Differentiating Warm vs. Drug-Induced AIHA Handouts June ...€¦ · • AABB Technical Manual, 17th ed., 2011 Drug-dependent antibodies reacting in the presence of drugpresence of

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Differentiating Warm vs. Drug-induced AIHA

South Central Association of Blood BanksPre-Meeting Symposium

Susan T. Johnson, MSTM, MT(ASCP)SBBBloodCenter of Wisconsin

Milwaukee, WI

June 6, 2012

Objectives• Describe the initial serologic results

observed in a patient with warm autoimmune hemolytic anemia and drug-induced immune hemolytic anemia

• Explain the serologic test results that differentiate WAIHA from DIIHA

• List drugs most commonly associated today with drug-induced immune hemolytic anemia.

SERUM/PLASMA COLOR

• Typical WAIHA Sample• Usually “normal” color

• Yellow/Straw etc…Yellow/Straw etc…

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Typical DIIHA Sample Serum/Plasma

Coke Colored?

INITIAL SEROLOGIC RESULTS

• Direct Antiglobulin Test• EluateEluate• Serum/Plasma Reactivity

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DIRECT ANTIGLOBULIN TEST

J. O’Connor

WAIHADAT RESULTS

Polyspecific AHG 3 - 4+Anti-IgG 3 - 4+Anti-C3 0 - 3+Anti-C3 0 - 3+Control 0

WARM AUTOANTIBODY IN PLASMA

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WARM AUTOANTIBODY COATING RBCs

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WARM AUTOANTIBODY IN PLASMA (1+)

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DIIHADAT Results

• Reactivity depends on time of testing compared to presentation

P l ifi AHG 3 4Polyspecific AHG 3-4+Anti-IgG 3-4+Anti-C3 3-4+Control 0

DAT RESULTS IN DIIHA

• AABB Technical Manual, 15th ed., 2005Drug-dependent antibodies detected by

the immune complex methodthe immune complex method…

“complement may be the only globulin easily detected on the red cells, but IgG may be present.”

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DAT RESULTS IN DIIHA

• AABB Technical Manual, 17th ed., 2011Drug-dependent antibodies reacting in the

presence of drugpresence of drug

“complement may be the only protein easily detected on the red cells, but IgG may be present.”

DAT STRENGTH & SPECIFICITY

IgG + C3 IgG Only C3 Only4+ 19 5 13+ 15 4

Transfusion 2007;47:697-702

2+ 6 11+ 2 5<1+ 4 2 4Total Total 46 46 17 17 553 of 71 patients had negative DAT

Typical DAT Results - DIIHA

On Day 3 Day 7 Day 14

Drug is discontinued

Presentationy y y

DAT 4+ 3+ 2+ 1+

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Typical DAT Results - WAIHA

On Presentation

Day 3 Day 7 Day 14

DAT 4+ 4+ 4+ 3+DAT 4+ 4+ 4+ 3+

ELUTION IN WAIHA

• On initial evaluation• Confirms autoantibody is coating RBCs• Rules-out or identifies presence ofRules out or identifies presence of

drug-dependent antibody

Performing an Acid Elution

J. O’Connor

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ELUATED C c E e K Fya Fyb Jka Jkb S s IAT IAT

1 + + 0 0 + 0 + 0 0 + 0 + 3 0√

2 0 0 + 0 + + 0 + + + + + 3 0√

3 + 0 + + 0 0 0 + + 0 + 0 3 0√

4 + 0 + 0 + 0 0 + 0 + 0 + 3

5 0 + + 0 + 0 + + + 0 + 0 3

Last WashEluate

6 0 0 + 0 + + 0 + 0 + + + 3

7 0 0 + 0 + 0 + 0 0 + 0 + 3

8 0 0 + 0 + 0 0 + + 0 + 0 3

Auto NT

ELUATE RESULTS IN DIIHA

• Rapid Acid• Most are negative

F di ti t l k• Few are disproportionately weaker as compared to strength of DAT


1 + + 0 0 + 0 + 0 0 + 0 + 0√ 0√

2 0 0 + 0 + + 0 + + + + + 0√ 0√

3 + 0 + + 0 0 0 + + 0 + 0 0√ 0√

4 + 0 + 0 + 0 0 + 0 + 0 + 0√

5 0 + + 0 + 0 + + + 0 + 0 0√

Last WashEluate

6 0 0 + 0 + + 0 + 0 + + + 0√

7 0 0 + 0 + 0 + 0 0 + 0 + 0√

8 0 0 + 0 + 0 0 + + 0 + 0 0√

Auto NT

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1 + + 0 0 + 0 + 0 0 + 0 + w-2 0√

2 0 0 + 0 + + 0 + + + + + w-2 0√

3 + 0 + + 0 0 0 + + 0 + 0 w-2 0√

4 + 0 + 0 + 0 0 + 0 + 0 + w-2

5 0 + + 0 + 0 + + + 0 + 0 w-2

Last WashEluate

6 0 0 + 0 + + 0 + 0 + + + w-2

7 0 0 + 0 + 0 + 0 0 + 0 + w-2

8 0 0 + 0 + 0 0 + + 0 + 0 w-2

Auto NT

INITIAL ELUATE REACTIVITY

Negative 49<2+ 13

Transfusion 2007;47:697-702

4+ 2Not Tested 3

WAIHA vs. DIIHA

• Eluate• WAA strongly positive• DDA is negative or weakg

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ANTIBODY DETECTION TEST IN WAIHATest Tube - Saline

IS 37C IATI 0 0 0 4+I 0 0 0-4+II 0 0 0-4+

ANTIBODY DETECTION TEST IN WAIHATest Tube - LISS

IS 37C IATI 0 0 0-4+II 0 0 0-4+

ANTIBODY DETECTION TEST IN WAIHATest Tube - PEG

IS 37C IATI 0 NH w-4+II 0 NH w-4+

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ANTIBODY DETECTION TEST IN WAIHAGel or Solid Phase

IATI w-4+II w-4+

Antibody Identification in WAIHA – Gel

D C c E e K Fya Fyb Jka Jkb S s IAT

1 + + 0 0 + 0 + 0 + 0 0 + 3

2 + + 0 0 + + 0 + + + + + 3

3 + 0 + + 0 0 0 + + 0 + + 3

4 + 0 + 0 + 0 0 + 0 + 0 + 3

5 0 + + 0 + 0 + + + 0 + 0 3

Gel

6 0 0 + 0 + + 0 + 0 + + + 3

7 0 0 + 0 + 0 + 0 0 + 0 + 3

8 0 0 + 0 + 0 0 + + 0 + 0 3

Auto 3-4

Antibody Identification in WAIHA – Solid Phase

D C c E e K Fya Fyb Jka Jkb S s IAT

1 + + 0 0 + 0 + 0 + 0 0 + 3

2 + + 0 0 + + 0 + + + + + 3

3 + 0 + + 0 0 0 + + 0 + + 3

4 + 0 + 0 + 0 0 + 0 + 0 + 3

SP

5 0 + + 0 + 0 + + + 0 + 0 3

6 0 0 + 0 + + 0 + 0 + + + 3

7 0 0 + 0 + 0 + 0 0 + 0 + 3

8 0 0 + 0 + 0 0 + + 0 + 0 3

Auto ND

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SERUM REACTIVITY IN DIIHA• Reactivity depends on time of testing

compared to presentation • Saline IAT - some positive• PEG or Ficin IAT - many positivey p• Gel – many positive

• http://www.aabb.org/development/awardsscholarships/scholarships/Documents/10kirchner.pdf

• SPRCA – similar to saline IAT• I.S. – few positive

ANTIBODY DETECTION TEST IN DIIHATest Tube - Saline

IS 37C IATI 0 0 0 4+I 0 0 0-4+II 0 0 0-4+

SERUM REACTIVITY WITHOUT ADDITION OF DRUG

• Drug-Dependent Antibody • Drug remains in patient’s circulation• Drug is present in test method

• Trimethoprim & Sulfamethoxazole (gel test)

• Drug-Independent “Autoantibody”• Serum is reactive after drug has cleared

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SERUM DIALYSIS

Drug

TESTING OF DIALYZED SERUM

IS 60’ 37C IAT

Pre-Dialysis Serum + RBCs 0 1 4

Di l d S RBC 0 0 0Dialyzed Serum + RBCs 0 0 0

Dialyzed Serum +RBCs + Drug 0 1 4

Typical IAT Results - DIIHA

On Day 3 Day 7 Day 14

Drug is discontinued

Presentationy y y

IAT 3+ 0 0 0

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Typical IAT Results - WAIHA

On Presentation

Day 3 Day 7 Day 14

IAT 3+ 3+ 3+ 2+IAT 3+ 3+ 3+ 2+

PATIENT HISTORY

• Thorough investigation • Signs & symptoms of hemolysis• Medication/Drug History

DRUG HISTORY

• Prescription drugs• Over-The-Counter drugs

Alt ti (h b l) di ti• Alternative (herbal) medications

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DRUG HISTORY

• Timing and dosage of medication

• History of surgical procedures requiring• History of surgical procedures requiring antibiotics

• History of pain requiring NSAIDS

“UNIFYING” THEORY(Habibi & Garratty)

HaptenHapten--dependent antibodydependent antibodyType IType I

*Drug*Drug--independent antibodyindependent antibodyType IIType II

*Drug*Drug--dependent dependent antibodyantibodyType IIIType III

Type I“Penicillin-Type”“Hapten-Type”

(Drug Adsorption)

• Drug binds covalently to membrane proteins and stimulates hapten-dependent antibodies

• Antibody reacts with normal RBCs pretreated with drug

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Type I – Drug Treated RBCsHapten-Dependent Antibody

Type I – Drug Treated RBCsHapten-Dependent Antibody

HaptenHapten--dependent antibodydependent antibody

Drug-independent antibody Drug-dependent antibody

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DDA REACTING WITH DRUG-TREATED RBCsCephalosporins 31

Cefotetan 26Cefuroxime 1Cefotaxime 1Cefoxitin 1Cefoxitin 1Ceftazidime 1Cefazolin 1

Penicillin/Penicillin Derivatives 7Piperacillin 5Ampicillin 1Nafcillin 1

Total 38

Cases/Fatalities of DIIHA over 10 years

Drug Number* PercentageCefotetan 36 (4) 43Ceftriaxone 17 (5) 21Piperacillin 14(1) 17β l t 6 7

G Garratty, Blood Reviews 24 (2010) 143–150

β-lactamaseinhibitors

6 7

Other Cephalosporins

11

Others 9# 11Total 83 (10) 100

* Columns contain number (fatalities) of cases associated with each drug.# Oxaliplatin (3), carboplatin (1), rifampin (1), diclofenac (1), cimetidine (1),sulfamethoxazole (1), and trimethoprime (1).

Type IIDrug-Independent Antibody

(Autoantibody)• Through an unknown mechanism, drug

induces autoantibodies specific for RBC duces au oa bod es spec c o Cmembrane proteins

• Antibody reacts with normal RBCs in the absence of drug

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Hapten-dependent antibody

DD i d d t i d d t DrugDrug--independent independent antibodyantibody

Drug-dependent antibody

Type III“Nonpenicillin-Type”“Neoantigen Type” (“Immune Complex”)

• Through an unknown mechanism, d i d tib di th t bi d tdrug induces antibodies that bind to RBC only when drug is present in soluble form

• Antibody reacts with RBCs when soluble drug is present

Type III – I.P.O.D.Drug-Dependent Antibody

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Type III – I.P.O.D.Drug-Dependent Antibody

Hapten-dependent antibody

Drug-independent antibodyDrugDrug--dependent dependent antibodyantibody

BCW EXPERIENCEDDA REACTING IN PRESENCE OF DRUG

Quinine/Quinidine 7

Others 15Ceftriaxone 5Ceftazidime 1Zosyn (piperacillin & tazobactum) 5Oxaliplatin 2Carboplatin 1Bactrim/Trimethoprim 1

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BCW EXPERIENCEDDA REACTING IN PRESENCE OF DRUG

NSAIDs 11Tolmetin 3Sulindac 2Ibuprofen 1

Others 7Probenecid 1Levafloxacin 1M f i 1

pDiclofenac* 2Etodolac* 1Nabumetone* 1Indomethacin* 1

*Urine-metabolite dependent

Mefoxin 1Carboplatin 1Oxaliplatin 2Bactrim 1

NON-IMMUNOLOGIC ADSORPTION OF PROTEIN ONTO RBCs

• Cephalosporins• Cisplatin/Carboplatin/Oxaliplatin• Diglycoaldehyde (INOX)• Suramin• β-lactamase inhibitors

• Sulbactam (in Unasyn)• Clavulanate (in Augmentin and Timentin) • Tazobactam (in Zosyn/Tazocin)

Garratty

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NON-IMMUNOLOGIC ADSORPTION OF PROTEIN ONTO RBCs

• Not thought to cause DIIHA when first described with cephalothin-treated RBCsRBCs.

• Since then, suggestions in the literature that certain drugs that cause nonspecific protein uptake (e.g., cisplatin, sulbactam, clavulanate) can cause HA

DRUG EVALUATION• Check for previous implications of causing

hemolysis• Physicians Desk Referencey• Drug Facts and Comparison• AABB Technical Manual• Judds Methods in Immunohematology• Published articles & abstracts

TESTING DRUG-TREATED RBCs

Patient Serum Pos Con Normal SerumNEAT 1:2 1:4 1:8 1:20 NEAT 1:2 1:4 1:8 1:20

15’RT 4 4 3 3 3 4 3 2 0 0 030 37C 4 4 3 2 1 4 0 0 0 0 030 37C 4 4 3 2 1 4 0 0 0 0 0IAT 4 4 4 4 4 4 0 0 0 0 0

15’RT 0

30 37C 0IAT 0

Unt

rtd

RB

Cs

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TESTING IN PRESENCE OF DRUGe+ RBCs 30’RT 60’ 37C IAT

Patient Serum + Drug 2 3 4Patient Serum + Drug + C 2 3 4Patient Serum + Diluent 0 0 0P ti t S Dil t C 0 0 0Patient Serum + Diluent + C 0 0 0C + Drug 0 0 0C + Diluent 0 0 0Diluent + Drug 0 0 0Eluate + Drug 0 0 3Eluate + Diluent 0 0 0Positive Control + Drug 4 4 4

TESTING IN PRESENCE OF DRUG Autoantibody Present

Patient Serum (No Drug Added) Patient Serum + DrugNEAT 1:2 1:4 1:8 1:16 NEAT 1:2 1:4 1:8 1:16

30’ RT 0 0 0 0 0 3 2 1 0 030 RT 0 0 0 0 0 3 2 1 0 060’ 37C 0 0 0 0 0 4 3 2 1 0IAT 2 1 0 0 0 4 4 4 3 2

b l i b dwww.belearning.bcw.edu

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TYPICAL INITIAL SEROLOGIC RESULTS

Polyspecific AHG 3 - 4+Anti-IgG 3 - 4+Anti-C3 0 - 3+Control 0

IS 37C IATI 0 0 0 - 4+II 0 0 0 - 4+

TYPICAL INITIAL SEROLOGIC RESULTS

Polyspecific AHG 3 - 4+Anti-IgG 0Anti-C3 3 – 4+Control 0

IS 37C IATI 2-4+ 0-2+ 0II 2-4+ 0-2+ 0

DIIHA vs AIHA• Eluate

• DDA is negative or weak• WAA strongly positiveWAA strongly positive

• Serum • DDA disappears within days if drug

is discontinued• WAA persists

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Objectives• Describe the initial serologic results

observed in a patient with warm autoimmune hemolytic anemia and drug-induced immune hemolytic anemia

• Explain the serologic test results that differentiate WAIHA from DIIHA

• List drugs most commonly associated today with drug-induced immune hemolytic anemia.

Thank You

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