ImmunohematologyTransfusion MedicineBlood Bank History 101 of Blood Bank
1492: first attempt at using blood for therapeutic use. • 1665: First animal to animal-Richard Lower • 1667: Jean Baptiste Denys, first successful IV transfusion of blood from animal to human • 1818: James Blundell First to transfuse human to human. • 1901: Karl Landsteiner: Discovered ABO blood grouping
1927: Landsteiner and Levine discovered M,N and P system • 1939,40: Levine, Stetson, Landsteiner and Weiner discovers Rh system and it’s role in erythroblastosis foctalis (HDN) • 1946-Kell system discovered by Coombs, Mourant and Race • 1950-51: Duffy, Kidd, Lutheran system discovered. • Landsteiner and Alexanders lead to the discovery of >800 Blood group systems.
Antigen and Antibody in blood banking • Antibody production is result of blood group antigen (foreign-transfusion or pregnancy), or can be naturally occurring. • Blood group antigens are integral part of the RBC membrane • B Cells produce antibody molecules that are specific for a target antigen (part of the surface of RBC) • Antigenic determinants: on RBC-elicit the production of different antibodies
Complement System: set or group of serum proteins that generates membrane attack that causes cell destruction. (Hemolysis) • Classical vs Alternative pathway • Antibody-Antigen complex activates Complement. • Complement activation • Anaphylatoxins • Vasoactive amines • Chemotactic • Opsonins • Receptors
Antigens-have different levels of immogenicity-cause immune response. • Factors effecting immunogenecity • Chemical composition and complexity • Degree of foreignness • Size • Dosage • Route of administration
Red Blood Cell Antigens • 23 blood group systems with more than 200 RBC antigens • RBC antigens are determined by genetic inheritance pattern • Antigen immunogenicity based on stimulation production upon exposure • Types of antigen RBC, HLA, Platelet
Antibodies-protein (immunoglobulin-Ig) • 5 Classification: • IgG- Clinically significant in BB • IgM- Can be Clinically significant in BB • IgA • IgE • IgD
IgM antibodies: • Produced initially in response to foreign antigen • Large pentamer structure • Contains 10 potential antigenic sites • In BB – reaction in saline procedures • 5-10 % of Ig class • Short half-life 5-6 days • Activates Complement with great efficiency.
IgG antibodies • Monomer • Accounts for 80% of Ig • Found in extravascular fluid • 2 antigen binding sites • In BB- reaction less apparent in saline procedures, need additive to show reaction. • Half-life 23 days • Cross placenta • Activates complement • Subclasses
Immune response (Primary vs. Secondary) • Immune response influenced by: • Age • Route of exposure • Genetic make-up • Health
Antigen-Antibody interaction: • Formation of antigen –antibody complex causes an immune reaction. • Amount is determined by: • Goodness to fit • Size • Shape • Charge
Antigen-Antibody reaction in Vivo vs. Vitro • Primary concern in Blood Bank-Vivo • Want to reduce the potential of exposure to foreign antigens which would result in antibody production. • Identify antigens present on the RBC • Identify any antibodies produced – found in the serum/plasma
Identify antigen-antibody complex by: • Hemolysis (Complement reaction associated with complex) • Agglutination (antigen/antibody complex), like clotting of the blood Don’t want agglutination- 2 stages of agglutination: • Sensitization- antibody binds to an antigen. Is influenced by amount of antigen and antibody present.
Sensitization enhanced by: • Serum to cell ratio • Temperature • Incubation • pH • Ionic strength
Lattice Formation stage (cell interaction, see visible agglutination, linkage between antigen and antibody) • Factors that influence Lattice formation • Zeta potential • Serum to cell ratio (zone of equivalence) • Prozone effect • Centrifugation Grading agglutination (lattice formation) 0(neg), 1+, 2+, 3+, 4+
Indicators of antibody-antigen complex • Hemolysis • Agglutination Hemolysis is an indication of antigen-antibody complex that results in cell destruction. Identified usually in the Antiglobulin Test.
Antiglobulin Test (Anti-humanglobulin or AHG) • Discovered by Coombs, Mourant and Race in 1945- found that RBC can become sensitized without visible agglutination. • Reagent base test • Identifies IgG antibodies and Complement proteins • Polyspecific and monospecific AHG reagent.
AHG reagent will react with bound and/or free IgG and Complement proteins. • Must get rid of the free to I.D. the bound forms that can cause reaction. • WASH cells (RBC for testing-cell suspension) • If cells wash properly and add AHG reagent and agglutination forms this is a positive reaction (NOT GOOD) • 2 Types of AHG test: • Direct antiglobulin test (DAT) • Indirect Antiglobulin test (IAT or antibody screen)
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 • 2 Step process: • Incubation at 37°C serum with donor cells False positive and False negative reaction for various reasons. Positive IAT indicates a specific reaction between antigen and antibody in serum of patient.
Potentiator Reagent: enhances antibody and antigen complex • A.K.A. as enhancement media • Enhances antibody uptake • Promotes agglutination • 4 Types potentiators • Low ionic strength solution (LISS) • Bovine serum albumin • Polyethylene glycol (PEG) • Proteolytic enzyme (3 Types) • Papain • Ficin • Bromelin
Chapter 2: Blood Bank Reagents • Basic blood banking reagents depends on the source of antigens and source of antibody in testing. (What are we looking for?) • Detect an antigen present or absent on RBC (donor/patient cells) • Detect antibody present or absent in serum (donor/patient serum) Have to have a known source of antigen to detect antibody or known antibody to detect antigen.
Source of Antigens: • Commercially prepared RBC suspension with known antigens to detect unknown antibodies. • In BB- usually patient RBC antigen type is unknown and we test the cells to identify the type of antigen present on the RBC by testing with a known antibody • I.D. blood type (forward typing)
Source of Antibody • Anti-sera commercially prepared or can be patient serum or plasma in BB. • Commercially prepared anti-sera contain known RBC antibodies to identify unknown RBC antigens. • Reverse Typing
Routine Testing procedure: • ABO/Rh typing • Antibody screen (IAT) • Antibody Identification • Cross match Only do antibody identification if IAT is positive. If IAT is negative-indicates there are no unknown antibodies present in patients sample.
Important to understand reagent so that you understand what you are looking for or identifying Antigen Antibody ABO/Rh Pt. RBC Commercial Anti-A, Anti-B, Anti-D Antibody Screen Screen cells Pt. Serum Antibody I.D. Panel Cells Pt. Serum Cross-match Donor cells Pt. Serum
Reagents – 4 basic groups • Reagent RBC- Known RBC antigens • Antisera- Known RBC antibodies • Antiglobulin (AHG) • Potentiators Reagents are regulated by the FDA: minimum standards for reagents used for testing. • Specificity • Potency
Reagent Quality Control • Technical procedures to determine analytical testing phase work properly. • Specific Q.C. requirements • Checks: • Reagents • Equipment
Reagents are: • Monoclonal (single clone of cells- specificity) • Advantage vs disadvantage 2. Polyclonal (human source: mixture of cells-contains multiple antibodies) Assist in identifying antigen present on patients RBC that may cause and reaction in vivo.
Anti-Sera for ABO typing (forward typing): • Anti-A and Anti-B determines the presence of A,B or no antigens on RBC. • Test performed using Donor/Patient RBC with known Anti-sera • (+) agglutination = antigen present • (=) agglutination = antigen absent • Identifies the 4 major blood groups A: posses A antigens B: posses B antigens AB: posses both A and B antigens O: lack antigens (has no antigens)
Anti-sera for Rh typing: • There are multiple antigens in the Rh system (c,C,D,E,e), most prevalent and most important to identify is D. • Identify the presence (+) or absence (=) of the D antigen on patient RBC. • Use anti-D reagent combined with testing RBC. • Agglutination: D present, D (+) • No agglutination: D absent, D(=)
Antiglobulin Reagent: (AHG) • Detects IgG antibodies and Complement protein that have attached to RBC. • 2 Types • Polyspecific • Monospecific
Check Cells (Coomb’s Control Cells) • Required control system by AABB standards • Control system: • RBC commercially prepared with IgG antibodies • Identified true negative reactions-false negative
Reagent RBC • Reagent A1- RBC with known antigen (A) mixed with serum to identify or confirm ABO typing • Reagent B- RBC with known antigen (B) mixed with serum to identify or confirm ABO typing • Testing phase known as Reverse typing or Back typing.
Patient posses the antibody directed against the antigen of their ABO group. • Example: Group A individual: A antigens on RBC; Lack B antigen; produce B antibodies (in serum or plasma). Serum agglutinates with B cell reagent. • Group B individual: B antigens on RBC; Lack A antigen; produce A antibodies (in serum or plasma). Serum agglutinates with A cell reagent
Screen Cells-Group of known reagents that contain known antibodies. • Looks for antibodies with specificity to RBC antigens in patient and donor sample (naturally occurring or from exposure) • Commercially available: Group O donor source • AABB standards states antibodies test performed on recipient specimen required • Blood group antigens expressed on or in screen cells: D, C, E, c, e, M, N, S, s, P1, Lewis, Lutheran, Kell, Duffy and Jka, Jkb
Panel Cells (10 + reagent based testing system) • Group of test to determine the specificity of RBC antibody that was Identified in antibody screen. • Antigenic profile is important. • Lectins which are useful in identifying certain antibodies through panel cells. • Lectins: pg 50
Testing procedure uses tube method and slide method. • New method include: • Gel technology • Microplate • Solid phase – serological method-automative
Chapter 3: Genetics • Blood type is determined by genetic inherited patterns. • Phenotype: observable trait • Genotype: actual genetic make-up • Predict genotype, if you know phenotype and can predict phenotype, if you know genotype. • Blood type is determined by the antigen present on the RBC. • Punnet Square
Genes: unit of inheritance on a chromosome. They are located on specific areas of the cells called genetic loci. • Alleles: Form or different forms of a gene of a given loci • Ex: A, B and O alleles on the ABO gene locus. • Polymorphic: having two or more alleles at a given locus. (Rh system)
Inheritance Pattern: • Co-dominant (equal expression of a gene on an individual) • Recessive or dominant ( only one alleles is expressed on the cell) • Amorphic expression: gene present, but does not express detectable product. Mandelian Principle: Independent segregation of traits 4. Mutations
Homozygous vs Heterozygous inheritance • Homozygous: 2 alleles for a given trait are the same-genotype are identical genes • Heterozygous: 2 alleles are inherited are different-genotype are different. • Agglutination reactions will be effected by inheritance pattern. • Homozygous pattern- stronger reactions • Heterozygous pattern- weaker reactions
Inheritance pattern with Cis and Trans position. (related to the Rh system and how it expresses itself) • Cis : gene expression is from the same chromosome • Trans: gene expression is from different or opposite Chromosome. • Can help determine agglutination reaction.
Silent Genes- “Amorph”, present and cause problems, but do not produce detectable antigen. • Result in unusual phenotype • Example is a “Null” type individual- blood type is not apparent or predictable- see with the Rh system.
Paternity Testing • Direct exclusion: child has a trait present that neither parent posses. • Indirect exclusion: child lacks a gene that should be inherited from the parent in question. • Inclusion: when the child has the predictable traits that are expected form the parent in question.