INTRODUCTION TO IMMUNOHEMATOLOGY

Immunohematology

 Studies antigen-antibody reactions (w/ regards to blood) and analogous phenomena as they
relate to the pathogenesis and clinical manifestations of blood disorders.
Antigen-antibody reaction – refer if an antibody will attach to antigen
Antigen – foreign that enters the body
Antibody – substances produce by immune system in response to antigen

Blood Banking

 Refers to the process of collecting, separating, and storing blood

Blood is biological specimen and considered as drug and cause adverse reaction
e.g. Traly – Transfusion Related Acute Lung Injury - nasobrahan ng transfuse ng blood sa
patient, it causes lung injury)
e.g. TACO – transfusion Associated Circulatory Overload – increase volume of blood being
pump

Historical Overview

 1492 – blood was from 3 young men and given to POPE INNOCENT VII
 Pope Innocent VII – first time a blood transfusion was recorded in history
o Transfusion is not successful, all of them died (4), and clotting was the principle obstacle
to overcome. 1st the attempt to find nontoxic coagulant, next, the devices for
performing transfusion is not available that time, and next, Development of preservative
solutions to enhance the metabolism of the RBC (120 days survival of RBC)

Find non-toxic anticoagulant

 1869 Braxton Hicks – Sodium phosphate

o 1st example of blood preservation research
 1901 Karl Landsteiner – discover ABO Blood Groups
o Improve the quality of blood transfusion
 Edward E. Lindemann – vein to vein transfusion
 Unger – designed his syringe-valve apparatus
 1914 Hustin – sodium citrate as an anticoagulant but can lead to citrate toxicity kung saan
nahihirapan mag clot yung blood
 1915 – Lewisohn – minimum amount/concentration of citrate needed for anticoagulation
Development of preservative solutions to enhance the metabolism of the RBC:
o Rous and Turner – introduced a citrate-dextrose solution for the preservation of blood
 Dextrose – different concentration of glucose; gives energy
o Dr. Charles Drew – developing techniques in blood transfusion and blood prevention
 Became the model for the national volunteer blood donor program of the
American Red Cross (world war II)
  February 1941 – Dr. Drew was appointed as the Director of the first American Red Cross blood
bank at the Presbyterian Hospital
 1943 Loutit and Mollison – introduced the formula for the preservative acid-citrate-dextrose
(ACD)
 July 1947 – Journal of Clinical Investigation was published – devoted nearly a dozen papers to
blood preservation; contains records of blood preservations
 1957 Gibson introduced an improved preservative solution called CPD (citrate-phosphate-
dextrose)
o Studies found out that acid citrate dextrose is very acidic, kaya pag na transfuse na sa
human hindi na nagiging balance yung katawan, kaya Gibson improve ACD to CPD
o CPD is less acidic and replace ACD as the standard preservative use for blood storage.
But there’s a problem with CPD, CIRCULATORY OVERLOAD (hindi na standardize yung
composition ng citrate, phosphate and dextrose)
 Due to Frequent transfusion and the massive use of blood resulted in new
problems
o Component Therapy - hindi pala dapat laging whole blood ang trinatransfuse, so there
is specific component na dapat kailangan ng patient
 E.g. Those suffering from severe chronic anemia, wherein there is decrease of
RBC, once na detect ng katawan, body will increase plasma, then when you
collate with blood bank, anong magandang component kaya ang ibibigay?
PACKED RED BLOOD CELL ONLY Anemia pxt
→

PRESENT OVERVIEW OF IMMUNOHEMATOLOGY

 The amount of whole blood in a unit has been 450mL +/- 10% of blood (1 Pint)
o In 450 mL kasama na ang anticoagulant with at least 63 mL – 70 mL of anticoagulant
present in 1 blood bag
 More recently 500 mL +/- 10% of blood is collected
 For a 110 lb donor, a maximum of 525 mL of blood can be collected
o 110lb = 50kgs
 Total blood volume for most adult is 10 to 12 pints
 Donors can be replenish the fluid lost from the donation of 1 pint in 24 hours
 The donors red cells are replaced within 1 to 2 months after donation
o In the Philippines, referral for donation of blood, wait for 3 months/12 weeks for
donation of blood
o Referral – term used if pwede/bawal bang mag donate si donor Plasma
Platelets
•

 A volunteer donor can donate blood every 8 weeks PRBC

•

 Units of whole blood can be separated into three components (FFT/plasma, cryoprecipitate,
platelets)
 The plasma can be converted by cryoprecipitate to a clotting factor concentrate that is rich
in antihemophilic factor
 A unit of whole blood-prepared RBCs may be stored for 21 to 42 days, depending on the
anticoagulant-preservative solution GDD / ACD shelf life 21dam :

35 days
CPDA Shelf-life
:

THE DONATION PROCESS DSD

: 42 days

Step 1: Educational Materials

 Contains information on the risks of infectious diseases transmitted by blood transfusion,

including the signs and symptoms of AIDS, is given to each prospective donor to read
o AIDS is transmitted through blood
  E.g. AABB pamphlet, “An Important Message to All Blood Donors”

Step 2: The Donor Health History Questionnaire

 A uniform donor history questionnaire designed to ask questions that protect the health of
both the donor and the recipient is given to every donor who have been exposed to other
diseases.
o Questionnaire is filled up with donors, and this tool is actually used to identify donors
who has been exposed to other diseases
 E.g. variant Creutzfeldt-Jakob, West Nile fever, Malaria, Babesiosis, or Chaga’s disease

Step 3: The abbreviated Physical Examination 60

-100 blm

t
 The abbreviated physical examination for donors includes blood pressure, pulse, and
)
not exceed

370C
(must
temperature readings, hemoglobin or hematocrit level, and the inspection of the arms for skin
lesions
o 1990s don nag boom ang HIV

SCREENING TESTS FOR INFECTIOUS DISEASE

 Syphilis
 Hepatitis B surface antigen (HBsAg)
 Hepatitis B core antibody (anti-HBc)
 Hepatitis C virus antibody (anti-HCV)
 HIV antibodies (anti-HIV-1/2)
 Human T-cell lymphotropic virus antibody (anti-HTLV-I/II)
 Human Immunodeficiency virus (HIV-1) (NAT*) idea virus
cytomeqal.vim.dz
 Hepatitis C virus (HCV) (NAT*)
 West Nile Virus (NAT*)
 Trypanosoma cruzi antibody (anti-T. cruzi)

(according to Harmening, currently there are 12 screening test (international standard) for
infectious disease that are performed on each unit of donated blood, but in Philippines it’s not 12,
only MALARIA, SYPHILIS, ANTI-HCV, HEP B SURFACE ANTIGEN, HIV)

The use of nucleic acid amplification testing (NAT) licensed by the FDA is one of the reason for the
increased safety of the blood supply.

What type of hepatitis is the most common cause of hepatitis disease? HEPA C

RBC BIOLOGY AND PRESERVATION

 3 areas of RBC biology are crucial for normal erythrocyte survival function:
o Normal chemical composition and structure of the RBC membrane
 Biconcave shape – normal
o Hemoglobin structure and function
 Adults – Hemoglobin A
 Heme – carry oxygen
o RBC metabolism cells
senescent
 RBC’s – 120 days in the circulation

RBC Membrane
- represents a semipermeable lipid bilayer supported by a meshlike protein cytoskeleton
structure
o this semipermeable membrane will allow only those who enter and go outside the RBC
- Phospholipids – main lipid component
- Integral and peripheral proteins:
o Ex: ankyrin and spectrin – gives RBC the flexibility especially when it goes to small
capillaries

- The biochemical composition of the RBC membrane is approximately:

o 52% protein, 40% lipid, and 8% carbohydrate.
- Deformability:
o Loss of RBC membrane is exemplified by the formation of spherocytes and bite cells
Spherocytes Bite cells

*bite cells – removal of a portion of a membrane that has left

& permanent indentation in
the remaining cell membrane. An effect of a particular poikilocyte (Heinz bodies –
denatured hemoglobin)
*spleen eat denatured or the Heinz bodies
*If we have anisocytes and poikilocytes, the integrity of RBC will lead to shortened
survival of RBC outside the body

*2 main function of RBC: Deformability and Permeability

- PERMEABILITY
o RBC membrane is freely permeable to water and anions
o RBC membrane is relatively impermeable to cation’s such as Na+ and K+
o Erythrocyte intracellular-to-extracellular ratios for Na+ and K+ are 1:12 and 25:1,
respectively
o Major intracellular – K+; major extracellular – Na+
 o PISO
o This permeability will allow liquids and gases to pass through

RBC Preservation
- Goal: To provide viable and functional blood components for patients requiring blood
transfusion.
- 2 criteria used to evaluate new preservation solutions and storage containers:
o An average 24-hour post transfusion RBC survival of more than 75%
o Red cell integrity be maintained throughout the shelf-life of the stored RBC’s
- To maintain optimum viability, blood is stored in the liquid state between 1 and 6 degrees
Celsius (ref temp)

The loss of RBC viability has been correlated with the “lesion of storage”.
1. % viable cells
2. Glucose
3. ATP
4. Lactic acid – increase in lesion
5. pH
6. 2,3 DPG
7. Oxygen Dissociation Curve
8. Plasma K+ - increase in lesion
9. Plasma Hemoglobin – increase in lesion

Approved Anticoagulant Preservative Solutions:

- ACP-A (Acid Citrate Dextrose Formula A); 21 days
- CPD (Citrate Phosphate Dextrose); 21 days
- CP2D (Citrate Phosphate Double Dextrose); 21 days
- CPDA-1 (Citrate Phosphate Dextrose Adenine); 35 days – most commonly used anticoagulant
o Adenine supplemented blood can be stored at 1 to 6 degrees Celsius for 35 days; other
anticoagulants are approved for 21 days

Chemicals in Anticoagulant Solutions

1. Citrate – chelates calcium; prevents clotting; main anticoagulant
2. Monobasic sodium phosphate – maintains pH during storage; necessary for maintenance of
adequate levels of 2,3-DPG
3. Dextrose – substrate for ATP production
4. Adenine – Production of ATP

Additive Solutions
- Preserving solutions added to the RBCs after removal of the plasma with or without platelets
- Currently, 3 additive solutions are licensed in the US:
1. Adsol – AS1 formula and developed by Baxter Healthcare
2. Nutricel – AS3 formula, Pal Corporation
3. Optisol – AS5 formula, Terumo Corporation
*these 3 additives contains saline, adenine and glucose
*Adsol and Optisol contains manitol – protects against storage related hemolysis
*AS3 contains phosphate and citrate
*these additive solutions extend rbc life for another 7 days more

Benefits of RBC Additive Solutions

- Extends the shelf-life of RBC’s to 42 days by adding nutrients
- Allows for the harvesting of more plasma and platelets from the unit
- Produces an RBC concentrate of lower viscosity that is easier to infuse
RBC Freezing
- Used for autologous units and storage of rare blood types.
- Glycerol is used to protect RBC from cold
o Advantages:
 Long-term storage process – last up to 10 years
 Maintenance of RBC viability and function
 Low residual leukocytes and platelets
 Removal of significant amounts of plasma proteins
o Disadvantages:
 A time-consuming – RBC that will undergo freezing is less than 6 day-old
 Higher cost of equipment and materials
 Storage requirements (-65 degrees C)
 Higher cost of product

Glycerol
- Glycerol is used most commonly and is added to RBC slowly with vigorous shaking
- Cryo protective agent
- 2 concentrations of glycerol have been used to freeze RBCs
o HIGH CONCENTRATION GLYCEROL – often used with 40% weigh in volume
o LOW CONCENTRATION GLYCEROL – not often used with 20% weigh in volume

Advantages High Glycerol Low Glycerol

1. Initial freezing temperature -80 degrees Celsius -196 degrees Celsius
2. Need to control freezing No Yes
rate
3. Type of freezer Mechanical Liquid nitrogen
4. Maximum storage -65 degrees Celsius -120 degrees Celsius
temperature
5. Shipping requirements Dry ice Liquid nitrogen
6. Effect of changes in storage Can be thawed and Critical
temperature refrozen

RBC Rejuvenation
- ATP and 2,3 DPG are restored or enhanced
o Increased level of 2,3 DPG will maintain oxygen dissociation curve into the right; less
affinity between the oxygen and the hemoglobin, therefore more oxygen will be
delivered to the tissues
- Rejuvesol – only FDA approved rejuvenation solution sold in the US
- It contains:
o Phosphate
o Inosine
o Glucose
o Pyruvate
o Adenine
o RBC in the liquid state can be rejuvenated at outdate or up to 3 days after outdate,
depending on the preservative used.
o Only RBCs prepared from 450 – mL collections can be rejuvenated. (Out of 525 mL we
collected)
- Rejuvenation is accomplished by
o Incubating RBC unit with 50 mL of the rejuvenating solution
o Wash with NSS or Normal saline solution
o Transfused within 24 hours
  Open system – mandated that’s it should be transfused within 24 hours of
thawing
 Mode of rejuvenation: open system (more prone to contamination)
- Blood pharming
o Creating RBCs in the laboratory
o Potential to increase the amount of blood available for transfusion

Notes
- Platelet concentrate – contain a minimum of 5.5 x 10^10/L plaeletes in a volume routinely
between 45 and 65 mL that is sufficient to maintain a pH of 6.2 or greater at the conclusion of
the 5-day storage period.
o Plate concentrate – component of blood that eaily expire
o Stored at room temp or 20-24C with continous agitation (to maintain the pH of 6.2)
- When platelet concentrate (usually 4 to 6) are pooled using an open system, the storage time
changes to 4 hour.
o Pooled or pooling – mixing platelet concentrate of different people
o 6 hour storage time for closed system because the blood didn’t penetrate unlike the
open system
- A new method of pooling that used a closed sytem that pool to be stored for 5 days from the
date of collection.
- Apheresis components contain 4 to 6 times as many platelets as a PC prepared from whole
blood. They should contain a minimum of 3.0x10 platelets (in 90% of the sampled units).
- Apheresis – a donor’s blood is removed, anticoagulated and transported directly to an
apheresis machine.
o the blood is separated into specific components by centrifugation within the machine
o desired component is removed
o remainong portion of the blood is returned to the donor.
- Platelet components are stored for up to 5 days at 20-24 degrees Celsius
- When necessary, as during shipping, platelets can be stored without continuous agitation for
up to 24 hours at 20-24C during a 5-day storage period.
- Platelets are rarely stored at 1 to 6 degrees Celsius
- If a platelet bag is broken or opened, the platelets must be transfused within 4 hours when
stored at 20 to 24 degrees Celsius

Basic Genetics
- Genetics:
o Study of transmission of inherited characteristics
o Important in the study of antigen inheritance and inherited disorders
o Normal number of human chromosomes
 46 chromosomes 22 pairs of autosomes, 1 pair of sex chromosome
- DNA
o Double helix
o Gene is a segment of DNA arranged along the chromosome at a specific position
called locus.
o Gene at a specific locus differ on their nucleotide sequence are called alleles.
- 75% post transfusion survival of RBC is necessary for a successful transfusion
Chromosomes: chromosomes are found in the cell nucleus and contain double stranded DNA that
have specific areas called genes that code for proteins

Inside the cell, we have the nucleus and inside the nucleus is chromosome X type structure will
determine the characteristics of the DNA and DNA will determine the characteristics of a gene and
gene can also lead to affirmation of protein

BASIC MORPHOLOGY OF CHROMOSOMES

Metacentric – location of the centromere

- Meta – centromere at the center
- Telomere – upper part of the chromosome
Submetacentric – the centromere is located near the top
Acrocentric – mas malapit sa taas
Telocentric – no centromere kasi nasa pinaka toktok ng telomere
Long arm – mas mahaba
Short arm – mas short
  Blood group system – groups of related RBC antigens inherited according to Mendelian
genetics
 Pedigree chart – visual map that displays a family history and can display inheritance patterns
for individual traits
 Gene – smallest unit of inheritance
 Genetic locus – site on chromosome where specific genes are located
 Alleles – alternative forms of a gene
 Antithetical – opposite form of a gene, different allele
 Polymorphic – having two or more possible alleles at a locus
 Codominant – equal expression of both alleles in phenotype
o Usually for ABO blood group, meaning pag na inherit mo si A gene from the parent and
B gene from the other parent, both the A and B can express
 Autosomal – genes expressed with equal frequency in males and females, on non-sex
chromosomes
o 22 pairs; basically 4 autosomal chromosomes
 Haplotype – set of genes inherited via one of the two parental gametes
 Amorph – genes that do not produce a detectable product
o E.g. Blood group O because they don’t have A or B gene inherited
 Homozygous – identical alleles at the same gene locus from both parent
o E.g. A gene from mother and father = AA
 Heterozygous – different alleles at the same gene locus from each parent
o E.g. A gene mother + B gene father = AB
 Dosage effect – agglutination reactions are generally stronger for homozygous cells and
slightly weaker for heterozygous cells
 Cis – genes are inherited on the same chromosome
 Trans – genes are inherited on separate chromosomes. Genes inherited in transposition can
weaken the trait’s expression
o Dapat ang code of inheritance ay nasa cis position

GENOTYPE

 Total genetic composition of an individual, representing maternally and paternally derived

genes
 The patient’s genotype is not determined in routine blood bank testing. DNA-based assays or
family studies are used for the determination of genotype
 Genes inherit from mother and father

PHENOTYPE

 Detectable or expressed characteristic of genes

 The patient’s red cell phenotype is determined by hemagglutination of red cell antigens using
specific antisera. Basically the forward typing

PUNNETT SQUARE

 Illustrates the probabilities of phenotypes from known or inferred genotypes

 It visually portrays the genotypes of the potential offspring or the probable genotypes of the
parents

ABO BLOOD GROUP SYSTEM

- ABO forward reverse grouping test must be performed on all donors and patients.
 o ABO blood group is considered to be the major blood group
o Most of the hemolytic transfusion reactions cause by ABO mismatched will be
deadly
o Leading cause of clerical error and causes death on patient because ABO blood
group is the only blood group wherein we have antibodies in our serum to antigens
that are absent from our RBCs
- Most frequently performed test in the blood bank.
- Most important of all blood groups in transfusion practice.
- Naturally occurring antibodies
- Only blood group system in which individuals have antibodies in their serum to antigens
that are absent from their RBCs.

Inheritance of the ABO Blood Groups

- First described by Bernstein in 1924
o An individual will inherit one ABO gene from each parent and that these two genes
will determine which ABO antigens are present on the RBC membrane of the
offspring
- The inheritance of ABO genes follows the Mendelian genetics
- ABO is codominant in expression
o Codominant: if you inherit both the A and B gene, therefore you will have AB
antigens
- One position or locus, on each chromosome 9 is occupied by an A, B, or O gene
- The O gene is considered an amorph
o Amorph: no detectable antigen is produced in response to the inheritance of these
genes
- The group O phenotype is an autosomal recessive trait with the inheritance of 2 O genes
- Phenotypes: Blood type A, Blood type B, Blood type O
o Characteristics that we will express
- Genotypes: AA, AO, BO, OO

Genotype Phenotype
A1A1 A
A1A2 A
A1O A
A2A2 A
A2O A
A1B AB
A2B AB
OO O
BB B
BO B

Karl Landsteiner
- Discovery of the first human blood group system
- He was the first individual to perorm forward and reverse grouping
- Landsteiner’s Laws
1. The antigen on the RBC determines the blood group
 ABO is on RBC membrane
2. The corresponding antibody is never found in the individuals serum
3. The opposite antibody is always present in the individual’s serum
ABO Antibodies
- “naturally occuring”
- Predominantly IgM
o Anti-AB is the exception of ABO antibody, it is IgG in nature
- Produce strong direct agglutination reactions during ABO testing
- Production: initiated at birth
o Never perform reverse grouping in newborn because what we search is the ABO
antibodies and the production of ABO is not yet high
- Detectable titers: 3 to 6 months
- Peaks at: 5 to 10 years
- ABO antibodies can cause rapid intravascular hemolysis
Blood group Antibody produced Characteristics
A Anti-B IgM
Reacts at cold temperature
Activates the omplement
Only one molecule to activate the
complement
B Anti-A IgM
Reacts at cold temperature
Activates the omplement
Only one molecule to activate the
complement
AB None
O Anti-A, Anti-B, Anti-AB IgM
Reacts at cold temperature
Activates the omplement
Only one molecule to activate the
complement
EXCEPT:
Anti-AB is IgG in nature, therefore it can
cross the placenta and reacts with
warm temperature
Cause of yellowing of baby (baby: A;
mother: O)

ABO Antigens
- Based on the combinations of 3 genes on chromosome 9: A, B and O
- Demonstrated as early as 2nd month of fetal life
o We can perform forward typing in babies
o ABO antigens is already available in fetus (37th day of fetal life)
- A and B antigens develop from precursor H substance
- Persist thru out life unaltered (hindi napapalitan ang blood type)
- May be found in: saliva, pancreatic secretions and gastric secretions
- It has been postulated that bacteria, pollen particles, and other substances present in
nature are chemically similar to A and B antigens – located in red blood cells
- Most common blood type: O and A
- Rarest: AB (royal blood)
 Blood type Antigen present
A A and H
B B and H
AB AB and H
O None and H

Formation of A, B and H Red Cell Antigens

- Results from the interaction of genes at three separate loci – ABO, H, and Se
- Produces specific glycosyltransferases that add sugars to a basic precursor substance
- A, B, H antigens are formed from the same basic precursor material – paragloboside or
glycan (will now exit the inherited gene)
- H antigen – precursor structure on which A and B antigens are made
- Inheritance of the H gene results in the formation of the H antigen
- H and Se gene (needed to have ABH antigen) are closely linked and are located on
chromosome 19
o H and Se gene are not part of the ABO system, however their inheritance does
infuence the A and B antigen expresion
o H gene will form ABO antigen in the red blood cells
o Se gene will form the ABO antigen in secretions
- Precursor substance on erythrocytes type 2
o Terminal galactose on the precursor substance is attached to the N-
acetylglucosamine in a beta 1-4 linkage
o ABH antigens: RBC
- A type 1 precursor substance refers to a beta 1-3 linkage between galactose and N-
acetylglucosamine
o ABH Antigens: secretions

Beta 1-4 linkage

 ABH antigen is constructed on the oligosaccharide chains of a type 2 precursor

substance

- Immunodominant Sugar
o sugars that occupy the terminal galactose of the precursor chain and confer blood
group specificity
 - The substance (L-fructose) must be formed for the other sugars to be attached in response
to an inherited A and/or B gene.

Formation of H antigen
*the enzyme here is alpha-2 L-
fucosyltransferase

Formation of A antigen

*the enzyme here is N-acetylgalactosaminyl

transferase
*sugar: N-acetyl d-galactosamine
 Formation of B antigen

*the enzyme here is alpha-3 galactosyl

transferase
*sugar: d-galactose

- O gene is amorph therefore, it does not elicit the production of catalytically active
polypeptide transferase thus the H substance will remain unmodified

INTERACTION OF Hh and ABO genes

Bombay Phenotype

Formation of ABH  precursor substance is the H antigen  H antigen has sugar L-fucose if nag add
ng another sugar  A-antigen ( N-acetylgalactosaminyl)  B-antigen (D-galactosyl)  AB-antigen
(Both; N-aceytl galactosamine and galactose)
Formation of A, B and H Soluble Substances

 Secretor gene products of alleles at ABO and Hh loci

o If Se gene is present, watery secretions contain water soluble substances with blood
group activity
o SeSe or Sese individuals (80% of the population) are secretors
o sese individuals are non-secretors
 no blood group substances in their saliva

if Se, ABH secretion is found

if se, no ABH secretion is found

Fluids in which A, B and H substances can be detected in secretors:

1. saliva
2. tears
3. urine
4. digestive juices (HCl)
5. bile
6. milk
7. amniotic fluid
8. pathological fluids
Formation of ABO antigens

GENE GLYCOSYLTRANSFERASE Immunodominant Ag

sugar
H a-2-L-fucosyltransferase L-fucose (L-F) H
A a-3-N- N- A
acetylgalactosaminyltransferase acetylgalactosamine
(N-A)
B a-3-D-galactosyltransferase D-galactose (D-G) B
AB a-3-N- Both (N-A) and (D-G) AB
acetylgalactosaminyltransferase
a-3-D-galactosyltransferase
O None L-fucose (L-F) H

Chemical structures of ABO antigens

Antigen on RBC Substance in saliva

A A, H
B B, H
AB A, B, H
H

Comparison of ABH antigens on RBCs with A, B, and H Soluble Substances

ABH ANTIGENS ON RBCs A, B and H soluble substances

 RBC antigens can be glycolipids,
glycoproteins, glycosphingolipids
 RBCs antigens are synthesized only
on RBCs
 Type 2 chain refers to a Beta-1-4
linkage
 The enzyme produced by the H-
gene acts primarily on type 2
chains, which are prevalent on the
RBC membrane
 Secreted substances are
glycoproteins
 Secreted substances are primarily
synthesized on secretions
 Type 1 chains refers to a beta-1-3
Linkage
 The enzyme produced by the Se-
gene Preferentially acts on type 1
chains in secretory tissues

Blood Group O

 Genotype: OO
 Antigen: H
o Lectin: seed extracts that agglutinate human cells with some degree of specificity
(dependent on the type of antigen)
 E.g. anti-H antisera lectin is Ulex europaeus
 Lectin is a reagent used to demonstrate antigen; ginagamit sa forward typing
yung antisera
 Antibodies: anti-A, anti-B, anti-A, B (IgG)
Blood Group A

 Genotype: AA, AO
 Antigen: A, H
o Subgroups of A
o Dolichos Biflorus (lectin): A1 cells
o Antibodies: anti-B (IgM)

Blood Group B

 Genotype: BB, BO
 Antigen: B, H
o Subgroups of B
o Bandeiraea simplicifolia (lectin) revised to Griffonia simplicifonia
 Antibodies
o Anti-A (IgM)

Blood Group AB

 Genotype: AB
 Antigens
o A, B, very little H (very little because 2 sugars na ang na add sa original structure)
 Antibodies
o None

ABO Reactivity with anti-H antisera

Greatest amount of reactivity to the least amount:

O>A2>B>A2B>A1>A1B

Bombay phenotype

 Recessive gene “hh”

 Also called “Oh phenotype”
 Total lack of H, A and B antigens due to lack of H and Se genes
 Develop strong anti-H, anti-A and anti-B
 Compatible only with Bombay individuals
 Appear as Group O on forward typing (result is no agglutination on both anti-A and anti-B)
 Discrepancy on reverse typing (result has agglutination on A-cells and B-cells; differentiate
using anti-H lectin w/c is the Ulex europaeus)

ABO typing

 Suspension of RBCs in saline solution + solution of known anti-A antiserum (stored at 2-8’C)
 Suspension of RBCs in saline solution + solution of known anti-B antiserum (stored at 2 – 8’C)
 Positive reaction: agglutination
 Negative reaction: absence of agglutination
 Forward grouping
o Defined as using unknown sources of commercial antisera to detect antigens on an
individual’s RBCs
o Reagent is antisera that contains antibodies
 Reverse grouping
 o Defined as detecting ABO antibodies in the patient’s serum by using known reagent
RBCs
o Reagent is antigen

Characteristics of Routine reagents used for ABO testing

Anti-A reagent Anti-B reagent

Forward grouping Monoclonal antibody Monoclonal antibody
(1 antibody present)
Highly specific Highly specific
IgM IgM
Clear blue colored
reagent (it is blue because Clear yellow colored
it contains trypan blue, reagent (it is yellow
patent blue, brown phenol because it contains
blue) Acriflatin yellow dye and
tartrazind)
Expected 3+ to 4+ reaction Expected 3+ to 4 +
reaction
Usually use 1 – 2 drops Usually use 1 – 2 drops
For antisera it contains
0.1% sodium acide as
preservative and stable for
2 years from the date of
manufacture
Reverse grouping Human source 4% - 5% red
cell suspension
Expected 2+ to 4+ reaction
Usually use 1 drop

Summary of Forward and Reverse Groupings

Forward group Reverse group

Patient’s cells with reagents Patient’s serum with reagents
Blood Anti-A Anti-B Antigen(s) A1 cells B cells Antibody
group on RBCs (ies) in
serum
O 0 0 None/H 4+ 4+ Anti-A,
Anti-B &
Anti-AB
A 4+ 0 A 0 4+ Anti-B
B 0 4+ B 4+ 0 Anti-A
AB 4+ 4+ AB 0 0 0

Important Facts: ABO and H Blood Group System Antigens

Widespread antigen
distribution
Biochemical composition
Common structures
Blood cells, tissues, body fluids, secretions
- One of the reason wherein the ABO system is regarded as
most important of all blood group system
Glycolipid/glycoprotein
Type 1 and type 2 oligosaccharide chains
- Type 2: will predict the production of ABO antigen in blood
cells

Gene products
Immunodominant sugars
Antigen expression
Genetic loci
Major alleles
Bombay phenotype
- Did not inherit H gene
therefore no precursor
and no production of
ABO Ag
Secretor status
Landsteiner’s rule
Antibody production
Antibody immunoglobulin class
In vitro reactions
Complement binding
Clinical significance
- Type 1: predict the antigen production in secretions
Glycosyltransferases
- For each antigen, we have specific glycosyltransferases
H antigen: 1 fructose
A antigen: N-acetylgalactosamine
B antigen: D-galactose
- Determine the specific antigen that will be present or
specific
Cord blood cells: weak
- Cord blood cells does not only consist of baby’s blood but
also the blood of the mother
ABO blood group system: chromosome 9
H system: chromosome 19
A1, A2, B, O
H, h
Genotype hh; no H or ABO antigen
Se allele; soluble H and ABO antigens
- Will determine the production of ABO antigens in the
secretions or body fluids
Serum possesses the ABO antibody directed toward the A or B
antigen that is absent from red cells
No antibodies detectable in first few months of life; decreases in
elderly adults
- No reverse typing for babies until it reached its 3-6 months
IgM and IgG
At or below room temperature
Yes; some hemolytic
- IgM: 1 molecule
- IgG: 2 molecules
- The hemolysis is intravascular (within blood vessels);
extravascular (within spleen and liver)
Yes when it comes to transfusion and transplantation

ABO Subgroups
- represents phenotypes that show weaker variable serologic reactivity with the commonly used
human polyclonal
o Anti-A (blue color)
o Anti-B (yellow)
o Anti-A, B reagents (colorless but the cover is pink)
- Weaker serologic reactivity of ABO subgroups
o Decreased number of A and B antigen sites on their red cells

- A subgroups
o More common than B subgroups
o Von Dungern
 Described two different A antigens based on reactions between group A RBCs
and anti-A (blue reagent) and anti-A1 (dolichos biflorus)
 Group A phenotype
  Differ slightly in their ability to convert H antigen to A antigen
o A1 – antigens are highly concentrated on branched and linear
oligosaccharide chains
o A2 – assembled on the simplified linear forms of the
oligosaccharide chains
o The A antigen is fewer than in A1 phenotype
- Classification into A1 and A2 phenotypes
o A1 – approximately 80% of all group A (or AB individuals) ++
o A2 – remaining 20%
o A1 versus A2 phenotypes:

Reactions of Patient’s RBCs with

Blood Group Anti-A Reactant Anti-A1 Lectin Reagent
A1 + +
A2 + -
+=Positive (agglutination)
0=Negative (no agglutination)
o The group A RBCs that will react with both anti-A and anti-A1, they are classified as A1
o Whereas, those will react with anti-A only but not with anti-A1 lectin, they are classified
as A2 individuals

- The differences between A1 and A2 are both quantitative and qualitative

Quantitative and Qualitative Differences of Subgroups A1 and A2
Quantitative Qualitative
- ↓ Number of antigen sites - Differences in the precursor
o A1 have more antigen sites oligosaccharide chains
than A2
- ↓ Amount of transferase enzyme - Subtle differences in transferase
enzymes
- ↓Amount of branching - Formation of anti-A1, in a
percentage of some subgroups

Comparison of A1 and A2 red cells

- The immunodominant sugar on both A1 and A2 RBCs is N-acetyl-D-galactosamine

 - Differentiation of A1 and A2 phenotypes can be determined by using a reagent made from
the seeds of a plant (LECTIN)
Lectins used in Blood Banking
1. Dolichos biflorus – A1 B cells
2. Bandeiraea simplicifolia – B cells
3. Ulex europaeus – agglutinates O cells (H specificity)
and other ABO blood groups depending on the
amount of H antigen available
o Strong reaction: O
o Weak reaction: A1B
 H antigen I found in greatest concentration on the RBCs of O individuals
 Reactivity of anti-H antisera or anti-H lectin with ABO groups: (greatest to least)

Characteristics of A1 and A2 Phenotypes

Reagents Antibodies in Serum Other
Phenotypes Anti-A Anti-B Anti- Anti-A1 Common Unexpected Substances Number
A,B present in of
the saliva antigen
of sites
secretors RBC x
103
A1 + - + + Anti-B None (-) A, H 810-
1170
A2 + - + - Anti-B Anti-A1 A, H 240-290

Weak A Subgroups
- Characteristics:
o Decreased number of A antigen sites per RBC
o Varying degrees of agglutination by human anti-A, B
o Increased variability in the detectability of H antigen resulting in strong reactions with
anti-H
o Presence or absence of anti-A1 in the serum
o Weak A phenotypes can be serologically differentiated using the following techniques:
1. Forward grouping of A and H antigens
o We will use Anti-A, Anti-B, Anti-AB, Anti-A1
2. Reverse grouping of ABO isoagglutinins and the presence of anti-A1
o We will use A cells and B cells
3. Adsorption – elution tests with anti-A
 ADSORPTION-ELUTION TEST
Adsorption – used to bind antibodies to red blood cells in order to remove
them from the plasma and better analyse the antibodies that might
remain behind
Elution – removing an antibody that is attached to the surface of a red
blood cell
o Ex: heating and cooling
4. Saliva studies to detect the presence of A and H substances
5. Additional special procedures such as molecular testing
 Serum glycosyltransferase studies for detecting the A enzyme – performed
for the differentiation of weak subgroups
- Weak A subgroups can be distinguished as:
o A3
o Ax
 o Aend
o Am
o Ay
o Ael
A3 RBCs

 Demonstrate a mixed field pattern of agglutination with anti-A and most anti-A, B reagents
o Mixed field – define as small agglutinates within predominantly unagglutinated RBCs
 Estimated number of A antigen sites is approximately 35,000 per RBC
o A1 = almost 3M; A2 = 500,000
 Weak a-3-N-acetylglactosaminyl transferase activity is detectable in the serum

Ax RBCs

 Characteristically are not agglutinated by anti-A reagent but do agglutinate with most
examples of anti-A, B.
 Estimated number of A antigen sites is approximately 4,000 per RBC
 Transferase: not detectable in the serum

Aend RBCs

 Characteristically demonstrate mixed-field agglutination with anti-A and anti-A, B but only a
very small percentage of the RBCs (10% or less) agglutinate
 Estimated number of A antigen sites on the few agglutinable RBCs is approximately *35,000
per RBC whereas no detectable A antigen are demonstrated on RBCs that do not agglutinate
 The phenotypes of Afinn and Abantu are considered by some investigators to represent
variants of the Aend subgroup
o Afinn and Abantu are also scientist who studied weak A subgorups

Am RBCs

 Characteristically not agglutinated, or are agglutinated only weakly, by anti-A or anti-A, B

 The estimated number of A antigen sites varies from *200-1900 per RBC
 Normal quantities of A and H substance are found in the saliva of Am secretors

Ay RBCs

 Ay RBCs are not agglutinated by anti-A or anti-A, B

 The Ay phenotype can be observed in siblings, implicating a recessive mode of inheritance
 This phenotype does not represent expression of an alternate allele at the ABO locus but
rather as a germline mutation of an A gene within a family

Ael RBCs

 Ael RBCS typically are unagglutinated by anti-A or anti-A, B; however, adsorption and elution
can be used to demonstrate the presence of the A antigen
o Adsorption/elution – remove antibodies to study it well
o ADSORPTION – use a media/membrane in order to separate antibodies from the
antigen
o ELUTION – can be done physically (heating/ cooling)
 Secretor studies demonstrate the presence of only H substance in the saliva of Ael secretors
 DIFFERENCE BETWEEN WEAK A SUBGROUP

What weak A subgroup does not agglutinate with anti-A or anti-A, B? Am, Ay, Ael (this 3 are only
demonstrated by adsorption or elution technique

NUMBER OF A ANTIGENS
Weak B subgroups

 Very rare and much less frequent than A subgroups

 Criteria used for differentiation of weak B phenotypes include the following serologic
techniques
o Strength and type of agglutination with anti-B, anti-A, B and anti-H
o Presence or absence of ABO isoagglutinins in the serum
o Adsorption-elution studies with anti-B
o Presence of B substance in the saliva
o Molecular testing
 Antisera use is Anti-B or the lectin for detecting B cells will be griffonia simplicifolia
 Weak B phenotypes or weak B subgroups include:
o B3
o Bx
o Bm and
o Bel phenotypes
1. B3 phenotype generally results from the inheritance of a rare gene at the ABO locus and is
characterized by a mixed field pattern of agglutination with anti-B an anti-A, B
o It is the most frequent weak B phenotype
2. Bx RBCs typically demonstrate weak agglutination anti-B and anti-A, B antisera. Family studies
suggest that Bx is a rare allele at the ABO locus.
3. Bm RBCs are characteristically unagglutinated by anti-B or anti-A, B
o The Bm RBCs easily adsorb and elute anti-B
o Reduced activity of B enzyme in hematopoietic tissue is clearly the defect causing the
formation of the Bm subgroup, since normal B plasma incubated with Bm RBCs and
UDP-galactose transforms this subgroup into a normal group B phenotype
o The Bm subgroup is reported to be more frequent in Japan
4. Bel RBCs are
o Unagglutinated by anti-B or anti-A, B
o A weak anti-B may be present in the serum of this subgroup. Only H substance is
demonstrated in saliva of Bel secretors

Importance of Subgroup identification:

Subgroup of A and B
o Academic interest – need to study all the subgroups to identify the incompatibility
o Failure to detect a weak subgroup could have serious consequences
i. If a weak subgroup is missed in a recipient the recipient would be classified as
group O
ii. Result in decreased survival of the transfused cells in a group O recipient
For group O recipient they possess ABO antibodies that is capable of
reacting with the weak subgroup antigen in vivo, this will result in the
decrease survival of this transfuse red cells in the recipient circulation

The Bombay Phenotype

General characteristics of Bombay Oh (Hnull) phenotypes

 First reported by Bhende in 1952 in Bombay, India

 Inherited as an autosomal recessive trait
 It represents the inheritance of a double dose of the h gene, producing the very rare
genotype hh.
  The Bombay anti-H can often be potent and reacts strongly at 37*C
 Absence of H, A and B antigens; no agglutination with anti-A, anti-B or anti-H lectin
o For true blood group O it will show a reaction with anti-H
 Presence of anti-A, anti-B, anti-A, B and a potent wide thermal range of anti-H in the serum
 Completely walang H antigen

A, B, H nonsecretor

 Absence of a-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 (Oh) will not react with the anti-H lectin
 RBCs on the Bombay phenotype (Oh) are compatible only with the serum from another
Bombay individual
o If you are considered Bombay hindi ka pwede kang salinan ng group O dahil meron
silang H antigen

The Parabombay Phenotypes

 Rare phenotypes in which the RBCs are completely devoid of H antigens or have small
amounts of H antigen present
 The genetic basis for the para-bombay is a mutated FUT1 (H gene) with or without an active
FUT2 gene (Se gene) or a silences FUT1 gene with an active FUT2 gene
 Completely na walang H antigen but some of them can still have small amount of H antigen
3 categories of H-deficient phenotypes:

1. Bombay phenotype

o hh sese (recessive)
o Antibodies in serum: Anti-A, anti-B, anti-H
o RBC H-deficient
o Nonsecretor
o Oh, OhA, OhB, OhAB
2. RBC H-partially deficient
o Nonsecretor
o Oh, Ah, Bh, ABh
o hh se
o proposed genes inherited: A and/or B
3. Para-Bombay Phenotype
o Red cell H-deficient
o hh Se
o secretor
o OhO, OhA, OhB, OhAB

ABH Antigens and Antibodies in Disease

Associations between ABH antigens and practically any disorder known to man can be found
throughout medical literature
o A-hangover
o B-criminality
o O-good teeth
ABO Discrepancies
- Unexpected reactions occur in the forward and reverse grouping.
o Forward: we are looking for ABO antigens
o Reverse: we are looking for ABO antibodies
- Due to problems with the:
o Patient’s serum
o Problem with the patient’s red cells (leukemia, paraproteinemia) or
o Problems with both the serum and cells
- Technical errors can also cause ABO discrepancies
o Errors in labelling the blood sample
o Failure to add reagents
 Forward: clear reagent is antisera
 Reverse: clear reagent is serum from patient
o Addition of incorrect reagents or sample

Common sources of technical errors resulting in ABO discrepancies:

- Incorrect or inadequate identification of blood specimens, test tubes and slides
- Cell suspension either too heavy or too light (concentration should be 2-5%)
- Clerical errors or incorrect recording of results (most commonly mistake in record)
- A mix-up in samples
- Missed observation of hemolysis
o Hemolysis: lighter in color and no agglutination
- Failure to add reagents
- Failure to add sample
- Failure to follow manufacturer’s instructions
- Uncalibrated centrifuge
- Overcentrifugation and undercentrifugation
- Contaminated reagents
- Warming during centrifugation

Resolution
- It is essential to acquire information regarding the patient’s:
o Age
o Diagnosis
o Transfusion history
o Medications
o History of pregnancy

Categories of ABO Discrepancies

1. Group 1 discrepancies
- Unexpected reactions in the reverse grouping due to weakly reacting or missing antibodies
o One of the reasons for the missing or weak isoagglutinins is that the patient has
depressed antibody production or cannot produce the ABO antibodies
- Common populations with Group I Discrepancies
o Newborns
o Elderly patients
o Patients with leukemia (e.g. CLL) or lymphoma (e.g. malignant lymphoma)
demonstrating hypogammaglobulinemia
o Patients with congenital or acquired agammaglobulinemia or immunodeficiency
diseases (no IgM, IgG, IgA, etc.)
o Patients with bone marrow or stem cell transplantations
 Patients develop hypogammaglobulinemia from therapy and start producing a
different RBC population from that of the transplanted bone marrow
 o Patient’s whose existing ABO antibodies may have been
 Diluted by plasma transfusion or
 Exchange transfusion
o ABO subgroups

- Resolution of Group I Discrepancies

o Incubate the patient serum with reagent A1 and B cells
 Room temperature for approximately 15-30 minutes or
 By adding one to two drops more plasma or serum to the test
o If there is still no reaction after centrifugation
 Serum-cell mixtures can be incubated at 4⁰C for 15 minutes
o An auto control (self) and O cell control
 Must always be tested concurrently with the reverse typing when trying to solve
the discrepancy
o The lower temperature of testing will most likely enhance the reactivity of other
commonly occurring cold agglutinins such as anti-I that react with all adult RBCs

- Example: Weak or Missing antibodies

Forward grouping reaction of Reverse grouping reaction of
patient’s cells patient’s serum
Anti-A Anti-B A1 cells B cells
Patient 0 0 0 0
Patient’s probable group: Blood type O because the problem is the
reverse – no agglutination

o Note: the absence of agglutination with reagents cells in the reverse type is because
the production of ABO antibodies can be weak or absent in the elderly
o Resolution:
 1. Check age of the patient
 2. Increase incubation time to 30 minutes
 3. Lower the temperature for 4degrees Celsius for 15 minutes (include O cells and
an autocontrol)
2. Group II Discrepancies
- Unexpected reactions in the forward grouping due to weakly reacting or missing antigens
o Subgroups of A (or B) may be present – A3 An Ax Al, B3, Bel Bx Bn
o Leukemias may yield weakened A or B antigens and Hodgkins disease has been
reported in some cases to mimic the depression of antigens found in leukemia
o “acquired B” phenomenon will show weak reactions with anti-B antisera and is most
often associated with diseases of the digestive tract (e.g. cancer of the colon)

- Acquired B antigen
o Arises when bacterial enzymes (deacetylase) modify the immunodominant blood
group A sugar (N-acetyl-D-galactosamine) into D-galactosamine which is sufficiently
similar to the blood group B sugar (D-galactose) and cross-reacts with anti-B sera.
 Gram-negative bacteria (especially those of colonic origin in cases of colon
cancer and gram-negative sepsis).
 E.g., E. coli, clostridium tertium, bacteroides fragilis – associated with GI
pathology – e.g., cancer or severe infection

- Rare Group II Discrepancies

o Weakly reactive or missing reactions in RBC grouping may be due to excess amounts of
blood-group specific soluble (BGSS) substances present in the plasma, which sometimes
occurs with certain diseases (e.g. carcinoma of the stomach and pancreas).
 o Resolution:
 Wash patient’s cell free of the BGSS substances with NSS

o Antibodies to low incidence antigens in reagent anti-A or anti-B

o Resolution:
 Repeat the forward type, using antisera with a different lot number
o Chimerism
 Defined as the presence of two cell populations in a single individual
 Discovered in twins who had a mixture of both B and O cells
o Artificial Chimeras occur, which yield mixed cell populations as a result of:
 Blood transfusions (e.g., group O cells given to an A or B patient)
 Transplanted bone marrows or peripheral blood stem cells of a different ABO
type
 Exchange transfusions
 Fetal-maternal bleeding

- Resolution to Group II Discrepancies

o Incubate the test mixture at room temperature for up to 30 minutes, which will increase
the association of the antibody with the RBC antigen
o If it is still negative, incubate the test mixture at 4 degrees Celsius for 15 minutes
o Include group O and autologous cells as controls
- Example: Acquired B antigen
Forward grouping reaction of Reverse grouping reaction of
patient’s cells patient’s serum
Anti-A Anti-B A1 cells B cells
Patient 4+ 2+ 0 4+
Patient’s probable group: blood type A

o Note: Patients RBCs have acquired a B-like antigen that reacts with the reagent anti-B
and is associated with cancer of the colon or other diseases of the digestive tract.
o Resolution:
 1. Acidify anti-B reagent to a pH of 6
 2. Run DAT (detect the presence of in vivo agglutination); mix the patient serum
or plasma together with patient’s rbc
 3. Run autocontrol

3. Group III Discrepancies

- Caused by protein or plasma abnormalities and result in rouleaux formation or
pseudoagglutination, attributable to:
o Elevated levels of globulin from certain disease states,
 Multiple myeloma – Bence jones proteins is present in the urine of patients having
multiple myeloma
 Waldenstroms macroglobulinemia
 Other plasma cell dyscrasias, and certain moderately advanced cases of
Hodgkin’s lymphoma
o Elevated levels of fibrinogen
o Plasma expanders
o Wharton’s jelly – seen in mother’s placenta
o Rouleaux – stacking of erythrocytes that adhere in a coin like fashion, giving the
appearance of agglutination
- Resolution of Group III Discrepancies
o Rouleaux: wash patient’s RBCs several times with saline
o Wharton’s jelly: Wash cord cells 6-8 times with saline
- Example: Rouleaux Formation
Forward grouping reaction of Reverse grouping reaction of
patient’s cells patient’s serum
Anti-A Anti-B A1 cells B cells
Patient 4+ 4+ 2+ 2+
Patient’s probable group: blood type is AB

o Note: agglutination with A1, and B cells in reverse type is due to rouleaux formation as a
result of increased serum protein or plasma abnormalities
o Resolution:
 1. Microscopic examination
 2. Saline replacement technique
 3. Wash cells with saline 3x
 4. Run antibody screen

4. Group IV Discrepancies
- due to miscellaneous problems:
o Cold reactive autoantibodies
 RBCs are so heavily coated with antibody that they spontaneously agglutinate,
idependent of the specificity of the reagent antibody
o Patient has circulating RBCs of more than one ABO group due to RBC transfusion or
marrow/ stem cell transplant
o Unexpected ABO isoagglutinins
 Include A2 and A2B individuals
o Unexpected non-ABO alloantibodies

- Rare Group IV Discrepncies

o Antibodies other than anti-A and anti-B may react to form antigen-antibody complexes
that may then be absorb onto patient’s RBCs
 E.g. individual who has an antibody against acrifavine
o The acriflavine (found in anti-B and gives yellow color) – anti-acriflavine complex
attaches to the patient’s RBCs, causing agglutination in the forward type
 Washing the patient’s cells 3x should resolve this discrepancy.
o Cis-AB
 Refers to the inheritance of both AB genes from one parent carried on one
chromosome and on an O gene inherited from the other parent
 This results in the offspring inheriting 3 AB genes instead of 2
- Resolution of Group IV Discrepancies
o patient’s RBCs could be incubated at 37 degrees Celsius for a short period, then
washed with saline at 37 degrees Celsius three times then retype
o If this is not successful, patients RBCs can be treated with 0.01 M DTT dithiothreitol to
disperse IgM agglutination
o As for the serum, the reagent RBCs and serum can be warmed to 37 degrees Celsius,
then mixed, tested, and read at 37 degrees Celsius

- Example: Cold Autoantibodies

Forward grouping reaction of Reverse grouping reaction of
patient’s cells patient’s serum
Anti-A Anti-B A1 cells B cells
Patient 2+ 4+ 4+ 2+
Patient’s probable group: blood type is B

o Note: Reaction of anti-A in forward type is due to spontaneous agglutination of

antibody coated cells; reaction with B cells in reverse type is due to cold autoantibody
(e.g. anti-I) reacting with I antigen on B cells
o Resolution:
 1. Wash patient cells with warm saline and retest
 2. Run DAT and autocontrol
 3. Run antibody screen
SELECTION OF ABO COMPATIBLE RED CELLS AND PLASMA PRODUCTS FOR TRANSFUSION

 In routine transfusion practices, donor products (RBCs and plasma) with identical ABO
phenotypes are usually available to the recipient. This transfusion selection is referred to as
providing ABO identical (ABO group-specific) blood for the intended recipient. In situations
where blood of identical ABO phenotype is unavailable, ABO-compatible (ABO group-
compatible) blood may be issued to the recipient.
 For RBC transfusions, ABO compatibility between the recipient and the donor is defined as the
serologic compatibility between the ABO antibodies present in the recipient’s serum and the
ABO antigens expressed on the donor’s red cells.
o For example, RBC transfusion, we remove plasma from the donor blood bag, packed
red blood cell remain, and in the first law of Landsteiner, the antigen on the red cell will
determine the blood type of the patient
o Donor red cell is of blood type O, no presence of A or B antigen
 Whole blood transfusion – ABO-identical donor units must be provided because both plasma
and red cells are present in the product
o Must be ABO compatible
 When plasma products are transfused, the selection of an ABO-identical phenotype is the
ideal situation. When identical BAO phenotype are unavailable, the rationale for compatible
plasma transfusions is the reverse of RBC transfusions.
o Recipient needs plasma, unfortunately patient is blood type A, and there is no A
available. The recipient has A antigens on his/her red cells, therefore, plasma that will
be transfused should not contain any anti-A.
 Persons with Group O red cells are called universal donors because the RBC product lacks
both A and B antigens and could be transfused to any ABO phenotype
 Group O donor RBCs can be used in times of urgency for emergency release of donor units
 Group AB recipients are considered universal recipients because these individuals lack
circulating ABO antibodies and can receive RBCs of any ABO phenotype
o No antibodies
 Universal donor for RBC transfusions is group O; universal donor for plasma transfusions is group
AB (antibodies are not present in serum)
 Universal recipient for RBC transfusions is group AB; universal recipient for plasma transfusions is
group O (absence of A/B antigen)

Practical application: ABO compatibility for whole blood, red blood cells, and plasma
transfusions
Recipient Donor
ABO phenotype Whole blood Red blood cells Plasma
Group A Group A Groups A, O Groups A, AB
Group B Group B Groups B, O Groups B, AB
Group AB Group AB Groups AB, A, B, Group AB
O
Group O Group O Group O Group O, A, B, AB
Rh Blood Group System

Rh

 Refers to a specific red blood cell antigen (presence of the D antigen)

 2nd most important blood group system in terms of transfusion because Rh are very
immunogenic means can stimulate our immune system to produce antibodies
 ABO 1st important
 Complex blood group system currently composed of over 50 antigenic specificities
o Specifically, 57 antigens are present in Rh
 Rh antibodies – produced only after exposure to foreign red blood cells
o ABO is naturally occurring antibodies
o Once present, they can produce:
 HDFN (Hemolytic disease of the newborn/eryhthroblastosis fetalis)
 HTR (hemolytic transfusion reaction)
 Anti-D testing  determining the presence of capital D (d denotes the absence of the capital
D)
 Rh positive – individual’s red blood cells possess one particular Rh antigen, the D antigen
 Rh negative – red blood cells lacks the D antigen
 Rh resides on proteins vs the carbohydrate antigens (ABO)

History

 Levine and Stetson – described a hemolytic transfusion reaction in an obstetrical patient

o How Rh blood group system discovered? HEMOLYTIC TRANSFUSION REACTION
o An antibody was isolated from the mother’s serum that reacted both at 37 degrees
Celsius and 20 degrees Celsius with the father’s RBC’s
 IgG antibodies in nature  37*C
o It was postulated that the father possessed a common factor the mother lacked
 Landsteiner and Wiener
o Reported on an antibody made by guinea pigs and rabbits when they were transfused
with Rhesus macaque monkey RBCs
o Named Rh
 Levine and coworkers
o Demonstrated that the agglutinin causing the HTR and the antibody defined by
Landsteiner and Wiener appeared to define the same blood group
 Rh was retained for the human-produced antibody (antibody seen in obstetrical patient)
 Anti-Rhesus formed by the animals was renamed anti-LW (in honor to Landsteiner and Wiener)
 Primary cause of hemolytic disease of the fetus and new born (HDFN) or erythroblastosis fetali s
 Significant cause of hemolytic transfusion reaction

Terminology

1. Fisher-Race: DCE terminology

o They postulated that the antigens of the system were produced by 3 closely linked set
alleles
o They named the antigens of the system D, d, C, c, E, e (5 antigens)
o “d” – absence of the D antigen
o Fisher-Race Theory
 Each person inherits a set of Rh genes from each parent (1 from mother and 1
from father)
 o Rh genes are codominant – each inherited gene expresses its corresponding antigen on
the RBC
o An individual’s Rh phenotype is reported as DCE rather than CDE
 Fisher postulated that the C/c locus lies between the D/d and E/e loci

o Combination of maternal and paternal haplotypes determines one’s genotype and

dictates one’s phenotype
o Probable genotype of a person exhibiting deletion phenotype: DC- or Dc- or D-
 A deletion of Cc with Ee has not been reported
o Absence of Rh antigens on the RBC: Rhnull  - - - / - - -
o Weakend expression of all Rh antigens: Rhmod phenotype  (D)(C)(e)

Frequency of common Rh antigens in Caucasians

Antigen Gene frequency (%)

D 85
No D (absence of D) 15
C 70
E 30
c 80
e 98

Fisher-Race Haplotypes of the Rh system

Prevalence (%)
Haplotypes white black Asian
DCe 42 17 70
dce 37 26 3
DcE 14 11 21
Dce 4 44 3
dCe 2 2 2
dcE 1 <0.01 <0.01
DCE <0.01 <0.01 1
dCE <0.01 <0.01 <0.01

2. Wiener: Rh-Hr Terminology

o Wiener believed that there was one gene responsible for defining Rh that produced an
agglutinogen containing a series of blood factors.
o This Rh gene produced at least 3 factors within an agglutinogen.
 The agglutinogen may be considered the phenotypic expression of the
haplotype. (haplotypes  DCE antigens)
o Each factor is an antigen recognized by an antibody. Antibodies can recognize single
or multiple factors (antigens)

o When describing an agglutinogen:

 R – presence of D antigen
 r – absence of D antigen
 C – 1 or single prime (‘)
 c – no 1 or single (‘)
 E – 2 or double prime (‘’)
 e – no 2 or double prime
 example:
 R1 = DCe
 Ro = Dce
 R2 = DcE
 r = dce
 r’ = dce
 r’’ = dcE
 when both C and E are uppercase: z (R) or y (r) is used
 Rz = DCE
 ry = CE/dCE
o when referring to Rh antigens (or factors) in wiener nomenclature: (Blood factors)
 C or c-‘
 E or e-“
 If r precedes h (i.e., rh’ or rh’’) – refers to C or E antigens respectively
 If h precedes r (ie., hr’ or hr’’) refers to c or e antigens respectively
 Rho is equivalent to D
 No designation for the absence of D antigen
 Example:
 Rhohr’hr’’ = Dce
 Rhorh’rh’’ = DCE
 rh’hr’’ = dCe
o Italics or superscripts – used when describing Rh genes in the wiener nomenclature
o Standard type – used to describe the gene product or agglutinogen. – Rho, hr’, rh’’
o Subscripts – used with the uppercase R (antigen)
o Superscripts – used with the lowercase r (allele)
o genotype for the Rhnull that arises from an amorphic gene at both Rh loci
 pronounced little r double bar
 Gene Agglutinogen Blood factors Shorthand Fisher-Race
designation antigens
Rho Rho Rhohr’hr’’ Ro Dce
Rh1 R1 Rhorh’hr’’ R1 DCe
Rh2 R2 Rhohr’rh’’ R2 DcE
Rhz Rz Rhorh’rh’’ Rz DCE
rh r hr’hr’’ r ce
rh’ r’ rh’hr’’ r’ Ce
rh’’ r’’ hr’rh’’ r’’ cE
rhy ry rh’rh’’ ry CE

 R1 is an antigen expression for DCe, which uses the subscript 1

 R1 is the allele for the antigen expression, which uses italics and the subscripts 1

Converting Fisher-Race Terminology to wiener Terminology

Fisher-Race antigen Wiener Terminology Examples

D R (D+) r (D-) DCe =
C 1 ‘ Ce = r’, DCe =R1
E 2 ‘’ cE = r’’, DcE = R2
CE Z y DCE = R2 CE = ry
ce 0 ce = r

3. Rosenfield and Coworkers: Alphanumeric Terminology

o System that assigns a number to each antigen of the Rh system in order of its discovery
or recognized relationship to the Rh system
o No genetic basis
o Simply demonstrates the presence or absence of the antigen on the RBC
o A minus sign preceding a number designates absence of the antigen
o For the 5 major antigens:
 D is assigned Rh1
 C: Rh2
 E: Rh3
 c: Rh4
 e: Rh5
 if an antigen has not been typed, its number will not appear in the sequence
 example:
 D+C+E+c-e- = 1, 2, 3, -4, -5
 D+C-E-c+e+ = 1, -2, -3, 4, 5
4. ISBT: updated Numeric Terminology

ISBT (International Society of Blood Transfusion): Updated Numeric Terminology

- Combination of wiener, fisher race, and rosenfield
- To establish a uniform nomenclature that is both eye and machine – readable and in keeping
with the genetic basis of blood groups
- Adopted a 6-digit number for each authenticated antigen belonging to a blood group
system
- First three numbers represents the system
- Remaining three – antigenic specificity
o Ex: 004001 – 001 is the antigenic specificity (Rh group)
o ABO Group – first to discover – 001
 o MNS Group – second to discover – 002

Rh Trminologies
Common Antigens in the Rh Blood Group System: Equivalent Notations
Numeric Fisher-Race Other Names ISBT No.
Rh 1 D Rh+ 004001
Rh 2 C 004002
Rh 3 E 004003
Rh 4 c 004004
Rh 5 e 004005
Rh 6 ce cis-ce or f 004006
Rh 7 Ce cis-Ce 004007
Rh 8 C w 004008
Rh 9 Cx 004009
Rh 10 ce s V 004010
Rh 12 G 004012

Order of frequency of the common Rh Blood Group System Haplotypes

White Black Rare (both races)
CDe (R )1 Highest cDe (R )
0 Ce (r’)
ce (r) ce (r) cE (r’’)
cDE (R )
2 CDe (R )
1 CE (ry)
cDe (R0) cDE (R2) CDE (RZ)
Lowest

Wiener Blood Factors Fisher Race Rosenfield

R1r Rh0 rh’ hr’’ DCe 1,2 -3, 4, 5
hr' hr’’ dce
R1R1 Rh0 rh’ hr’’ DCe 1,2 -3, 4, 5
Rh0 rh’ hr’’ DCe
rr
R1R2
R2r
R2R2

Overview of Rh Terminologies
*the differences must be remembered when trying to locate compatible blood for recipients with an
usual or multiple Rh antibodies. But remember when it comes to blood system, the Rh antibodies are
not naturally occurring unlike our ABO
- Relationship Testing
o Determining probable or predicted genotypes (parentage testing)
o For population studies
- Zygosity testing (also known as Molecular testing)
o Performed to confirm whether the father possesses one or two copies of the RHD gene
- D+C-E-c+e+
o Most commonly seen in blacks but is considered relatively rare in whites
- For consistency of use:
o RHD and RHCE – genes
ISBT (International Society of Blood Transfusion): Updated Numeric Terminology
- Combination of wiener, fisher race, and rosenfield
- To establish a uniform nomenclature that is both eye and machine – readable and in keeping
with the genetic basis of blood groups
- Adopted a 6-digit number for each authenticated antigen belonging to a blood group
system
- First three numbers represents the system
- Remaining three – antigenic specificity
o Ex: 004001 – 001 is the antigenic specificity (Rh group)
o ABO Group – first to discover – 001
o MNS Group – second to discover – 002

Rh Trminologies
Common Antigens in the Rh Blood Group System: Equivalent Notations
Numeric Fisher-Race Other Names ISBT No.
Rh 1 D Rh+ 004001
Rh 2 C 004002
Rh 3 E 004003
Rh 4 c 004004
Rh 5 e 004005
Rh 6 ce cis-ce or f 004006
Rh 7 Ce cis-Ce 004007
Rh 8 C w 004008
Rh 9 Cx 004009
Rh 10 ce s V 004010
Rh 12 G 004012

Order of frequency of the common Rh Blood Group System Haplotypes

White Black Rare (both races)
CDe (R )1 Highest cDe (R )
0 Ce (r’)
ce (r) ce (r) cE (r’’)
cDE (R )
2 CDe (R )1 CE (ry)
cDe (R )
0 cDE (R )
2 CDE (RZ)
Lowest

Wiener Blood Factors Fisher Race Rosenfield

R1r Rh0 rh’ hr’’ DCe 1,2 -3, 4, 5
hr' hr’’ dce
R1R1 Rh0 rh’ hr’’ DCe 1,2 -3, 4, 5
Rh0 rh’ hr’’ DCe
rr
R1R2
R2r
R2R2

Overview of Rh Terminologies
*the differences must be remembered when trying to locate compatible blood for recipients with an
usual or multiple Rh antibodies. But remember when it comes to blood system, the Rh antibodies are
not naturally occurring unlike our ABO
 - Relationship Testing
o Determining probable or predicted genotypes (parentage testing)
o For population studies
- Zygosity testing (also known as Molecular testing)
o Performed to confirm whether the father possesses one or two copies of the RHD gene
- D+C-E-c+e+
o Most commonly seen in blacks but is considered relatively rare in whites
- For consistency of use:
o RHD and RHCE – genes (correct genes that will be producing antigens)
o RhD, RhCe, RhcE, Rhce, and RhCE – designate proteins on which the Rh antigens reside

Molecular Genetics
- Wiener – hypothesized that a single gene produces a single product that contains separately
recognizable factors.
- Fisher and Race – proposed that Rh locus contains 3 distinct genes that control the production
of their respective antigens
- Tippett correctly proposed 2 RH genes:
o RHD
o RHCE

Rh Genes
- Two closely linked genes located on chromosome 1 control the expression of Rh proteins.
- RHD: codes for the presence or absence of RhD protein
- RHCE: codes for either RhCe, RhcE, Rhce or RhCE proteins
- RHAG (Rh Associated Glycoprotein): resides on chromosome 6
o Acts as co-expressor and must be present for a successful expression of a Rh antigen,
however by itself this glycoprotein does not express any Rh antigens
o Combines with RHD or RHCE to produce antigen
o The product of this gene is RhAG – coexpressor
o Mutations – missing or altered RhD/RhCE

Rh-Positive Phenotypes
- Rh genes are inherited as codominant alleles.
- Rh-positive individuals
o Inherit one or two RHD genes which results in expression of RHD antigen and are typed
Rh-positive
o RHD gene mutations – weakened expression of the RhD antigen
 Weak D/ weak expression of D

Rh-Negative Phenotypes
- Can arise from three different mutations
- These mutations are most often found among three different ethnic backgrounds:
o European
 Deletion of the RHD gene – they possess no RHD gene but have inherited two
RHCE genes
o African
 RHD pseudogene
 do not produce the RhD protein
o Asian
 Alteration of the RHD gene
 Del
Biochemistry
- Rh antigens reside on transmembrane proteins
o Integral part of the RBC membrane
- The greatest number of D antigen sites
o Rare Rh phenotype D
o We have 3 million antigen sites
o D – 110,000-202,000 antigen sites
- Commonly encountered Rh genotypes: R 2R2 cells – possess the largest number of D antigen
sites

Rh function
- RhD and RhCE along with RhAG
o Exclusively on RBC’s
- Play a role in:
o Maintaining the structural integrity of red cells
o Transporters based on their structure
- Westhoff and colleagues
o Role in transporting ammonia
o CO2 transporters

Weak D: Variations of D Antigen Expression (Du)

- RhD antigen was altered
- Categorized into different phenotypes defined as weakened D due to:
1. C in trans to RHD
o due to a position effect or gene interaction effect.
o The allele carrying the RHD is trans to the allele carrying C
o Example: Dce/dCe
 Normally, the Rh antigen on the RBC is normal but the steric arrangement of the
C antigen in relationship to the D antigen will appear to interfere with the
expression of the D antigen. And this interference with the D expression does not
occur when the C gene is inherited in cis.
2. Weak D
o Results from the inheritance of RHD gene that codes for the weakened expression of
the D antigen
o D-appear to be complete but fewer in number
o Mutations in the RHD gene
 Cause changes in amino acids present in the transmembrane
 Causing conformational changes in the protein
3. Partial D
o D antigen expression can be weakened when one or more D epitopes within the entire
D protein is missing or altered
o Wiener and Unger
 D antigen is made of antigenic subparts, genetically determined, that could be
absent in rare instances
 This is what we call the D mosaic
o Tippett and Sanger
 Worked with RBCs and sera of partial D individuals, based on testing anti-D sera
from D-positive people
4. Del
o Occurring in individuals whose RBCs possess an extremely low number of D antigen sites
 Most reagent anti-D are unable to detect
o Adsorption and elution
 Only way to detect the D antigen
 o Common in Asian ethnicity (10-30%)
o Rare in whites

Detection of Rh Antibodies and Antigens

Rh Antibodies
o Most Rh antibodies are IgG
o Not naturally occurring and warm antibodies
o Produced following exposure through either HDFN (hemolytic disease of fetus or newborn) or
HTR (hemolytic transfusion reaction)
o May show Dosage effect
o Dosage effect: the anti-D reagent will react better or stronger with genotype that has a
double dose of D antigen
o Enhanced when tested with enzyme-treated RBCs
o Do not bind the complement (prone to extravascular hemolysis)
o Persist in the circulation for years
 Enhanced when tested with enzyme-treated RBCs
 Do not bind the circulation for years
o For Rh antibodies causing hemolytic transfusion reaction or hemolytic disease of the
new born madals na type of hemolysis will happen is extravascular hemolysis. Whereas
ABO, since they are IgM they are capable of fixing the complement and because of
they cause intravascular hemolysis
 Persist in the circulation for years
 IgG1, IgG2, IgG3, IgG4 – reported Rh Antibodies subclasses
 IgG1 and IgG3 – greatest clinical significance
o RES (Reticulo Endothelial system) rapidly clears RBCs coated with IgG1 and IgG3 from
the circulation
o Extravascular hemolysis usually happen in RES for example involving the spleen or liver

Rh Antigens

 Highly immunogenic – they are capable of stimulating our immune system to produce
antibodies
 Exposure to less than 0.1mL of Rh-positive RBCs can stimulate antibody production in an Rh-
negative person
o Rh positive individual – capital D
o Rh negative individual – no D
o Blood component used for emergency transfusion is actually group O negative packed
red blood cells (why Rh negative? For example, the individual is Rh negative, therefore
hindi siya ma eexpose sa D antigen.
o Why Rh positive? May D antigen, and if exposed to Rh negative person there will be no
effect.
 Immunogenicity of common Rh antigens:
o D>c>E>C>e (most immunogenic D, less immunogenic e)

Rh Typing Reagents

 The reagents may be:

o High-protein-based
o Low-protein-based
o Saline-based
o Chemically modified
o Monoclonal
 o Blends of monoclonals
 Saline anti-D has the advantage of being low-protein-based and can be used to test cells that
are already coated with IgG antibody
o Saline based anti-D – the first typing reagent that is available to test for the D antigen
(first manufactured)
 High-protein anti-D consisted primarily of IgG anti-D
o The goal is to use a reagent anti-D that will allow for typing individual’s RBCs as quickly
and accurately as typing for ABO
 Antisera is the anti-D, this is to check for the presence of the D antigen
 Color of lalagyan is GRAY

Clinical considerations

 Transfusions Reactions
o Primary exposure – circulating antibody appears within 120 days
o Secondary exposure – within 2 to 7 days
 Hemolytic Disease of the Newborn
o Levine and Stetson – postulated that the antibody causing the transfusions reaction also
crossed the placenta and destroyed the RBCs of the fetus, causing its death

Rh Deficiency Syndrome

 Rhnull syndrome – inherited in one of two ways:

o Amorphic – mutation in each RHCE gene inherited and a common deletion of the RHD
gene (abnormal). RHAG gene is normal
o Regulator – mutation in the RHAG gene: no RhAG protein expression
 Rhmod syndrome – exhibit a severely reduced expression if all Rh antigens

Unusual Phenotypes and Rare Alleles

1. Cw
o Antithetical to the high-prevalence antigen MAR.
 Antithetical – a term used to describe a pair of antigens that are coated by
different alleles of a single gene
o Results in a single amino acid change most often found on the RhCe protein
o May show dosage effect
 If both expressed in two haplotypes mas better ang reaction
2. f (ce)
o expressed on the RBC when both c and e are present on the same haplotype. It has
been called a compound antigen
o anti-f is generally a weakly reactive antibody often found with other antibodies
3. rhi (Ce)
o considered a compound antigen
o anti-rhi shows positive reactivity only with DCe/dce RBCs
4. G
o Antigen present on most D-positive and all C positive RBCs

Other unusual phenotypes and rare alleles:

 Rh13, Rh14, Rh15, Rh16

 Rh17 (Hro)
 Rh23, Rh30, and Rh40
  Rh33 (Har)
 Rh32
 Rh43 (Crawford)
 e Variants
 V and VS

Deletions

 Exalted D
o Lack all Cc or Ee but often have an unusually strong D antigen expression
o Indicated by the use of dash, example: D-
o Some deletion phenotypes are missing E/e only and are indicated as Dc- or DC-
o A deletion of only the C/c has not been reported

LW BGS (Landsteiner-Wiener Blood Group System)

 Anti-LW
o Reacts strongly with most D-positive RBCs
o Weakly with Rh-negative RBCs and
o Never with Rhnull cells
 Anti-LW shows equal reactivity with cord cells regardless of their D type
  Rh33 (Har)
 Rh32
 Rh43 (Crawford)
 e Variants
 V and VS

Deletions

 Exalted D
o Lack all Cc or Ee but often have an unusually strong D antigen expression
o Indicated by the use of dash, example: D-
o Some deletion phenotypes are missing E/e only and are indicated as Dc- or DC-
o A deletion of only the C/c has not been reported

LW BGS (Landsteiner-Wiener Blood Group System)

 Anti-LW
o Reacts strongly with most D-positive RBCs
o Weakly with Rh-negative RBCs and
o Never with Rhnull cells
 Anti-LW shows equal reactivity with cord cells regardless of their D type