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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

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

• Type .2

• Type .1

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

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 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).

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.

Possible Blood group Genotypes Parent Allele A A AA B AB O AO B O AB AO BB BO BO OO

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 present on the rbc.

fewer are • Subgroups of A Subgroups of B .

are more common than

Subgroups of A

• The two principle subgroups of A are: 1. A 1 and A 2 a) Both react strongly with reagent anti-A.

b) To distinguish A 1 from A 2 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 A 1 and A 1 B.

d) The remaining 20% are A 2 and A 2 B.

ABO Subgroups

• A 2 persons

A

2

Phenotype

produce anti-A 1 allo-antibodies • A 2 B persons produce anit-A 1 allo antibodies • Allo-Anti-A 1 can cause ABO

It is not considered clinically significant if it does not react at 37 o

Discrepancies

C.

Number of A antigen

• A1=800000 • A2=250000 • A3=35000 • Ax=4800 • Aend=3500 • Am=700

A2 +++ 200000 TYPE2 لآ هدیا و مک تیلاعف PH=7 رد Mg اهنت 6-7 A1 ++++ 800000 +++ TYPE1,2 لآ هدیاو رت لاعف PH=6 رد Mn,Mg 9-10 تایصوصخ A یتنآ اب شنکاو دادعت هد ش قیقر نیتکلاب شنکاو نژ یتنآ نامتخاس تیلاعف یزارفسنارت زایندروم زلف یکی رتکلاوزیا گنیسوکوف هطقن

Amount of H Antigen according to ABO Blood Group

• Blood Group O people 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

O > A 2 > B > A 2 B > A 1 > A 1 B Lectin

lectin-H

O cells

4+ Lectin-A 1 negative

A 2 cells

3+ negative

A 2 B cells

2-3+ negative

B cells

2+ negative

A 1 cells

weak to negative positive

Least

Amount of H

A 1 B cells

weak to negative positive

Bombay cells

negative negative

PS2 Formation Of ABO Antigens In Secretions

Hh

H Antigen ABO

ABO on Cells

PS1

Se se

H Antigen ABO

ABO in secretions

Bombay (O

h

) Phenotype

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

ParaBombay (Ah) Phenotype

A Mystery….Why “preformed” ?

Ab titer 3-6 mo 5-10 yr

ABO Blood Grouping Reagents

• Forward Grouping – Reagent Anti-A and Anti-B • IgM class Monoclonal antibody reagent • Reverse Grouping – Reagent A must 1 and B cells (3-5% suspension) • Routine tests on donors and patients 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 • Rh null

Weak D Phenotype (D

u

)

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 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



BEFORE BIRTH

 Antibodies cause destruction of the red cells  Anemia  heart failure  fetal 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 incompatibility

: نیبور یلیب

  (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

 Amniocentesis

 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 necessary  Baby  ABO - Rh - Du - DAT for IgG antibodies - elute DAT positive and identify antibody  CBC  Imaging studies



TREATMENT

 Exchange transfusion  Phototherapy

Phototherapy

 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

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.

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

HISTORY

Harvey Discovered Circulation of Blood

1628 1665 ’66 1667 1818

Wilkins & Lower Transfusions from dog to dog

Jean-Baptiste Denis

Performed first recorded blood transfusions from animals to humans James Blundell , Obstetrician First transfusion of human to human

James Blundell

Animal to Human Transfusion Early lamb blood transfusion

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.

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 donation  Blood dilution  Intraoperative blood salvage  Post operative blood collection

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

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 – 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 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 screening  Cross-matching  Cross-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

Play a game on Blood grouping for blood transfusion

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