TM Methods Flashcards
Define primary vs secondary sensitization
Primary: Ab binds antigen on cells (can’t see this)
Secondary: Ab coated cells bind each other (lattice/cross-linking); requires slight Ab excess; results in visible hemagglutination
factors that affect sensitization
- concentration
> antibody
> antigen (dosage, individual variation) - temperature
> 4C - room temp for IgM
> 37C for IgG - time (longer time = more time for rxn but too long = popping off so rxn starts to decrease)
- pH (used to our advantage to elute Ab)
> react best at 7.0 - 7.5
> less reactive <6 or >8
zeta potential
- force of repulsion between red cells
- red cells carry a net negative charge at their surface (sialic acid)
- when suspended in saline, Na+ form ionic cloud around cells (like charges repel)
enhancement media (additives to decrease ZP)
- Low Protein (6%) Albumin
- similar to plasma concentration
- IgM will bind at 6% but not IgG
- used as a control instead of autocontrol - LISS*:
- reduces positive charge bc sodium is decreased
*NOTE: Low Ionic Strength Solution
PEG
- ideal charge helps Ab uptake
- dehydrates cell (removes ionic water from cell surface)
- this assists with hydrophobic interactions of Ab/Ag
disadvantages of PEG
- must wash away really well once Ab:Ag complex is attained
- can cause BINDING OF CLINICALLY INSIGNIFICANT Abs
T or F: PEG is more sensitive than LISS
TRUE; PEG is more sensitive than LISS
enzymes increase sensitivity of TM testing by..
- removes sialic acid (decreasing Z potential) AND steric hindrance to allow Ab to bind
3 natural enzyme treatments in TM
- Ficin from figs
- Papain from papayas
- Bromelin from pineapples
method to detect IgG antibodies
Antiglobulin test
- uses LISS, PEG, enzymes for Ab uptake at 37°C
- anti-IgG Ab (IgM) as 2° Ab
- increases sensitivity
IAT principle
- IgG antibody (plasma or antisera) mixed with cells (screen cell, panel cell donor cell or patient cell) and enhancement media
- 37°C incubation: IgG binds cell
- WASH removes unbound Ab (in plasma)
- anti-IgG binds sensitized cells = agglutination
- if no agglutination is present = add Coombs cells to ensure AHG is working
DAT vs IAT
Direct Antiglobulin Test:
- used to detect immunoglobulin, complement, or both on the surface of red blood cells (RBCs)
Indirect Antiglobulin Test:
- used to detect RBC antibodies in patient serum
NOTE: immunoglobulin = antibodies
DAT procedure
- add one drop PATIENT RED CELLS (3%)
- WASH three times
- 2 drops poly AHG + spin
- If negative: add 1 drop COOMBS control
causes of a positive DAT
- AIHA (warm, cold)
- hemolytic TRANSFUSION RXNS
- HDFN
- DRUG-related hemolytic anemia
NOTE: sensitized cells = pos DAT
what are the Coombs control cells?
- IgG sensitized cells
- added to negative IAT and DAT tests
- ensures that AHS was added; is working; adequate washing to remove unbound Abs
- expected = 1-2+
- neg result = invalid test and must be repeated
