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
No further testing if
- No unexpected antibodies
present in serum/plasma. - Only autoantibody detected in
eluate. - No recent transfusion
Further studies
Confirm specificity of _______ if present
Alloantibodies
If DAT is positive but serum and elution studies are negative suspect _______
drug induced
hemolysis – reference lab
ABO incompatible components transfused test for
Anti-A and Anti-B
Further studies
If patient is an infant perform
appropriate
testing on maternal
sample and elution on
cord cells.
Two Types of Cold Autoadsorptions
RABBIT ERYTHROCYTE
STROMA TEST (REST)
COLD AUTO
ADSORPITON
Two Types of Warm Autoadsorptions
W.A.R.M
WARM AUTO
ADSORPITON
Rabbit Erythrocyte Stroma Test
(REST)
For patients who have been recently transfused.
Rabbit cells have I antigen
REST procedure
-Incubate patient serum/plasma with rabbit
stroma at 4C.
-I antibody absorbed out.
-Remove (harvest) serum/plasma
-Test absorbed serum against screen cells.
Rabbit Erythrocyte Stroma Test
(REST)
Use with caution because
may adsorb out clinically significant antibodies
to D, E, Vel antigens and IgM antibodies regardless of specificity.
Cold Autoadsorption Test
Cant be performed on
recently transfused patients
Cold Autoadsorption Test
Collect ______
EDTA sample, keep warm
Cold Autoadsorption Test
Seperate _____ from RBCs
wash _____ with saline
Separate plasma from RBCs.
Wash RBCs with warm saline
Cold Autoadsorption Test
Ass aliquot of
Add aliquot of plasma/serum to RBCs incubate at 4C for 1 hour.
Cold Autoadsorption Test
Harvest serum/plasma and
test against screen cells
Negative – no alloantibody.
Positive and negative – alloantibody present, run panel.
All positive, unsuccessful, repeat with 1X adsorbed sample.
Cold Autoabsorption test
Supernatant used for
- Ab screen
- Reverse grouping cells
- Autocontrol
- Limitation: Will not remove a high titer cold agglutinin completely
Autoimmune hemolysis
Immune hemolytic anemia
shortened RBC survival mediated
through the immune response, specifically by humoral antibody
Autoimmune Immune hemolytic anemia types
- Warm autoimmune hemolytic anemia (WAIHA)
- Cold agglutinin syndrome (CAS)
- Mixed type – both warm and cold autoantibodies present
- Paroxysmal cold hemoglobinuria (PCH)
Alloimmune Immune hemolytic anemia types
- Hemolytic transfusion reaction
- Hemolytic disease of the fetus and newborn
Drug induced Immune hemolytic anemia types
- Drug dependent
- Drug independent
Characteristics of Immune Red Cell
Destruction
Diagnosis of hemolysis :
- Possible decreased H&H
- Increased reticulocytes
- Increased LDH
- Decreased haptoglobin
- Increased unconjugated bilirubin
- Hemoglobinemia and/or hemoglobinuria may indicate acute hemolysis or
intravascular RBC destruction.
Autoantibodies definition
Antibodies that are directed against the individual’s own RBCs
are termed autoantibodies or autoagglutinins.
Autoantibodies
Studies in animal models suggest
that autoantibodies occur because of
a failure of the immune regulatory responses
Autoantibodies
RBC survival may be
Shorten
Autoantibodies
identification of a autoantibody may explain
decreased RBC survival
in vivo.
Autoantibodies
If a patient’s RBCs are coated with autoantibody, the patient may
present with:
- ABO discrepancy
- positive Rh control
- positive DAT
Autoantibodies
A positive DAT, a positive
autocontrol, or serum
autoantibody does not
necessarily confer the
diagnosis of AIHA.
- Approximately 0.1% of normal blood donors and up
to 15% of hospitalized patients have positive DATs
with no evidence of hemolytic anemia
compensated anemia
the rate of RBC production
will nearly equal the rate of RBC destruction.
uncompensated anemia
the rate of RBC
destruction exceeds the rate of RBC production.
Serologic Testing
Diagnostic tests in a symptomatic patient include
- DAT using polyspecific and monospecific antiglobulin reagents
- Characterization of the autoantibody in the serum and/or eluate
using standard antibody detection and identification procedures
AIHA may be diagnosed and classified as
cold reactive,
warm reactive, or drug-induced.
Autoantibodies can be characterized by
their
optimal temperature of reactivity
Autoantibodies optimal temperature of reactivity
- Warm Temperatures (30°C to 37°C) - About 70%
of cases - Cold reactive (4°C to 30°C) - About 18% of cases
- Drug-induced - About 12% of cases
Cold reactive autoantibodies are
Most are not
are
frequently encountered in serologic
testing.
* Most are not clinically
significant, but occasionally they
are clinically significant and cause
immune hemolytic anemia.
Cold reactive autoantibodies
When testing is performed at
4°C, the most commonly
encountered autoantibody is a
benign cold agglutinin that may
be found in the serum of most
normal, healthy individuals
Cold Reactive
Autoantibodies
The typical cold agglutinin has a
relatively _____ titer
low titer (<64 at 4°C)
Cold Reactive
Autoantibodies
Cold agglutinins can interfere
with routine
serum and cell
testing performed at RT,
potentially affecting
* ABO/Rh typing
* Direct Antiglobulin Test (DAT)
* Antibody detection and
identification
* Compatibility testing
Anti-I, Anti-i
Most cold reactive
autoantibodies have anti-I
specificity.
The I antigen is fully
expressed on RBCs of most
adults but only weakly
expressed on cord RBCs.
Anti-i is a relatively
uncommon autoantibody.
This antibody reacts in an
antithetical manner to
anti-I.
Anti-H, Anti-IH
Cold agglutinins found in
the sera of group A 1 and
A 1B individuals (and
occasionally group B) may
have anti-H specificity.
Group O and A 2 cells react
best because they have
the largest amounts of H
antigen.
Group A 1 and A 1 B cells
have the least H antigen so
they react weakly.
Short cold panel
Incubate patient serum/plasma with
- Type O adult cells (I)
- Type O cord cells (i)
- Type A1/B adult cells
- Autologous
Other Cold Reactive
Autoagglutinins
Less commonly encountered cold autoagglutinins
have been described, such as
anti-Pr, anti-Gd, and
anti-Sdx (anti-R x).
Pathologic Cold
Autoagglutinins types
Chronic condition- no known cause
Acute condition
= Mycoplasma pneumoniae- Anti-I
= Infectious mononucleosis- Anti-i
= Lymphoproliferative disorder- Anti-i
Paroxysmal Cold Hemoglobinuria
Acute condition
Pathologic Cold Autoagglutinins
Mycoplasma pneumoniae- Anti-I
Infectious mononucleosis- Anti-i
Lymphoproliferative disorder- Anti-i
Cold Hemagglutinin
Disease
(Idiopathic Cold AIHA
or CHD) (cont’d)
Antibody specificity is almost always
anti-I, less
commonly anti-i, and rarely anti-Pr
Cold Hemagglutinin
Disease
(Idiopathic Cold AIHA
or CHD) (cont’d)
Laboratory findings in CHD
include reticulocytosis and a
positive DAT due to complement only.
Cold Hemagglutinin
Disease
(Idiopathic Cold AIHA
or CHD) (cont’d)
The peripheral smear may show agglutinated
BCs,
polychromasia, mild to moderate anisocytosis, and
poikilocytosis
Cold Hemagglutinin
Disease
(Idiopathic Cold AIHA
or CHD) (cont’d)
May cause false positive
- forward and reverse typing
- Forward – cells so heavily coated spontaneously
agglutinate. - Reverse cells have I antigen and will agglutinate
Cold Hemagglutinin
Disease
(Idiopathic Cold AIHA
or CHD) (cont’d)
Pre-warmed testing may eliminate
Reactivity
Cold
Hemagglutinin
Disease
DAT
Only what is detected on the
RBCs
Cold
Hemagglutinin
Disease
Why is complement the only protein present
- Antibody is IgM, which in-vivo binds to
RBCs in peripheral circulation where
temperature falls to 32C or less. - IgM causes binding of complement in
the cold. - When RBCs return to warmer
circulation IgM dissociates leaving
RBCs coated with complement only
Cold
Hemagglutinin
Disease
Eluate will be
will be negative as only complement
is binding to the RBC
Cold Hemagglutinin Disease
(Idiopathic Cold AIHA or CHD)
MOST PATIENTS REQUIRE NO
TREATMENT AND ARE INSTRUCTED
TO AVOID THE
COLD, KEEP WARM,
OR MOVE TO A MILDER CLIMATE.
Cold Hemagglutinin Disease
(Idiopathic Cold AIHA or CHD) (cont’d)
CONSIDERATIONS FOR TRANSFUSION THERAPY
- Rarely require transfusion.
- Use prewarmed technique for
compatibility tests or use cold
autoadsorbed serum. - Transfuse blood through blood warmer
Paroxysmal Cold
Hemoglobinuria
(PCH)
Different from
Paroxysmal Nocturnal
Hemoglobinuria in which hemolysis is caused by
acid produced during sleep
Paroxysmal Cold
Hemoglobinuria
(PCH)
RAREST form of
DAT positive AIHA
Paroxysmal Cold
Hemoglobinuria
(PCH)
Presents as acute transient condition secondary to
viral infections particularly in young children
Paroxysmal Cold
Hemoglobinuria
(PCH)
Caused by a
biphasic hemolysin which induces
hemolysis after exposure to cold
Paroxysmal Cold
Hemoglobinuria
(PCH)
Results in what symptoms
hemoglobinuria and hemoglobinemia
PCH test
Classic antibody
produced: the Donath-
Landsteiner antibody
(an autoantibody with
anti-P specificity)
Treatment for PCH
- Chronic: Protection from cold exposure is the only useful
therapy. - Acute: postinfection forms are transient and usually
terminate spontaneously after the infection resolves.
Steroids and transfusion may be required, depending on
the severity of the attacks.
PCH –
DAT and
Eluate
DAT
- Autoantibody is IgG which acts as cold agglutinin.
- IgG binds to RBCs in colder parts of body.
- Causes complement to be bound irreversibly.
- IgG elutes off of RBCs in warmer parts of the body.
- ONLY COMPLEMENT is detected
PCH
Eluate
Since only complement coats cells eluate is
negative
PCH
Antibody specificity
- Anti-P
- Not necessary to transfuse rare P negative blood
PCH –
Serum/Plasma
Testing
Autoantibody described as
biphasic
hemolysin”.
* Binds to RBCs at low temperatures.
* Binds complement.
* As cells warm up hemolysis occurs.
* Basis of Donath-Landsteiner test
Donath-Landsteiner
- Usually add fresh complement
- Antibody binds at cold temperatures
- Test is warmed to 37C.
- As warming occurs complement is activated and
lysis of RBCs occurs.
Warm
Autoimmune
Hemolytic
Anemia
(WAIHA)
Most common type of
Most common type of AIHA and most
difficult problem encountered in the
transfusion service.
Warm Autoantibodies
React best at
37C
Warm Autoantibodies
Not found as often in the random population as
the almost universal cold
autoanti-I
Warm Autoantibodies
Some are apparently
Some are apparently harmless.
Harmless autoantibodies are
serologically indistinguishable from
the harmful ones.
A significant percentage of cases
suffer from an anemia of sufficient
severity to require transfusion.
Warm
Autoantibodies
Most patients with warm autoimmune hemolytic
anemia (WAIHA) have both
IgG and complement
on their RBCs (67%)
Warm
Autoantibodies
The IgG immunoglobulins react best by the
indirect antiglobulin technique
WARM AUTOANTIBODIES (CONT’D)
Peripheral blood symptoms
- polychromasia and
macrocytosis - reticulocytosis - possilbe nucleated RBCs -
hyperactive bone marrow - spherocytosis and occasionally
RBC fragmentation, indicating
extravascular hemolysis
WARM AUTOANTIBODIES (CONT’D)
Products of hemolysis
- Increased LDH
- increased bilirubin (particularly
the unconjugated indirect
fraction) - Increased urinary urobilinogen
- depleted serum haptoglobin
- hemoglobinemia and
hemoglobinuria
Rh typing
There can be false positives; however,
with the introduction of monoclonal
antisera, the incidence has decreased
DAT testing
The DAT can be EGA-treated or CDP-
treated RBCs are negative, it is possible
to use these cells for testing with the
Anti-D, -C, -E, -c, -e, -K, -Jk(a), -Jk(b)
and others
* A positive DAT is expected.
* The majority of the cases have both IgG
and complement coating the cells.
* In rare cases, the DAT may be negative
because it is coated with IgA or IgM
Serum/Plasma testin
- May have little free autoantibody, may all be
on RBCs - Once all antigen sites coated, detectable
- Approximately 50% of WAIHA will have
autoantibody reactive with ALL CELLS TESTED. - DANGER – alloantibody may be present also
Eluate
- Usually reactive with all cells tested.
- Negative reactions due to complement only
OR presence of drug induced antibody
WAIHA – Antibody Specificity
May be very
often intially directly against
-May be very complex.
-Often initially directed against Rh antigen complex
WAIHA – Antibody Specificity
Specificity against simple
Rh or other blood group
antigens occasionally seen
WAIHA – Antibody Specificity
If simple specificity, give antigen negative and if high
frequency or complex antibody
Antigen negative not practical
Warm
Autoantibodies mechinisms
Extravascular immune RBC destruction
occurs. Sensitized RBC removed by
reticuloendothelial system (RES) cells of liver and
spleen.
Warm
Autoantibodies
A minority have either
IgG only (20%) or
complement only (13%)
Warm
Autoimmune
Hemolytic
Anemia
(WAIHA)
DAT
67% of the
cases, RBCs
are coated
with both IgG
and
complement.
20% of the
cases, RBCs
are coated
with IgG
alone.
13% of the
cases, RBCs
are coated
with
complement
alone
