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

IMMUNOHEMATOLOGY

By

E. Salehi Ph.D.

Assistant prof.

Department of Immunology

Karl Landsteiner (1868-1943)

• Discovered ABO blood groups, 1900

• Nobel Prize, 1930

Red Blood Cell Membrane

Components

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

3

*PS = oligosaccharide chain attached to either glycosphingolipid (RBC) or glycoprotein (secretions).

• Type .2

• Type .1

Type 2 Precursor Chain

Formation of H Antigen

ABO Antigen Genetics

LOCATION a) The presence or absence of the ABH

antigens on the red blood cell membrane is controlled by the H gene

b) The presence or absence of the ABH antigens in secretions is indirectly controlled by the Se genes.

H gene acts on a Precursor substance(PS)* by adding Fucose

H Antigen

*PS = oligosaccharide chain attached to either glycosphingolipid (RBC) or glycoprotein (secretions).

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.)

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.

Parent

Allele

A B O

A AA AB AO

B AB BB BO

O AO BO OO

Possible Blood group Genotypes

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.

Subgroups of A

• The two principle subgroups of A are: 1. A1 and A2

a) Both react strongly with reagent anti-A.b) To distinguish A1 from A2 red blood cells test

with plant lectin: Dolichos biflorus

c) Approximately 80% of Group A and Group AB persons red cells are agglutinated by Dolichos biflorus and can be designated A1 and A1B.

d) The remaining 20% are A2 and A2B.

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.

Number of A antigen

• A1=800000

• A2=250000

• A3=35000

• Ax=4800

• Aend=3500

• Am=700

A2 A1 خصوصیات

+++ ++++ آنتی با Aواکنش

200000 800000 تعداد

- +++ رقیق بالکتین واکنششده

TYPE2 TYPE1,2 ژن آنتی ساختمان

آل ایده و کم فعالیتPH=7در

آل وایده تر فعالPH=6در

فعالیت ترانسفرازی

Mgتنها Mn,Mg موردنیاز فلز

6-7 9-10 فوکوسینگ نقطهایزوالکتریکی

 

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!

Greatest Amount of H

LeastAmount of H

O > A2 > B > A2B > A1 > A1B

Lectin O cells A2 cellsA2B

cellsB cells A1 cells

A1B

cellsBombay cells

lectin-H 4+ 3+ 2-3+ 2+weak to negative

weak to negative

negative

Lectin-A1 negative negative negative negative positive positive negative

H Antigen

Se se

Hh

PS1

PS2 ABO ABO on Cells

H AntigenABO

ABO in secretions

Formation Of ABO Antigens In SecretionsSecretions

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

A Mystery….Why “preformed” ?

3-6 mo 5-10 yr

Ab titer

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

Frequency of ABO Blood Groups

• Group O 47%

• Group A 42%

• Group B 8%

• Group AB 3%

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

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

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

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)

Hemolytic Disease of the Newborn (HDN)

(Erythroblastosis fetalis)

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.

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.

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 sites—hence the low incidence of significant hemolysis in affected neonates.

Causes

Common causes for HDNRh system antibodies

ABO system antibodies

Uncommon causes Kell system antibodies

Rare causesDuffy system antibodies

MNS and s system antibodies

No occurrence in HDNLewis system antibodies

P system antibodies

BEFORE BIRTH

Antibodies cause destruction of the red cellsAnemiaheart failurefetal death

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

Kernicterus due to hyperbilirubinemia due to erythroblastosis fetalis due to Rh incompatibilityKernicterus due to hyperbilirubinemia due to erythroblastosis fetalis due to Rh incompatibility

: روبین بیلی (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).

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

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 1:32 rise or change in titer

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

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

AmniocentesisAmniocentesis

Intrauterine transfusions Bilirubin Hb is below 11 g/dL

Usually O and compatible with mother’s 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

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

Post Natal Laboratory Studies

Mother ABO - Rh - Du (micro) - Antibody screen - Antibody

identification if necessaryBaby

ABO - Rh - Du - DAT for IgG antibodies - elute DAT positive and identify antibody

CBC Imaging studies

TREATMENT

Exchange transfusion

Phototherapy

PhototherapyPhototherapy

The following are requirements for exchange transfusion :

Severe anemia (Hb <10 g/dL)

Rate of bilirubin rises more than 0.5 mg/dL/hr despite optimal phototherapy

Hyperbilirubinemia

DAT

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

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

Blood banking & transfusion

China, 1000 BCThe 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 cascadedfrom a sacrificial bull, was practiced by the Romans.

Blood in History

Pope Innocent VIII

“…a Jewish daring innovator,whose name has not come down to us in memory ofhis deed, proposed to find the pontiff a fountain ofjouvenance in the blood of three youths who died asmartyrs to their own devotion and the practitioners zeal.”

Drinkard, 1870

HISTORY

1628

1665-’66

1667

Harvey Harvey Discovered Circulation of Blood

Wilkins & LowerWilkins & Lower Transfusions from dog to dog

Jean-Baptiste Denis Performed first recorded blood transfusions from animals to humans

1818

First transfusion of human to humanJames BlundellJames Blundell, Obstetrician

James Blundell

Animal to Human Transfusion

Early lamb blood transfusion

The Kimpton-Browntransfusion apparatus wascommonly used beforecitration. It consisted of aparaffin-coated gradient glasscylinder with a horizontalside tube for suction. It was in use until approximately 1918.

Lewisohn’s Method of Transfusion

Blood is collected in a citrated flask….…...and immediately transfused.

Early transfusion: Paris, France

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

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 

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.

Autologous transfusion

Preoperative donationBlood dilutionIntraoperative blood salvagePost operative blood collection

Experienced mild side effects by a donor

Stinging during insertion the needleUpset stomachDizzinessA small amount of bruising

A donor may faintHaving muscle spasmSuffering damage

No Whole blood BUT blood components

Plastic Blood Bags and Component Separation

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

Plasma

Contain albumin – fibrinogen – globulinsUsually 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 Willbrand’s disease.

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.

White blood cells

Transfused within 24 h after collection. Used for infections that are unresponsive to

antibiotic therapy. The effectiveness is still being investigated.

Compatibility testing

ABO-Rh blood typing Antibody screeningCross-matchingCross-matching is performed to determine

if the patient has antibodies that react with the donor’s cells

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

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.

Several types of transfusion reactions like allergic and febrile(characterized by fever)

Treatment will depend on type of reaction and patient’s symptoms.

Fully automated grouping and antibody screening

http://nobelprize.org/medicine/educational/landsteiner/index.html

Play a game on Blood grouping for blood transfusion