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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