Post on 11-Mar-2019
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
Kell antigens -K, k -Kpa, Kpb
-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
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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
Kell antigens -K, k -Kpa, Kpb
-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
Kell antigens -K, k -Kpa, Kpb
-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
Kell antigens -K, k -Kpa, Kpb
-Jsa, Jsb
Kx
Normal
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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
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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
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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 +
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Kell, Duffy, Kidd
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 +
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?
Cell K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb
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
Cell K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb
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
© 2017 Last Chance Review SCABB / BloodCenter of Wisconsin
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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
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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
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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
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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
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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
© 2017 Last Chance Review SCABB / BloodCenter of Wisconsin
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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?
© 2017 Last Chance Review SCABB / BloodCenter of Wisconsin
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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
Strength of Reaction?
≤1+ 2-4+
Acute or Delayed
HTR
WAIHADIIHA
Anti-IgG
Strength of Reaction?
≤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
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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
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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
Autologous Adsorption
ZZAP RBCs Pt Serum + ZZAP RBCs
Mix & Incubate at
37C
= Anti-Jka
= WAA
Autologous Adsorption
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+
© 2017 Last Chance Review SCABB / BloodCenter of Wisconsin
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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
© 2017 Last Chance Review SCABB / BloodCenter of Wisconsin
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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
© 2017 Last Chance Review SCABB / BloodCenter of Wisconsin
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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?
sue.johnson@bcw.edu
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