Download pdf - Kell and Duffy in 30 minutes …you’ve got to be Kidd-ing! · Kell and Duffy in 30 minutes …you’ve got to be Kidd-ing! (Ok, we’ll talk about Kidd, too) Jessica Drouillard,

Kell and Duffy in 30 minutes …you’ve got to be Kidd-ing!

(Ok, we’ll talk about Kidd, too)

Jessica Drouillard, SBB(ASCP)CM

Heartland Blood Centers, part of Versiti Aurora, IL

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

• Discuss implications of null phenotypes

• List the characteristics of antibodies directed against each blood group

• Discuss the use of chemicals in antibody identification

• Identify diseases related to blood groups

Kell Blood Group System (KEL) ISBT 006

Inheritance

– XK gene – on X chromosome • Xk protein

• Kx antigen

– KEL gene – on Chromosome 7 • Kell glycoprotein

• Kell antigens

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

Expression

Kell antigens -K, k -Kpa, Kpb

-Jsa, Jsb

Kx

Weak Kell antigens

No Kx

Kx

No Kell Ku- Km-

Normal Kell Expression


-Jsa, Jsb

Kx

Antigen ISBT Whites (%)

Blacks (%)

K KEL1 9 2

k KEL2 99.8 100

Kpa KEL3 2 Rare

Kpb KEL4 100 100

Ku KEL5 100 100

Jsa KEL6 0.01 20

Jsb KEL7 100 99

© 2017 Last Chance Review SCABB / BloodCenter of Wisconsin

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Kell, Duffy, Kidd

Ko phenotype

-SNP of KEL*02 -Homozygous -No Kell glycoproteins -Null phenotype -Cells type Kx+s

Ku- Km- -Can form anti-Ku and anti-Km

Kx No Kell Ku- Km-

Ko phenotype


-Jsa, Jsb

Kx

Normal

McLeod Phenotype

• No XK gene

– No Kx

– No Km

– Can form anti-Kx and –Km

• Other Kell typings weak

– Ku

– K, k, Kpa, Kpb, Jsa, Jsb


-Jsa, Jsb

Kx

Weak Kell antigens

No Kx

McLeod phenotype

Normal

McLeod Syndrome

• X linked, occurs almost exclusively in males – Some patients also have X-linked Chronic

Granulomatous Disease (CGD)

• Range of neurological and muscular defects – Muscle wasting, reduction in deep tendon

reflexes

• Hematologic abnormalities – Decreased RBC survival, acanthocytosis,

reticulocytosis, reduced serum haptoglobin, increased bilirubin, compensated anemia

– Difficult to find blood for transfusion

Antibodies

• Primarily IgG, do not bind complement

• Usually immune stimulated

• Clinically significant (HTR and HDFN)

Phenotype Antibody Compatible blood

Anti-Ku Ko

Anti-Km Ko and McLeod

Anti-Km Ko and McLeod

Anti-Kx McLeod

Anti-Kx, -Km McLeod

Kx No Kell Ku- Km-

Ko phenotype

Weak Kell antigens

No Kx

McLeod phenotype (no CGD)

Weak Kell antigens

No Kx

McLeod phenotype with CGD

Weakened Kell Antigen Expression

• Kmod phenotypes

• Kpa in cis

– k, Kpa, Jsb

• Gerbich null types

– Ge:2,3,4 is normal

– Ge:-2,3,4 = Yus - Kell normal

– Ge:-2,-3, 4 = Gerbich – Kell expression weakened

– Ge:-2,-3,-4 = Leach (true null) – Kell expression depressed

Possible Kell haplotypes

k, Kpb, Jsb

K, Kpb, Jsb

k, Kpa, Jsb

k, Kpb, Jsa

Kmod

• Arises from SNP at KEL*02

• Weakened expression of Kell glycoproteins – Often need

adsorption/elution studies to detect

• Strong Kx expression

• Some may make anti-Ku-like antibody that reacts with all cells but other Kmod

Kmod


-Jsa, Jsb

Kx

Normal


38

Kell, Duffy, Kidd

Duffy Blood Group System ISBT 008

Inheritance

• FY*01 and FY*02 genes on Chromosome 1 – Syntenic to Rh

– Produce Duffy glycoproteins: Fy3, Fy5, Fy6, and Fya/Fyb

• Duffy Antigen Receptor for Chemokines – Binds cytokines, especially IL-8

– Role in inflammation

– Also receptor for malaria • Plasmodium vivax

• Plasmodium knowlesi

Expression

• Well developed at birth

• Destroyed by enzymes and ZZAP

• Antigens found on RBCs and other tissues

– Endothelial cells, brain, colon, lung, spleen, kidney, etc.

• Antigens reported to weaken when stored

Fy6 Fya/Fyb

Fy3

Duffy Phenotypes

RBC Phenotype Whites (%) Blacks (%) Asians (%)

Fy(a+b-) 20 10 91

Fy(a+b+) 48 3 9

Fy(a-b+) 32 20 <1

Fy(a-b-) 0 67 0

Fy3 100 32 99.9

Fy5 99.9 32 99.9

Fyx 1.4 0 0

Fy(a-b-) type

• Rare in whites, but usually true null – Duffy antigens not expressed on RBCs or tissues

– Can make anti-Fy3

• Most frequently found in blacks – Arises from SNP mutation in GATA-1 erythroid

promoter region of FY*01 (Fya) or FY*02 (Fyb) – more common

– Duffy antigens not expressed on RBCs, but are expressed on tissues

Duffy genotypes

Genotype

RBC Phenotype Comments

Fya Fyb Fy3

FY*A / FY*A + 0 + Normal, homozygous Fya expression

FY*A / FY*B + + + Normal, heterozygous Fya/Fyb expression

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) Should not make anti-Fyb

FY*B / FY*B_GATA 0 + + Fyb expressed on RBC and tissues Can make anti-Fya

FY*B_GATA / FY*B_GATA 0 0 0 Fy(a-b-) phenotype Fyb expressed on tissues (not RBC) Can make anti-Fya Should not make anti-Fyb

Fymod / Fymod w+ w+ w+ Weak Fy6 antigen expression


39

Kell, Duffy, Kidd

Duffy antibodies

• Usually IgG, do not bind complement well

• Show dosage

• Anti-Fya is 20 times more common than anti-Fyb

• Clinically significant (HTR and HDFN, usually mild)

Antigen Reaction with enzymes

Fya / Fyb Destroyed

Fy3 Resistant

Fy5 Resistant

Fy6 Destroyed

Other Duffy antibodies

• Anti-Fy3 – Found only in Fy(a-b-) individuals – Reacts like an inseparable anti-Fya, -Fyb, but enzymes will

not eliminate reactivity

• Anti-Fy5 – Found only in Fy(a-b-) individuals – Reacts with all cells except

• Fy(a-b-) • Rh null cells, regardless of Fya/Fyb typing

– D-- cells have weak Fy5 expression

• Anti-Fy6 – Murine antibody – not found in humans

Kidd Blood Group System

ISBT 009

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 renal medulla

• Jk3 antigen absent/weak in Jk(a-b-) individuals – Dominant inhibitor gene In(Jk)

• Reported in Japanese families

• No Jka or Jkb expression

• Weak Jk3 expression

– Homozygous for Jk gene – true JKnull (No Jka, Jkb or Jk3) • Polynesians and Finns

Expression

• Well developed at birth

• Antigens cluster on RBC surface

• Transmembrane protein (like Rh)

– Not destroyed by DTT/AET or enzymes

– Enhanced by enzymes

• Poor immunogens

Kidd Phenotypes

Phenotype Whites (%) Blacks (%)

Jk(a+b-), Jk3+ 26 52

Jk(a+b+), Jk3+ 50 40

Jk(a-b+), Jk3+ 24 8

Jk(a-b-), Jk3- Polynesians, Finns

Jk(a-b-), Jk3+ (weak) Japanese


40

Kell, Duffy, Kidd

Jk(a-b-) Phenotype

• When caused by dominant inhibitor gene – Need only 1 copy of In(Jk) gene

– Cells type Jk(a-b-) and Jk3-, but antigens may be detected by adsorption/elution studies

– Since trace amounts of antigens are present, cannot make anti-Jk3

• When caused by inheritance of JK gene – Need 2 copies of JK gene (must be homozygous)

– Cells type Jk(a-b-) and Jk3-. Kidd antigens completely absent

– Can make anti-Jk3

2M Urea Lysis Test

• Cheap, effective screening test to identify Jk(a-b-) individuals

• Cells with normal Kidd antigen expression have normal HUT11, will swell and lyse

• Jk(a-b-) cells resist lysis by 2M urea, but shrink and shrivel

2M Urea Lysis Test

• 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

Kidd Antibodies

Review Questions

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 + +

4 + 0 0 + 0 + + + + +

5 0 + 0 + + + 0 0 + +

6 0 + 0 + 0 + + 0 0 0

7 0 + 0 + 0 + 0 + 0 +


41

Kell, Duffy, Kidd


1 0 + 0 + 0 + 0 0 0 +

2 + + 0 + 0 + + + + 0

3 0 + + 0 0 + + 0 + +

4 + 0 0 + 0 + + + + +

5 0 + 0 + + + 0 0 + +

6 0 + 0 + 0 + + 0 0 0

7 0 + 0 + 0 + 0 + 0 +

Can you guess each donor’s likely ethnicity? Answers

1, 5 – African American / Black 2, 3, 4 – White 6 – Japanese, Finnish, Polynesian (?) 7 – Unknown

Which cell likely has HOMOZYGOUS expression of the Fya antigen?


1 0 + 0 + + + + 0 0 +

2 0 + 0 + + 0 + 0 + 0

3 0 + + 0 0 + + 0 + +

4 + + 0 + 0 + + + + +

5 0 + 0 + + + 0 + + +

6 0 + 0 + 0 + 0 0 0 0

7 0 + 0 + 0 + 0 + 0 +

Which cell likely has HOMOZYGOUS expression of the Fya antigen? ANSWER


1 0 + 0 + + + + 0 0 +

2 0 + 0 + + 0 + 0 + 0

3 0 + + + 0 + + 0 + +

4 + + 0 + 0 + + + + +

5 0 + 0 + + + 0 + + +

6 0 + 0 + 0 + 0 0 0 0

7 0 + 0 + 0 + 0 + 0 +

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

Cell K k Kpa Kpb Jsa Jsb Anti-k (weak)

Anti-Jsb (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

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

Cell K k Kpa Kpb Jsa Jsb Anti-k (weak)

Anti-Jsb (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


42

Kell, Duffy, Kidd

MNS and Other Blood Groups

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

This presentation highlights selected systems; it is not intended to be a comprehensive review.

Objectives

• Discuss the antigens, gene location, protein, nomenclature, and phenotype distribution.

• Describe the serological characteristics of antibodies to antigens in these systems.

• Discuss investigational techniques for identifying the antibodies.

• Describe the clinical significance of antibodies in transfusion and in HDFN

MNS Blood Group System (ISBT 002)

Chromosome 4

Genes GYPA, GYPB Gene products Glycophorin A (GPA) & Glycophorin B (GPB)

Glycophorin function > sialic acid contributes to the negative charge on red cells

GPA and GPB are type 1 transmembrane sialoglycoproteins, cleaved by proteolytic enzymes

GYPE – adjacent to GYPB, no RBC membrane product, believed to cause the variant/hybrid alleles

Gylcophorin A and B Glycophorin Comparison

Glycophorin A

1 million copies per RBC 131 amino acids M: Ser-Ser-Thr-Thr-Gly N: Leu-Ser-Thr-Thr-Glu

Glycophorin B

200,000 copies per RBC 72 amino acids S: 48 Methionine(previously 29) s: 48 Threonine(previously 29) “N” first 26 aa same as N GPA

Not cleaved by trypsin


43

MNS and Others

Null Phenotypes

Deletion of GYPA and/or GYPB results in the silencing of the genes; no gene products are made.

Deficient Glycophorin

Phenotype Deletion of exons

En(a-) No GPA M-N-En(a-) GYPA (exon 2-7) and GYPB (exon 1)

U- No GPB S-s-U- GYPB (exon 2-6) and GYPE (exon 1)

Mk No GPA or GPB

MkMk M-N-En(a-) S-s-U-

GYPA (exon 2-7) GYPB (exon 1-6) GYPE (exon 1)

En(a-) (MNS28)

• En means Ag carried on the envelope of RBC, high-prevalence antigen

• En(a-) cells lack GPA or have variant form • GPA is closely associated with Band 3, required for expression

of Wrb • Type as Wr(a-b-)

• Enzyme testing can determine antibody specificity • Resistant to DTT and Chymotrypsin • No to severe HTR and HDFN

U (MNS5) and U variants

• High prevalence antigen, 99% of AA are U+ • Result from the absence of GPB (S-s-) or an altered

(hybrid) form of GPB (He, Dantu, SAT and Sta) • Dantu+, S- s+weak • 49% of S-s- are Uvar

• 37% of these are He+ • Ficin resistant • Molecular testing better for detecting Uvar

Low Prevalence Antigens Hybrid gene: crossing over between GPA and GPB give rise to rare, low-prevalence variant alleles. • Mur is low, but more common in Southeast Asia Up to 90% in certain regions of Taiwan Anti-Mur can cause severe HTRs and HDFN • Anti-Mur most common after anti-A and anti-B • Mur+ red cell important on screening cells in SE Asia • Others: Mg – MN allele; previously used in paternity

MNS Antibodies Anti-M more common, anti-N rare • Show dosage • Anti-M enhanced at pH <6.5/acidified serum • Anti-N reagent may be Vicia graminea lectin • Anti-N associated with dialysis equipment formaldehyde treatment • Most anti-M and -N are not clinically significant If PW+ at 37C or IgG: give antigen neg and do IAT XM • Anti-S, -s, -U: usually IgG, AHTRs/DHTRs, HDFN

Lutheran Blood Group System (ISBT 005)

Chromosome 19; linked to Se • 24 antigens; four antithetical pairs: Lua(LU1)/Lub(LU2) Lu6/Lu9 Lu8/Lu14 Aua (LU18)/Aub (LU19) • Sensitive to trypsin, AET, DTT • Resistant to ficin and papain


44

MNS and Others

LU Phenotypes

Reaction with anti-Lua

Reaction with anti-Lub

Phenotype Incidence (%)

+ 0 Lu(a+b-) 0.2

+ + Lu(a+b+) 7.4

0 + Lu(a-b+) 92.4

0 0 Lu(a-b-)* RARE

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

The Nulls

Most of the red cells are normal, but may be acanthocytic • May be due to Lu gp binding to spectrin

• Three Types

• Recessive – Silent allele at the Lu locus • Dominant – Suppressor gene at a separate locus • X-linked – Suppressor gene on the X chromosome

Recessive Lu(a-b-) Recessive silent allele; amorphic Lu gene inherited from both parents • LuLu cells are Lu(a-b-) • Only form that can make anti-Lu3 and/or -Lua, -Lub

Dominant Lu(a-b-) Dominant inhibitor In(Lu), most common (1 in 3000 or 0.03%) • Cells are Lu(a-b-) but can be detected by adsorption - elution • No antibody production • Decreased expression of P1, i, AnWj, In, Knops, Cost and MER2 antigens

X-linked Lu(a-b-) aka Lu mod

X-linked gene – daughters are carriers • Daughters will have normal expression if father’s

expression is normal (XS2/X vs. XS2/XS2)

• Sons are affected (XS2/Y) – No antibody production

Lutheran Antibodies – “Loose” or “stringy” mixed-field agglutination – Naturally occurring, IgM and IgA – Most are immune: IgG • Anti-Lua and anti-Lub have caused mild DHTRs; anti-Lu8

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


45

MNS and Others

Diego Blood Group System (ISBT 010)

DI gene located on Band 3 or Anion Exchanger 1 (AE1) – Maintains the structural integrity of the red cell. – Allows anion (HCO3- and Cl-) exchange across red cell membrane.

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

22 Antigens Assigned to Diego High Prevalence

(only 3 Ag) Low Prevalence (19

Ag’s)

Dib

Wrb

DISK

Dia

Wra

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

Anti-Dia or Anti-Dib Anti-Wra Anti-Wrb

•IgG1 and IgG3 •Anti-Dia: DHTR and HDFN •Anti-Dib rare HTR; can be an autoantibody •Anti-Dib

demonstrates dosage

•RT (IgM), IAT (IgG1) •Common antibody •Naturally occurring in 1-2% of donors •Severe HDFN and HTRs •Common in AIHA

•Alloantibody: rare •Autoab: common and may be implicated in AIHA •Cases of acute & delayed HTRs •HDFN DAT+ not clinical finding

YT Blood Group System (ISBT 011)

Two antigens on acetylcholinesterase (AChE) Yta (high prevalence) and Ytb (8%) • Chemicals: – Ficin variable – DTT and chymotrypsin sensitive – Trypsin resistant • Anti-Yta ; questionable clinical significance (IgG1 & IgG4) • No HDFN

XG Blood Group System (ISBT 012)

Gene on X chromosome

Two antigens: • Xga 66%males and 89% females • CD99 (high prevalence) Chemicals: • Ficin, trypsin, and chymotrypsin sensitive • DTT resistant

Xg Antibodies & Use

Anti-Xga • IgG • Some are naturally occurring • No HTRs or HDFN (weak expression on cord RBCs) Genetic uses • Disproved Lyon hypothesis of one X chromosome being

inactivated early in embryonic life


46

MNS and Others

Colton Blood Group System (ISBT 015) Coa - High-prevalence antigen

Cob - Antithetical antigen, prevalence of about 8% in Whites, lower in other ethnic groups Co(a-b-) null phenotype makes anti-Co3 • Resistant to chemicals (ficin and DTT) • Anti-Coa /Cob have caused HTRs and HDFN • Anti-Cob occurs in sera that contain other antibodies

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

Gerbich Blood Group System (ISBT 020)

• 12 antigens: 7 high prevalence and 5 low prevalence antigens • Carried on glycophorin C and D (GPC, GPD) • Interact directly with protein band 4.1 and p55, • Contributes to RBC membrane stability 4.1-deficient RBCs can be associated with elliptocytosis • GE:2,3,4 in >99% population • RBC receptor for Influenza A and Influenza B

Gerbich Phenotypes

Phenotype Name Nucleotide

Change

Ethnicity

Occurrence

Can make

Antibody

Kell and

Vel

typing

GE: -2, 3, 4 Yus Deletion exon 2 altered GPC

Hispanic, Israeli, Mediterranean (rare)

Anti-Ge2 Normal

GE: -2,-3, 4

Gerbich Deletion exon 3 altered GPC

Melanesians (50%)

Anti-Ge2 or Anti-Ge3

Weak

GE: -2,-3,-4 Leach Deletion exon 3 & 4

Rare Anti-Ge2 or Anti-Ge3 or Anti-Ge4

Weak

Gerbich Antibodies • Mostly IgG; may have IgM component • Do not bind complement • Generally not considered clinically significant, but clinically significant

antibodies include Anti-Ge2 and Anti-Ge3 in HDFN • Autoanti-Ge2, -Ge3 reported in AIHA cases • Ficin treatment: differentiates anti-Ge3

Antigen Destroyed by Ficin and/or Papain

Ge2 Yes

Ge3 NO

Ge4 Yes

Cromer Blood Group System (ISBT 021) 18 antigens on complement-regulatory glycoprotein (DAF, decay acceleratory factor, or CD55) • DAF deficiency is associated with PNH • 15 high prevalence antigens • 3 low prevalence antigens: Tcb, Tcc, Wesa Antithetical pairs:

Tca/Tcb/Tcc WESa/WESb

•Null phenotype = Inab phenotype can make anti-IFC

Cromer Antigens and Antibodies

Antigens present in serum/plasma, urine, platelets, WBC and placental tissues • Depressed during pregnancy, and poorly expressed on

cord cells • Chemicals: Ficin resistant and weakened with DTT • None to moderate HTR • Does not cause HDFN – DAF on surface of trophoblasts

in the placenta


47

MNS and Others

Indian Blood Group System (ISBT 023)

Indian glycoprotein CD44 Two Antigens: Ina (low), Inb (high) • Sensitive to ficin, DTT, trypsin, chymotrypsin • Weak on cord cells, pregnant woman and In(Lu) RBCs Antibodies: • HTR: anti-Ina none; anti-Inb none to severe/delayed and

hemolytic • HDFN: no, DAT may be positive

VEL Blood Group System (ISBT 034) Vel- RBCs found in 1:4000 people and 1:1700 Norwegians and Swedes Chemicals: • Ficin, trypsin, chymotrypsin resistant (enhanced) • DTT: variable/resistant Anti-Vel: • IgM and IgG, bind complement, some hemolytic • HTR: mild to severe/hemolytic and HDFN: rare • May be an autoantibody

The effect of enzymes and DTT on antigens

Ficin/Papain DTT Possible Specificty

Negative Positive M,N,S,s*; Ge2, Ge4; Xga

Negative Negative Indian

Positive Weak Cromer, Lutheran

Variable Negative Yta

Positive Positive Diego; Colton; Ge3; Vel

*s variable expression with ficin/papain

Thank you!

I appreciate all the help from the various blood bank leaders who have studied these systems and determined the information I have shared with you. Any questions?


48

MNS and Others

Autoimmune Hemolytic Anemias

Sue Johnson, MSTM, MT(ASCP)SBB Director, Clinical Education BloodCenter of Wisconsin

Milwaukee, WI

Objectives

• Describe the serologic findings that characterize WAIHA and cold agglutinin syndrome.

• Discuss the laboratory investigation and methods used to evaluate patients with autoimmune hemolytic anemia.

• Discuss the role of blood transfusion in autoimmune disorders, problems encountered in obtaining "compatible 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-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

http://www.bbguy.org/2017/02/27/028/

Positive Polyspecific

DAT

Perform DAT with Monospecific Reagents

and Controls

What is Positive?

Anti-C3

Strength of Reaction?

2-4+≤1+

Acute or Delayed

HTR

DIIHA CAD

Both Anti-IgG and C3


≤1+ 2-4+

Acute or Delayed

HTR

WAIHADIIHA

Anti-IgG


≤1+ 2-4+

Acute or Delayed

HTR

ABO HDFN

Rh HDFN

WAIHADIIHAOther HDFN

Passively Acquired Antibody

PCH

*Serological results must be correlated with

clinical findings.

Courtesy of C Feldman & J O’Connor

AIHA Serologic Types

Serologic Type %

Warm, DAT – IgG only 43

Warm, DAT - IgG & C3 17

Cold, DAT - C3 27

Mixed, DAT - IgG & C3, Warm & Cold Autoantibody

8

Atypical 5

Total Patients

Barcellini et al. Blood. 2014 Nov 6;124(19):2930-6


49

AIHA



Warm Autoimmune Hemolytic Anemia

Incidence and Cause

– 1º - idiopathic

– 2 º - lymphoma, SLE, carcinoma

– Most common type of immune hemolysis

– IgG antibody reactive at 37ºC

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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Warm Autoantibody in Plasma (3+)

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Antibody Identification – Saline or LISS

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


50

AIHA

Warm Autoantibody Common Specificities – Rh-related

• Broad: Negative with Rhnull cells

• Single: -c, -e, -D, -C, -E, -f

• Relative: c-like, e-like

Uncommon Specificities

• M, N, S, U, EnaFS, EnaFR, Wrb

• K, Kpb, K13

• LWa, LWab, Jka, Jkb, Jk3

• A, B, IT

• Ge, Vel, AnWj, Sc1, Sc3, Rx

Relative Specificity: -e-like

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 + + 1

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

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 1

Auto 4

Antibody Identification – Saline

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

1 + + 0 0 + 0 + 0 + 0 0 + 2 2

2 + + 0 0 + + 0 + + + + + 1 2

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

4 + 0 + 0 + 0 0 + 0 + 0 + 0 2

5 0 + + 0 + 0 + + + 0 + 0 2 2

6 0 0 + 0 + + 0 + 0 + + + 0 2

7 0 0 + 0 + 0 + 0 0 + 0 + 0 2

8 0 0 + 0 + 0 0 + + 0 + 0 2 2

Auto 4

Autologous Adsorption

• Prepare DAT Negative RBCs

–ZZAP

• Ficin or Papain

• 2-ME or DTT

–W.A.R.M.TM

–Enzymes

• Ficin or Papain

ZZAP Treat Patient RBCs

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After ZZAP Treating

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AIHA


ZZAP RBCs Pt Serum + ZZAP RBCs

Mix & Incubate at

37C

= Anti-Jka

= WAA


Centrifuge Pt Serum +

ZZAP RBCs

Adsorbed

Serum

= Anti-Jka

= WAA

Allogeneic Adsorption

Other Methods

• Gluteraldehyde -Treated RBCs

• Stroma

• PEG

Other Considerations

• Perform cell separation & phenotype or genotype

• Select phenotype matched RBCs

• Treat RBCs with ZZAP or enzymes

Allogeneic Adsorption Patient’s Phenotype is Unknown

RBC Phenotype Antibodies Remaining

R1R1; Jk(a-) -c, -E, -Jka

R2R2; Jk(b-); S- -e, -C, -Jkb, -S rr; K-; s-

-D, -C, -E, -s

Allogeneic Adsorption

Pt Serum +

R2R2, Jk(a+) RBCs

Pt Serum +

R1R1, Jk(a-) RBCs

Pt Serum +

rr, Jk(a-) RBCs

= Anti-Jka

= Jk(a+) RBCs

= WAA

Allogeneic Adsorption After 37C Incubation

Pt Serum + R2R2, Jk(a+) RBCs

Pt Serum + R1R1, Jk(a-) RBCs

Pt Serum + rr, Jk(a-) RBCs

= Anti-Jka

= Jk(a+) RBCs


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AIHA

Testing Adsorbed Serum/Plasma Positive Results

• Adsorption not complete

– Reactivity is weakened but consistent with RBCs tested

– Perform additional adsorptions

• Antigen destroyed by enzymes or ZZAP

– No change in reactivity post adsorption

– Test raw serum with enzymes or ZZAP treated RBCs

– Perform adsorptions with untreated RBCs

• Reactivity due to alloantibody

– Some positive, some negative results

– Test adsorbed serum with DAT negative RBCs

– Test adsorbed serum with phenotypically similar RBCs

– Identify alloantibody

Alloadsorbed Serum Saline Tube

R1R1, Jk(a-)

IAT

R2R2,

Jk(a+)

IAT

rr,

Jk(a-)

IAT

I Jk(a+) 2+ 0√ 2+ II Jk(a-) 0√ 0√ 0√ III Jk(a+) 2+ 0√ 2+

Serum Adsorbed with

R1R1, Jk(a-)

IAT

R2R2,

Jk(a+)

IAT

rr,

Jk(a-)

IAT

I Jk(a+) 2+ 2+ 2+ II Jk(a-) 2+ 2+ 2+ III Jk(a+) 2+ 2+ 2+

Serum Adsorbed with

R1R1, Jk(a-)

IAT

R2R2,

Jk(a+)

IAT

rr,

Jk(a-)

IAT

I Jk(a+) 1+ 1+ 1+ II Jk(a-) 1+ 1+ 1+ III Jk(a+) 1+ 1+ 1+

Serum Adsorbed with

Adsorption not complete Antigen on adsorbing RBCs denatured No adsorption

Underlying alloantibody

ELUATE D 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

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

Last Wash Eluate WAIHA vs. DIIHA

• Eluate

• WAA strongly positive

• DDA is negative or weak

• Serum

• WAA persists

• DDA disappears within days if drug is discontinued

Management of WAIHA

• Steroids • Rituximab • Immunosuppressive drugs - Cytoxan • Intravenous Immune Globulin (IVIG)

• Plasmapheresis

• Others, Cyclosporine

Avoid transfusion unless life-threatening hemolysis!

Unusual AIHAs

• ~13% of AIHAs* are Unusual Types

• ~ 8% Mixed Type AIHA*

– DAT positive with both IgG & C3

– Warm & Cold Autoantibody present

• DAT-Negative AIHA – IgA warm autoantibody-induced AIHA

– IgM warm autoantibody-induced AIHA

• IgM warm-reactive autoantibody

*Barcellini et al. Blood. 2014 Nov 6;124(19):2930-6

IS 37C IAT I 0 2-4+ 0 - 1+ II 0 2-4+ 0 –1+


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AIHA

WAIHA Associated With a Negative DAT

• 1-5% of patients with signs & symptoms of hemolysis

• Low affinity IgG autoantibodies

• Small amount of bound IgG

• IgM and IgA autoantibodies

Cold Saline Wash Cold LISS Wash Direct PEG Direct Polybrene ELAT Flow Cytometry

Enhanced DAT Methods

Cold-Hemagglutinin Disease (CHD)

Acute Lymphoproliferative

disorders Mycoplasma

pneumonia infection Infectious

mononucleosis

Chronic More severe symptoms Elderly, during cold

weather Hemolysis mild-

moderate

Serology • DAT shows complement

only • Antibody characteristics

Increased thermal amplitude

IgM antibody Reacts up to 37ºC in

albumin Titer > 1000 at 4ºC

Cold-Reactive Autoantibodies Phase & Strength of Reactivity

IS 37C IAT

I 4+ 3+ 0-3+

II 4+ 3+ 0-3+

Antibody specificity Most commonly anti-I Occasionally anti-i associated with infectious mononucleosis

Clinical Manifestations of CAD

• Mild chronic anemia • Occasional jaundice and pallor

• Some patients have increased episodes of hemolysis associated with hemoglobinuria and hemoglobinemia upon cold exposure

• Hemolysis usually self-limited if associated with mycoplasma or other viral infection

Management of CAD

• Avoid cold

• Plasmapheresis - improvements only temporary.

• Steroids and splenectomy of little benefit.

• IVIG of little benefit.

Drug-Induced Immune Hemolytic Anemia

• DAT - Reactivity depends on time of testing compared to presentation

Polyspecific AHG 3-4+

Anti-IgG 3-4+

Anti-C3 3-4+

Control 0

• Eluate

– Rapid Acid

– Most are negative

– Few are disproportionately weaker as compared to strength of DAT


54

AIHA

DIIHA Serum Reactivity

• Reactivity depends on time of testing compared to presentation

• Saline IAT - some positive

• PEG or Ficin IAT - many positive

IS 37C IAT

I 0 0 0 - 4+ II 0 0 0 - 4+

Drug Binds to RBCs (Hapten) Drug Adsorption

• Penicillin/penicillin derivatives & cephalosporins

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

• Antibody reacts with normal RBCs pretreated with drug

Drug-Dependent Drug Does Not Coat RBCs

“Immune Complex”

• Quinidine, quinine, NSAIDs

• Through an unknown mechanism, drug induces antibodies that bind to RBC only when drug is present in soluble form

• Antibody reacts with RBCs when soluble drug is present

Drug-Induced Autoantibody

• Alpha methyldopa, procainamide

• Through an unknown mechanism, drug induces autoantibodies specific for RBC membrane proteins

• Antibody reacts with normal RBCs in the absence of drug

Nonimmunologic Protein Adsorption (NIPA) Membrane Modification

• Cephalosporins

• Tazobactum, Clavunate

• Drug coats RBCs and causes them to become “sticky”

• DAT - weakly positive

• Rarely associated with DIIHA

Testing Drug-Treated RBCs

Patient Serum Pos Con Normal Serum

15’RT 4 4 0

30 37C 4 4 0

IAT 4 4 0

15’RT 0

30 37C 0

IAT 0 Un

trtd

RB

Cs


55

AIHA

Testing in Presence of Drug

e+ RBCs 30’RT 60’ 37C IAT

Patient Serum + Drug 4+ 4+ 4+

Patient Serum + Diluent 0 0 0

Diluent + Drug 0 0 0

Eluate + Drug 0 0 0/3+

Eluate + Diluent 0 0 0

Positive Control + Drug 4+ 4+ 4+

Drug-Induced Hemolytic Anemia Treatment

• STOP the DRUG!!!!

• Treat the symptoms

• Don’t take drug in the future

AIHA & DIIHA - Things to remember…

• Always look for medication history in a question but don’t assume it’s the culprit

• Look at strength of reactivity of DAT

• Correlate DAT reactivity with patients clinical information

• DAT strongly positive, Eluate negative is key finding in DIIHA BUT patient should be showing clinical signs of hemolysis

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

21 Case Studies – all with positive DATs

22 Case Studies – all antibody problems, simple to complex

Questions & Answers covering broad range of TM topics

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: 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.

• 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.

Questions?

[email protected]


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AIHA

