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
> react best at 7.0 - 7.5
> less reactive <6 or >8
** we can use this to our advantage to elute Ab **
zeta potential
- force of repulsion between red cells suspended in solution
- 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
albumin
- high protein (22-30%)
> reduce the dielectric constant by attracting NaCl ions (think of rouleaux)
> this is no longer used today as there are better methods and high protein can cause interferences - low protein (6%) albumin is similar to plasma concentration
> IgM will bind at 6% but not IgG
> used as control instead of autocontrol
LISS
- enhancement
- reduces positive charge bc sodium is decreased
PEG
- organic polymer in a low ionic strength solution
- ideal charge to help antibody uptake
- dehydrates cell (removes ionic water from cell surface)
- 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
T!
enzymes increase sensitivity by:
- removing sialic acid (decreasing Z potential)
- removing steric hindrance to allow Ab to bind
enyme treatment
- Ficin from figs
- Papain from papayas
- Bromelin from pineapples
method to detect IgG antibodies
Antiglobulin test
- uses LISS, PEG, enzymes for Ab uptake at 37C
- anti-IgG Ab (IgM) as 2ry Ab
- increases sensitivity
IAT prinicple
- IgG antibody (plasma or antisera) mixed with cells (screen cell, panel cell donor cell or patient cell) and enhancement media
- 37C incubation - IgG binds cell
- wash step removes unbound Ab (in plasma)
- anti-IgG binds sensitized cells - forms agglutination
- if no agglutination is present, addition of Coombs cells ensure AHG is working
what is an antihuman globulin (AHG)
- mouse, rabbit anti-human Ab to IgG or complement
monospecific
- anti-IgG or anti-C3d (needs 5 min RT inc)
- used in IAT & DAT testing
polyspecific
- mixture of both anti-IgG and anti-C3d
- used in DAT testing
DAT vs IAT
The DAT is used to detect immunoglobulin, complement, or both on the surface of red blood cells (RBCs). The indirect antiglobulin test (IAT) is used to detect red cell antibodies in patient serum. In certain diseases or conditions, their RBCs are coated with immunoglobulin
DAT procedure
- add one drop patient 3% red cells
- wash three times
- 2 drops poly AHG
- add 1 drop Coombs control cells to negative IAT tests
causes of a positive DAT
- autoimmune hemolytic annemia (warm, cold)
- hemolytic transfusion rxns
- hemolytic disease of the fetus and newborn
- drug-related hemolytic anemia
- passive antibodies due to transfusion
what are the Coombs control cells?
- IgG sensitized cells
- added to negative IAT and DAt tests
- ensures that AHS was added + working, adequate washing to remove unbound Abs
- expected = 1-2+
- neg result = invalid test and must be repeated
causes of false negatives for antiglobulin tests
- poor wshing - unbound IgG neutralized AHG
- reagent deterioration
- interruption of testing
- failure to decant wash saline (dilutes AHG)
- undercentrifugation
- overshaking tubes
- plasma not added
- incorrect incubation time or temperature
- contaminated glassware
causes of false positives for antiglobulin tests
- failue to wash before adding AHG (PEG)
- overcentrifugaiton
- undershakig
- IAT performed using DAT positive RBC
- clotted, old, refrigerated specimen
- Wharton’s jelly (if doing DAT on cord cells)
