ImmunohematologyImmunohematologyTonang Dwi ArdyantoFK UMS Mei 2009

19-2

Composition of BloodComposition of Blood

19-3

HematopoiesisHematopoiesis

Basic Immuno-hematology Basic Immuno-hematology Immunohematology: Science that deals with

antigens of the blood and their antibodies

Antigen: A substance, enzyme or toxin, capable of stimulating the production of an antibody and then reacting with

that antibody in a specific way. Antibody: A protein in the plasma that reacts with a

specific antigen, serving to neutralize the antigen, thus creating immunity.

Agglutinin= Antibody: An antibody that causes particulate antigens, such as other cells, to adhere to one another, forming clumps

Agglutinogen = Antigen: A antigenic substance that stimulated the formation of a particular antibody

Alloimmunization: Development of a immune response to alloantigens; this occurs during pregnancy, blood transfusions and organ transplantation

Antigens: On RBC’s, protrusions on the surface of the cell that could cause an antibody to form against it

Antibodies: Proteins in the plasma that form in response to a foreign antigen

Antigens in Blood Antigens in Blood TransfusionsTransfusionsFirst important Antigen

◦ Situated on the surface of the red blood cells

◦ A antigen and B antigen◦ Determine blood group (ABO)

Second important antigen◦ Rh system – D (strongest factor), C,E◦ Located on the red cell◦ Inherited

HLA ◦ Alloimmunization

ABO AntigensABO Antigens

Immunology, 5th Edition, 2003

The Blood Group Systems

                                                                                                

                            Inheritance and Genetics

19-9

Blood GroupingBlood Grouping

Determined by antigens (agglutinogens) on surface of RBCs

Antibodies (agglutinins) can bind to RBC antigens, resulting in agglutination (clumping) or hemolysis (rupture) of RBCs

Groups◦ABO and Rh

              

       

 

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

Experiments with blood transfusions have been carried out for hundreds of years. Many patients have died and it was not until 1901, when the Austrian Karl Landsteiner discovered human blood groups, that blood transfusions became safer.

He found that mixing blood from two individuals can lead to blood clumping. The clumped RBCs can crack and cause toxic reactions. This can be fatal.

History of Blood Groups and Blood Transfusions

Karl Landsteiner discovered that blood clumping was an immunological reaction which occurs when the receiver of a blood transfusion has antibodies against the donor blood cells.

Karl Landsteiner's work made it possible to determine blood types and thus paved the way for blood transfusions to be carried out safely. For this discovery he was awarded the Nobel Prize in Physiology or Medicine in 1930.

History of Blood Groups and Blood Transfusions (Cont.)

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

19-20

ABO Blood GroupsABO Blood Groups

19-21

Agglutination ReactionAgglutination Reaction

Blood GroupABO

Antigens on RBCs

Antibodies in Plasma

Onone Anti-A , Anti-

B

A A Anti-B

B B Anti-A

AB A and B Neither

Illustration of the forward and reverse grouping reaction patterns of the ABO groups using a blood group tile

http://www.bh.rmit.edu.au/mls/subjects/abo/resources/genetics1.htm

http://www.lanecountyblood.org/images/other/bloodfacts.jpg

45%

40%

11%

4%

How common are the 4 blood types?

Group OGroup O

Approximately 45% of the population is group O.

No A or B antigens present, think of as “0” antigens present.

These individuals form potent anti-A and anti-B antibodies which circulate in the blood plasma at all times.

Group AGroup A

Approximately 40% of the population is group A.

No B antigens present.These individuals form

potent anti-B antibodies which circulate in the blood plasma at all times.

Group BGroup B

Approximately 11% of the population is group B.

No A antigens present.These individuals form

potent anti-A antibodies which circulate in the blood plasma at all times.

Group ABGroup AB

Approximately 4% of the population is group AB.

Both A and B antigens present.

These individuals possess no ABO antibodies.

NOTE: This slide is in error as it only illustrates presence of one antigen not 2.

The ABO blood groups

Blood Group

Antigens on RBCs

Antibodies in Serum

Genotypes

A A Anti-B AA or AO

B B Anti-A BB or BO

AB A and B Neither AB

O Neither Anti-A and anti-B OO

19-32

Rh Blood GroupRh Blood Group

First studied in rhesus monkeysTypes

◦Rh positive: Have these antigens present on surface of RBCs

◦Rh negative: Do not have these antigens present

Hemolytic disease of the newborn (HDN)◦Mother produces anti-Rh antibodies

that cross placenta and cause agglutination and hemolysis of fetal RBCs

Another antigen on RBC surfaces is called Rh, also known as D.◦ If a person that is Rh negative is transfused

with Rh positive blood, it is very likely that they will make an antibody to the Rh, called anti-D

◦ In U.S., 85% are Rh positive, 15% Rh negative

When a person gets a blood type, both the ABO and the RH are tested◦Group O Rh positive is usually abbreviated

as O Pos◦Group A Rh negative is called A Neg

• Rh antigens are transmembrane proteins with loops exposed at the surface of red blood cells, for the transport of carbon dioxide and/or ammonia across the plasma membrane.

• They are named for the rhesus monkey in which they were first discovered.

• RBCs that are "Rh positive" express the antigen designated D.

• 85% of the population is RhD positive, the other 15% of the population is running around with RhD negative blood.

The Rhesus (Rh) System

Rh Blood Group and Rh Incompatibility

BloodType

Genotype

AllelesProduced

Rh positive

RR R

Rr R or r

Rh negative

rr r

Blood Group

Antigens Antibodies Can give blood to

Can receive

blood from

AB A and B None AB AB, A, B, O

A A B A and AB A and O

B B A B and AB B and O

O None A and B AB, A, B, O O

Blood Group and Incompatibility

HemolysisHemolysisIf an individual is transfused with an

incompatible blood group destruction of the red blood cells will occur.

This may result in the death of the recipient.

Hemolytic Disease of the NewbornHemolytic Disease of the Newborn

HDN occurs when the Mother has an antibody capable of crossing the placental barrier that is specific to an antigen present on the red blood cells of the fetus.

Fetal red cells become coated with the IgG alloantibody and undergo accelerated destruction both before and after birth.◦ Where does the baby get an antigen that is foreign to the

Mom?

It’s the Dads fault!

Pathophysiology of HDNPathophysiology of HDNAccelerated red cell destruction: leads fetus

to increase production of RBCs therefore there are increased numbers of nucleated RBCs. Also called Erythroblastosis fetalis.

macrophages have receptors for the Fc of IgG promoting the the phagocytosis of IgG coated erythrocytes

Severe cases of HDN can result in:Generalized edema of the fetus: Hydrops

fetalisSevere anemia that can lead to

cardiovascular failure and tissue hypoxia, both of which can lead to fetal death.

Pathophysiology of HDNPathophysiology of HDN

Bilirubinemia Results from increased RBC destruction Fetus in utero: Not a problem because

Mom’s liver conjugates the bilirubin Newborn: Problem

◦ Newborn liver not yet able to conjugate the bilirubin. Can build up to toxic levels and cause Kernicterus.

1. Cold reacting

2. Good complement activator

3. Pentamer

4. ABO, Ii, Lewis, MN, & P

1. Warm Reacting

2. Poor complement activators

3. Monomer

4. Rh, Kell, Kidd, Duffy, and Ss

IgG

Antibody’s

IgM Antibody’s

19-43

Erythroblastosis FetalisErythroblastosis Fetalis

Categories of HDNCategories of HDN

Rh System Antibodies

Other Blood Group Antibodies

ABO Antibodies

Most severe form of HDN. • Anti-D is #1. • Less common due to RhIg

Anti-K, -Fya, -s, etc. Page 424, Table 20-1

Least severe. Group O mom with A or B fetus. Most common form of HDN.

ABOABO vs. vs. RhRh HDNal HDNalRh ABO

Mother Negative Group O

Infant Positive A or B (AB)

Occurrence in first born 5% 40-50%

Stillbirth and or hydrops Frequent Rare

Severe Anemia Frequent Rare

DAT PositivePos or

Negative

Spherocytes None Present

Exchange Transfusion Frequent Infrequent

PhototherapyAdjunct to exchange

Often only treatment

HDNBHDNB

AIHAAIHA

OverviewOverviewImmune-mediated

◦ Autoimmune◦ Alloimmune

Hemolytic disease of the newborn◦ Drug-related

Microangiopathic◦ Thrombotic thrombocytopenic purpura

(TTP)◦ Hemolytic-uremic syndrome (HUS)◦ Pregnancy-related (HELLP)

Macrovascular◦ Prosthetic heart valves

Autoimmune Hemolytic Autoimmune Hemolytic Anemia (AIHA)Anemia (AIHA)Result of generalized immune

system dysfunction1-3 cases/100,000 yearlyDiagnosis

◦Positive direct antigen test (DAT) and antibody identification

Types◦Warm Antibody◦Cold Antibody

Autoimmune Hemolytic Autoimmune Hemolytic Anemia (AIHA)Anemia (AIHA)Warm Antibody (near 37° C)

◦ 70% AIHA cases◦ Chronic lymphocytic leukemia◦ Treatment

First line High-dose corticosteroids – prednisone PO 1 – 1.5 mg/kg QD

for 1-3 weeks Splenectomy

Removes site of hemolysis and of antibody production 65 – 70% response rate Should receive pneumococcal and meningiococcal

vaccinations b/c of risk of infection of encapsulated bacteria

Cytotoxic Drugs 40 – 60% response rate Failed steroid and splenectomy treatment

RBC Transfusion (use the least incompatible) Life-threatening anemia High-risk cardiac or cerebrovascular

More AIHAMore AIHACold Antibody

◦ Most hemolytic at 0 – 4° C, leading to clumping of RBCs on the smears

◦ Cold Agglutinin Syndrome (CAS) Typically mild symptoms Hgb 9 – 12 Tx – avoid cold weather, symptomatic tx

◦ Paroxysmal Cold Hemoglobinuria (PCH) Hemolysis at cold & normal temps High fever, chills, abdominal pain with cold

weather Tx – keep warm, consider steroids

http://www.meddean.luc.edu/lumen/MedEd/medicine/Rheumatology/Hands/Hand9/Hand9.jpeg

Even more AIHAEven more AIHAAlloimmune Hemolytic Anemia

◦ Hemolytic disease of the newborn (HDN) RhD – negative mom developing antibodies to

RhD – positive fetal RBCs. Mild anemia to fatal results Hydrops fetalis – seen in severe HDN Treatment

RhoGAM with any fetomaternal hemorrhage event or post-delivery

Intrauterine and intravascular fetal transfusion with plasma exchange and/or IV IG

http://www.yamagiku.co.jp/pathology/image/219/1.jpg

CLASSIFICATIONCLASSIFICATION

AIHA

Primary AIHA

Warm-reactive autoantibodies Paroxysmal cold hemoglobinuria Cold agglutinin disease

Secondary AIHA

SLEImmunodeficiency Malignancy Drug exposure Infections

Test LaboratoriumTest Laboratorium

Antiglobulin Test (2)Antiglobulin Test (2)Direct AHG test: detect in-vivo RBC

sensitization◦ Autoimmune hemolytic anemia◦ Drug-induced hemolysis◦ Hemolytic disease of newborn◦ Alloimmune reactions

Indirect AHG test: detect in-vitro reaction of RBCs and sensitizing antibodies◦ Blood group phenotyping◦ Antibody detection and identification◦ AHG phase of the crossmatch

3-Coomb's Test (Antiglobulin Test):

a. Direct Coomb's Test When antibodies bind to erythrocytes, they do not always result in agglutination. These antibodies that bind to but do not cause agglutination of red blood cells are sometimes referred to as incomplete antibodies. In order to detect the presence of non-agglutinating antibodies on red blood cells, one simply adds a second antibody directed against the immunoglobulin (antibody) coating the red cells. This anti-immunoglobulin can now cross link the red blood cells and result in agglutination.

3-Coomb's Test (Antiglobulin Test):

b. Indirect Coomb's Test If it is necessary to know whether a serum sample has antibodies directed against a particular red blood cell and to be sure that it also detect potential non- agglutinating antibodies in the sample, an Indirect Coomb's test is performed.This test is done by incubating the red blood cells with the serum sample, washing out any unbound antibodies and then adding a second anti-immunoglobulin reagent to cross link the cells.

Direct antiglobulin Test:•Detects antibodies bound to RBC in

vivo•Results in clinical event or illness•(+) DAT indicates an immune

response; patients cells have attached IgG and/or Complement)

•EDTA is sample choice for DAT

Indirect Antiglobulin Test (IAT)• Detects in vitro sensitization of RBC

False positive and False negative reaction for various reasons.

Positive IAT indicates a specific reaction between antigen and antibody in serum of patient.

Coombs (Antiglobulin)TestsCoombs (Antiglobulin)Tests

• Direct Coombs Test– Detects antibodies on erythrocytes

+ ↔

Patient’s RBCs Coombs Reagent(Antiglobulin)

Coombs (Antiglobulin)TestsCoombs (Antiglobulin)Tests

Indirect Coombs Test◦Detects anti-erythrocyte antibodies in serum

Patient’s Serum

TargetRBCs

+ ↔Step 1

+ ↔

Coombs Reagent(Antiglobulin)

Step 2

Compatibility TestsCompatibility Tests