History

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IMMUNOHEMATOLOGY By E. Salehi Ph.D. Assistant
prof. Department of Immunology

• History
• ABO System Phenotype
• ABO System Genotype
• Rh system
• Other Blood Groups
• Blood Group detection and incompatibility
• Hereditary Newborn Disease HDN
• Blood Transfusion

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Karl Landsteiner (1868-1943)

• Discovered ABO blood groups, 1900
• Nobel Prize, 1930

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Red Blood Cell Membrane Components
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Biological Functions of Blood group Systems

• Functional Diversity
• Transporters/Channels
• Transporting Water-Soluble molecules/compounds
• Rh, Colton, Diago, Kx, Kidd
• Receptors
• Biological
• Duffy, Knops, Indian
• Microbial
• MNS, P, Lewis, Duffy, Cromer
• Adhesion Molecules
• Leuthran, Xg, L-W, Indian
• Role in Complement Pathway
• Chido/Rodgers, Cromer, Knops
• Enzymes
• ABO, P, Lewis, H
• Structural Proteins
• Maintain Shape
• MNS, Diago, Gerbich

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• Type .2
• Type .1

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PS oligosaccharide chain attached to either
glycosphingolipid (RBC) or glycoprotein
(secretions).
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Type 2 Precursor Chain
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Formation of H Antigen
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ABO Antigen Genetics

• LOCATION
• The presence or absence of the ABH antigens on
  the red blood cell membrane is controlled by the
  H gene
• The presence or absence of the ABH antigens in
  secretions is indirectly controlled by the Se
  genes.

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H Antigen
The H gene codes for an enzyme that adds a sugar
(Fucose) to the terminal sugar of a Precursor
Substance (PS). The biochemical structure below
constitutes the H Antigen. (h gene is an amorph.)
H gene acts on a Precursor substance(PS) by
adding Fucose
PS oligosaccharide chain attached to either
glycosphingolipid (RBC) or glycoprotein
(secretions).
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The H antigen is found on the rbc when you have
the Hh or HH genotypes but NOT from the hh
genotype.
The A antigen is found on the rbc when you have
the Hh, HH, and A/A, A/O or A/B genotypes.
The B antigen is found on the rbc when you have
the Hh, HH, and B/B, B/O or A/B genotypes.
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Possible Blood group Genotypes
Parent Allele A B O
A AA AB AO
B AB BB BO
O AO BO OO
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ABO Subgroups

• ABO subgroups differ in the amount of antigen
  present on the red blood cell membrane,
  specifically, they have less - it is
  quantitative.
• Subgroups are the results of less effective
  enzymes! Not as efficient at converting H
  antigens to A or B antigens so fewer are present
  on the rbc.
• Subgroups of A are more common than Subgroups of
  B.

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Subgroups of A

• The two principle subgroups of A are
• A1 and A2
• Both react strongly with reagent anti-A.
• To distinguish A1 from A2 red blood cells test
  with plant lectin Dolichos biflorus
• Approximately 80 of Group A and Group AB
  persons red cells are agglutinated by Dolichos
  biflorus and can be designated A1 and A1B.
• The remaining 20 are A2 and A2B.

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ABO Subgroups

• A2 Phenotype
• A2 persons produce anti-A1 allo-antibodies (1-8)
• A2B persons produce anit-A1 allo antibodies
  (22-35)
• Allo-Anti-A1 can cause ABO Discrepancies (How?)
  and incompatibility in crossmatching. It is not
  considered clinically significant if it does not
  react at 37oC.

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Number of A antigen

• A1800000
• A2250000
• A335000
• Ax4800
• Aend3500
• Am700

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A2 A1 ???????
????? ?? ????A
200000 800000 ?????
- ????? ??????? ???? ???
TYPE2 TYPE1,2 ??????? ???? ??
?????? ?? ? ???? ?? ??PH7 ???? ?? ????? ?? ??PH6 ?????? ??????????
????Mg Mn,Mg ??? ????????
6-7 9-10 ???? ???????? ????????????
 
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Amount of H Antigen according to ABO Blood Group

• Blood Group O people have red blood cells rich in
  H antigen. Why?

Neither the A or B genes have converted the H
antigens to A or B antigens - just a whole bunch
of H!
LeastAmount of H
Greatest Amount of H
O gt A2 gt B gt A2B gt A1 gt A1B
Lectin O cells A2 cells A2B cells B cells A1 cells A1B cells Bombay cells
lectin-H 4 3 2-3 2 weak to negative weak to negative negative
Lectin-A1 negative negative negative negative positive positive negative
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Formation Of ABO Antigens In Secretions
Hh
ABO
PS2
ABO on Cells
H Antigen
ABO
PS1
ABO in secretions
H Antigen
Se se

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Bombay (Oh) Phenotype

• Results from the inheritance of hh genotype
• Red blood cells lack H, A and B antigens
• First discovered in Bombay, India
• Red cells are NOT agglutinated with anti-A,
  Anti-B or Anti-H (Ulex europaeus - lectin)
• Serum has strong anti-A, Anti-B and anti-H so
  they agglutinate ALL ABO blood groups
• ParaBombay (Ah) Phenotype

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A Mystery.Why preformed ?
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ABO Blood Grouping Reagents

• Forward Grouping
• Reagent Anti-A and Anti-B
• IgM class Monoclonal antibody reagent
• Reverse Grouping
• Reagent A1 and B cells (3-5 suspension)
• Routine tests on donors and patients must include
  both the forward and reverse grouping

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Frequency of ABO Blood Groups

• Group O 47
• Group A 42
• Group B 8
• Group AB 3

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The Rh Blood Group System

• Described by Landsteiner in 1940
• Antibodies produced as a result of pregnancy or
  transfusion
• Immune antibodies - IgG
• Can cause haemolytic disease of the newborn and
  transfusion reactions

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Inheritance of Rh genes

• Fisher-Race theory of inheritance
• Rh antigens produced by three closely linked
  alleles C or c, D or d, E or e. (these alleles
  are located in 2 locus RHD RHCE
• We inherit these genes in groups of three from
  each parent
• A common combination is CDe/cde
• Other individuals have combinations of cDE, cde,
  Cde, cdE

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Rh System

• D Positive are either D/D or D/d
• D Negative are d/d
• 85 of the population are D Positive
• 15 of the population are D Negative
• Other Rh antigens discovered and named C,c,E and
  e
• Weak D phenotype
• Rhnull

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Weak D Phenotype (Du)

• The weak D phenotype is thought to occur by one
  of three mechanisms
• (a) inheritance of an RHD gene encoding for a
  weakened expression of D (DCe or DcE)
• (b) interaction of the D gene with other genes
  (Dce/Ce)
• (c) inheritance of an RHD gene missing some
  epitopes. (lack of part of D)

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Hemolytic Disease of the Newborn (HDN)

• (Erythroblastosis fetalis)

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Background

• A French midwife was the first to report
  hemolytic disease of the newborn (HDN) in 1609.
• In 1932, Diamond and colleagues described the
  relationship of fetal hydrops, jaundice, anemia,
  and erythroblasts in the circulation, a condition
  later called erythroblastosis fetalis.
• Levine later determined the cause after
  Landsteiner and Weiner discovered the Rh blood
  group system in 1940.
• In 1953, Chown subsequently confirmed the
  pathogenesis of Rh alloimmunization to be the
  result of passage of Rh-positive fetal red blood
  cells after transplacental hemorrhage into
  maternal circulation that lacked this antigen.

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Rh Incompatibility

• Expression is limited to RBCs
• Rh positive 45 are homozygous and 55 are
  heterozygous
• Rh incompatibility is a condition which develops
  when there is a difference in Rh blood type
  between that of the pregnant mother (Rh negative)
  and that of the fetus (Rh positive).
• After the initial exposure to a foreign antigen,
  the maternal immune system produces antibodies of
  the immunoglobulin M (IgM) isotype that do not
  cross the placenta, and later it produces
  antibodies of the IgG isotype that traverse the
  placental barrier.

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ABO incompatibility

• ABO incompatibility is limited to type O mothers
  with fetuses who have type A or B blood
• in type O mothers, the antibodies are
  predominantly IgM in nature
• Because A and B antigens are widely expressed in
  a variety of tissues besides RBCs, only small
  portion of antibodies crossing the placenta is
  available to bind to fetal RBCs. In addition,
  fetal RBCs appear to have less surface expression
  of A or B antigen, resulting in few reactive
  siteshence the low incidence of significant
  hemolysis in affected neonates.

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Causes

• Common causes for HDN
• Rh system antibodies
• ABO system antibodies
• Uncommon causes
• Kell system antibodies
• Rare causes
• Duffy system antibodies
• MNS and s system antibodies
• No occurrence in HDN
• Lewis system antibodies
• P system antibodies

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• BEFORE BIRTH
• Antibodies cause destruction of the red cells
• Anemia
• heart failure
• fetal death

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• AFTER BIRTH
• Antibodies cause destruction of the red cells
• Anemia
• Heart failure
• Erythroblastosis
• General edema Called hydrops fetalis and
  erythroblastosis fetalis
• Build up of billirubin
• Kernicterus
• Severe retardation

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Kernicterus due to hyperbilirubinemia due to
erythroblastosis fetalis due to Rh incompatibility
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• ???? ?????
• (1) interruption of normal neurotransmission
  (inhibits phosphorylation of enzymes critical in
  release of neurotransmitters)
• (2) mitochondrial dysfunction
• (3) cellular and intracellular membrane
  impairment (billirubin acid affects membrane ion
  channels and precipitates on phospholipid
  membranes of mitochondria
• (4) interference with enzyme activity (binds to
  specific billirubin receptor sites on enzymes).

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• PREVENTION
• Before birth
• Work up mother for risk and evaluation of
  complications
• After birth
• Rh immune globulin - IgG anti-D given to prevent
  primary immunization

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• Before birth workup
• Identify women at risk
• ABO - Rh -(Du) - Antibody screen
• based on results continue testing (Handout)
• IgM antibodies are insignificant
• IgG antibodies - titer - freeze and store -
  retiter with a second sample - looking for a 132
  rise or change in titer

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• Before birth workup
• titer identifies mothers who need amniocentesis
• titer every 4 week until 24th week - then every 2
  weeks
• amniocentesis is performed after 21st week on
  high titer - high mortality

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• Amniocentesis
• Analyze pigment that indicates increased
  hemolysis
• Measure OD from 350 - 700 and plot as a function
  of wavelength
• Draw straight line and obtain difference in OD at
  450

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• Amniocentesis

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• Intrauterine transfusions
• Bilirubin
• Hb is below 11 g/dL
• Usually O and compatible with mothers antibody
• CMV, Hb S, and leukocyte negative
• immediate correction of anemia and resolution of
  fetal hydrops, reduced rate of hemolysis and
  subsequent hyperinsulinemia, and acceleration of
  fetal growth for nonhydropic fetuses who often
  are growth retarded

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• After birth
• Rh Immune Globulin
• Give antenatal 28 -32 weeks
• also after amniocentesis - IUT - abortion -
  ectopic pregnancy - miscarriage
• Each vial contains 300 ugm and will prevent
  sensitization by 15 ml RBC or 30 ml whole blood

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• Post Natal Laboratory Studies
• Mother
• ABO - Rh - Du (micro) - Antibody screen -
  Antibody identification if necessary
• Baby
• ABO - Rh - Du - DAT for IgG antibodies - elute
  DAT positive and identify antibody
• CBC
• Imaging studies

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TREATMENT

• Exchange transfusion
• Phototherapy

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Phototherapy
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• The following are requirements for exchange
  transfusion
• Severe anemia (Hb lt10 g/dL)
• Rate of bilirubin rises more than 0.5 mg/dL/hr
  despite optimal phototherapy
• Hyperbilirubinemia
• DAT

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• Exchange Transfusions Objectives
• Decrease serum billirubin and prevent kernicterus
• Provide compatible red cells to provide oxygen
  carrying capacity
• Decrease amount of incompatible antibody
• Remove fetal antibody coated red cells

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• Potential complications of exchange transfusion
  include the following
• Cardiac - Arrhythmia, volume overload, congestive
  failure, and arrest
• Hematologic - Overheparinization, neutropenia,
  thrombocytopenia, and graft versus host disease
• Infectious - Bacterial, viral (CMV, HIV,
  hepatitis), and malarial
• Metabolic - Acidosis, hypocalcemia, hypoglycemia,
  hyperkalemia, and hypernatremia
• Vascular - Embolization, thrombosis, necrotizing
  enterocolitis, and perforation of umbilical
  vessel
• Systemic - Hypothermia

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Blood banking transfusion
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Blood in History
China, 1000 BC The soul was contained in the
blood. Egyptians bathed in blood for their
health. Pliny and Celsus describe Romans
drinking the blood of fallen gladiators to gain
strength and vitality and to cure
epilepsy. Taurobolium, the practice of bathing
in blood as it cascaded from a sacrificial bull,
was practiced by the Romans.
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Pope Innocent VIII
a Jewish daring innovator, whose name has not
come down to us in memory of his deed, proposed
to find the pontiff a fountain of jouvenance in
the blood of three youths who died as martyrs to
their own devotion and the practitioners
zeal. Drinkard, 1870
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HISTORY
Harvey Discovered Circulation of Blood
1628
1665-66
Wilkins Lower Transfusions from dog to dog
1667
Jean-Baptiste Denis Performed first recorded
blood transfusions from animals to humans
James Blundell, Obstetrician
1818
First transfusion of human to human
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James Blundell
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Animal to Human Transfusion
Early lamb blood transfusion
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The Kimpton-Brown transfusion apparatus
was commonly used before citration. It consisted
of a paraffin-coated gradient glass cylinder with
a horizontal side tube for suction. It was in
use until approximately 1918.
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Lewisohns Method of Transfusion
Blood is collected in a citrated flask....and
immediately transfused.
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Early transfusion Paris, France
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Donors must be 

• 17 years of age
• in good health
•  
• weigh at least 40 kg
• pass a physical and health history examination
  prior to donation

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Who should not donate blood?

• Anyone who has ever used illegal intravenous (IV)
  drugs 
•  
• Hemophiliacs 
• Anyone with a positive test for HIV 
• Anyone who has had hepatitis since his or her
  eleventh birthday 

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Transfusion

• Autologus transfusion it refers to those
  transfusions in which the blood donor
  transfusion recipient are the same.
• Allogeneic transfusion It refers to blood
  transfused to someone other than the donor.

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Autologous transfusion

• Preoperative donation
• Blood dilution
• Intraoperative blood salvage
• Post operative blood collection

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Experienced mild side effects by a donor

• Stinging during insertion the needle
• Upset stomach
• Dizziness
• A small amount of bruising

• A donor may faint
• Having muscle spasm
• Suffering damage

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No Whole blood BUT blood components
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Plastic Blood Bags and Component Separation
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Red blood cells

• For chronic anemia resulting from disorders
• For acute blood loss
• resulting from trauma or surgery
• Shelf life of RBC 42 days
• Frozen for up to 10 years

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Plasma

• Contain albumin fibrinogen globulins
• Usually separated into specific products.
• Fresh frozen plasma stored for 1 7 years.
• Cryoprecipated AHF, rich in certain clotting
  factors.( factor VIII , fibrinogen, von Willbrand
  factor, factor XIII
• AHF prevent or control bleeding in individuals
  with hemophilia and von Willbrands disease.

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Platelets

• Prevent massive blood loss resulting from trauma.
• Maybe obtained from donor by a process known as
  APHERESIS.
• Stored at room temperature for up to 5 days.
• used to treat thrombocytopenia.

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White blood cells

• Transfused within 24 h after collection.
• Used for infections that are unresponsive to
  antibiotic therapy.
• The effectiveness is still being investigated.

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Compatibility testing

• ABO-Rh blood typing
• Antibody screening
• Cross-matching
• Cross-matching is performed to determine if the
  patient has antibodies that react with the
  donors cells

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The risk of infection from transfusion

• About 1 in 600,000 units for hepatitis B
• About 1 in 2 million units for HIV and hepatitis C

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The greater concern is an ABO incompatibility and
transfusion reactions.ABO incompatibility occurs
when blood samples from two people with different
ABO blood types are mixed.
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• Several types of transfusion reactions like
  allergic and febrile(characterized by fever)
• Treatment will depend on type of reaction and
  patients symptoms.

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Fully automated grouping and antibody screening
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Play a game on Blood grouping for blood
transfusion
http//nobelprize.org/medicine/educational/landste
iner/index.html
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