1. Immunohematology Transfusion Medicine Blood Bank
History 101 of Blood Bank

2. 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

3. 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
: Duffy, Kidd, Lutheran system discovered.
Landsteiner and Alexanders lead to the discovery of >800 Blood group systems.

4. 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

5. 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

6. Antigens-have different levels of immogenicity-cause immune response.
Factors effecting immunogenecity
Chemical composition and complexity
Degree of foreignness
Size
Dosage
Route of administration

7. 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

8. Antibodies-protein (immunoglobulin-Ig)
5 Classification:
IgG- Clinically significant in BB
IgM- Can be Clinically significant in BB
IgA
IgE
IgD

9. 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.

10. 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

11. Immune response (Primary vs. Secondary)
Immune response influenced by:
Age
Route of exposure
Genetic make-up
Health

12. Antigen-Antibody interaction:
Formation of antigen –antibody complex causes an immune reaction.
Amount is determined by:
Goodness to fit
Size
Shape
Charge

13. 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

14. 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.

15. Sensitization enhanced by:
Serum to cell ratio
Temperature
Incubation pH
Ionic strength

16. 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+

17. 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.

18. Antiglobulin Test (Anti-humanglobulin or AHG)
Discovered by Coombs, Mourant and Race in found that RBC can become sensitized without visible agglutination.
Reagent base test
Identifies IgG antibodies and Complement proteins
Polyspecific and monospecific AHG reagent.

19. 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)

20. 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

21. 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.

22. 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

23. 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.

24. 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)

25. 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

26. 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.

27. 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

28. 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

29. Reagent Quality Control
Technical procedures to determine analytical testing phase work properly.
Specific Q.C. requirements
Checks:
Reagents
Equipment

30. 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.

31. 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)

32. 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(=)

33. Antiglobulin Reagent: (AHG)
Detects IgG antibodies and Complement protein that have attached to RBC.
2 Types
Polyspecific
Monospecific

34. 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

35. 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.

36. 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

37. 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

38. 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

39. Testing procedure uses tube method and slide method.
New method include:
Gel technology
Microplate
Solid phase – serological method-automative

40. 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

41. 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)

42. 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

43. 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

44. 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.

45. 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.

46. 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.