Immunohematology

Tonang Dwi Ardyanto FK UMS Mei 2009

Composition of Blood

192

Hematopoiesis

193

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 RBCs, 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 Transfusions

First 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 Antigens

Immunology, 5th Edition, 2003

The Blood Group Systems

Inheritance and Genetics

Blood 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
199

History of Blood Groups and Blood Transfusions

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.
http://nobelprize.org/medicine/educational/landsteiner/readmore.htm l

History of Blood Groups and Blood Transfusions (Cont.)

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.
http://nobelprize.org/medicine/educational/landsteiner/readmore.ht ml

ABO Blood Groups

1920

Agglutination Reaction

1921

Blood Group O A B
AB

ABO Antigens on RBCs none

A B A and B

Antibodies in Plasma Anti-A , Anti-B

Anti-B Anti-A 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

How common are the 4 blood types?

4%

11%

45%

40%

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

Group 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 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 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 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 Antibodies in Serum Genotypes on RBCs A B A and B Anti-B Anti-A Neither AA or AO BB or BO AB

A B AB

Neither

Anti-A and anti-B

OO

Rh Blood Group
First studied in rhesus monkeys Types

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
1932

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

The Rhesus (Rh) System 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.

Rh Blood Group and Rh Incompatibility

Blood Type

Genotype

Alleles Produced R R or r

RR Rh positive Rr

Rh negative

rr

Blood Group and Incompatibility

Blood Group Antigens Antibodies Can give blood to Can receive blood from

AB

A and B

None

AB

AB, A, B, O

A and AB

A and O

B and AB

B and O

None

A and B

AB, A, B, O

Hemolysis
If 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 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?

Its the Dads fault!

Pathophysiology of HDN
Accelerated 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 fetalis Severe anemia that can lead to cardiovascular failure and tissue hypoxia, both of which can lead to fetal death.

Pathophysiology of HDN
Bilirubinemia

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

IgM Antibodys

3. Pentamer 4. ABO, Ii, Lewis, MN, & P 1. Warm Reacting

IgG Antibodys

2. Poor complement activators 3. Monomer 4. Rh, Kell, Kidd, Duffy, and Ss

Erythroblastosis Fetalis

1943

Categories of HDN
Rh System Antibodies Most severe form of HDN.
Anti-D is #1. Less common due to RhIg

Other Blood Group Antibodies ABO Antibodies

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.

ABO vs. Rh HDNal

Mother Infant Occurrence in first born Stillbirth and or hydrops Severe Anemia DAT Rh Negative Positive 5% Frequent Frequent Positive ABO Group O A or B (AB) 40-50% Rare Rare Pos or Negative Present Infrequent Often only treatment

Spherocytes
Exchange Transfusion Phototherapy

None
Frequent Adjunct to exchange

HDNB

AIHA

Overview

Immune-mediated
Autoimmune Alloimmune Drug-related
Hemolytic disease of the newborn

Microangiopathic
Thrombotic thrombocytopenic purpura (TTP) Hemolytic-uremic syndrome (HUS) Pregnancy-related (HELLP)

Macrovascular
Prosthetic heart valves

Autoimmune Hemolytic Anemia (AIHA)

Result of generalized immune system dysfunction 1-3 cases/100,000 yearly Diagnosis

Positive direct antigen test (DAT) and antibody identification

Types
Warm Antibody Cold Antibody

Autoimmune Hemolytic 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 AIHA

Cold 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/Ha nd9.jpeg

Even more AIHA

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

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

CLASSIFICATION

AIHA
Primary AIHA Secondary AIHA

Warm-reactive autoantibodies Paroxysmal cold hemoglobinuria Cold agglutinin disease

SLE Immunodeficiency Malignancy Drug exposure Infections

Test Laboratorium

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 antiimmunoglobulin 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 nonagglutinating 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 antiimmunoglobulin 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)Tests

Direct Coombs Test

Detects

antibodies on erythrocytes

Patients RBCs

Coombs Reagent (Antiglobulin)

Coombs (Antiglobulin)Tests

Indirect Coombs Test

Detects anti-erythrocyte antibodies in serum

Step 1 Patients Serum Step 2

+

Target RBCs

Coombs Reagent (Antiglobulin)

Compatibility Tests