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