Immunohematology Unit 1

Started on slide 71 of ABO + Hh Blood Group System

A2 phenotype and genotype

Larger protein with reduced activity (less functional) due to a nucleotide deletion

¼ of the sites as A1, less active enzyme

A1 vs A2

A1 transferase adds GalNAc to type 4 chains (Super-branching, aka Globo-A) (A1 have lots of Globo-A)

A2 can make anti-A1

A1 and A2 can be a part of Type 3 chain repetitive structures

Quantitative difference in the number of antigen sites

Qualitative difference in A2 can make Anti-A1 and A1 transferase activity can lead to Globo-A

What is the number one reason for an ABO discrepancy

an A2 individual making Anti-A1

difference between A1 and A2 Ag

there is no A2 Ag!

it is all A antigen with slightly different biochemical structures containing the same epitope and immunodominant sugar

ABO Blood Group System Abs

Anti-A, -B, -A,B, -A1

Naturally occurring

what antibodies cause the most serious form of acute immune hemolysis

ABO Abs

intravascular (complement activated)

Anti-A and -B

Naturally occurring, highest titers in O persons

Mostly IgM in group A or B, mostly IgG in Group O

IgM > IgG > IgA

activate complement, intravascular hemolysis, HTR cause, and HDFN cause

IgM optimal temperature

4C

IgG optimum temperature

4C and 37C

Anti-A,B

found in group O persons

Absorbed by either A or B RBCs

reacts with all non-O blood groups

Anti-A1

found in 1-8% of A2 individuals

found in 1/3 of A2B individuals

considered insignificant

Dolichos biflorus

Lectin

proteins that bind to carbohydrates, found in plants/seeds

Anti-H

Ulex europaeus

very rare, insignificant unless truly H deficient (nonsecretors)

cold-reactive

Can cause HTR

H Deficient phenotypes

Bombay Phenotype

Para-Bombay

Bombay

1952 by Bhende

no ABH detected on RBCs

described as “O_h” type (O without H)

No H (FUT1) or Se (FUT 2) gene

Para-Bombay

Weak ABH detected on RBCs (A or B genes can be present, A_h or B_h)

No detectable H on RBCs

No H (FUT 1) gene

Se (FUT 2) gene is present!

What is an ABO discrepancy

Present and historical ABO do not match

Forward and Reverse ABO do not match

Must be resolved before issuing type specific products

How to transfuse those with an unresolved ABO discrepancy

Group O RBCs and AB plasma

Steps to solving ABO Discrepancies

Repeat testing (technical errors)

See history (pregnancy, transfusion, transplant)

Assume F or R is correct

Perform testing to resolve the discrepancy

Initial Testing of RBCs to Resolve ABO Discrepancies

Type with Anti-A,B, Anti-A1 lectin, and try different manufacturers of -A, -B, and -A,B,

Test O cells (screening cells) at IS

Causes of ABO discrepancies

extra antigens (F)

extra antibodies (R)

missing antigens (F)

missing antibodies (R)

mixed field agglutination (F, most often caused by blood transfusion or fetal/maternal bleed)

A3 subgroup

an inherited mutation in A gene

causes Anti-A and Anti-A,B Forward type to be 4+mf

weaker than A2 with anti-A1

Chimerism

Forward testing with 4+mf

Fraternal twins

mosaicism from dispermy

What is mixed field agglutination

First, small and tightly agglutinated clumps of cells that chip off the button

Followed by a stream of free cells creating a smoky looking background

(or vice versa)

Unagglutinated RBCs may appear as a halo

B(A) type

Extra A Antigen

Active B gene transferase adds some GalNac

Typed as a B and can be given to Bs

What is Acquired B type

Found in A1 persons

Deacetylase enzyme removes the acetylase from GalNac (A immunodominant sugar) to make it B-like Immunodominant sugar (Galactosamine rather than galactose)

What is the significance of Antibody Coated RBCs

May cause spontaneous agglutination of RBCs following centrifugation

leads to a positive control, invalidating all responses because it agglutinated regardless of reagent

Causes of Extra Antigen reactions

Type B(A), Acquired B, and Antibody Coated RBCs

Causes of Missing Antigen reactions

Subgroups of A or B

Serological characteristics of A subgroups

A1; A2; A3 has an Anti-A and -A,B mf; Ael types as an O

33 and counting well defined alleles

Mutations mainly in exon 6 and 7

Serological characteristics of B subgroups

B3 has mixed field reactions for Anti-B and Anti-A,B

much less common

other weak Ag possible

Causes of Extra Antibody

“Ab reacts with an Ag different than A or B Reverse type cells”

A2 with Anti-A1 (the most common ABO discrepancy)

Cold Autoantibody

Cold reactive Alloantibodies

Rouleaux (all red cells rested with pt plasma will be +)

What are Cold Autoantibodies

less specific

makes whole screen 3+

Autoanti-I, -IH, -i,

What are cold reactive Allo antibodies

more specific (ex. cell II screen 3+ only)

Alloanti-M, -P₁, -Le^a, etc…

Rouleaux effect

caused by abnormal serum/plasma proteins in multiple myeloma for example

SALINE REPLACEMENT of plasma fixes

Causes of Missing antibody

infants, old, immunodeficiency

Group A or B transplanted with group O

What are advanced testing of RBCs to resolve ABO discrepancies

Acidified Anti-B

Lectins

Treat with chemicals to remove Abs

addditional testing of serum/plasma to resolve ABO discrepancies

prewarm reverse typing (for cold reactive Abs)

What to consider based on patient therapy

Transfusion —> mixed field

infused with ABO compatible plasma products —> extra Abs

HPC transplant —> mf and missing Ab

Hodgkin’s —> weak or missing antigens

Multiple myeloma —> extra Ab and rouleaux

What type of antigen is Rh

Protein antigen, transmembrane protein

Very immunogenic

History of Rh

1940: Landsteiner and Wiener ran experiments in guinea pigs/rabbits were injected with Rhesus monkey cells, hence “Rh”

1941: Levine linked HDFN with anti-Rh; Fisher described CcE

Frequency of Rh

White: 85% RhD Positive and 15% RhD Negative

Black: 92% + and 6.8% =

Asian: 99.9% + and 0.1% =

Native American: 99% + and 1% =

Immunogenicity of D

D is the second most important antigen

VERY Immunogenic

Abs cause HDFN and HTR

Most D= individuals lack the entire RhD protein

Large number of amino acid changes (32-35 aa) seen as foreign and cause a potent immune response

What is the Weiner Theory of Rh Inheritance?

Single gene at Rh locus

Gene product is an agglutinogen (haplotype) [agglutinogen is composed of factors/epitope

Haplotypes: R¹, r, R², R^o, r’, r’’, R^z, r^y

Fisher-Race theory of Rh Inheritance

3 closely linked loci, each with a set of allelic genes (D/d, C/c, E/e)

rare crossing over

3 genes inherited as complex

R¹

D, C, e

r

c, e

R²

D, c, E

R^o

D, c, e

r’

D, c, e

r’’

c, E

R^z

D, C, E

r^y

C,E

What are the most common Weiner Haplotypes in Whites

R¹, R², r

What are the most common Weiner haplotypes in Blacks

R¹, R², r, and R^o

What is Rosenfield Nomenclature?

A system based on serologic observations

Ags numbers in order of discovery

Ag present ex is Rh:1 (D+)

Ag absent ex is Rh:-1 (D=)

What are the RH genes

RHD, RHCE, and RHAG

RHD and RHCE

on Chromosome 1 and is polymorphic

RHD codes for the presence or absence of D

RHCE codes for Ce, CE, cE, or ce

Both transport Ammonia and maybe CO2

Structural role in RBC membrane

RHAG

On Chromosome 6 and monomorphic

An ancestral gene required for D, C, and E expression

“the gene product is required for the expression of the Rh protiens”

Carry ABO structures

Help assemble the mature red cell cytoskeleton

Rh_null cells are fragile, spherocytic and stomatocytic

RhD Negative reasons for African Background

RHD deletion, inactivating mutation of RHD psi (3%), or Hybrid RHD-CE-D (3-9%, an insertion of RHCE in RHD resulting in no D Ag and weak c)

Rh Proteins

2 distinct polypeptides (D and G, C and G or c and E or e)

Major fatty acid acylated protein in lipid membrane

transmembrane protein that crosses membrane 12 times

hydrophobic

RhCE

E is found in the Rh vestibule, C on the top edge

Anti-D

“Rock Star” of Antibodies

Most studied of all antibodies to RBC antigens

memory lasts for years, strong IgG response within 24 hours of re-stimulation

Most are IgG 1 or 3

Implicated in HDN and HTR

Always come in a gray reagent bottle

Weak Expression of RhD

D^u: a test for weak D Ag

Negative with anti-D at IS and Positive with IgG anti-D at IAT

Described by Stratton in 1946

Serologic Weak D Definition

Identified by weak reactivity or by discordant typing results

2.9% of RhD= individuals in a mixed population

Not at risk of making anti-D

C in trans with RHD (Ceppellini effect, r’ haplotype), most common with unknown cause

Weak D “Types”: amino acid change, usually a single change [Reese’s pieces but with less in the box depending on type] (types 1,2, and 3 are most common in whites; because no outer Ag/epitope changes, only number; Type 4.0 and 4.1)

At risk of making Anti-D

Partial Ds: hybrid RHD alleles (Missing one of the Reese’s pieces colors, lack of exofacial epitopes or have altered exofacial epitopes due to missense mutations; Partial DVI is the most famous in European Ancestry “DVI” due to 2 reports of fatal hydrops fetalis; DIIIa and DIVa most common in African American populations and type as RhD positive at IS which is a problem

DEL: Type as D=, change due to deletion on exon 9 and common in Korea