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Continuing Education
• PACE, Florida and California DHS
• 1.0 Contact Hours
• Each attendee must register to receive CE at: https://www.surveymonkey.com/r/DATAutoControlsAndEluates
• Registration deadline is 9 August, 2019
• Certificates will be sent via email only to those
who have registered 23 August, 2019
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• Course content is for information and
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used for clinical or maintenance evaluations.
• The opinions contained in this presentation
are those of the presenter and do not
necessarily reflect those of Immucor.
2019 10
DAT, Auto Controls & Eluates:
They are Not Just for the Reference Lab
Presented by
Jayanna Slayten, MS, MT(ASCP)SBB
Supervisor, Indiana University Health Transfusion Service and
Adjunct Faculty, University of Texas Medical Branch SBB Program
Objectives
• Compare and contrast the indirect antiglobulin
test (IAT) and direct antiglobulin test (DAT)
• DAT reagents
– Polyspecific, IgG with C3
– Monospecific, IgG or C3
• Possible causes of a positive DAT
• Elution types with application
– Lui
– Acid
2019 11
History of AHG Testing
• It started with anti-D Hemolytic Disease of the
Fetus and Newborn (HDFN)
• In 1945 Coombs, Mourant and Race described
the use of antihuman globulin, AHG/IgG, for
the detection of Rh antibodies in serum to
investigate HDFN.
– Indirect Antiglobulin Test - IAT
Coombs RRA, Mourant AE, and Race RR: (1945) A new test for the detection of
weak and ‘incomplete’ Rh agglutinins. Br J Exp Pathol 26: 255
2019 12
History of AHG Testing
• In 1946, Coombs and associates described the
use of anti-human globulin (AHG) to detect in
vivo cell sensitization of babies with hemolytic
disease of the fetus and newborn (HDFN).
–Direct Antiglobulin Test = DAT
Coombs RRA, Mourant AE, and Pace RR: (1946) In vivo isosensitization of
red cells in babies with haemolytic disease. Lancet i: 264
2019 13
History of AHG Testing
• AHG testing was implemented for antibody
identification procedures.
• Because Coombs and associates described
the test, the AHG tests is often referred to as
the Coombs Test.
• However, Coombs was not the first to
describe the principle of the test.
2019 14
History of AHG Testing
• Moreschi in 1908
– Studies involved the use of rabbit anti-goat serum to
agglutinate rabbit red cells which were sensitized with
low non-agglutinating doses of goat anti-rabbit red cell
serum.
– Demonstrating that immune serum may be used for
enhancing agglutination
– Could have been called the Moreschi Test
AHG Testing was Re-discovered by Coombs.
Moreschi, C: Neue Tatsachen über die Blutkorperchen Agglutinationen. Zentralbl
Bakteriol 46:49, 1908.
2019 15
Anti-Complement
• Dacie, JV, et al: Br J Haematol 3:77, 1957. – Reported antibody types demonstrated differing reaction patterns
– Lewis antibodies did not react at 37C and IAT-IgG method but reacted
well in IAT-IgM method
– They suggested components of complement may be involved and
coated on the red cells
• Polley MJ, Mollison PL, and Soothill JF: Br J Haematol 8: 149
– Reported these antibodies react best with anti-complement
• Jenkins et al 1960, Pondman et al 1960 and Harboe et al
1963
– Verified the work form Dacie et al
– The main component being detected were C3 and C4
2019 16
Definition
• Anti-Human Globulin Test (AGT) or (AHG)
– Test to ascertain the presence or absence of
red cell coating by immunoglobulin (IgG)
and/or complement.
– This test uses a xenoantibody (rabbit anti-
human serum) to act as a bridge between
sensitized cells; thus; yielding agglutination as
a positive result.
2019 17
AABB Technical Manual, current edition.
Polyspecific AHG Reagents
• Polyspecific regents
– Rabbit polyclonal
•Contains anti-IgG and anti-C3d
– Rabbit/murine monoclonal blend
•Contains monoclonal blend of rabbit polyclonal
antihuman anti-IgG and murine anti-C3bd
– Murine monoclonal
•Contains murine monoclonal anti-IgG,-C3d, -C3b
Polyspecific AHG Reagents
• Used in DAT and IAT testing
• Contains human source anti-IgG and -C3d
• Other anti-complement (anti-C4d, -C4b and -
C3b may be present
• Other antiglobulins may also be present (anti-
IgM, - IgA, IgD)
Polyspecific AHG Reagents
• Polyspecific reagents are prepared to detect
IgG and complement
• FDA requires that products marked as
polyspecific-AHG have anti-C3d reactivity which
is greater than or equal to the FDA anti-C3d
reference sample.
IgG AHG Reagents
• Anti-IgG
– Rabbit polyclonal
•Contains anti-IgG with no anti-complement
activity (not only Gamma chain specific)
– IgG Heavy Chain
•Contains only antibodies reactive against human
gamma chains
– Monoclonal IgG
•Contains murine monoclonal IgG only
IgG AHG Reagents
• Prepared by Hybridoma technique to produce
monospecific reagent
ANTICORPI MONOCLONALI . slideplayer.it/slide/579877/2/images/1/ANTICORPI+MONOCLONALI.jpg . Title"ANTMONOCLONALI“, Adapted from Milstein (1980) Scientific American, Oct. p.58
IgG AHG Reagents
• Used more than Poly AHG for IAT and DAT
• Able to detect IgG for interpretation of
clinical significance
• Does not react with anti-complement bound
to red cells.
– Cold reactive antibodies not as detectable
– Cold reactive antibodies are not considered
clinically significant.
Anti-complement AHG
• Anti-C3d and anti-C3b
– Contains only antibodies
reactive against the
designated complement
component with no
immunoglobulin (anti-IgG)
reactivity
Anti-complement AHG
• Prepared by animal immunization or
hybridoma technique
• Anti-complement reagents are helpful in the
evaluation of
– Clinical hemolysis
– Cold reactive antibodies (allo or auto antibodies)
– Complement dependent antibodies (Kidd system)
Definition
• Indirect Antiglobulin Test (IAT)
– Used to detect antigen-antibody reactions that
occur in vitro
– Sensitization and Lattice Formation (2 step)
•Antibody Screen
•Antibody Identification
•Antigen Typing
•Crossmatching
2019 26
AABB Technical Manual, current edition.
Definition
• Direct Antiglobulin Test (DAT)
–Used to detect in vivo cell
sensitization (1 step)
•Immunoglobulins (IgG)
•Complement
2019 27
Image from: https://www.med-ed.virginia.edu/courses/path/innes/rcd/antibody.cfm
AABB Technical Manual, current edition.
Positive DAT
Positive IAT
2019 28
Principle of DAT
• DAT will detect
– 100-500 molecules of IgG/red cell
– 400 - 1100 molecules of C3d / red cell
• Detection of lower levels of antibody may
require more sensitive methods
– Flow Cytometry
– ELISA
2019 29
AABB Technical Manual, current edition.
Principle: Specimen
• EDTA anticoagulated blood specimen
– EDTA chelates Ca++ and Mg++ preventing the in
vitro binding of complement
– Using EDTA specimens with not interfere with
complement bound in vivo
• A clotted sample is not ideal
– For Column Agglutination Test – EDTA or Red Top Tube
– For Tube DAT
•If a clotted tube (red top tube) is used for DAT the
results should be confirmed with an EDTA tube.
2019 30
AABB Technical Manual, current edition.
Principle: Reagents
• Polyspecific AHG
– Contains rabbi/murine polyclonal or monoclonal
anti-IgG and anti-C3d
• IgG AHG
– Contains rabbit or murine
polyclonal/monoclonal anti-IgG only.
No source of complement
• Anti-C3bd
– Contains antibodies reactive to complement
2019 31
AABB Technical Manual, current edition.
Principle : Tube Reagents
• Coombs Control Cells
– IgG Sensitized
•Weakly IgG Coated Cells
•Used as a control to validate that IgG or Poly
was added to the test system
– Complement Sensitized
• Weakly Complement Coated cells
•Used as a control to validate that Anti-C3d
was added to the test system.
2019 32
AABB Technical Manual, current edition.
Tube Method
• Wash the 3% cell suspension 4 times
manually with saline or automatically using a
cell washer.
• After washing, add the appropriate reagent.
• Spin and read using an agglutination viewer.
– Agglutination = Positive
– No agglutination = Negative
2019 33
DAT Control?AABB Technical Manual, current edition.
Poll the Audience
If one is testing a IgG-DAT and achieve the
following results, IgG DAT= 3+, Saline DAT control
= 2+), then what is the interpretation?
• Valid the DAT is positive
• Invalid the DAT is positive and the control is
positive
2019 34
Testing a DAT Control
• Run one tube for each AHG reagent, in parallel
– Reason for control is to verify the positive DAT
detected is not spontaneous agglutination
Example: a cold agglutinin
• Control is valid
– Testing is valid and may be interpreted
appropriately
• Control is invalid
– If patient and control are positive, then the
testing cannot be interpreted
2019 35
Poll the audience
• Do you use a microscope to evaluate DAT
results?
– Yes, all DATs are evaluated microscopically
– Yes, but only when the results are questionable
– No, we do not use the microscope to evaluate
DAT testing
– No, we only use automated platform for DAT
testing
2019 36
Tube Methods
• Is it advisable to examine DAT testing using a microscope?
– Yazer and Triuzli. TRANSFUSION 2009;49:2395-2399, reported that this may have limited value
• Why it may be helpful
– Due to the nature of the testing to detect in vivo binding of antibody
– One may one detect this interaction using a microscope more easily
– With generalist blood bankers, this may add additional confidence to interpretation for DAT
2019 37
Tube DAT Method
• To validate that the appropriate antihuman
globulin has been added to the test system
• Add the appropriate Coombs control cell
– IgG Coated cells for Polyspecific and IgG
– Complement coated cells for anti-C3d
• If reactivity is not detected after the addition of
the Coombs control cell the test is invalid and
should be repeated.
2019 38
AABB Technical Manual, current edition.
Tube DAT Results C’= complement
Polyspecifi
c
Anti-IgG Anti-
C3d,C3b
Saline
Control
Interpretation
+ + 0 0 IgG + only
+ 0 + 0 C’+ only
+ + + 0 IgG + & C’ +
+ + + + Test Invalid
0 0 0 0 Neg DAT
2019 39
AABB Technical Manual, current edition.
Source of False Positive DAT
• Cells agglutinate prior to washing
– Cold reactive antibodies
• Particle or contaminants from dirty glassware
• Over-centrifugation may pack the red cells so tightly
that the button cannot be dispersed
• If sample collected in a serum separator tube the
silicon gel has been documented to cause up to
13% false reactivity
2019 40
AABB Technical Manual, Current Edition.
Source of False Positive DAT
• Cells are polyagglutinable (rare disorder)
• Complement components may bind at lower
temperatures if the sample is stored at 4C
– Segments
– Samples
• Complement may attach if the sample is drawn from
a line with dextrose containing solutions
2019 41
AABB Technical Manual, Current Edition
Resolving a False Positive
• Warm wash DAT
– Use if cold reactive antibody detected in the serum
testing.
– Repeat same method with control and pre-warmed 37C
saline
• 0.01 M DTT treat cells and repeat testing
– Standard method
– Dilute 0.2M DTT
– Treat the cells and a parallel control
– Repeat DAT
2019 42
Judd's Methods in immunohematology, 3rd Edition / W. John Judd, Susan T. Johnson, Jill Storry. AABB Press.2008.
Source of False Negative DAT• Failure to wash cells adequately
– AHG neutralize the unbound components
• Interrupted testing
– If AHG reagents are not added quickly after
washing, previously bound IgG and complement
may dissociate.
• Improper technique
– 5-60% of positive tests were incorrectly
interpreted as negative in one study
2019 43
AABB Technical Manual, Current Edition
Source of False Negative DAT
• False reactivity with anti-C3bd may be
indicated if package insert is not followed for
additional incubations.
• Cells may have IgG and complement bound is
very few numbers
– Polyspecific and IgG reagents may detect 200-
500 IgG per cell
2019 44
AABB Technical Manual, Current Edition
Resolving a False Negative DAT
• Training, training, training
• Increase sensitivity of DAT method
– DAT negative hemolytic anemia investigation
•DAT Anti-IgA
•Elution
– Flow cytometry
•Detection of IgG coated cells
2019 45
Segal and Litchman, Blood Cells Mol Dis. 2014 Apr;52(4):152-60
Poll the Audience
• Does your facility complete DAT testing with an
automated platform?
– Yes
– No
– We may consider it
2019 46
Automated DAT Testing –Investigation Option for Tube False Negative Results
• Column Agglutination Test
– 0.8% cell suspension in IgG or C3bd well
– Detects a lower level of IgG coated cells
– Difficult to run a control
• Solid Phase Test
– Select-R Strip is used
– Well is coated with red cells and tested with IgG
– Increased sensitivity
2019 47
Parker, V and Tormey, CA. Arch Pathol Lab Med. 2017;141:305–310
How does the DAT
correlate with the
Autocontrol?
2019 48
Autocontrol Testing
• In general, autocontrol testing
is added when the patient’s
IAT is reactive
• The autocontrol may be tested
along with a panel
• The autocontrol and DAT
should reflect similar reactions
– The autocontrol and DAT;
however, are not testing for
exactly the same reactivity
2019 49
Image from: http://mymagicdog.com/4521/man-mans-best-friend-similar-think/
Autocontrol compared to DAT
Autocontrol DAT
1) Serum and Patient’s Red Cells tested
by IAT – Indirect
2) Serum antibody tested in IAT method
which indicates antibody in the
serum, not on the red cells.
3) Results may be mixed-field, aiding
the investigation of transfusion
reactions
1) Patient Red Cells evaluated with IgG
or C3 reagents by DAT – DIRECT
2) IgG and Complement Coating the
patient’s red cells
3) Results may be mixed-field,
indicating two cell populations
coated with IgG
2019 50
Subtle Differences but Important
for Serologic Investigation
Why Test a DAT?
2019 51
Indications for a DAT order
• Transfusion Reaction Investigation
– Positive DAT indicates donor blood has been coated with
patient antibody “in vivo”
– Positive DAT could also indicate incompatible donor
plasma (with ABO Abs) has been transfused and is
coating the patient’s red cells
– Negative DAT indicates either no incompatibility between
donor and patient OR coated cells have already been
removed from circulation
2019 52
AABB Technical Manual, Current Edition
Indications of a DAT order
• HDFN/Cord Blood Work-up
– Positive DAT indicates maternal IgG has crossed the
placenta and attached to antigen positive fetal cells in
utero. This is Hemolytic Disease of the Fetus & Newborn
(HDFN).
– Negative DAT indicates no incompatibility between
mother and baby blood
2019 53
AABB Technical Manual, Current Edition
Indication of DAT order
• Autoantibody investigation
– Has the patient made antibodies to “self” antigens?
– Can cause positive DAT with IgG and/or C’ coating
– Usually associated with a disease state
• Drugs/Medication
– Drugs can cause a positive DAT seen in patients and
donors.
2019 54
AABB Technical Manual, Current Edition
Reflex Testing for
Positive IgG DAT is an
Eluate
Acid Eluate
And
Lui-Freeze Thaw Eluate
2019 55
Poll the Audience
• If you perform eluates in your lab, what type of
eluate is your primary method?
– Lui-Freeze Thaw
– Acid Eluate
– Other
2019 56
Elution Techniques
• Freeze-Thaw (Lui) IgG ABO Antibodies
• Digitonin Acid (Acid Eluate Kit) IgG
• Heat – 56C or 45C
• Cold Citric Acid
• Chloroform
• Xylene
• Ether
Judd's Methods in immunohematology, 3rd Edition / W. John Judd, Susan T. Johnson, Jill Storry. AABB Press.2008.
Eluate Process
Y
Y
Y
YY
Y
YY
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
YYY
Y
YY
YY Y=
Eluate Method
to Remove
antibody for
testing
Recovered
Antibody to
Test
Lui Freeze-Thaw Eluate
Wash
• Wash red cells to remove any excess bound antibody
• Retain the last wash.
Freeze
• Add drops of washed red cells with saline
• Place the tubes in the -18C freezer
Test
• Remove the frozen tubes and allow to thaw
• Spin the tubes down
• Recover the supernatant/hemolysate
• Test against ABO cells and O cell as a control.
2019 59
Case Study
• 30-year-old female presents for delivery of her
5th child. The delivery was uneventful and the
cord blood was submitted to the blood bank for
routine blood type and DAT.
• Results
– Baby ABO/Rh A, D Positive
– Baby IgG DAT 2+
– Maternal ABO/Rh O, D Positive
– Heal Stick
ABO Antibody Screen Weak Anti-A detected
2019 60
Case Study
• ABO antibodies detected so reflex to LUI-eluate
A1 Cells A1 Cell #2 B Cells O Cells
LUI Eluate 2+ 2+ 0 0
Last Wash 0 0 0 0
Eluate study agrees with ABO screen, anti-A coating
the baby’s cells at delivery
Consistent with ABO HDFN.
2019 61
Digitonin Acid
2019 62
Digitonin Acid Eluate Preparation
Wash
• Wash cells once with saline
• Wash cells 4x with Working Wash
• Save Last Wash Aliquot
Prep
•Drop washed cells into clean, labeled tube
•Add equal drops of Eluting Reagent
•Spin 60 seconds
•Remove supernatant – red cells will be destroyed.
•Using Buffering Solution, add dropwise until a blue color change
•Spin to remove RBC debris
•Transfer eluate in clean, labeled tube
Test• Test eluate and last wash in parallel
• Testing with our without enhancement
• Interpret results
2019 63
Case Study• A 10-year-old male with a primary diagnosis of
Evans Syndrome has presented in the
Emergency Room with pallor and weakness.
• His H/H are 6/18
• The physician ordered a T and S, and requested
one unit for transfusion
• Type and Screen
– A, D Positive
– Solid Phase IAT = Positive with all cells 4+
– Manual IgG DAT = 4+ with valid control
2019 64
Case Study PanelRh Kell Duffy Kidd MNS P Lewis
Cell D C c E e f V C
w
K k F
ya
F
yb
Jk
a
Jk
b
M N S s P
1
L
e
a
L
e
b
LISS-
AHG
1 + + 0 0 + 0 0 0 + + + + + 0 + + + + + + 0 2+
2 + + 0 0 + 0 0 + 0 + + + + 0 + 0 0 + + + 0 2+
3 + 0 + + 0 0 0 0 0 + 0 + 0 + 0 + 0 + + 0 + 2+
4 + 0 + 0 + + 0 0 + + + 0 + 0 + + 0 0 + + 0 2+
5 0 + + 0 + + 0 0 0 + + 0 0 + + + + + + 0 + 2+
6 0 0 + + + + 0 0 0 + + + + + 0 + 0 + + 0 + 2+
7 0 0 + 0 + + 0 0 + + 0 + + + 0 + 0 + + + 0 2+
8 0 0 + 0 + + 0 0 0 + + 0 + + + 0 0 + 0 + 0 2+
9 0 0 + 0 + + + 0 0 + + + 0 + + + + 0 0 + 0 2+
10 0 0 + 0 + + 0 0 0 + 0 0 0 + 0 + 0 + + 0 0 2+
Auto Control 2+
Case Study – Eluate
Rh Kell Duffy Kidd MNS P Lewis
Cell D C c E e f V C
w
K k F
ya
F
yb
Jk
a
Jk
b
M N S s P
1
L
e
a
L
e
b
Eluate
- AHG
1 + + 0 0 + 0 0 0 + + + + + 0 + + + + + + 0 3+
2 + + 0 0 + 0 0 + 0 + + + + 0 + 0 0 + + + 0 3+
3 + 0 + + 0 0 0 0 0 + 0 + 0 + 0 + 0 + + 0 + 3+
4 + 0 + 0 + + 0 0 + + + 0 + 0 + + 0 0 + + 0 3+
5 0 + + 0 + + 0 0 0 + + 0 0 + + + + + + 0 + 3+
6 0 0 + + + + 0 0 0 + + + + + 0 + 0 + + 0 + 3+
7 0 0 + 0 + + 0 0 + + 0 + + + 0 + 0 + + + 0 3+
8 0 0 + 0 + + 0 0 0 + + 0 + + + 0 0 + 0 + 0 3+
9 0 0 + 0 + + + 0 0 + + + 0 + + + + 0 0 + 0 3+
10 0 0 + 0 + + 0 0 0 + 0 0 0 + 0 + 0 + + 0 0 3+
Last Wash 0√
Case Study Summary
• A delay in provision of blood was called along
with a request for an order for least
incompatible blood
• Correlation of Testing and Diagnosis
– A Pos Positive IAT
– Positive IgG DAT
– Panreactive Panel and Eluate
– Further adsorption studies were pursued and all
underlying alloantibodies were excluded.
2019 67
Correlation is Key
2019 68
Eluate Pitfalls
• Improper technique
– Stromal debris
– Incomplete removal of solvent
– Failure to adjust pH
• Incomplete washing
– Ensure that antibody in eluate is not from solution
– Test last wash
• Adherence of protein to glass surface
– Transfer to clean tube after washing
Eluate Pitfalls
• Storage changes in organic solvents
– Change in pH
• Dissociation of antibody from cells prior to
elution
– Problem with digitonin elution
• Instability of eluate
– Suggestion to albumin for storage or freeze if
not tested soon
Summary of Presentation
• Compare and contrast the indirect
antiglobulin test (IAT) and direct
antiglobulin test (DAT)
• DAT reagents
– Polyspecific, IgG with C3
– Monospecific, IgG or C3
• Possible causes of a positive DAT
• Elution types with application
– Lui
– Acid
2019 71
References not listed in presentation
1. Jenkins, GC, et al: Role of C4 in the antiglobulin reaction.
Nature 186:482, 1960.
2. Pondman KW, Rosenfield RE, and Tallal L et
al.: (1960) The specificity of the complement antiglobulin
test. Vox Sang 5: 297
3. Harboe, M, et al: Identification of the component of
complement participating in the antiglobulin reaction.
Immunology 6:412, 1963.
2019 72
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Continuing Education
• PACE, Florida and California DHS
• 1.0 Contact Hours
• Each attendee must register to receive CE at:
https://www.surveymonkey.com/r/DATAutoControlsAndEluates
• Registration deadline is 9 August, 2019
• Certificates will be sent via email only to those who have registered 23 August, 2019
78 All Content © Immucor, Inc.
Thank you!