AB Screening & ID Flashcards
detection & ID of AB against RBC AG; critical in PRE-TRANSFUSION SETTINGS
Antibody Screening & ID
principal tool for investigating potential HTR, AIHA, HDFN
Ab Screening & ID
_______aids in detecting and monitoring patients who are at risk of delivering infants with HDFN; The ony ig that can cross the placenta, associated with this disease is _______.
Ab Screening/ID
IgG
Focus of Ab Screening & iD
Detection of UNEXPECTED IRREGULAR ALLOANTIBODIES
4 types of unexpected irregular alloantibodies
- Ab reacting to transfused blood.
- Naturally occurring
- Acquired Ab
- AutoAb
Explain occurrence of
Ab reacting to transfused blood.
in response to RBC STIMULATION through transfusion, transplantation, pregnancy
Explain occurrence of
Naturally occurring unexpected immune allonantibodies
no RBC stimulation
through exposure to environmental surfaces/sources that have similar structure to RBC Ag
examples of environmental surfaces/sources causing naturally occurring unexpected immune allonantibodies
pollen
fungus
bacteria
Explain occurrence of
acquired unexpected immune allonantibodies
from one individual to another via plasma-containing blood components/derivatives.
example of plasma-containing bloodcomponents/derivatives causing naturally occurring unexpected immune allonantibodies — which is a treatment of choice for AB DEFICIENCIES
Intravenous Immunoglobulin [IVIG]
Explain occurrence of
Autoantibodies
against own antigens & generally react with all RBCs tested
it involves the reaction between px serum/plasma with screening cells.
Ab screening
Purpose is to detect RBC unexpected alloantibodies other than the expected anti-A and anti-B
Ab screening
Method of AB Screening
Indirect Antihuman globulin test
4 components of IAT
IS phase
AHG phase
Monospecific anti-IgG AHG reagent
37C
RBC reagent of IAT
[explain chracteristics]
Screening Cells
-Group O Ab + clinically unique combi of clinically important RBC Ag: 2-3 phenotypes
2-3 phenotypes combines with Group O Ab for screening cells
2:
R1R1 DCe
R2R2 DcE
3:
R1R1
R2T2
rr dce
why is Group O antigen used as a component of screening cells?
has no Ag in rBC no reaction and anti-a/b will not interfere
the homozygous expression w/in the screening cells allows Ab show ____.
dosage
Each set of screen cells is accompanied by:
Ag profile sheet/antigram
Antibodies that react strongly w/ cells having homozygous antigen expression
Ag profile sheet/antigram
antigen expression is from an individual who inherited 2 different alleles at a given genetic locus.
Heterozygous
The alleles share the available antigen sites on the cell surface.
Heterozygous
antigen expression is from an individual who inherited only one allele at a given genetic locus
Homozygous
Common Blood Group Systems
With Antibodies That Exhibit Dosage (Homozygous blood group)
Rh [except D[
Kidd
Duffy
MNS
Lutheran
Explain the process of IAT for AB Screening
if there is no sensitization nor agglutination in IAT, what do we add?
Check cells/Coomb’s reagentI
If after the addition of coomb’s reagent or check cells, what are the usual problems and what do we do about it>
During testing problems/ error [AHG. not added]
REPEAT
RBCs become “IgG-coated RBC’’ due to the
Addition of an enhancement reagent that triggers the px’s serum containing [IgG] to target the known RBC reagent/Screen cell which possesses a known antigen making it the targetq
Effect of AHG reagent addition after NSS washing in IAT
The anti-IgG in it will react w/ the IgG-coated RBC resulting to cross-linking and forming a BRIDGE where observable AGGLUTINATION occurs
OTHER METHODS USED FOR ANTIBODY SCREENING:
Gel Method
Solid phase adherence method
Gel Method Principle
If sensitization occurred, anti-IgG IN THE GEL will REACT with the sensitized rbcs resulting in agglutination
Agglutinated cells will be TRAPPED within the gel because agglutinates are too large to pass the spaces between gel particles
Agglutinated cells in the Gel Method will be TRAPPED within the gel because
they are too large to pass the spaces between gel particles
Grade?
cell top: SOLID BAND of agglutinates
bottom: #red cells visible
4+
Grade?
LOWER-half gel column: agglutinates
bottom: red cell [above red cell pallet: few agglutinates]
1+
Grade?
top of gel column: ag DISPERSED
bottom: few agglutinates
;distributed through upper/lower halves of gel
2+
Grade?
top gel column: LAYER of red cell agglutinates
bottom: PELLET of UNAGGLUTINATED CELLS
Mixed-field
Grade?
bottom: rbc forming a WELL-DELINEATED PALLET
[Above pallet] CLEAR/FREE agglutinates
NEGATIVE
Grade?
top gel column: #amount of agglutinates
bottom: few agglutinates
3+
Components of Gel method
6 chambers/gel
Dextran Acrylamide gel
Screen Cells + Px serum/plasma
37C 15mins
Principle of Solid Phase Adherence Method
- RBC Ag-COATED microtiter wells
- Px serum/plasma + LISS added
37C incubation - Washed to remove unbound Ab
4.
AHG-coated RBCs [indicator] added - POSITIVE= AHG react w/ Ab-Ag bound
NEGATIVE=AHF form a PELLET in the bottom
Done when the antibody screening test is positive to _____ the alloantibody
AB ID
Identify
IAT may or may not include this as a detection for the AB reacting at room temp; not required as it leads to clinically [significant/insignificant] [cold/warm] Ab
Immediate spin phase
Insignificant
Cold
AB ID uses what:
cells:
material:
methods:
panel cells [group O, 11-20 extended screening cells]
Ag profile sheet/antigram
DAT
autocontrol
Steps in AB ID
- Know the Px HISTORY
- Check RXN phase w/ positive agglutination
- Check AUTOCONTROL
- Exclusion
- Inclusion
- Perform ADDT’L techniques
[Selected Panel cells, Neutralization, Adsorption, Elution]
7.CONFIRM result by 3 & 3 RULE
Enumerate what to consider in Px history
Age
Sex
Race
Diagnosis
Pregnancy history
Medications
IV solutions
Race: can be beneficial because some antibodies are associated with a particular race.
anti-__ – blacks
anti-__ – Africans)
Fya
U
Transfusion and pregnancy: exposure can cause ______ of immune antibodies
production
Medications to take into consideration as they may transfer PASSIVE AB [anti-A/B/D]
IVIG
RhIg
Anti-lymphocyte globulin
administer to Rh negative mother with an Rh positive baby
rhogam
animal antibody against human T cells that is used in treatment for acute rejection in organ transplantation
Anti-lymphocyte globulin
Infections and autoimmune disorders can cause production of ______
autoantibodies
Patients transfused in the last 3 months (DHTR) can also cause a _______
POSITIVE DAT
3 and 3 rule meaning
3 positive
3 negative
________
Additional panel cells that are added to help in identifying antibodies
2 or 3 can be used but we can still add. _____ panel cells can be used
Selected Panel Cells
11-20
Treating the panel cells with enzymes may help separate the __________ and allow antibody ________
specificities
identification
Examples of enzymes used as an additional technique for AB ID
[general]
[specific]
Proteolytic enzymes
[papain, bromelin, trypsin]
Proteolytic enzymes modify the rbc surface by removing the ________ and by denaturing or removing _______.
sialic acid residues
glycoprotein
antigens enhanced by enzymes
Rh
Kidd
Lewis
P1
I
ABO
antigens activated by enzymes
Duffy
MNS
Xga
Other substances in the body and in nature have antigenic structures similar to antigens
Neutralization
Soluble substances can be used to _______ antibodies in serum, allowing ________ of antibodies or confirmation that a particular ______ is present.
neutralize
seperation
antibody
inhibit reaction between antibody and antigens on panel cells
Soluble substances
Anti-P1
Source of neutralizing substances
Hydatid cyst fluid
Pigeon droppings
Turtledoves egg whites
Anti-Lewis
Source of neutralizing substances
Plasma/Serum
Saliva
Anti-Chido/Rogers
Source of neutralizing substances
Serum [+complement]
Anti-Sda
Source of neutralizing substances
Urine
Antii-I
Source of neutralizing substances
Human breast milk
Removal of antibodies from serum by adding the target antigen allowing antibody to bind to the antigen
Adsorption
In adsorption, Antigen-antibody complexes is removed from the test system by ______.
centrifugation
________ is tested against panel cells for the presence of unabsorbed alloantibodies
Adsorbed serum
Adsorbed serum is TESTED AGAINST PANEL CELLS for the presence of unabsorbed alloantibodies. THROUGH? [application steps]
- remove non-specific ab from serum
- seperate mixtures of ab [to aid their [ID]
- determine [+] alloantibody/autoantibody in a px w/ HEMOLYTIC ANEMIA
Types of Adsorption
Autologous
Homologus Adsorption
Differential Adsorption
simplest method of absorption
AUTOADSORPTION
In autoadsoprtion,
warm ab is adsorbed at what temp?
cold ab is adsorbed at what temp?
37C
4C
Autoadsorption steps
- Autologous washed [remove unbound ab]
- Cells treated [remove any Ab-coating RBC]
- Cells incubated w/ px serum for up to 1 hour [30-60 mins]
[Temp: thermal-range of removed-Ab]
[During incubation: mix periodically]
[X AB @serum –> ADSORBED @RBC]
4.
Adsorbed serum then harvested
& Tested against Panel cells
5.
DAT[-] autologous cells–
If REACTIVE = further adsorption
using a new aliquot of autologous required.
If NON-reactive=
autoantibody completely adsorbed + adsorbed serums can be used in antibody identification testing.
DAT[-] autologous cells–
If REACTIVE = _______
If NON-reactive=
___________
further adsoption [using new aliquot of autologous cells]
Autologous completely adsorbed
+Adsorbed serums can be used in AB ID TESTING
used to remove, concentrate, purify AB from RBCs
Elution
This is being performed when DAT is positive
Elution
diluent where removed ab from RBCs are harvested/tested against panel cells
Eluate
ab removed from RBCs
& RBC Ag are destroyed
Total elution
ab removed
RBC Ag remain intact [RBC ______ & used for autoadsorption techniques.
Partial elution
Elution Application:
—Demonstrate & Identify Ab on the RBC of HDN, HTR,AIHA
— Seperate & Identify Ab IN A MIXTURE OF AB
Elution Methods
Temperature
pH
Organic solvents
Elution Temperature Method:
HEAT
45C:
56C:
FREEZE
RBCs are washed/suspended in ______ and frozen at [temp].
Mixture is _____ rapidly bursting _____ freeing the ab
Gentle heat method/Partial Elution
Total elution/ elute ABO Ab from RBC
saline
18C
thawed
RBCs
Elution method
pH
[Acid/Alkaline] elution:
washed ______rbcs are mixed w/ _________ [ph__]–> then released into the ______.
Acid elution
Ab-coated
glycine acid
3
supernatant
Elution method
Organic solvents
[Partial/Total] elution; best for detecting non-ABO Ab: [3]
Total
dichloromethane
xylene
ether
Done to determine the relative
amount of antibody in the serum
Ab Titration
expressed as the reciprocal of the highest dilution with macroscopic agglutination
Titer
Elution Method
Ig TYPING
Uses ________ reagents which inactivates ____ leaving IgG intact.
Used to break agglutination by strong _________
DTT, 2-ME, AET
sulfhydryl reagents
IgM
IgM cold autoantibodies
Reagents used in Ig Typing method of elution
ZZAP
DTT
2-ME
AET
