2019 advanced track webinars - immucor, inc. program... · vivo cell sensitization of babies with...

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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

https://www.surveymonkey.com/r/DATAutoControlsAndEluates


Questions?

• You are all muted

• Q&A following session - Type in questions


• Course content is for information and

illustration purposes only. Immucor makes

no representation or warranties about the

accuracy or reliability of the information

presented, and this information is not to be

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


• 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


• 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


• 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


• 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 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


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


https://www.med-ed.virginia.edu/courses/path/innes/rcd/antibody.cfm

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


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


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


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


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


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


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


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


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

https://www.ncbi.nlm.nih.gov/pubmed/24411920

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


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


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


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


Questions?




Continuing Education

• PACE, Florida and California DHS

• 1.0 Contact Hours

• Each attendee must register to receive CE at:


• Registration deadline is 9 August, 2019

• Certificates will be sent via email only to those who have registered 23 August, 2019



Thank you!

2019 advanced track webinars - immucor, inc. program... · vivo cell sensitization of babies with...

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