T4 - Serological Techniques Flashcards
Techniques to Enhance IgG Antibody Detection
RBC ζ-potential or surface charge is altered to enhance IgG aby binding and/or agglutination
Use anti-human globulin (AHG), potentiators/enhancement media, and enzymes
- low ionic strength saline (LISS)
- polyethylene glycol (PEG)
Polybrene test
Anti-Human Globulin (AHG) Test
Used to detect
- IgG antibodies bound to RBCs that are not detectable by other methods
- complement proteins bound to RBCs as a consequence of antibody binding
Use anti-human IgG and/or anti-C3d antibodies to cross link IgG or C3d already bound to RBCs, which causes haemagglutination
AHG Reagent
A.k.a ‘Coombs’ reagent
Two types:
- polyspecific
- monospecific
Polyspecific contains:
- anti-human IgG antibody
- anti-C3d antibody
Monospecific contains only one of the above components
Polyspecific reagent is widely used in routine tests
- adv: detects antibodies that bring complement to the RBC surface, but might be undetectable themselves
- disadv: detects complement bound by antibodies considered to be clinically insignificant
Direct and Indirect AHG Test
Direct
- a.k.a Direct Antiglobulin Test (DAT), Direct Coombs’ Test (DCT)
- detects antibody and/or C3d bound to patient RBCs in vivo:
- haemolytic transfusion reactions
- haemolytic disease of the newborn
- autoimmune haemolytic anaemias
Indirect
- a.k.a. Indirect Antiglobulin Test (IAT), Indirect Coombs’ Test (ICT)
- detects antibody bound to RBCs in vitro:
- antibody screening and identification, cross-matching, phenotyping
Washing in the AHG Test
Washing is crucial when performing in tubes
Removes IgG that is not bound to RBCs (i.e. free in plasma)
If sample not thoroughly washed, AHG reagent will bind to IgG that is free in plasma => false negative result
Causes of False Negative Results in the AHG Test
Delay in washing or adding AHG reagent
Not removing residual saline
Non-reactive AHG reagent due to deterioration
Patient plasma or AHG reagent not added
AHG Test - Controls
When the AHG test is performed in tubes, AHG control cells must be added to EVERY negative reaction
- A.k.a. Coombs’ control cells, sensitised cells
- these are RBCs that have IgG antibody bound to them
- these bind AHG reagent that should be present and free in solution in negative tests → agglutination
Performed to ensure that:
- you added AHG reagent to your tubes
- the AHG reagent works
- you have correctly performed your washes
If you don’t see agglutination after the addition of AHG control cells to negative reactions, the test is invalid
Potentiators/Enhancement Media
Decrease the RBC ζ potential which increases antibody binding and decreases the distance between adjacent RBCs
Some IgG antibodies can directly agglutinate RBCs in the presence of potentiators
Potentiators are widely used to decrease the time taken to perform the AHG test
Examples:
- low ionic strength saline (LISS)
- polyethylene glycol (PEG)
- albumin
Potentiators/Enhancement Media - Low Ionic Strength Saline
RBCs are diluted in 0.2% saline OR
Low ionic additive (LIA) is added to RBCs suspended in isotonic saline
Glycine is present maintain iso-osmotic conditions
Reduces ζ potential by decreasing the ionic strength of the reaction medium
Increases antibody uptake during sensitisation
Allows the incubation time in the AHG test to be significantly shortened
Potentiators/Enhancement Media - Polyethylene Glycol
Linear polymer
Accelerates antibody binding to RBCs by steric exclusion of water molecules
Concentrates antibody around RBCs
Allows use of shortened incubation time in AHG test
- 10-30 mins
PEG can give non specific reactions
Potentiators/Enhancement Media - Albumin
Increases the dielectric constant of the medium, leading to a decrease in ζ potential
Used at 22-30%
Allows use of shortened incubation period in AHG test
- 15-45 mins
No advantage compared to LISS
More expensive
May miss clinically significant antibodies
Potentiators/Enhancement Media - Enzymes
E.g. ficin, papain, trypsin, bromelase, neuraminidase
Modify antigens on the RBC surface
Decrease ζ potential by removing sialic acid residues
Enhance the reactivity of some blood group antigens
- e.g. Rh, Kidd
Decrease the reactivity of other blood group antigens
- e.g. MNS, Duffy
Performed in one or two stages
Potentiators/Enhancement Media - Enzyme Stages
1 stage:
- incubate patient plasma, RBCs, and enzyme together
- centrifuge and read result
- faster, but can destroy antibody in patient plasma
2 stage:
- incubate RBCs and enzyme together
- wash cells
- incubate patient plasma with enzyme treated cells
- centrifuge and read result
- more sensitive because exposure of antibody to enzyme is avoided
Can be extended to AHG technique
Detect many clinically insignificant antibodies
Useful in antibody identification
Polybrene
Cationic ammonium polymer that causes reversible aggregation of RBCs
Patient plasma is mixed with RBCs in a low ionic environment
Polybrene is added to aggregate cells
Sodium citrate is added to neutralise the polybrene
Check for agglutination
- if an antibody is present, agglutination will persist after neutralisation
- if no antibody is present, agglutination will disperse
Column Aggregation Technique (Gel/Card)
Plastic column contains glass or sephadex beads/matrix
Reagent antibody can be incorporated into the bead
High density solution is laid over top of bead
- traps plasma proteins
- AHG tests don’t require washing
Use 1% cell suspension
Cells (+/- sera) are added into the top of the column
Card is centrifuged
Agglutinated cells are trapped at the top of the gel matrix
Cells not agglutinated can move through the gel and form a button at the bottom of the column
