AHG/Coombs (Lab) Flashcards
AHG is obtained from ___________
immunized non human species (rabbits/guinea pigs) that binds to IgG or complement from Px serum or RBC
The type of Ab generated and used nowadays for AHG is ___________
monospecific AHG/monoclonal Abs
A monospecific AHG contains ________________
a single component AHG (either Anti-IgG, Anti-C3). Only one reagent per vial = cannot be both IgG and complement
Monospecific Ab is produced through _____________
hybridoma technology (spleen and cancer cells)
A polyspecific AHG contains __________________
multiple components (Anti-IgG + Anti-C3)
In AHG testing, this is employed to aid visualization of Ag-Ab reaction
AHG sera
What are the two methods of AHG test?
Direct and Indirect AHG test
This method of AHG test combines known Ag/Ab with either plasma/cells w/ known component
Indirect method
In indirect method, it detects _____________
either Ag or Ab depending what is needed and detects in-vitro sensitization
What is the specimen for indirect AHG test?
Donor Red cell and Patient’s Serum
What is the enhancement medium used for indirect AHG test?
22% bovine albumin
TRUE OR FALSE:
POSITIVE reaction indicates the absence of Abs in Px serum
FALSE
POSITIVE reaction indicates presence of circulating Abs causing sensitization to red cell
TRUE OR FALSE:
NEGATIVE results require addition of check cells/coomb’s cells
TRUE
What are the uses of indirect AHG test?
employed during crossmatching detection/odentification of unexpected Abs red cell phenotyping investigation of transfusion reaction detecting weak D (Du)
What is the difference between direct and indirect AHG test?
Direct AHG test only has a single step procedure (no incubation)
detects Abs coating red cells in vivo (in vivo sensitization)
test for certain clinical conditions
The specimen used for direct AHG test is _________________
RBC
What are the uses of direct AHG test?
diagnosis of HDN
diagnosis of AIHA, CAIHA, WAIHA
investigation of transfusion reaction
detection of red ell sensitization reactions caused by drugs
What are the examples of drugs that can cause red cell sensitization?
Penicillin
Methyldopa
Cephalothin
In detecting Weak D, ______ portion of Ab attach to ______ portion of IgG or complement
Fab, and Fc
What are the factors in the sensitization phase (in vitro) affecting AHG test?
temperature - must be 37 deg C
incubation time - 30-120mins (saline/NSS) or 10-15 (LISS)
ratio of serum to cells - 40:1 or 133:1
reaction medium - (Albumin/LISS/PEG)
TRUE OR FALSE:
In the sensitization phase, INC ratio leads to DEC sensitivity
FALSE
INC ratio = INC sensitivity
This reaction medium affects the 2nd step of reaction and allows Ab-coated cells to come closer w/ each other
Albumin
This reaction medium affects the sensitization stage that enhances Ab uptake and removed negativity
LISS
This reaction medium is a water soluble linear polymer and is also used for increase Ab uptake
PEG
In the washing phase, what is the ideal solution for washing?
Fresh/buffered saline
DEC pH during storage = acidic = affect
What is the relative centrifugal force and time when observing hemagglutination reaction?
1000 rcf for 20secs
500 RCF for 15 to 20 secs.
3400 RPM for 15-20 secs.
If the tapping of the tube is vigorously the result may be interpreted as ______________
FALSE +
Over centrifugation is cause false ____________
positive
Use of dirty glassware causes false ____________
positive
If the red cells agglutinated before washing step and addition of AHG, it causes false ___________
positive
AHG reagent not immediately added causes false ____________
negative
Loss of reagent activity causes false ____________
negative
Inappropriate red cell concentrations causes false ___________
negative
Bacterial contamination causes false ___________
negative
In DAT/IAT, how long will the tube be centrifuged?
15 secs at 3400rpm
In IAT, the unknown test tube contains _________
2 drops of unknown serum and 2 drops 3% RCS
In IAT, the negative control test tube contains _________
2 drops of 22% BSA and 2 drops 3% RCS
What is the anticoagulant used for DAT?
EDTA to prevent complement activation
At what time and temperature will the tubes be incubated in IAT?
37 deg C water bath for 15mins
Which AHG test method is used to detect Du?
IAT
The presence of weak D can be demonstrated by ____________________
incubating red cell suspension with Anti-D followed by antiglobulin testing
In detecting Du, it must be incubated at 37 deg C for 15mins to _____________
facilitate sensitization
In detecting weak D, the agglutination must be a grade of _____________
2+ to be considered as true negative
if 1+/mf = invalidate result
