Transcript RHD

Fetal RHD a RHCE status determination from
maternal circulation, alloimmunisation
Assoc. Prof. Ilona Hromadnikova, PhD.
Department of Molecular Biology and Cell
Pathology
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Alloimmunisation – production of maternal
antibodies against Ags on fetal erythrocytes
Placental transfer of IgG
Destruction of fetal erythrocytes
→ Erythroblastosis fetalis and HDN is most often
caused by incompatibility in RhD system
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Anti-c, anti-E, anti-C, anti-e
Alloantibodies x other blood group
antigens
Lewis (Lea, Leb)
Kidd (Jka, Jkb)
Diego (Dia, Dib)
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Duffy (Fya, Fyb)
MNSs (M, N, S, s, U)
Lutheran (Lua, Lub)
AB0 incompatibility (women 0, man A
or B; anti-A, anti-B IgG production, rare
intrauterine hemolysis,
hyperbilirubinemia postnatally,
risk of icterus
E. Sjoberg-Wester; Jill Storry
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RHD gene – many variants
In the Czech republic – most common variants:
D VI, DFR, D VII, DCS
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Rearrangement between RHD and RHCE genes, point
mutations
Frequency – about 1% in Caucasian population
RhD variant protein – absence of 1 or more epitopes
→ women is laboratorally positive but can produce
anti-D antibodies against „missing epitopes“
Women with variant RhD protein – considered as RhD
negative, can cause HDN
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Lower expression of RhD protein (weak RhD, Du) on
erythrocyte surface, but with more or less full D
epitope „repertoire“
(could be serologically negative with weak Rh
positivity)
Patients with weak RhD antigen – no production of
anti-D Abs, no risk of HDN
Prophylaxis not necessary
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Incompatible blood transfusion
In previous pregnancy –passage of fetal
cells into maternal circulation
Invasive procedure(CVS, AMC, cordocentesis)
 Miscarriage
 Delivery
 Bleeding during pregnancy
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Colour Atlas of Immunology
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IgG active transport (all 4 subclasses of
alloantibodies) across the placenta
Abs transport– low till the 20th week of gestation, then
exponential increase
30th week – ½ of serum maternal concentration [IgG]
At time of delivery: [IgG] in fetal circulation about 10%
higher than in maternal serum
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IgG – transcytosis via syncytiotrophoblast cells
1. active process using receptor
2. pinocytosis
Vesicle fusion, in endosomes↓ pH – binding to FcRn
(IgG unbound – lysosomal degradation)
Exocytosis on the basal surface, diffusion to fetal
circulation
Colour Atlas of
Immunology
Immunopathological reaction type II – cytotoxic Abs
 ADCC (antibody-dependent cell-mediated
cytotoxicity ) → destruction of fetal erythrocytes by
splenic macrophages
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2.
3.
3 stages:
Anemia neonatorum – low levels of Hb and
hematocrit
Icterus neonatorum (more serious anaemia,
hepatosplenomegaly, without treatment – bilirubin
encephalopathy)
Hydrops fetalis(generalized edema of the fetus
with fluid accumulation in the body cavities, very
bad prognosis)
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Danger of anaemia – positive indirect antiglobulin
(Coombs) test
Detection of 1 or more Abs associated with HDN in
maternal serum
Colour Atlas of
Immunology
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First screening between 10th and 12th week of
gestation, all pregnant women
Determination of maternal blood group
Negativity – again in the 24th and 32nd week
Positivity – determination of Ab specificity and titer
1:8 and higher – repetition every month
titre 1:16 and higher for anti-D Ab; 1:8 for Kell Ab and
higher – risk of HDN
Antibodies present (Indirect Coombs test positive)
Father homo/heterozygosity in RHD gene
homozygote
Noninvasive RhD status determination
Heterozygote,
50% probability of fetal
RhD positivity
RhD negative
RhD positive
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At anti-D alloimmunized RhD negative
pregnant women at risk of HDN
RhD negativity – deletion of RHD gene in
Caucasian population
RHD (pseudogene)
Complete inactive RHD gene, 37-bp insertion in
exon 4 (PCR) + 1-2 stop codons in exon 6,
earlier termination of translation, 0 HON
66% of Africans, 27,7% Japaneses and11% of
Brazilian
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Hybrid RHD-CE-D gene
RhD negative phenotype: 3´ end of exon 3 a
exons 4-8 of RHCE gene
RHD exon 10 +, exon 7 – (PCR)
Weak C, VS+, Africans (3%)
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RHD genotyping– necessary to analyse more
regions of RHD gene
Most often combination of exon 7 and 10 or exon
7 and 5
Interpretation of results together with ethnic group
(incidence of RHD gene alterations)
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Our laboratory – combination of exon 7 and 10
with 100 % specificity a 100 % sensitivity
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RhD negative foetuses at alloimmunized
pregnancies - not endangered by HDN
RhD positive foetuses – important information for
clinicians
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At anti-c alloimmunized CC homozygous
pregnant women at risk of HDN
Determination of fetal Rhc allele
At anti-C alloimmunized cc homozygous pregnant
women at risk of HDN
Determination of fetal RhC alelle
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At anti-E alloimmunized ee homozygous pregnant
women at risk of HDN
Determination of fetal RhE allele
At anti-e alloimmunized EE homozygous pregnant
women at risk of HDN
Determination of fetal Rhe alelle
SNP exon 2 (Rhc)
Specific insertion in intron 2 (RhC)
SNP exon 5 (RhE/Rhe)
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RHD exon 7 and exon 10, RHCE - C allele
detection with 100 % specificity and 100 %
sensitivity
RHCE - c allele and E allele genotyping (SNP) –
100 % specificity and 95 % sensitivity, more difficult
– most of cell-free DNA is of maternal origin
RhcCE negative foetuses at alloimmunized
pregnancies – not endangered by HDN,
positive foetuses – early information for clinicians
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Stage of sensitisation
 Titer, specificiy, concentration, avidity and
subclass of IgG
 Expression of antigen on erythrocyte surface
(weak RhD antigens)
 Gestational age
 Presence of „blocking“ antibodies in maternal
serum
→ other examination necessary: ultrasound, Doppler,
cordocenthesis
Spectrophotometric measurement of bilirubin in
amniotic fluid – serial AMC
 Not used anymore – intensify immunization
Ultrasound examination
 Compensatory reaction
symptoms
(hepatosplenomegaly),
occurrence of ascites,
hydrops)
Queenan et al., 1993
Doppler measurement of the fetal
middle cerebral artery peak
systolic velocity - arteria cerebri
media
 In the anemic fetus, low blood
viscosity and increased cardiac
output contribute to an
increased blood velocity,  PI
Doubek a kol., 2005
Kenneth, 2004
Cordocenthesis
 Mostly used for access to fetal
circulation
 Often continue from diagnostic to
therapeutic procedure –
intraumbilical transfusion
 Possibility to determine fetal Rh
status and blood count
 From the 20th week of gestation,
not convenient after 34th week of
gestation
2 therapeutic alternatives:
1.
Intrauterine transfusion(5% risk)
2.
Preterm delivery
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Gestational age
Maturity of lungs
General fetal condition
↑ gestational age - ↓ postnatal risk
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Firstly at 1963
Usually intraumbilical transfusion, refill an/or
exchange blood transfusion under ultrasound
control
No consensus about fetal hematocrit– 40 – 65%
But when hematocrit over 50% - ↑ blood viscosity,
hypoxia in some organs
Complications – severe bradycardia
Hájek et al. recommendation
erythrocyte transfusion preparates Hct 80 – 85%
→ final fetal Hct 40 – 45%
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From the 20th till the
34th week
7-10 days interval
(shorter in cases of
hydrops fetalis)
Pregnancy termination
in 2-3 week after last
transfusion
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Repeated intraumbilical transfusions, latest at 34th
– 35th week of gestation, usually followed by SC
between 37th – 38th week
Intrauterinely bilirubin excreted by placenta
x lower conjugation of higher levels of bilirubin in
fetal liver – increase of indirect bilirubin,
accumulation in basal ganglia
→ icterus – bilirubin encephalopathy
Application of phenobarbital (30 mg per day, 10
days before delivery) – improves bilirubin
metabolism in fetal liver
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Immediately after delivery do tests for:
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pH and blood gases
Blood group and Rh status
Hemoglobin a hematocrit
Control of bilirubin levels
Anti-D Abs – direct Coombs test
Phototherapy, transfusion
After repeated IUT – newborn usually has mild or middle
anaemia, icterus is usually curable only with phototherapy
without transfusion
The biggest problem – patients with repeated hydrops fetalis
before the 20th week of gestation (no effective treatment)
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In 1950s, every 2nd alloimunized woman lost her
baby, very serious problem till 1970s
RhD prophylaxis in RhD negative women –
decrease of perinatal morbidity and mortality
Still Rh alloimmunization is problem because of
No prophylaxis after delivery
 No prophylaxis after some other invasive procedures
 Low prophylaxis dosage after intensive bleeding
during delivery
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Application of anti-D Abs, half-time ~16 days
From the 1960s
Decrease sensibilisation from 8% to 0,8% of all
pregnancies
Only for non-sensitized women (no anti-D Abs in
maternal blood)
Partobulin, Rhega
Administration necessary 72h post-partum or after
sensibilisation event
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After delivery: usually 250 – 300 μg intramuscularly
(20 μg anti-D – neutralisation of 1 ml RhD positive blood)
Intensive bleeding (SC)– higher dosage - 500 μg i.m.
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Prevents sensibilisation of maternal IS
Application of anti-D Abs – blocks Ag
Foetus (D)
Mother (d)
IgG-anti-D
(prophylaxis)
Prevents Abs production, hemolysis
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Next to elimination of fetal erythrocytes (no B cell
activation)
Active supression of B lymphocytes by
immunocomplexes of RhD antigens with binded Abs
BCR a Fc receptors aggregation
Hořejší a Bartůňková, 2001
Necessary application of RhD prophylaxis after:
 Delivery of RhD positive child
 After miscarriage or abortion
 After ectopic pregnancy
 After procedures connected with increased
fetomaternal bleeding (invasive procedure)
 After bleeding generally (blunt hits into belly)
 After transfusion of RhD positive blood
No maternal complication
 Still blood preparate – usually from donors
with ↑ anti-D Abs levels
 Risk – haemolytic anaemia in foetus,
passage of Abs via placenta and
destruction of fetal erythrocytes
But application of low doses, low risk of fetal
damage
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In the case of RhD negative foetus –
no RhD prophylaxis necessary