Test 5 COPY COPY Flashcards
DAT test freshly washed cells with
antiglobulin reagents (anti-
IgG and/or anti-C3d) to detect IN-VIVO coated of RBCs.
DAT
The ___ Fab sites on the antiglobulin molecule bind to the Fc
a portion of sensitizing antibody or complement on ____
adjacent RBCs, bridging gap and causing visible
agglutination
2 and 2
DAT
The stength of agglutination is proportional to the
amount of bound
protein.
Direct AHG
whats all added
Washed (3xs) pateint red cells + coombs sera ———-> visual Red cell agglutination
Reasons to
Perform a
DAT
-SCREEN FOR
CLINICALLY
UNEXPECTED
AUTOIMMUNE
PHENOMENA
-DETECT EARLY
MANIFESTATION
OF IMMUNE
RESPONSE TO
RECENT
TRANSFUSION.
-ASSIST IN
DIAGNOSIS OF
HDFN.
Collection of Blood Sample for DAT
-To verify in-vivo sensitization
EDTA sample should be used.
-EDTA sample will provide RBCs
for elution if necessary.
-If cold hemagglutinin
suspected keep sample at 37C.
Significance of Positive DAT
Positive DAT does not mean that RBCs will
have shortened survival
Positive DAT without clinical problems occurs in
1:1,000-1:14,000
blood donors and 1-15% of hospital patients.
Significance of positive DAT
what is coated on RBCs
IgG and C3d
Healthy individuals can have ___________ molecules of IgG/RBC and C3d/RBC, this is
below threshold of detection
5-90
DAT can detect ________ IgG/RBC and _________ C3d/RBC
100-500
400-1100
Causes of a positive DAT
Autoantibodies Alloantibodies - HTR
Passively acquired
alloantibodies (plasma,
derivatives)
Maternal alloantibodies
Nonspecifically adsorbed
proteins or membrane
modification
Drug induced antibodies Antibodies produced by
passenger lymphocytes
Complement activation
due to bacterial
infection, autoantibodies
or alloantibodies
Autocontrol (AC) run as part of
antibody
work up and is not the same as DAT
DAT cells are taken directly to
AHG, no
adding of serum or enhancement, no
incubation detects IN-VIVO sensitization
False
Positive
DAT
-Want to detect
IN-VIVO
sensitization
not in-vitro.
-False positive
most often
associated with
using
refrigerated or
clotted samples.
-Any positive
obtained on
clotted sample
should be
confirmed with
EDTA sample.
-EDTA
will provide
RBCs for elution
if necessary.
Patient history
Crucial to investigation
- History of recent transfusion.
- Administration of drugs previously
associated with immune hemolysis. - History of hematopoietic
progenitor cell or organ
transplantation. - Administration of IVIG or IV anti-D
Test serum/plasma to detect and identify clinically significant antibodies to
may have to distinguish between
red cell antigens,
may have to distinguish auto-
from allo-antibodies, if present.
Prepare and test eluate from DAT positive RBCs to define whether
Prepare and test eluate from DAT
positive RBCs to define whether
coating protein has red cell antibody
specificity.
Eluate from complement only coated cells
______________ if present, which may not be
present in patient serum/plasma.
- Eluate from complement only coated cells should be
tested if clinical evidence of hemolysis. - Concentrates IgG, if present, which may not be
present in patient serum/plasma.
Elution definition
Removes antibody from sensitized RBCs and recovers antibody in
usable form.
Elution
Thorough washing of the RBCs essential to ensure that
that antibody
detected in eluate is only RBC bound antibody.
Elution
Last wash will detect proper
washing and should be negative with
all cells tested.
Elution when to preform
whenever DAT is positive
for IgG)
Elution techniques free antibodies form the
sensitized red cells so that the antibodies can
be identified