Reagents and Methods for
Testing in the Blood Bank
Major Focus
• Constructed around detecting Antigens and
Antibodies
• Historically viewed by agglutination: now
includes
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Gel
Solid phase
Molecular techniques
Automation
With the exception of molecular, they are still
Antibody-Antigen reactions
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
ABO and Rh Antisera
• Where to the Antigens come from?
Antibody Screen Antigens
• Where do the Antibodies come from?
Reagent cells for reverse typing
• Where do the Antibodies come from?
Anti-Human Antiglobulin
• Our new best friend
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
Polyspecific vs Monospecific AHG
• Polyspecific
▫ 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
Used with all negative AHG reactions
• Coombs Check Cells
• Check cells are added to see if AHG is present
and functioning
This methodology is vital to blood
bank testing
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
Antibody Screen Limitations
• Low frequency antibodies may not be detected
• Antibodies with low titers may not be detected
• ABO antibodies will not be detected
Direct Antiglobulin Test (DAT)
• Detects in Vivo sensitization
• Examples when this would be used would be
HDN, Hemolytic Anemia, Auto Immune
Hemolytic Anemia (AIHA)
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Usually would use polyspecific AHG
Need to consider if there is in vivo hemolysis
Patient recently transfused
Medications
Unexpected Allo antibody
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
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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
Example of Antigram
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
Alternate Test Methods
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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.
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.