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Transcript of 3.inmuno hematología.inmunologia.2011.dr hilario
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Immunohematology
Dr. Julio Hilario VargasDepartment of Physiology
School of MedicineNational University of Trujillo
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Es la parte de la hematología que estudia los procesos inmunitarios que tienen lugar en el organismo en relación con los elementos sanguíneos.
Uno de los aspectos más importantes de la inmunohematología es el estudio y cuantificación de los grupos sanguíneos eritrocitarios que son componentes antigénicos presentes en la superficie de los hematíes, ya que se relaciona directamente con la terapéutica transfusional y la prevención de accidentes hemolíticos graves.
DEFINICION
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En primer termino se identificaron sobre los hematíes, pero luego se describieron determinantes antigénicos plaquetarios, leucocitarios y séricos.
Los genes determinantes de los grupos sanguíneos transmiten, generalmente, caracteres codominantes ( se expresan en homocigotos y heterocigotos).
Existen genes “amorfos” que no generan productos que puedan ser identificados como antígenos ( ej: gen d)
Todos los antígenos de los grupos sanguíneos han sido definidos serológicamente por la presencia de sus anticuerpos correspondientes.
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Merges aspects of hematology, immunology & genetics
Serologic, genetic, biochemical and molecular study of antigens associated with membrane structures on the cellular constituents of the blood
Immunologic reactions involving all blood components and constituents
Primary immunological components: antigens & antibodies provides basis for blood bank testing and reactions
Immunohematology
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CARDINAL RULE IN BLOOD BANK:
Antigens are found on the surface of red blood cells and the antibodies
are found in serum or plasma
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IMMUNOLOGIC PRINCIPLES
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Substances that have the capability to stimulate the production of an antibody
Characteristics:
1. Chemical nature – protein, CHO, nucleic acid or lipopolysaccharide
2. Molecular weight > 10,000 daltons
3. Complexity – more complex, > antibody stimulation
4. Stability – if unstable degrade less Ab stimulation
5. Foreign
ANTIGENS
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1. Glycoproteins & lipoproteins – most potent
Glycolipids
2. Pure polysaccharides – not immunogenic except in humans and mice
3. Pure lipids & nucleic acids – not immunogenic but can be antigenic serve as haptens
Chemical composition of antigens
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GRUPOS SANGUÍNEOSANTÍGENOS MAS IMPORTANTES
ABO A, B, AB, O
Rh D, C, c, E, e
MNS M, N, S, s, U
Lewis Lea, Leb
P P1, P2
Lutheran Lua, Lub
Kell K, k, Kpa, Kpb
Duffy Fya, Fyb
Kidd Jka, Jkb
Grupos Eritrocitarios y sus Antígenos
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SISTEMAS DE GRUPOS SANGUINEOS
Grispan S. Rev. Medica Hondur. 51:103-14. 1983
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A, B and D (Rho) – most immunogenic
Kell (K)
Duffy: Fya
Fyb
Kidd:Jka
Jkb
Immunogenicity of Blood Group Antigens
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Also called immunoglobulins
Characteristics:
1. Protein
2. Produced in response to stimulation by an antigen
3. Specific for the stimulating antigen
- Consists of 2 heavy chains & 2 light chains held
together by disulfide bonds
- Produce 3 fragments when cleaved by enzymes 2 Ag- binding fragments (Fab) & 1 crystallizable fragment (Fc)
ANTIBODIES
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Alloantibodies
Reacts with foreign Ag not present on patient’s own RBC
Most produced as result of immune stimulation via transfusion or pregnancy (usually during delivery)
Autoantibodies
Reacts with an Ag on patient’s own cells & with that same Ag on the cells of other individuals
Classification of Blood Group Antibodies
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Discovered by Karl Landsteiner; locus on chr 9
Single most important blood group for the selection and transfusion of blood
Widely expressed tissues & body fluids including red cells, platelets & endothelial cells
Three antigens: A, B, H
Two major antibodies: anti-A and anti-B
Four phenotypes: A, B, AB, O A & B Ag’s autosomal co-dominant (expressed on grp A, B and AB red cells; O phenotype autosomal recessive (most frequent)
ABO BLOOD GROUP SYSTEM
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ABO BLOOD GROUP SYSTEM
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ABO Antigens
Present on the surface of red cells as well as tissue and endothelial cells in the body
Found in soluble form in plasma & other body secretions in people known as secretors
Inherited in simple Mendelian fashion from an individual’s parents
3 possible genes that can be inherited: A, B, O
A and B genes produce a detectable product
O gene does not produce a detectable product
ABO BLOOD GROUP SYSTEM
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ABO System
ABO BLOOD GROUP SYSTEM
Phenotype Antigen Natural antibody
Genotype
A A only Anti-B AA or AO
B B only Anti-A BB or BO
AB A and B None AB
O NoneAnti-A,
Anti-BOO
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ABO BLOOD GROUP SYSTEM A and B genes do not directly produce antigens enzymatic reaction products of enzymes called glycosyltransferases attaches a sugar molecule to the chemical structure of the antigen sugar molecule responsible for specificity
O antigen no transferase no antigen produced
A and B antigens on surface of RBC protrude from outermost layer of cell membrane
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ABO BLOOD GROUP SYSTEM
Red blood cell precursor structure
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ABO BLOOD GROUP SYSTEMAntigen formation
H antigen B antigen B antigen
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http://www.ncbi.nlm.nih.gov/gv/rbc/xslcgi.fcgi?cmd=bgmut/systems_info&system=abo
ABO Blood Group System
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ABO BLOOD GROUP SYSTEM
H Antigen
Required to produce either A or B antigens
possible genetic combinations: HH, Hh, or hh
HH or Hh (+) produce H Ag 99.99% of Caucasians
hh does not produce H Ag Bombay phenotype (Oh)
Anti-H antibodies rare – found only in individuals with Bombay phenotype
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ABO BLOOD GROUP SYSTEMExample of determining offspring blood types from known or suspected genotypes:
Genotype parent #1 (AO)
A O
Genotype parent A AA AO
#2 (AB) B AB BO
Phenotypes of possible offsprings: A, AB, B
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ABO BLOOD GROUP SYSTEM
Frequencies of ABO Blood Groups:
Blood Group Frequency
O 45%
A 41%
B 10%
AB 4%
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ABO BLOOD GROUP SYSTEM
ABO Subtypes:
1. A variants (A1, A2)
A1 most common (80%) & most antigenic
A1 and A2 differentiated using antisera specific for A1 Ag (anti-A1 lectin) prepared from seed known as Dolichos biflorus (+) reaction with A1 but not A2
Anti-A reacts with both A1 & A2 but more strongly with A2
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ABO BLOOD GROUP SYSTEM
ABO Subtypes:
2. Weak A and weak B phenotypes
3. Null phenotypes:
(a) Bombay (Oh)
No A, B or H Ag on red cells & secretions
With anti-A, anti-B & anti-H in their sera
(b) para-Bombay
Absent or only trace A,B & H Ag’s detected on rbc w/ normal expression in secretions & body fluids
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ABO BLOOD GROUP SYSTEM
ABO Antibodies
Natural antibodies antigenic stimulus is environmental exposure occurs from birth
Newborns without ABO antibodies of their own; begin to produce Ab with detectable titer at 6 months of age
Other characteristics of ABO antibodies:
- IgM
- Reacts at room temp. after an immediate spin
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ABO ROUTINE TESTING
(slide or test tube method)
DIRECT TYPING
- test for antigens
- patient’s cells containing unknown antigens tested with
known antisera
- antisera manufactured from human sera
- antisera used:
Antisera Color Source
Anti-A Blue Group B donor
Anti-B Yellow Group A donor
Anti-A,B Clear Group O donor
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ABO ROUTINE TESTING
Reaction Patterns for ABO Groups
Blood groupAgglutination
with Anti-AAgglutination
with Anti-B
A + -
B - +
AB + +
O - -
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ABO ROUTINE TESTING
INDIRECT/REVERSE TYPING
- Known antigen (cell) vs. unknown antibody (patient’s serum)
- Serum is combined with cells having known Ag content in a 2:1 ratio
- Uses commercially prepared reagents containing saline-suspended A1 and B cells
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ABO ROUTINE TESTING
Reaction Patterns for ABO Groups
Blood GroupAgglutination with A cells
Agglutination with B cells
A - +
B + -
AB - -
O + +
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ABO ROUTINE TESTING
Causes of Discrepancies in ABO Testing
Technical
1. Incorrect ID/recording
2. Patient/donor serum not added
3. Reagent contamination
4. Under-/over-centrifugation
5. Hemolysis
6. Warming of test mixture
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ABO ROUTINE TESTING
Causes of Discrepancies in ABO Testing
B. Red Blood Cells
1. Missing or weak A/B antigen
2. Acquired B Ag – colon or gastric CA, intestinal obstruction
3. Polyagglutinable RBC
4. Ab-coated RBC – post-transfusion incompatibility; autoimmune hemolytic anemia
5. Maternal-fetal agglutination – mismatched transfusion
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ABO ROUTINE TESTING
Causes of Discrepancies in ABO Testing
C. Serum
1. Roleaux formation – presence of plasma expanders, monoclonal gamma globulins
2. Anti-A1
3. Unexpected alloantibodies
4. Expected antibody absent – hypogammaglobulinemia, extreme ages, immunosuppression
Roleaux
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Rh BLOOD GROUP SYSTEM
- Discovered in 1940 by Landsteiner & Wiener
- Most complex erythrocyte antigen system; located on chromosome 1
- Found exclusively on surface of rbc integral part of red cell membrane
- Primary antigen if present, consider Rh (+)
- Lack corresponding naturally-occurring antibodies in serum
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ANTIGENO Rh Frecuencia ( %)
D (+) 85
D(-) 15
C 70
c 80
E 30
e 98
FRECUENCIAS FENOTIPICAS DEL SISTEMA RH
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Rh BLOOD GROUP SYSTEM
CLASSIFICATION/NOMENCLATURE SYSTEM
Wiener
Multiple allele hypothesis
5 antigens: Rho, rh’, rh”, hr’, hr”
Single locus inheritance system with 8 alternate common alleles coding for agglutinogens 1 individual produces 2 agglutinogens inherited from both parents
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Rh BLOOD GROUP SYSTEM
CLASSIFICATION/NOMENCLATURE SYSTEM
Fischer & Race
Three alleles: D/d, C/c and E/e
Five antigens: D, C, E, c, e
d no D locus no antigenic products
Rosenfeld
Numerical system
Rh1 to Rh5
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Wiener Fisher –
RaceRosenfield
Rho D Rh1rh` C Rh2rh” E Rh3h`r c Rh4hr” e Rh5
Comparación de las nomenclaturas para los Ag del Sistema Rh
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Rh BLOOD GROUP SYSTEM
Presence of D = presence of Rho factor Rh (+)
Absence of D Rh (-)
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Rh BLOOD GROUP SYSTEM
Testing for Rho (D) Antigen
- Use antisera originating from human source
- Antisera with different constituents use of high protein media necessary to produce agglutination since antigens are an integral part of the red cell membrane less numerous than ABO antigens
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Rh BLOOD GROUP SYSTEM
Testing for Du Variant
Use bovine or albumin-suspended anti-D reagent
Incubate at 37oC for 15-60 minutes to facilitate formation of Ag-Ab complex
Interpretation: (+) Du consider Rh (+)
Person who appear to be Rh (-) should be proven to be Du (-) before they are considered to be eligible to receive transfusion
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Rh BLOOD GROUP SYSTEM
Rh Antibodies
- Not naturally-occurring immune antibodies produced upon sensitization IgG isotype
- Reactive at 37oC enhanced with enzyme-treated red cells
- Can cross the placenta
- Associated with hemolytic transfusion reaction and hemolytic disease of the newborn (HDN)
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Rh BLOOD GROUP SYSTEM
Rh Typing – slide or test tube method
False (+) results
a. Drying
b. Roleaux formation
c. Auto-agglutination
d. Patient’s red cells heavily coated with Ab’s
e. Presence of cold agglutinins
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Rh BLOOD GROUP SYSTEM
Rh Typing
False (-) results
a. Use of old cells
b. Wrong cell concentration
c. Hemolysis
d. Inadequate mixing of cells
e. Inactive typing sera
f. Incorrect temperature
g. Existence of Du variant
h. High concentration of blocking antibodies
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HEMOLYTIC DISEASE OF THE NEWBORN
Involves hemolysis of red cells in the fetus and neonate
Antibody is present in the mother that corresponds to an antigen on the surface of the red cells of the fetus Ab crosses placenta attaches to fetal Ag hemolyze red cells of fetus
Differential diagnosis: physiologic jaundice, septicemia, CID, toxoplasmosis, congenital syphilis
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HEMOLYTIC DISEASE OF THE NEWBORNComparison of ABO versus Rh HDN
Characteristic ABO HDN
First pregnancy
Disease predicted by titers
Antibody IgG
Bilirubin at birth
Anemia at birth
Phototherapy
Exchange transfusion
Intrauterine transfusion
Spherocytosis
Yes
No
Yes (anti-A,B)
Normal range
No
Yes
Rare
None
Yes
Rare
Yes
Yes (anti-D)
Elevated
Yes
Yes
Common
Sometimes
Rare