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9 January 2019 ADAMTS-13 Testing, TTP, & MORE 6 March 2019 Advanced Antibody Work-Up Techniques: MMA & Molecular Testing

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6 All Content © Immucor, Inc. 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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8 All Content © Immucor, Inc. • 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.

9 All Content © Immucor, Inc. 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

2019 10 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. AABB Technical Manual, current edition.

2019 17 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 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

• Prepared by Hybridoma technique to produce monospecific reagent 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

AABB Technical Manual, current edition.

2019 26 Definition

• Direct Antiglobulin Test (DAT) –Used to detect in vivo cell sensitization (1 step) •Immunoglobulins (IgG) •Complement

Image from: https://www.med-ed.virginia.edu/courses/path/innes/rcd/antibody.cfm AABB Technical Manual, current edition.

2019 27 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

AABB Technical Manual, current edition.

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.

AABB Technical Manual, current edition.

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

AABB Technical Manual, current edition.

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. AABB Technical Manual, current edition.

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 DAT Control?

AABB Technical Manual, current edition. 2019 33 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.

AABB Technical Manual, current edition.

2019 38 Tube DAT Results C’= complement

Polyspecifi Anti-IgG Anti- Saline Interpretation c C3d,C3b Control + + 0 0 IgG + only + 0 + 0 C’+ only + + + 0 IgG + & C’ + + + + + Test Invalid 0 0 0 0 Neg DAT

AABB Technical Manual, current edition.

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

AABB Technical Manual, Current Edition. 2019 40 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

AABB Technical Manual, Current Edition

2019 41 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 Judd's Methods in immunohematology, 3rd Edition / W. John Judd, Susan T. Johnson, Jill Storry. AABB Press.2008.

2019 42 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

AABB Technical Manual, Current Edition

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

AABB Technical Manual, Current Edition

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 Segal and Litchman, Blood Cells Mol Dis. 2014 Apr;52(4):152-60

2019 45 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

Parker, V and Tormey, CA. Arch Pathol Lab Med. 2017;141:305–310

2019 47 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

Image from: http://mymagicdog.com/4521/man-mans-best-friend-similar-think/ 2019 49 Autocontrol compared to DAT

Autocontrol DAT 1) Serum and Patient’s Red Cells tested 1) Patient Red Cells evaluated with IgG by IAT – Indirect or C3 reagents by DAT – DIRECT

2) Serum antibody tested in IAT method 2) IgG and Complement Coating the which indicates antibody in the patient’s red cells serum, not on the red cells.

3) Results may be mixed-field, aiding 3) Results may be mixed-field, the investigation of transfusion indicating two cell populations reactions coated with IgG Subtle Differences but Important for Serologic Investigation

2019 50 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

AABB Technical Manual, Current Edition

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

AABB Technical Manual, Current Edition

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.

AABB Technical Manual, Current Edition

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 Eluate Method Y Y Y Y Y Y to Remove Y Y antibody for = Y Y Y Y testing Y Y Y Y Y Y Y Y Recovered Y Y Y Y Y Antibody to Test Lui Freeze-Thaw Eluate

• Wash red cells to remove any excess bound antibody Wash • Retain the last wash.

• Add drops of washed red cells with saline Freeze • Place the tubes in the -18C freezer

• Remove the frozen tubes and allow to thaw • Spin the tubes down Test • 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 cells once with saline • Wash cells 4x with Working Wash Wash • Save Last Wash Aliquot

•Drop washed cells into clean, labeled tube •Add equal drops of Eluting Reagent •Spin 60 seconds •Remove supernatant – red cells will be destroyed. Prep •Using Buffering Solution, add dropwise until a blue color change •Spin to remove RBC debris •Transfer eluate in clean, labeled tube

• Test eluate and last wash in parallel Test • 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 Panel

Rh Kell Duffy Kidd MNS P Lewis

Cell D C c E e f V C K k F F Jk Jk M N S s P L L LISS- w ya yb a b 1 e e AHG a b 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 K k F F Jk Jk M N S s P L L Eluate w ya yb a b 1 e e - AHG a b 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? • You are all muted • Q&A following session - Type in questions

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76 All Content © Immucor, Inc. 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

77 All Content © Immucor, Inc. Thank you!

78 All Content © Immucor, Inc.