Indirect Antiglobulin testing Flashcards
What are other names for indirect antiglobulin testing?
AHG, coombs, enhanced ab detection
How do you confirm negative results?
use a saline control
What must you do for a forwards rxn that is all positive?
Check with saline and confirm positives
Frequencies:
Antigen
D ag
C ag
c ag
E ag
e ag
D 85 lower white
C 70 lower black
c 80 lower asian
E 30
e 98
What do you do to enhance weak rxn?
put in the fridge for IgM
Describe the overview of Anti-D briefly
ig
stim
IgG immune stimulated, warm reactive (incomplete Ab)
Describe the coombs test
detects?
detects weak/incomplete (IgG) ab using IgM to complete hemagglutination
indirect test
Describe the coombs rxn agglutination test
what forms a lattice
must have removal of?
incubation?
method?
IgG ab forms a lattice w/ IgM Anti-IgG rgn
must have removal of unbound material
incubate at body Temp
INDIRECT
Describe the immediate spin agglutination test
What forms a lattice
centrifuge
Plasma to cell ratio
Incubation?
binding?
IgM ab lattice w/o additional rgnt
Direct test
Centrifuge brings reactants close
RT
Plasma 2:1 cells 3% sol
37C
(Compliment and IgG ab more likely to bind at body temp)
When is the enhancement added in the coombs test?
added after immediate spin and before 37C incubation
this reduces zeta potential
reduces steric hinderance
Incomplete agglutinins
usually refers to IgG ab non reactive at RT
Coombs
researcher who proposed antiglobulin test method
Tube Test
classic serological method preformed with glass tubes
AHG
anti human globulin
IAT
indirect antiglobulin test
Enhanced ab detection
test method requireing more steps than immediate spin to see macroscopic agglutination
Briefly cover how the immediate spin test looks
mix cells and plasma
centrifuge
observe agglutination
Describe how the coombs test is done
mix cells and plasma
incubate 30-60 min
wash away non attached ab
add anti-ab “coombs rgn”
centrifuge
What happens if non attached antibodies are improperly washed?
false negatives
If antibody didnt attach to ag at incubation?
add red cells and plasma
incubate
wash
add anti-ab
centrifuge and read for agg
add check cells to agglut w anti-ab
Main keys of check cells
have anti-IgG coating the cell
if system isnt washed correctly, anti-ab will attach to ab left over
no binding sites left for binding w check cells
How do you confirm a negative reaction in the wash of the coombs test?
check cells
What are the 3 possible outcomes of a coombs test
all pos/all neg/ or invalid if check cells are negative
When is there an invalid test in coombs?
When the ab in the system is bound to the rgnt not the red cell, causing negative rxn in the check cells
START OVER
False negatives in Coombs
No agg in AHG
exc
incor
left
No agg in CC
left ov
insuff
iinsuff
looks like no agglut in AHG step
aged cells
excessive washing
incorrect centrifuge
left over wash dilutes
No agglut w check cells
left over ab abound check cells
insuff washing
insuff incubation
False positives in coombs test
agg in AHG
p
f
ab
b
develop
looks like agg with AHG step
proteins in plasma
fibrin strands
ab attached in vivo to cells (DAT)
bacterial contam
pt devel ab to preservative
Standardization
cell washer
monoclonal rgnt
gel test
solid phase
cell washer: wash step for vol and number of washes, reduces false negatives
Monoclonal: binding site, reduces false pos
Gel: test and rgnt vol, centrif, removes wash step
Solid phase: test/rgnt vol, test ag, interpret
LISS
Reduce zeta potential
PEG
remove water vol
Albumin
increase collisions
Bromelin
reduce steric hindrance
Polybrene
polymer nonspecific binding
Describe gel automated method
precipitation through polyacrylamide gel matrix
add red cells and plasma
incubate no wash
centrifuge no check cells
Rouleax looks like what kind of rxn on gel?
looks like 2+ rxn
Describe solid phase automated method
adherence of ab to rbc ag on solid substance
prone to have false +
If gel is available, why tube methods?
Trouble shooting - everyone goes back to tube
ref lab method - gold standard
tube method is most clinically relevant
harder but cheaper
When do you do coombs testing?
with w
sb
cross
what kind of test? cells in?
with weak D, ab screen, extended cross match, autocontrol
Indirect test what has attached to the cell in VITRO
Describe direct testing
what is on the cell in the body
In VIVO
is there already ab on the cell?
Requirements for finding compatible blood
pt will rx poor if?
females?
pt with clin sign ab?
pt will react poorly if given incompatible blood
females of childbearing age should not be exposed to Rh incomp blood
if pt has made clinically sign immune stim ab, they should recive ag neg blood
“Blood type” and test
ABO type + Rh type on pt red cells (IS)
“Ab screen” and test
test for the presence of clinc sign imm stim ab in pt plasma (IAT)
“type and cross match”
ABO + RH+ AB screen + sero between donor cell and pt plasma (IS-IAT)
“extended crossmatch”
sero test between
sero test between donor cell and pt plasma util at body temp (IAT ONLY)
‘Ab ID”
logical problem utilizing
logical problem utilizing known cells and pt plasma to ID source of unexcpet reactivity in ab screen (IAT)
“coombs test”
test for ab attached to cells in VIVO (DAT)
Reverse cells
mixture of donors, blood type A or B in preservative (no plasma)
Screen cells
single donor type O known ag in preservative no plasma
Antigram sheet
package insert that lists all interpretations of ag for screen cells
AB screen
each cell represents a single donor (no mf)
if any cells in screen rx + more work needs to be done and pt will need extended crossmatch to determ compatibil w donor blood
