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Fetal RHD a RHCE status determination from maternal circulation, alloimmunisation 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) Kidd (Jka, Jkb) Diego (Dia, Dib) 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 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 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 1. 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) 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 homozygote Noninvasive RhD status determination 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 HDN Determination 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 HDN Determination 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., 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 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. recommendation erythrocyte 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 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 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