Immunohematology of inherited and acquired blood group alterations

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Immunohematology of inherited and acquired blood
group alterations

• Günther Körmöczi
• Department of Blood Group Serology
• and Transfusion Medicine
• Medical University of Vienna, Austria

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Unusual blood group phenotypes

• inherited
• blood group variants (A3, weak D, Kmod )
• present at birth
• chimerism (more than 1 zygote)
• acquired
• genetic alteration gt mosaicism

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Rh a complex example

• RhD variants
• partial D types
• weak D types
• DEL types
• acquired Rh phenotype changes

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The RH genes RHD and RHCE
1p36.11
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The Rh polypeptides RhD and RhCcEe
extracellular
intracellular
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The D antigen (Rh factor)
extracellular
Europe 83 D 17 D-
consists of gt30 D epitopes
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D most immunogenic red cell antigen

• anti-D immunization of
• D- patients by D transfusion - 80
• D- pregnant women by D fetus 15
• (without Anti-D prophylaxis)
• hemolytic transfusion reaction
• hemolytic disease of the fetus and newborn
• reliable D typing !

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The serologic spectrum of D antigen expression
red cells with anti-D in indirect antiglobulin
test (IAT)
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Dweak

• weak D types
• intracellular RhD alterations
• 1) RHD point mutations
• no D epitope loss
• D antigen density reduced

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Dweak

• weak D types
• intracellular RhD alterations
• 1) RHD point mutations
• no D epitope loss
• D antigen density reduced

• partial D
• extracellular RhD alterations
• 1) RHD point mutations
• 2) RH gene hybridizations
• RHD-CE-D
• D epitope loss
• alloanti-D possible

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Quantitative and qualitative differences
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Weak D types reduced D antigen numbers
without D antigen alteration (all D epitopes)
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D antigen density determination

• monoclonal IgG anti-D
• indirect immunofluorescence with anti-IgG
  Fab-FITC
• flow cytometry
• median fluorescence intensities
• comparison with standard cell

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Typical D antigen densities
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Qualitative analysis of the D antigen
monoclonal anti-D antibodies against single D
epitopes gt D epitope mapping
partial D with epitope loss
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Anti-D panel for routine serology
6 different monoclonal anti-D antibodies (in IAT)
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D epitope profiles of partial D types
Scott 2002
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The upper end of the spectrum

• Austrian female, 74a
• routine serology D
• anti-D (titer 4)
• exclusion of anti-LW
• direct antiglobulin test negative
• point mutation RHD(M358T)

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An almost normal partial D DWI

• 8.300 D antigens/red cell
• only 1 out of 79 anti-D monoclonals not reactive
• minor D epitope loss gt alloanti-D (D
  pregnancies!)

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Crossmatch with DWI-positive relatives
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Immunogenicity of very weak RhD variants

• anti-D immunization of D- transfusion
• recipients by
• weak D type 2 450 D/cell
• novel weak D type 26 29-70 D/cell
• DEL types lt 30 D/cell

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DEL extremely weak RhD variants

• routine serology (including IAT) D-
• detection only by adsorption/ELution of anti-D
  reagent
• discovery
• molecular genetic screening
• lookback examination

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DEL RHD missense and splice site mutations
all DEL alleles are associated with Ce
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Anti-D immunization by DEL transfusion

• RHD(IVS5-38del4) - primary/secondary
• Wagner T et al. Transfusion 200545520
• RHD(K409K) - secondary
• Yasuda H et al. Transfusion 2005451581
• gt extremely low D antigen dose required

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DEL antigen expression
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DEL red cells with anti-D in IAT
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Serologic detection of DEL by adsorption/elution
red cells anti-D adsorption ? multiple
washing ? red cell sediment acid pH ? testing
of eluate (in IAT with D cells)
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Epitope mapping of RHD(IVS31ggta) with IgG anti-D
monoclonal antibodies
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Adsorption/elution with IgM anti-D
adsorption at RT gt heat elution gt testing (in
neutral gel)
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DEL epitope mapping by adsorption/elution
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Alloanti-D in partial DEL individuals

• all cases were RHD(IVS31ggta)
• female, 21a anti-D titer 32
• no transfusion or (known) pregnancy
• female, 24a anti-D titer 256
• no transfusion or (known) pregnancy
• male, 65a anti-D titer 4
• numerous transfusions gt30a ago

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A continuum Dweak ? DEL

• RHD(M295I)
• with ce (in cis) weak D type 11 (200 D/cell)
• with Ce (in cis) DEL
• weak D type 32 (Ce in cis)
• no C in trans 49 D/cell
• with C in trans DEL
• weak D type 26 (29-70 D/cell)

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The spectrum of D expression

• normal D
• partial D weak D types
• partial DEL complete DEL

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Clinical implications

• normal D
• partial D weak D types
• partial DEL complete DEL

elicit anti-D
can be anti-D immunized epitope loss gt only D-
transfusions, anti-D prophylaxis
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(No Transcript)
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Acquired Rh phenotype changes
no transfusion or HSCT
genotype CcDdee
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Mixed-field agglutination in blood grouping

• transfusion
• hematopoietic stem cell transplantation
• fetomaternal transfer
• technical error
• chimerism
• genetic alterations gt mosaicism

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Spontaneous mixed-field Rh typing

• Nine patients
• 4 male, 5 female (median age 63 years)
• 3 with hemato-oncologic disease
• D red cell fraction 22-85
• median follow-up 33 months
• RhD status over time
• stable mixed field to complete loss

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RhD phenotype splitting
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D and D-red cells
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Haplotypic Rh antigen loss ?

• normal
• ABO
• MNS
• P
• Lu
• Kell
• Fy
• Jk

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Haplotypic Rh phenotype splitting
DC DC D-C-
D-C-
DC
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zygosity
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Microsatellite marker analysis no chimerism
max. 2 alleles each
Patient 1
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Microsatellite markers on chromosome 1
finger nail hair root
blood
gt loss of heterozygosity (LOH) in blood cells !
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LOH onchromosome 1
only in myeloid cells
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Variable expansion of LOH
chromosome 1
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Nucleated erythroid precursors BFU-E
DNA isolation from single clones
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RH-PCR of single BFU-E colonies
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LOH only in RHD- BFU-E colonies
Blood
BFU-E CcDe
BFU-E only ce (C-D-)
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FISH 1p (RH, green) and 1q (red)
1x RH
2x RH
myeloid
lymphoid control
deletion
somatic recombination with duplication
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Myeloid stem cells with LOH
homozygous cde/cde all other patients
hemizygous cde/- - - patient 3
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Gene dose and Rh antigen expression
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Spontaneous Rh phenotype splitting

• mosaics (only rarely chimeras)
• LOH on chromosome 1
• myeloid stem cells
• duplication or deletion
• oncogenesis
• tumor suppressor genes on 1p
• LOH ? cancer
• genetic index for aging ?
• anti-D immunization by mosaic blood donors

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Contributing scientists

• Shenzhen (China)
• Chao-Peng Shao
• Bristol (UK)
• Geoff Daniels
• Tokyo (Japan)
• Makoto Uchikawa
• Graz (Austria)
• Thomas Wagner

• Vienna (Austria)
• Günther F. Körmöczi
• Wolfgang R. Mayr
• Simon Panzer
• Göttingen (Germany)
• Tobias J. Legler
• Innsbruck (Austria)
• Christoph Gassner
• Diether Schönitzer