1. Reagents and Methods for Testing in the Blood Bank

2. Major Focus Constructed around detecting Antigens and Antibodies
Historically viewed by agglutination: now includes
Gel
Solid phase
Molecular techniques
Automation
With the exception of molecular, they are still Antibody-Antigen reactions

3. Routine Testing Typing of the ABO and Rh Antigens
Typing for Antigens of Other Blood Group Systems
Antibody Screen
Antibody Identification
Compatibility Test (Crossmatch)
Direct Antiglobulin Test

4. ABO and Rh Antisera
Where to the Antigens come from?

5. Antibody Screen Antigens
Where do the Antibodies come from?

6. Reagent cells for reverse typing
Where do the Antibodies come from?

7. Anti-Human Antiglobulin
Our new best friend

8. Principle: Antihuman globulins (AHG) from immunized animals bind to human globulins whether free in serum or attached to RBC’S
IgM is so large it usually reacts at room temperature spin
IgG needs a little help- a bridge molecule to agglutinate RBC’s
AHG acts as a bridge molecule

9. Polyspecific vs Monospecific AHG
Antibodies to both human IgG and to C3d
Advantage is that they may detect complement dependent antibodies on RBC’s (Anti-Jka)
Disadvantage is it may cause more nuisance positives
Mono Specific
Antibodies to Human IgG or C3d only
May miss some antibodies but has less nuisance positives

10. Used with all negative AHG reactions
Coombs Check Cells
Check cells are added to see if AHG is present and functioning

11. This methodology is vital to blood bank testing

12. Antibody Screen (Indirect)
Purpose is to detect In Vitro sensitization
Detection of unexpected Antibody
Uses O cells with antigens represented on 2-3 different cells of DCEce, MNSsP, Lea Leb, Kk Fya, Fyb, Jka, Jkb.
Homozygous expression of Antigens is valued over heterozygous (may show dosage effect, greater antigen density per cell the greater the sensitivity
Need to be sure to run Auto Control

14. Antibody Screen Limitations
Low frequency antibodies may not be detected
Antibodies with low titers may not be detected
ABO antibodies will not be detected

15. Direct Antiglobulin Test (DAT)
Detects in Vivo sensitization
Examples when this would be used would be HDN, Hemolytic Anemia, Auto Immune Hemolytic Anemia (AIHA)
Usually would use polyspecific AHG
Need to consider if there is in vivo hemolysis
Patient recently transfused
Medications
Unexpected Allo antibody

17. Antibody Identification
Use multiple cell panels, enhancement processes and varying incubations to help identify the Antibody(ies).
What you need to consider in the process is
Patient Medical History
Antigen profile of Panel cells
Results of Auto control
What phase, what strength of agglutination
Crossing out procedure
Does this antibody match the reaction pattern

18. Example of Antigram

19. Enhancement Media
Enhancement media assists the attachment of an antibody to the specific antigen on the red cell
Bovine Albumin ( used during incubation)
Low-Ionic Strength Saline (what commercial test cells are in)
Polyethylene Glycol (concentrates Antibody)
Enzymes (Ficin and Papain) Reduces negative charge. Some Antibodies are enhanced like Rh, Lewis others are destroyed like Duffy and M,N,S

20. Alternate Test Methods
Automation
Gel Technology
Microplate testing
Solid Phase Testing
Solid and Microplate have exactly the opposite interpretations of reaction
Molecular Biology (above my pay grade)
Other than Automation and Gel, these other processes are used in Donor centers or large Reference Labs.

21. Lab Assignment We will be following Procedure seen on the video
We will be using manual wash for the AHG testing, this will be demonstrated for you in class
Try to limit the centrifuge spin time to 20 seconds especially on the wash cycle.
We need to learn a good technique on manual wash, it will mean the right result, not just any result.
This project was funded at $3,000,000 (100% of its total cost) from a grant awarded under the Trade Adjustment Assistance Community College and Career Training Grants, as implemented by the U.S. Department of Labor’s Employment and Training Administration. Rogue Community College is an equal opportunity employer/program. Auxiliary aids and services, alternate form and language services are available to individuals with disabilities and limited English proficiency free of cost upon request.
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