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,
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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
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
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
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
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
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
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?
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
• 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.