffy Antigen Enzyme Treatment

Proteolytic enzymes destroys Duffy antigens, resist neuraminidase & purified trypsin
Anti-Fy^a & Anti-Fy^b can’t react to enzyme treated RBCs, helps ID multiple Abs in serum containing them

Kidd Blood Group System (009)

JK gene makes urea transporter molecule on RBC membrane
JK(a-b-) RBCs lysis resistant to 2M urea due to defect, screens blood units to ID rare JK(a-b-) donors

Kidd (JK) Locus

3 alleles: Jk^a & Jk^b codominant, Jk³ found in all Jk^a/Jk^b RBCs (Jk^a/JK³, Jk^b/Jk³) but not on the JK(a-b-) RBCs

Jk(a-b-) Phenotype

Inherited via genetic mutations or independently of dominant suppressor ln(Jk) gene, found in Polynesian/Chinese populations
Expresses ↓quantities of weak Jk^a/Jk^b/Jk³ antigens to its abs

Kidd System Antigen Characteristics

Jk^a frequent in 77% Caucasians, 91% African Americans, Jk^b frequent in 73% Caucasians, 43% in African Americans
Mature at birth, poor immunogens, can’t be destroyed by enzymes

Anti-Jk^a & Anti-Jk^b Antibodies

Mostly IgG, efficient complement activation, poor immunogen= often combined w/other alloabs, rarely a lone ab

Anti-Jk^a & Anti-Jk^b Characteristics

Difficult detection: very weak reactions & display dosage, deteriorate fast in storage & few months in circulation=neg in ab test
Appears pos 1wk post transfusion in Kidd pos blood= DHTR
Represent 23% of abs in AHG testing

Kidds Ab Role in DHTRs

Made in response to pregnancy/transfusions, difficult detection=gradual titer decline in vivo, commonly cause DHTRs & infrequent mild HDFN

Anti-Jk³ Antibodies

Made by Jk(a-b-) individuals, react w/all JK^a+/Jk^b+ cells, inseparable from Anti-Jk^a/Anti-Jk^b

Autoantibody JK^a

Made by Jk^a antigen due to LISS preservative Paraben altering its ability to recognize itself,
seen in: Warm AHAs, Methyldopa DIHA, pts w/recent viral/bacterial infections, & pts receiving meds preserved w/Paraban

Lutheran Blood Group System (005)

LU locus linked to SE (secretor) locus, 19 antigens, 1^st ex of autosomal linkage in humans, makes Lutheran glycoprotein, changes in amino acids= variation in Luther antigens

Lutheran Antigens

Low freq Lu^a, high freq Lu^b, Antithetical to each other & differ by 1 amino acid
Lu(a-b-) Null Phenotype inherited via Dominant lnLu suppressor gene, Recessive LuLu gene, & Recessive X-Linked gene

Dominant lnLu Suppressor Gene

Located outside LU locus, suppresses Lutheran antigen expression but makes small amounts= no abs if exposed to Lutheran pos blood
suppresses P₁ antigen expression

Recessive LuLu gene

Inherits 2 null genes, makes anti-Lu³ Ab against Lu^a/Lu^b pos RBCs

Recessive Lutheran X-Linked Gene

Inherited recessive suppressor gene on X chromosome, inhibits Lutheran antigen expression= Lu(a-b-) phenotype, doesn’t form Abs when given Lutheran pos blood

Anti-Lu^a Antibody

Uncommon clinically insignificant naturally-occurring saline reactive Ab, react better at RT (22°C) than 37°C (except a few w/IAT)
Characterized loose mixed-field reaction in-vitro, show dosage due to variable antigen expression

Anti-Lu^b Antibody

Rare ab due to most RBC being Lu^b+, made after transfusion/pregnancy, clinically significant IgG, reacts optimally at 37°C & cause mild HTRs

Anti-Lu³ Antibody

Rare ab made by Lu(a-b-) pts inheriting LuLu, reacts w/AHG phase

Blood Banking Significance of Other BG Abs

Clinically Insignificant: M, P₁, I react at RT
Clinically Significant: K, S/s, Fy^a/Fy^b, & Jk^a/Jk^b react w/AHG phase
Silent, Regulator, & Inhibitor Genes can affect antigen expression & make panel reactions inconclusive, require Immunohematology Lab Reference Assistance