fetal rhd a rhce status determination from maternal circulation, alloimmunisation assoc. prof. ilona...
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Fetal RHD a RHCE status determination from maternal circulation, alloimmunisation
Assoc. Prof. Ilona Hromadnikova, PhD.
Department of Molecular Biology and Cell Pathology
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
Anti-c, anti-E, anti-C, anti-e
Alloantibodies x other blood group antigens
Lewis (Lea, Leb) Duffy (Fya, Fyb)Kidd (Jka, Jkb) MNSs (M, N, S, s, U)Diego (Dia, Dib) 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
RHD gene – many variants In the Czech republic – most common variants:
D VI, DFR, D VII, DCS 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
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
Incompatible blood transfusion In previous pregnancy –passage of fetal
cells into maternal circulation Invasive procedure(CVS, AMC, cordocentesis) Miscarriage Delivery Bleeding during pregnancy
Colour Atlas of Immunology
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
IgG – transcytosis via syncytiotrophoblast cells1. active process using receptor2. pinocytosis
Vesicle fusion, in endosomes↓ pH – binding to FcRn(IgG unbound – lysosomal degradation)
Exocytosis on the basal surface, diffusion to fetal circulation
Immunopathological reaction type II – cytotoxic Abs ADCC (antibody-dependent cell-mediated
cytotoxicity ) → destruction of fetal erythrocytes by splenic macrophages
Colour Atlas of Immunology
3 stages:1. Anemia neonatorum – low levels of Hb and
hematocrit2. Icterus neonatorum (more serious anaemia,
hepatosplenomegaly, without treatment – bilirubin encephalopathy)
3. Hydrops fetalis(generalized edema of the fetus with fluid accumulation in the body cavities, very bad prognosis)
Danger of anaemia – positive indirect antiglobulin (Coombs) test
Detection of 1 or more Abs associated with HDN in maternal serum
Colour Atlas of Immunology
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 Noninvasive RhD status determination
homozygote Heterozygote, 50% probability of fetal RhD positivity
RhD negative
RhD positive
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
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%)
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)
Our laboratory – combination of exon 7 and 10
with 100 % specificity a 100 % sensitivity
RhD negative foetuses at alloimmunized pregnancies - not endangered by HDN
RhD positive foetuses – important information for clinicians
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 HDNDetermination of fetal RhC alelle
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 HDNDetermination of fetal Rhe alelle
SNP exon 2 (Rhc) Specific insertion in intron 2 (RhC)
SNP exon 5 (RhE/Rhe)
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
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., 2005Kenneth, 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
Gestational age Maturity of lungs General fetal condition
↑ gestational age - ↓ postnatal risk
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. recommendationerythrocyte transfusion preparates Hct 80 – 85%→ final fetal Hct 40 – 45%
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
Repeated intraumbilical transfusions, latest at 34th – 35th week of gestation, usually followed by SC between 37th – 38th week
Intrauterinely bilirubin excreted by placentax 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
Immediately after delivery do tests for: 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)
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
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 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.
Prevents sensibilisation of maternal IS Application of anti-D Abs – blocks Ag
Foetus (D) Mother (d)
IgG-anti-D (prophylaxis)
Prevents Abs production, hemolysis
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
In the case of RhD negative foetus – no RhD prophylaxis necessary