Immunohematology Transfusion Medicine Blood 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.

ABO/Rh Pt. RBC Commercial 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 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 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.