BLOOD BANK METHODS Flashcards
memorization
The traditional method uses what reaction chamber?
Tube
The gel method uses what reaction chamber?
Microtube card
The solid phase method uses what reaction chamber?
Microplate wells
Reaction patterns of
Traditional method (tube):
Gel method (microtube card):
Solid phase method (microplate wells):
Reaction patterns of
Traditional method (tube): Hemagglutination
Gel method (microtube card): Hemagglutination
Solid phase method (microplate wells): Immune adherence
Reaction matrix of gel method:
Dextran acrylamide gel
Reaction matrix of solid phase method:
Modified polystyrene wells
Which blood bank method does not require washing?
Gel method
What us the purpose of the Gel?
serves as a filter to trap agglutinated RBCs
Developed in Europe by Dr. Yves Lapierre; uses dextran-acrylamide gel that contains pre dispensed reagents:
Column Agglutination Technology (CAT)/ Gel Test Method
Capture immunoassays are available to detect antibodies to RBCs, platelets, and CMV; it uses microplate wells coated with the target antigen; plasma or serum is then added as well as LISS incubated at 37C then washed; Finally, indicator red cells (anti-IgG-coated RBCs) are added and centrifuged
Solid Phase Red Cell Adherence (SPRCA) by Immucor
Positive and negative results in SPRCA
Positive =
Negative =
Positive and negative results in SPRCA
Positive = Diffuse pattern/ RBC monolayer
Negative = RBC pellet at the bottom
Uses microplate wells that are coated with Protein A:
Solid Phase Protein A Technology by BioRad
Formats inSolid Phase Protein A Technology by BioRad\
Erytype S -
Solidscreen II -
Formats inSolid Phase Protein A Technology by BioRad\
Erytype S - for ABO, Rh, and Kell typing
Solidscreen II - for antiglobulin testing
Factors that influence agglutination reactions:
- High speed centrifugation
- Effect of antigen-antibody ratio
- Dosage
- Effect of pH
- Effect of temperature
- Effect of immunoglobulin type
- Effect of enhancement media and potentiators
Simplest and most common technique to enhance agglutination:
High speed centrifugation
Prozone (Ab excess) and Postzone (Ag excess) reactions will lead to _______ results
FALSE-NEGATIVE
Remedies
Prozone =
Postzone =
Remedies
Prozone = serum dilution techniques
Postzone = increase serum to cell ratio
Strength of agglutination reaction of heterozygous alleles
weak agglutination upon testing (1+ or 2+ or WK)
Strength of agglutination reaction of homozygous alleles
stronger agglutination upon testing (4+ or 3+)
Blood groups that shows dosage effect:
- Duffy
- MNSs
- Kidd
- Rh (C, c, E, e)
- Lutheran
Ideal pH for agglutination reactions:
pH 6.5 or 7.5
Which immunoglobulin reacts best at cold temperature (4-22C)
IgM (cold-reactive)
Which immunoglobulin reacts best at warm temperature (37C)
IgG (warm-reactive)
Which immunoglobulin is capable of agglutinating red cells even in NSS (0.85%)
IgM (pentamer)
Which immunoglobulin cannot agglutinate red cells in saline and requires enhancement media or potentiators?
IgG (monomer)
Primarily used to help detect IgG antibodies; aimed at reducing the zeta potential of the red cell membrane
Enhancement media and potentiators
It is the expression of the difference in electrostatic potential at the surface of the rec cells and the ionic cloud of positive cations that are attracted to the negative charges on the surface
Zeta potential
Enhancement media/ potentiators:
- Protein Media
- LISS (low ionic strength solution)
- Enzymes
- Anti0Human Globulin (AHG)
An enhancement media that increases the dielectric constant, which reduces the zeta potential:
Protein media
Examples of protein media:
22% albumin
Polyethylene glycol (PEG)
An enhancement media that decrease the inonic strength of a reaction medium and so reduce the zeta potential; increase the attraction between positively charged Abs and negatively charged red cells; contain 0.2% NaCl and often used because of increase rate of Ab uptake during sensitization:
LISS (low ionic strength solution)
Red cells incubated with LISS incubation temp. and time:
37C for 10-15 minutes
Color of LISS solution:
Clear/ colorless
Examples of enhancement media enzymes:
- Ficin (fig plant)
- Papain (papaya)
- Trypsin (pig stomach)
- Bromelin (pineapple)
Effects of enhancement media enzymes
ENHANCES reactivity to:
DESTROYS reactivity to:
Effects of enhancement media enzymes
ENHANCES reactivity to: Rh, Kidd, Lewis, I, P1 antigens
DESTROYS reactivity to: MNS, Duffy, Chido-Rodgers, Xg antigens
Cross-links sensitized cells, resulting in visible agglutination; designed to detect cells coated with antibody or complement, or both
Anti-Human Globulin (AHG)
Dyes present in AHG:
Patent blue + Tartrazine yellow = GREEN solution
AHG color = GREEN
Methods for Red Cell Antibody Detection
Reaction Phase: IMMEDIATE SPIN (20-30 sec)
Detects:
Process/Mechanism:
Visible result:
Methods for Red Cell Antibody Detection
Reaction Phase: IMMEDIATE SPIN (20-30 sec)
Detects: IgM
Process/Mechanism: Test mixture is centrifuged at Room temperature
Visible result: Hemagglutination
Methods for Red Cell Antibody Detection
Reaction Phase: 37C INCUBATION
Detects:
Process/Mechanism:
Visible result:
Methods for Red Cell Antibody Detection
Reaction Phase: 37C INCUBATION
Detects: IgG
Process/Mechanism: Sensitization of red cells occur; formation of Complement
Visible result: Hemolysis (due to Cā activation)
Methods for Red Cell Antibody Detection
Reaction Phase: ANTIGLOBULIN TEST
Detects:
Process/Mechanism:
Visible result:
Methods for Red Cell Antibody Detection
Reaction Phase: ANTIGLOBULIN TEST
Detects: IgG
Process/Mechanism: Contains antibodies that bind the Fc portion of IgG, cross-linking red cells
Visible result: Hemagglutination
