Kell and Duffy in 30 Minutes

Kell, Duffy, Kidd

Objectives: Kell, Kidd and Duffy

For each blood group discussed, the learner will: • State blood group antigen frequencies among the general population and within specific ethnic groups • Appreciate the genetics and biochemistry Kell and Duffy in 30 minutes • Discuss implications of null phenotypes • List the characteristics of antibodies directed against …you’ve got to be Kidd-ing! each blood group (Ok, we’ll talk about Kidd, too) • Discuss the use of chemicals in antibody identification • Identify diseases related to blood groups CM Jessica Drouillard, SBB(ASCP) Heartland Blood Centers, part of Versiti Aurora, IL

Inheritance

– XK gene – on X chromosome • Xk protein • Kx antigen

Kell Blood Group System (KEL) – KEL gene – on Chromosome 7 • Kell glycoprotein ISBT 006 • Kell antigens

– KEL and XK genes interact to form normal Kell antigen expression

Expression Normal Kell Expression

Weak Kell antigens Antigen ISBT Whites Blacks (%) (%)

Kell antigens Kell antigens K KEL1 9 2 -K, k -K, k -Kpa, Kpb -Kpa, Kpb k KEL2 99.8 100 No Kx -Jsa, Jsb -Jsa, Jsb a Kp KEL3 2 Rare Kpb KEL4 100 100

No Kell Ku KEL5 100 100 Kx Ku- Kx Km- Kx Jsa KEL6 0.01 20

b Js KEL7 100 99

Ko phenotype McLeod Phenotype

Ko phenotype McLeod Weak Kell -SNP of KEL*02 No Kell • phenotype antigens Kx Ku- No XK gene -Homozygous Km- – No Kx -No Kell glycoproteins – No Km No Kx -Null phenotype – Can form anti-Kx and –Km -Cells type s Kell Kx+ Normal Normal Kell antigens Ku- antigens • Other Kell typings weak -K, k -K, k -Kpa, Kpb Km- a b – Ku -Kp , Kp -Jsa, Jsb a b -Can form anti-Ku and anti-Km -Js , Js – K, k, Kpa, Kpb, Jsa, Jsb

Kx Kx

McLeod Syndrome Antibodies

• X linked, occurs almost exclusively in • Primarily IgG, do not bind complement males • Usually immune stimulated – Some patients also have X-linked Chronic Granulomatous Disease (CGD) • Clinically significant (HTR and HDFN) • Range of neurological and muscular Phenotype Antibody Compatible blood defects No Kell Ko phenotype Kx Anti-Ku Ko – Muscle wasting, reduction in deep tendon Ku- reflexes Km- Anti-Km Ko and McLeod

• Hematologic abnormalities McLeod phenotype Weak Kell Anti-Km Ko and McLeod – Decreased RBC survival, acanthocytosis, antigens (no CGD) Anti-Kx McLeod reticulocytosis, reduced serum haptoglobin, No Kx increased bilirubin, compensated anemia McLeod phenotype Weak Kell Anti-Kx, -Km McLeod antigens – Difficult to find blood for transfusion with CGD No Kx

Weakened Kell Antigen Expression Kmod

Kmod • Arises from SNP at KEL*02 • Kmod phenotypes Possible Kell haplotypes a • Weakened expression of • Kp in cis k, Kpb, Jsb – k, Kpa, Jsb Kell glycoproteins K, Kpb, Jsb – Often need k, Kpa, Jsb adsorption/elution studies Kell • Gerbich null types Normal k, Kpb, Jsa to detect antigens – Ge:2,3,4 is normal • Strong Kx expression -K, k – Ge:-2,3,4 = Yus - Kell normal -Kpa, Kpb • Some may make anti-Ku- -Jsa, Jsb – Ge:-2,-3, 4 = Gerbich – Kell expression weakened like antibody that reacts – Ge:-2,-3,-4 = Leach (true null) – Kell expression depressed Kx with all cells but other Kmod

Inheritance

• FY*01 and FY*02 genes on Chromosome 1 – Syntenic to Rh – Produce Duffy glycoproteins: Fy3, Fy5, Fy6, and Fya/Fyb Duffy Blood Group System • Duffy Antigen Receptor for Chemokines ISBT 008 – Binds cytokines, especially IL-8 – Role in inflammation – Also receptor for malaria • Plasmodium vivax • Plasmodium knowlesi

Expression Duffy Phenotypes

RBC Phenotype Whites (%) Blacks (%) Asians (%) • Well developed at birth Fy6 Fy(a+b-) 20 10 91 • Destroyed by enzymes Fya/Fyb and ZZAP Fy(a+b+) 48 3 9 Fy3 • Antigens found on RBCs Fy(a-b+) 32 20 <1 and other tissues Fy(a-b-) 0 67 0

– Endothelial cells, brain, Fy3 100 32 99.9 colon, lung, spleen, kidney, etc. Fy5 99.9 32 99.9 • Antigens reported to Fyx 1.4 0 0 weaken when stored

Fy(a-b-) type Duffy genotypes

RBC Phenotype Genotype Comments • Rare in whites, but usually true null Fya Fyb Fy3 FY*A / FY*A + 0 + Normal, homozygous Fya expression – Duffy antigens not expressed on RBCs or tissues FY*A / FY*B + + + Normal, heterozygous Fya/Fyb expression – Can make anti-Fy3 FY*B / FY*B 0 + + Normal, homozygous Fyb expression FY*A / FY*B_GATA + 0 + Fya expressed on RBC and tissues Fyb expressed on tissues (not RBC) • Most frequently found in blacks Should not make anti-Fyb – Arises from SNP mutation in GATA-1 erythroid FY*B / FY*B_GATA 0 + + Fyb expressed on RBC and tissues promoter region of FY*01 (Fya) or FY*02 (Fyb) – more Can make anti-Fya common FY*B_GATA / FY*B_GATA 0 0 0 Fy(a-b-) phenotype Fyb expressed on tissues (not RBC) – Duffy antigens not expressed on RBCs, but are Can make anti-Fya expressed on tissues Should not make anti-Fyb Fymod / Fymod w+ w+ w+ Weak Fy6 antigen expression

Kell, Duffy, Kidd

Duffy antibodies Other Duffy antibodies • Anti-Fy3 – Found only in Fy(a-b-) individuals • Usually IgG, do not bind complement well – Reacts like an inseparable anti-Fya, -Fyb, but enzymes will • not eliminate reactivity Show dosage • Anti-Fy5 • Anti-Fya is 20 times more common than anti-Fyb – Found only in Fy(a-b-) individuals – Reacts with all cells except • Clinically significant (HTR and HDFN, usually mild) • Fy(a-b-) • Rh null cells, regardless of Fya/Fyb typing Antigen Reaction with enzymes – D-- cells have weak Fy5 expression Fya / Fyb Destroyed Fy3 Resistant • Anti-Fy6 Fy5 Resistant – Murine antibody – not found in humans

Fy6 Destroyed

Inheritance

• JK*01 and JK*02 found on Chromosome 18 • Human Urea Transporter 11 (HUT11) – Allows uptake of urea – Prevents RBC shrinkage in hypertonic environment of Kidd Blood Group System renal medulla • Jk3 antigen absent/weak in Jk(a-b-) individuals ISBT 009 – Dominant inhibitor gene In(Jk) • Reported in Japanese families • No Jka or Jkb expression • Weak Jk3 expression a b – Homozygous for Jk gene – true JKnull (No Jk , Jk or Jk3) • Polynesians and Finns

Expression Kidd Phenotypes

• Well developed at birth Phenotype Whites (%) Blacks (%) Jk(a+b-), Jk3+ 26 52 • Antigens cluster on RBC surface Jk(a+b+), Jk3+ 50 40

Jk(a-b+), Jk3+ 24 8 • Transmembrane protein (like Rh) Jk(a-b-), Jk3- Polynesians, Finns – Not destroyed by DTT/AET or enzymes – Enhanced by enzymes Jk(a-b-), Jk3+ (weak) Japanese

• Poor immunogens

Jk(a-b-) Phenotype 2M Urea Lysis Test

• When caused by dominant inhibitor gene • Cheap, effective screening test to identify – Need only 1 copy of In(Jk) gene Jk(a-b-) individuals – Cells type Jk(a-b-) and Jk3-, but antigens may be detected by adsorption/elution studies – Since trace amounts of antigens are present, cannot • Cells with normal Kidd antigen expression

make anti-Jk3 have normal HUT11, will swell and lyse • When caused by inheritance of JK gene – Need 2 copies of JK gene (must be homozygous) • Jk(a-b-) cells resist lysis by 2M urea, but – Cells type Jk(a-b-) and Jk3-. Kidd antigens completely absent shrink and shrivel – Can make anti-Jk3

2M Urea Lysis Test Kidd Antibodies • Disappear quickly from circulation and don’t store well – Associated with DHTR • Primarily IgG1, IgG3 • Can activate compliment – Antigens clustered together on RBC surface • Clinically significant – can cause HTR and rarely HDFN • Anti-Jk3 made by true Jk(a-b-) individuals – Looks like inseparable anti-Jka and anti-Jkb – Confirm with adsorption/elution studies

Can you guess each donor’s likely ethnicity?

Cell K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb 1 0 + 0 + 0 + 0 0 0 + 2 + + 0 + 0 + + + + 0 3 0 + + 0 0 + + 0 + + Review Questions 4 + 0 0 + 0 + + + + + 5 0 + 0 + + + 0 0 + + 6 0 + 0 + 0 + + 0 0 0 7 0 + 0 + 0 + 0 + 0 +

Kell, Duffy, Kidd

Can you guess each donor’s likely ethnicity? Which cell likely has HOMOZYGOUS expression Answers of the Fya antigen? Cell K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb Cell K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb 1 0 + 0 + 0 + 0 0 0 + 1 0 + 0 + + + + 0 0 + 2 + + 0 + 0 + + + + 0 2 0 + 0 + + 0 + 0 + 0 3 0 + + 0 0 + + 0 + + 3 0 + + 0 0 + + 0 + + 4 + 0 0 + 0 + + + + + 4 + + 0 + 0 + + + + + 5 0 + 0 + + + 0 0 + + 5 0 + 0 + + + 0 + + + 6 0 + 0 + 0 + + 0 0 0 6 0 + 0 + 0 + 0 0 0 0 7 0 + 0 + 0 + 0 + 0 + 7 0 + 0 + 0 + 0 + 0 + 1, 5 – African American / Black 2, 3, 4 – White 6 – Japanese, Finnish, Polynesian (?) 7 – Unknown

Which cell likely has HOMOZYGOUS expression What is the most likely cause of the reaction of the Fya antigen? ANSWER pattern observed below?

Cell K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb Cell K k Kpa Kpb Jsa Jsb Anti-k Anti-Jsb (weak) (weak) 1 0 + 0 + + + + 0 0 + 1 0 + 0 + 0 + 1+ 1+ 2 0 + 0 + + 0 + 0 + 0 2 + + + + 0 + 1+ 1+ 3 0 + + + 0 + + 0 + + 3 0 + 0 + 0 + 1+ 1+ 4 + + 0 + 0 + + + + + 4 0 + + + 0 + 0 1+ A. Anti-k antisera is expired. 5 0 + 0 + + + 0 + + + B. Cell 4 is from a patient with McLeod phenotype 6 0 + 0 + 0 + 0 0 0 0 C. Cell 4 is from a Kx patient D. Cell 4 is from a Kmod patient 7 0 + 0 + 0 + 0 + 0 + E. Kpa effect F. Cell 4 is from a Ge: -2, -3, -4 patient

What is the most likely cause of the reaction pattern observed below? ANSWER

Cell K k Kpa Kpb Jsa Jsb Anti-k Anti-Jsb (weak) (weak) 1 0 + 0 + 0 + 1+ 1+ 2 + + + + 0 + 1+ 1+ 3 0 + 0 + 0 + 1+ 1+ 4 0 + + + 0 + 0 1+ A. Anti-k antisera is expired. B. Cell 4 is from a patient with McLeod phenotype C. Cell 4 is from a Kx patient

D. Cell 4 is from a Kmod patient E. Kpa effect F. Cell 4 is from a Ge: -2, -3, -4 patient

MNS and Others

This presentation highlights selected systems; it is not intended to be a comprehensive review. MNS and Other Blood Groups

Cindy Piefer, MT(ASCP)SBB Manager, Immunohematology Reference Laboratory

Objectives MNS Blood Group System (ISBT 002)

Chromosome 4 • Discuss the antigens, gene location, protein, nomenclature, and phenotype distribution. Genes GYPA, GYPB Gene products Glycophorin A (GPA) & • Glycophorin B (GPB) Describe the serological characteristics of antibodies to antigens in these systems. Glycophorin function > sialic acid contributes to the negative charge on red • Discuss investigational techniques for identifying the cells

antibodies. GPA and GPB are type 1 transmembrane sialoglycoproteins, cleaved by • Describe the clinical significance of antibodies in proteolytic enzymes transfusion and in HDFN GYPE – adjacent to GYPB, no RBC membrane product, believed to cause the variant/hybrid alleles

Gylcophorin A and B Glycophorin Comparison

Glycophorin A Glycophorin B 1 million copies per RBC 200,000 copies per RBC 131 amino acids 72 amino acids  M: Ser-Ser-Thr-Thr-Gly  S: 48 Methionine(previously 29)  N: Leu-Ser-Thr-Thr-Glu  s: 48 Threonine(previously 29)  “N” first 26 aa same as N GPA  Not cleaved by trypsin

Null Phenotypes En(a-) (MNS28)

Deletion of GYPA and/or GYPB results in the silencing of • En means Ag carried on the envelope of RBC, high-prevalence the genes; no gene products are made. antigen • En(a-) cells lack GPA or have variant form Deficient Phenotype Deletion of exons Glycophorin • GPA is closely associated with Band 3, required for expression of Wrb En(a-) No GPA M-N-En(a-) GYPA (exon 2-7) and GYPB • Type as Wr(a-b-) (exon 1) • Enzyme testing can determine antibody specificity U- No GPB S-s-U- GYPB (exon 2-6) and GYPE • Resistant to DTT and Chymotrypsin (exon 1) • No to severe HTR and HDFN Mk No GPA or MkMk GYPA (exon 2-7) GPB M-N-En(a-) GYPB (exon 1-6) S-s-U- GYPE (exon 1)

U (MNS5) and U variants Low Prevalence Antigens

Hybrid gene: crossing over between GPA and GPB give • High prevalence antigen, 99% of AA are U+ rise to rare, low-prevalence variant alleles. • Result from the absence of GPB (S-s-) or an altered • Mur is low, but more common in Southeast Asia (hybrid) form of GPB (He, Dantu, SAT and Sta) Up to 90% in certain regions of Taiwan • Dantu+, S- s+weak Anti-Mur can cause severe HTRs and HDFN • 49% of S-s- are Uvar • Anti-Mur most common after anti-A and anti-B • 37% of these are He+ • Mur+ red cell important on screening cells in SE Asia • Ficin resistant • Others: Mg – MN allele; previously used in paternity • Molecular testing better for detecting Uvar

MNS Antibodies Lutheran Blood Group System (ISBT 005)

Anti-M more common, anti-N rare Chromosome 19; linked to Se • Show dosage • Anti-M enhanced at pH <6.5/acidified serum • 24 antigens; four antithetical pairs: a b • Anti-N reagent may be Vicia graminea lectin Lu (LU1)/Lu (LU2) • Anti-N associated with dialysis equipment formaldehyde Lu6/Lu9 treatment Lu8/Lu14 • Most anti-M and -N are not clinically significant Aua (LU18)/Aub (LU19)  If PW+ at 37C or IgG: give antigen neg and do IAT XM • Sensitive to trypsin, AET, DTT • Anti-S, -s, -U: usually IgG, AHTRs/DHTRs, HDFN • Resistant to ficin and papain

LU Phenotypes The Nulls

Reaction with Reaction with Phenotype Incidence (%) Most of the red cells are normal, but may be acanthocytic a b anti-Lu anti-Lu • May be due to Lu gp binding to spectrin

+ 0 Lu(a+b-) 0.2 • Three Types + + Lu(a+b+) 7.4 • Recessive – Silent allele at the Lu locus 0 + Lu(a-b+) 92.4 • Dominant – Suppressor gene at a separate locus • X-linked – Suppressor gene on the X chromosome 0 0 Lu(a-b-)* RARE

* Lu(a-b-): Three different types

Recessive Lu(a-b-) Dominant Lu(a-b-) Dominant inhibitor In(Lu), most common (1 in 3000 or 0.03%) Lu Recessive silent allele; amorphic gene inherited from both parents • Cells are Lu(a-b-) but can be detected by adsorption - elution • LuLu cells are Lu(a-b-) • No antibody production • Only form that can make anti-Lu3 and/or -Lua, -Lub • Decreased expression of P1, i, AnWj, In, Knops, Cost and MER2 antigens

X-linked Lu(a-b-) aka Lu mod Lutheran Antibodies – “Loose” or “stringy” mixed-field agglutination X-linked gene – daughters are carriers – Naturally occurring, IgM and IgA • Daughters will have normal expression if father’s – Most are immune: IgG expression is normal (XS2/X vs. XS2/XS2) • Anti-Lua and anti-Lub have caused mild DHTRs; anti-Lu8 • Sons are affected (XS2/Y) – No antibody production AHTRs • Do not cause HDFN; antigens not fully developed at birth. • AET/DTT sensitive (Lu ag located in the disulfide-bonded domains) • Ficin resistant • Capillary testing: pine tree-like appearance

Diego Blood Group System (ISBT 010) 22 Antigens Assigned to Diego

DI gene located on Band 3 or Anion Exchanger 1 (AE1) High Prevalence Low Prevalence (19 – Maintains the structural integrity of the red cell. (only 3 Ag) Ag’s) – Allows anion (HCO3- and Cl-) exchange across red cell membrane. Dib Dia b a Adapted from Reid ME, Lomas-Francis C Wr Wr The Blood Group Antigen Facts Book, 2nd ed. 2004 DISK Wu 16 others

Dia is low in Caucasians and Blacks, but higher in: • South American Indians ~36% • Japanese 12%, U.S. Mexicans 10%, Chinese 5% • Wr(b-) lacks GPA = Ena negative • Resistant to enzymes and DTT/AET

Diego System Antibodies YT Blood Group System (ISBT 011)

Anti-Dia or Anti-Dib Anti-Wra Anti-Wrb Two antigens on acetylcholinesterase (AChE) •IgG1 and IgG3 •RT (IgM), IAT (IgG1) •Alloantibody: rare Yta (high prevalence) and Ytb (8%) •Anti-Dia: DHTR and •Common antibody •Autoab: common • Chemicals: HDFN •Naturally occurring and may be – •Anti-Dib rare HTR; in 1-2% of donors implicated in AIHA Ficin variable can be an •Severe HDFN and •Cases of acute & – DTT and chymotrypsin sensitive autoantibody HTRs delayed HTRs – Trypsin resistant •Anti-Dib •Common in AIHA •HDFN DAT+ not • Anti-Yta ; questionable clinical significance (IgG1 & IgG4) demonstrates clinical finding • No HDFN dosage

XG Blood Group System (ISBT 012) Xg Antibodies & Use

Gene on X chromosome  Anti-Xga  Two antigens: • IgG • Xga 66%males and 89% females • Some are naturally occurring • CD99 (high prevalence) • No HTRs or HDFN (weak expression on cord RBCs)

 Chemicals:  Genetic uses • Ficin, trypsin, and chymotrypsin sensitive • Disproved Lyon hypothesis of one X chromosome being • DTT resistant inactivated early in embryonic life

Colton Blood Group System (ISBT 015) Gerbich Blood Group System (ISBT 020)

Coa - High-prevalence antigen Cob - Antithetical antigen, prevalence of about 8% in Whites, lower in • 12 antigens: 7 high prevalence and 5 low prevalence antigens other ethnic groups • Carried on glycophorin C and D (GPC, GPD) Co(a-b-) null phenotype makes anti-Co3 • Interact directly with protein band 4.1 and p55, • Resistant to chemicals (ficin and DTT) • • Anti-Coa /Cob have caused HTRs and HDFN Contributes to RBC membrane stability • Anti-Cob occurs in sera that contain other antibodies  4.1-deficient RBCs can be associated with elliptocytosis • GE:2,3,4 in >99% population • RBC receptor for Influenza A and Influenza B

Adapted from Reid ME, Lomas-Francis C The Blood Group Antigen Facts Book, 2nd ed. 2004

Gerbich Phenotypes Gerbich Antibodies

Phenotype Name Nucleotide Ethnicity Can make Kell and • Mostly IgG; may have IgM component Change Occurrence Antibody Vel • Do not bind complement typing • Generally not considered clinically significant, but clinically significant GE: -2, 3, 4 Yus Deletion Hispanic, Anti-Ge2 Normal antibodies include Anti-Ge2 and Anti-Ge3 in HDFN exon 2 Israeli, • Autoanti-Ge2, -Ge3 reported in AIHA cases altered GPC Mediterranean • Ficin treatment: differentiates anti-Ge3 (rare) GE: -2,-3, 4 Gerbich Deletion Melanesians Anti-Ge2 or Weak Antigen Destroyed by Ficin exon 3 (50%) Anti-Ge3 and/or Papain altered GPC Ge2 Yes GE: -2,-3,-4 Leach Deletion Rare Anti-Ge2 or Weak exon 3 & 4 Anti-Ge3 or Ge3 NO Anti-Ge4 Ge4 Yes

Cromer Blood Group System (ISBT 021) Cromer Antigens and Antibodies

18 antigens on complement-regulatory glycoprotein Antigens present in serum/plasma, urine, platelets, WBC (DAF, decay acceleratory factor, or CD55) and placental tissues • DAF deficiency is associated with PNH • Depressed during pregnancy, and poorly expressed on • 15 high prevalence antigens cord cells • 3 low prevalence antigens: Tc b, Tc c, Wesa • Chemicals: Antithetical pairs:  Ficin resistant and weakened with DTT  Tc a/Tc b/Tc c • None to moderate HTR  WESa/WESb • Does not cause HDFN – DAF on surface of trophoblasts •Null phenotype = Inab phenotype can make anti-IFC in the placenta

Indian Blood Group System (ISBT 023) VEL Blood Group System (ISBT 034)

Vel- RBCs found in 1:4000 people and 1:1700 Norwegians CD44 Indian glycoprotein and Swedes a b Two Antigens: In (low), In (high) Chemicals: • Sensitive to ficin, DTT, trypsin, chymotrypsin • Ficin, trypsin, chymotrypsin resistant (enhanced) • Weak on cord cells, pregnant woman and In(Lu) RBCs • DTT: variable/resistant Antibodies: Anti-Vel: • a b HTR: anti-In none; anti-In none to severe/delayed and • IgM and IgG, bind complement, some hemolytic hemolytic • HTR: mild to severe/hemolytic and HDFN: rare • HDFN: no, DAT may be positive • May be an autoantibody

The effect of enzymes and DTT on antigens Thank you!

Ficin/Papain DTT Possible Specificty I appreciate all the help from the various blood bank leaders who have studied these systems and determined Negative Positive M,N,S,s*; Ge2, Ge4; the information I have shared with you. Xga

Negative Negative Indian Any questions? Positive Weak Cromer, Lutheran

Variable Negative Yta

Positive Positive Diego; Colton; Ge3; Vel *s variable expression with ficin/papain

AIHA

Objectives • Describe the serologic findings that characterize WAIHA and Autoimmune Hemolytic Anemias cold agglutinin syndrome. • Discuss the laboratory investigation and methods used to Sue Johnson, MSTM, MT(ASCP)SBB evaluate patients with autoimmune hemolytic anemia. Director, Clinical Education • Discuss the role of blood transfusion in autoimmune BloodCenter of Wisconsin disorders, problems encountered in obtaining "compatible Milwaukee, WI blood" and approaches to transfusion. • Describe serologic findings in the following unusual cases of autoimmune hemolytic anemia: – DAT negative WAIHA – Mixed Type associated with both warm and cold-reactive autoantibodies – IgM warm-reactive autoantibodies

Objectives • Describe initial serologic results observed in a patient with drug- http://www.bbguy.org/2017/02/27/028/ induced immune hemolytic anemia. • Discuss methods used to detect drug-dependent red cell antibodies in serum and eluates. – Drug treated red blood cells – In the presence of drug – Drug metabolites (urine and serum) • Describe proposed immunological "mechanisms" of DIIHA & common drugs associated with each category. – Hapten-dependent antibody (Drug adsorption) - drug binds firmly to RBC membrane – Drug-dependent antibody binds to untreated RBCs in presence of drug. – Nonimmunologic protein adsorption – Drug-independent autoantibody – autoantibody induced by drug

Positive Polyspecific DAT AIHA Serologic Types

Perform DAT with Barcellini et al. Blood. 2014 Nov 6;124(19):2930-6 Monospecific Reagents and Controls Serologic Type % What is Anti-C3 Both Anti-IgG and C3 Positive? Warm, DAT – IgG only 43 Warm, DAT - IgG & C3 17 Strength of Strength of ≤1+ 2-4+ ≤1+ 2-4+ Reaction? Reaction? Anti-IgG Cold, DAT - C3 27

Acute or Acute or Mixed, DAT - IgG & C3, Warm & 8 DIIHA PCH CAD DIIHA WAIHA Delayed Delayed HTR Strength of HTR ≤1+ 2-4+ Cold Autoantibody Reaction?

*Serological results must Atypical 5 be correlated with Acute or ABO Rh Other Passively DIIHA WAIHA clinical findings. Delayed Acquired HDFN HDFN HDFN HTR Antibody Total Patients

Courtesy of C Feldman & J O’Connor

Warm Autoimmune Hemolytic Anemia Warm Autoantibody in Plasma Incidence and Cause – 1º - idiopathic . . – 2 º - lymphoma, SLE, carcinoma ...... – Most common type of immune . . . hemolysis . . . – IgG antibody reactive at 37ºC . . . . .

Warm Autoantibody Coating RBCs Warm Autoantibody in Plasma (1+) ......

Antibody Identification – Saline or LISS

Warm Autoantibody in Plasma (3+) D C c E e K Fya Fyb Jka Jkb S s IAT 1 + + 0 0 + 0 + 0 + 0 0 + 2

2 + + 0 0 + + 0 + + + + + 2 . 3 + 0 + + 0 0 0 + + 0 + + 2 ...... 4 + 0 + 0 + 0 0 + 0 + 0 + 2 ...... 5 0 + + 0 + 0 + + + 0 + 0 2 . . . . . 6 0 0 + 0 + + 0 + 0 + + + 2 . . . 7 0 0 + 0 + 0 + 0 0 + 0 + 2

. . . . 8 0 0 + 0 + 0 0 + + 0 + 0 2 . Auto 4

AIHA

Warm Autoantibody Relative Specificity: -e-like Common Specificities – Rh -related D C c E e K Fya Fyb Jka Jkb S s IAT 1 + + 0 0 + 0 + 0 + 0 0 + 3 • Broad: Negative with Rhnull cells 2 + + 0 0 + + 0 + + + + + 3

• Single: -c, -e, -D, -C, -E, -f 3 + 0 + + 0 0 0 + + 0 + + 1

4 + 0 + 0 + 0 0 + 0 + 0 + 3 • Relative: c-like, e-like 5 0 + + 0 + 0 + + + 0 + 0 3

Uncommon Specificities 6 0 0 + 0 + + 0 + 0 + + + 3

• M, N, S, U, EnaFS, EnaFR, Wrb 7 0 0 + 0 + 0 + 0 0 + 0 + 3

• K, Kpb, K13 8 + 0 + + 0 0 0 + + 0 + 0 1 Auto 4 • LWa, LWab, Jka, Jkb, Jk3 • A, B, IT

• Ge, Vel, AnWj, Sc1, Sc3, Rx

Antibody Identification – Saline

D C c E e K Fya Fyb Jka Jkb S s IAT IAT Autologous Adsorption 1 + + 0 0 + 0 + 0 + 0 0 + 2 2 • Prepare DAT Negative RBCs 2 + + 0 0 + + 0 + + + + + 1 2

3 + 0 + + 0 0 0 + + 0 + + 2 2 –ZZAP

4 + 0 + 0 + 0 0 + 0 + 0 + 0 2 • Ficin or Papain 5 0 + + 0 + 0 + + + 0 + 0 2 2

6 0 0 + 0 + + 0 + 0 + + + 0 2 • 2-ME or DTT TM 7 0 0 + 0 + 0 + 0 0 + 0 + 0 2 –W.A.R.M.

8 0 0 + 0 + 0 0 + + 0 + 0 2 2 –Enzymes Auto 4 • Ficin or Papain

ZZAP Treat Patient RBCs After ZZAP Treating ......

Autologous Adsorption Autologous Adsorption Mix & Pt Serum + Adsorbed ZZAP RBCs Pt Serum + Incubate at Centrifuge Serum ZZAP RBCs 37C ZZAP RBCs

= Anti-Jka = Anti-Jka = WAA = WAA

Allogeneic Adsorption Allogeneic Adsorption Other Methods Patient’s Phenotype is Unknown • Gluteraldehyde -Treated RBCs • Stroma RBC Phenotype Antibodies Remaining a • PEG R1R1; Jk(a-) -c, -E, -Jk b Other Considerations R2R2; Jk(b-); S- -e, -C, -Jk , -S • Perform cell separation & phenotype or rr; K-; s- -D, -C, -E, -s

genotype • Select phenotype matched RBCs • Treat RBCs with ZZAP or enzymes

Allogeneic Adsorption Allogeneic Adsorption After 37C Incubation

Pt Serum + Pt Serum + Pt Serum + Pt Serum + Pt Serum + Pt Serum +

R1R1, Jk(a-) RBCs R2R2, Jk(a+) RBCs rr, Jk(a-) RBCs R1R1, Jk(a-) RBCs R2R2, Jk(a+) RBCs rr, Jk(a-) RBCs

= Anti-Jka = Anti-Jka = WAA

= Jk(a+) RBCs = Jk(a+) RBCs

Testing Adsorbed Serum/Plasma Alloadsorbed Serum Positive Results Saline Tube • Adsorption not complete Serum Adsorbed with Serum Adsorbed with – Reactivity is weakened but consistent with RBCs tested R1R1, R2R2, rr, R1R1, R2R2, rr, Jk(a-) Jk(a+) Jk(a-) Jk(a-) Jk(a+) Jk(a-) – Perform additional adsorptions IAT IAT IAT IAT IAT IAT I Jk(a+) 2+ 2+ 2+ I Jk(a+) 1+ 1+ 1+ • Antigen destroyed by enzymes or ZZAP II Jk(a-) 2+ 2+ 2+ II Jk(a-) 1+ 1+ 1+ – No change in reactivity post adsorption III Jk(a+) 2+ 2+ 2+ III Jk(a+) 1+ 1+ 1+ – Test raw serum with enzymes or ZZAP treated RBCs Antigen on adsorbing RBCs denatured Adsorption not complete No adsorption – Perform adsorptions with untreated RBCs Serum Adsorbed with • Reactivity due to alloantibody R1R1, R2R2, rr, Jk(a-) Jk(a+) Jk(a-) – Some positive, some negative results IAT IAT IAT √ – Test adsorbed serum with DAT negative RBCs I Jk(a+) 2+ 0 2+ II Jk(a-) 0√ 0√ 0√ – Test adsorbed serum with phenotypically similar RBCs III Jk(a+) 2+ 0√ 2+ – Identify alloantibody Underlying alloantibody

Last ELUATE Eluate Wash WAIHA vs. DIIHA D C c E e K Fya Fyb Jka Jkb S s IAT IAT 1 + + 0 0 + 0 + 0 0 + 0 + 3 0√ • Eluate 2 0 0 + 0 + + 0 + + + + + 3 0√ • WAA strongly positive 3 + 0 + + 0 0 0 + + 0 + 0 3 0√ • DDA is negative or weak 4 + 0 + 0 + 0 0 + 0 + 0 + 3 • Serum 5 0 + + 0 + 0 + + + 0 + 0 3 • 6 0 0 + 0 + + 0 + 0 + + + 3 WAA persists

7 0 0 + 0 + 0 + 0 0 + 0 + 3 • DDA disappears within days

8 0 0 + 0 + 0 0 + + 0 + 0 3 if drug is discontinued

Auto NT

Unusual AIHAs Management of WAIHA • ~13% of AIHAs* are Unusual Types • Steroids *Barcellini et al. Blood. 2014 Nov 6;124(19):2930-6 • Rituximab • ~ 8% Mixed Type AIHA* • Immunosuppressive drugs - Cytoxan – DAT positive with both IgG & C3 • Intravenous Immune Globulin (IVIG) – Warm & Cold Autoantibody present • Plasmapheresis • DAT-Negative AIHA • Others, Cyclosporine – IgA warm autoantibody-induced AIHA – IgM warm autoantibody-induced AIHA • IgM warm-reactive autoantibody Avoid transfusion unless life-threatening hemolysis! IS 37C IAT I 0 2-4+ 0 - 1+ II 0 2-4+ 0 –1+

WAIHA Associated With a Negative DAT Cold-Hemagglutinin Disease (CHD) • 1-5% of patients with signs & symptoms of hemolysis Acute Serology Lymphoproliferative • DAT shows complement • Low affinity IgG autoantibodies disorders only Mycoplasma • Antibody characteristics • Small amount of bound IgG pneumonia infection Increased thermal • IgM and IgA autoantibodies Infectious amplitude mononucleosis IgM antibody Chronic Cold Saline Wash Reacts up to 37ºC in Cold LISS Wash More severe symptoms albumin Direct PEG Elderly, during cold Titer > 1000 at 4ºC Enhanced DAT Methods weather

Direct Polybrene Hemolysis mild- ELAT moderate Flow Cytometry

Cold-Reactive Autoantibodies Clinical Manifestations of CAD Phase & Strength of Reactivity IS 37C IAT • Mild chronic anemia I 4+ 3+ 0-3+ • Occasional jaundice and pallor II 4+ 3+ 0-3+ • Some patients have increased episodes of hemolysis associated with hemoglobinuria Antibody specificity and hemoglobinemia upon cold exposure Most commonly anti-I • Hemolysis usually self-limited if associated Occasionally anti-i associated with infectious with mycoplasma or other viral infection mononucleosis

Management of CAD Drug-Induced Immune Hemolytic Anemia • DAT - Reactivity depends on time of testing • Avoid cold compared to presentation • Plasmapheresis - improvements only Polyspecific AHG 3-4+ temporary. Anti-IgG 3-4+ • Steroids and splenectomy of little Anti-C3 3-4+ benefit. Control 0 • Eluate • IVIG of little benefit. – Rapid Acid – Most are negative – Few are disproportionately weaker as compared to strength of DAT

Drug Binds to RBCs (Hapten) DIIHA Serum Reactivity Drug Adsorption • Reactivity depends on time of testing • Penicillin/penicillin derivatives & compared to presentation cephalosporins • Saline IAT - some positive • Drug binds covalently to membrane proteins • PEG or Ficin IAT - many positive and stimulates hapten-dependent antibodies • Antibody reacts with normal RBCs pretreated IS 37C IAT with drug I 0 0 0 - 4+ II 0 0 0 - 4+

Drug-Dependent Drug Does Not Coat RBCs Drug-Induced Autoantibody “Immune Complex” • Alpha methyldopa, procainamide • Quinidine, quinine, NSAIDs • Through an unknown mechanism, drug induces • Through an unknown mechanism, drug autoantibodies specific for RBC membrane induces antibodies that bind to RBC only proteins when drug is present in soluble form • Antibody reacts with normal RBCs in the • Antibody reacts with RBCs when soluble absence of drug drug is present

Nonimmunologic Protein Adsorption (NIPA) Testing Drug-Treated RBCs Membrane Modification Patient Serum Pos Con Normal Serum • Cephalosporins • Tazobactum, Clavunate 15’RT 4 4 0 30 37C 4 4 0 • Drug coats RBCs and causes them to IAT 4 4 0 become “sticky”

• DAT - weakly positive 15’RT 0

• Rarely associated with DIIHA 30 37C 0

RBCsUntrtd IAT 0

Testing in Presence of Drug Drug-Induced Hemolytic Anemia e+ RBCs 30’RT 60’ 37C IAT Treatment Patient Serum + Drug 4+ 4+ 4+

Patient Serum + Diluent 0 0 0 • STOP the DRUG!!!!

Diluent + Drug 0 0 0 • Treat the symptoms

Eluate + Drug 0 0 0/3+ • Don’t take drug in the future

Eluate + Diluent 0 0 0

Positive Control + Drug 4+ 4+ 4+

AIHA & DIIHA - Things to remember… 21 Case Studies – all with positive DATs • Always look for medication history in a question but don’t assume it’s the culprit • Look at strength of reactivity of DAT 22 Case Studies – all antibody problems, • Correlate DAT reactivity with patients clinical simple to complex information • DAT strongly positive, Eluate negative is key finding in DIIHA BUT patient should be Questions & Answers covering showing clinical signs of hemolysis broad range of TM topics

http://marketplace.aabb.org/ebuspprod/Marketplace/AllProducts/ProductDetail.aspx?productId=12811191

Reading List

• AABB Technical Manual, 18th ed., 2014, Chapter 17, p. 425-451, Methods, Section 4 (Flash Drive). • Petz L.D., Garratty G. Acquired Immune Hemolytic Anemias. Philadelphia: Questions? Churchill Livingstone, 2004. • Judd W.J., Johnson S.T., Storry J.R. Judd’s Methods in Immunohematology, 3rd ed., p. 407-472, Section XI, 2008. [email protected] • Lechner K, Jager U. How I treat autoimmune hemolytic anemias in adults. Blood. 2010;116(11):1831-1838. • Barcellini W., et al. Clinical heterogeneity and predictors of outcome in primary autoimmune hemolytic anemia: a GIMEMA study of 308 patients. Blood. 2014;124(19):2930-2936. • Immunohematology 2014;30 (2). Special Edition on Drug-Induced Immune Cytopenias. • WJ Judd, ST Johnson, JR Storry. Judd’s Methods in Immunohematology, 3rd ed. 2009. AABB Press.