Obstetrics Flashcards
What is FMH
Cross-placental transfer of foetal red cells into the maternal circulation
Antibodies usually associated with HDFN
Anti-K often causes erythroblastopenia and cord DAT may be negative.
Antibodies occasionally associated with HDFN
Usually IgM (do not cross placenta)
Usually cold-reacting
Often not fully developed on neonatal red cells (e.g. Lewis system)
When to test for antibodies in HDN?
First booking (8-12 weeks)
Repeat at 26-28 weeks (prior to any anti-D prophylaxis in RhD negative mothers given at 28 & 34 weeks)
For mothers with established allo-antibodies:
- 4 weekly until 28 weeks
- Then 2 weekly until delivery
Levels of concern (refer to MFM):
- Titre >=1:32
- “clinically significant rise” for non-D antibodies,
- >=1:16 or Quant >15IU/mL for D antibodies
- any level of anti-K antibodies ,
- c- antibodies= 7.5
Further testing to stratify risk once antibodies in HDFN are established
- paternal blood phenotype and genotype is determined to predict the foetal risk of inheriting the antigen the maternal antibody is directed against
- Direct foetal genotyping can establish foetal blood group expression, either by CVS or foetal DNA obtained from the maternal serum (NIPA)
- Serial testing
Monitoring once established high risk of HDFN
Maternal antibody titres: increasing antibody strength indicates ongoing and increasing antibody strength, except in cases of previously-affected pregnancies; there is no strong correlation between titre strength and degree of foetal anaemia
RCOG suggests MFM referral for non- anti-D antibodies once they reach a titre of 1:32
All new maternal allo-antibodies and doubling of titres are reportable at the RWH
Cerebral MCA Doppler velocity assessments: performed 2-weekly after 16-24/40 as a validated surrogate for foetal anaemia.
+/-Weekly CTGs may also be performed.
Invasive testing : Cordocentesis is performed to determine foetal haematocrit and determine need for IUT.
Selection of blood for IUT
group O, RhD matched, irradiated, CMV-negative, less than 5 days old, and antigen-negative for maternal red cell antibodies
Usually also Rh C, c, E, e and K matched
Risk of IUT - infection, rupture of membranes
What is NIPA testing?
Non invasive prenatal analysis for fetal RhD.
Can be performed > 12/40
Molecular blood group genotyping assay to predict RhD status of fetus in RhD negative mothers.
Uses maternal PB whoe blood for extraction of cell free fetal DNA and analysed for presence of RhD Gene.
Performed at Red Cell Reference Lab in QLD.
Specificity >98% and sensitivity >99%
Who is offered NIPA testing?
- RhD negative pregnant women who are RhD alloimmunised
- RhD negative pregnant women with obstetric indications such as severe FMH during pregnancy or IU fetal death.
- Or in non sensitised RhD women in which there is a relatiev contraindication to anti-D prophylaxis (religious beliefs, prior reaction)
Anti-D administration - principle
Risk of anti-D sensitization in a pregnancy is up to 20% if anti-D is not used when there is a post-natal FMH, leading alloimmunization and risk of neonatal jaundice and/or anemia requiring IUT in future pregnancies
Also risk of alloimmunization earlier in pregnancy with other sensitizing event eg. clinical haemorrhage or other sensitizing event
Anti-D prophylaxis in the post-natal setting reduces this risk to 1-1.5%
Anti-D prophylaxis in the post-natal and pre-natal setting further reduces this risk to 0.2%
When Anti-D is not recommended
RhD positive woman
Baby is known to be D negative
Mother is already alloimmunised
Anti-D dosing
28/40: 625 IU RhD Immunoglobulin-VF for IMI*
34/40: 625 IU RhD Immunoglobulin-VF for IMI
Delivery of baby - given atleats within 72 h
Anti-D lasts 6 weeks; count backwards from 40/40
100 IU of Rh(D) Immunoglobulin-VF protects against a FMH of 1mL of foetal Rh(D) positive red cells (2mL of whole blood)
Sensitising event
- first trimester 250IU
- second/third trimester 625IU
RhD Ig products available
Dosing of anti-D in large FMH
For FMH >15mL (designated large-volume), a follow-up FMH should be performed 48h post anti-D administration and further anti-D given if FMH is still positive and RhD Ig is not detected by IAT in maternal plasma
FMH testing and subsequent actions
What is haemolytic disease of the fetus and newborn?
haemolysis in fetus and newborn due to maternal antibody
-antigen inherited from father
-IgG implicated isotype (IgM and IgA do not cross placenta)
Not all result in clinically significant disease
-1/2 mild and deliver at term
-1/4 moderate disease (top up Tx or exchange at birth)
-1/4 severe, require intrauterine transfusion, early delivery, exchange transfusion
Incidence of clinically relevant antigens in FMH
D antigen expressed by D38 of gestation
Clinical features of FMH
Mild:
-early onset jaundice (unconjugated bilirubinaemia within 24 hours of birth)
-symptomatic anaemia without circulatory collapse (lethargy, tachycardia, poor feeding)
-thrombocytopenia (up to 25% due to suppression of thrombopoiesis, in response to increased erythropoiesis)
Severe (hydrops fetalis):
-two or more of: diffuse skin oedema, pleural/pericardial effusions and ascites
-when fetal Hb deficit 7g/dL or more below mean for gestational age (or eg// Hb <5g/dL, Hct <15%)
-concomitant thrombocytopenia and neutropenia
ABO
-usually not clinically significant disease
-hyperbilirubinaemia within 24 hours of birth if affected
Investigations of FMH
Neonatal Testing:
-maternal and infant group and maternal Ab screen
-bilirubin, retic count, FBE
-DAT
-positive: consistent with HDFN (false positives –> Wharton’s jelly if cord blood)
-negative: does not exclude HDFN esp in ABOi (poorly developed antigens), elution + IAT
-IUT can give false negative DAT
*haemolysis screen may be negative in anti-K due to erythroblastopenia
FMH treatment - postnatal considerations
-delayed cord clamping (assoc with lower incidence of anaemia and exchange transfusion)
Postnatal transfusion
-for hydrops fetalis
-volume restriction due to fluid overload state
-exchange transfusion recommended for HF, severe anaemia/hyperbilirubinaemia
-simple transfusion preferred for non-severe anaemia and hyperbilirubinaemia
Hyperbiliruinaemia (general)
-oral hydration
-phototherapy to prevent neurotoxicity
Kleihaur Betke test - principle
Relies on the fact that HbF is resistant to acid elution from the cells more than adult haemoglobin (HbA)
“Screening” test
Principle: acid-elution cytochemical method of quantifying HbF
Kleihaur Betke METHOD
After ethanol fixation, hydochloric acid at pH of 3.3 is applied to the maternal blood sample –> the HbA is denatured and the HbF remains intact
Using Shepard’s method, the smear is counterstained with eosin or erythrosin, leaving the foetal RBC pink and maternal red cells ‘ghost-like’ with absent staining
10,000 cells (using miller optical field) are counted and the % of foetal cells is determined using Mollison’s formula.
Mollisons formular assumes that the maternal red cell volume is 1800 mL, fetal cells are 22% larger than maternal cells and only 92% of fetal cells stain darkly.)
% fetal cells x 18 x 1.22 = Estimated volume of FMH in mL.
Controls in Kleihaur Betke
Positive control: fresh EDTA cord blood diluted 1:100 in adult EDTA blood
Negative control: adult EDTA blood
False positives in HbF quantification
Thalassaemias
Sickle cell anaemia
Hereditary persistence of foetal haemoglobin (HPFH)
HbF quantification via Flow
Principle - Ab to intracellular HbF detectable by flow cytometric methods
-Propidium iodide included in assay to exclude contaminating leukocytes
FMH flow cytometry measures foetal red cells using either (a) an antibody against HbF or (b) an antibody against ‘D’ positive antigens on foetal red cells and is more sensitive than the Kleihauer-Betke acid elution test as the count variability (CV) is reduced
MP lab - HbF is used.
SS vs HbF-PE to gate out % of HbF cells.
>50,000 events
QA/QC
-FETALtrol control run with every batch (contains negative, low level and high level positive FBCs)
Benefits: rapid, sensitive and reproducible
Immune vs passive D
Immune D is usually stronger
Immune D will get stronger on repeat/serial testing
Nipocalimab - clinical trial therapy in obstetric patients with known alloAb and high risk of HDFN
Blocking maternal to fetal IgG transport across the placenta.
Decreased systemic IgG by blocking maternal FcRN-dependent IgG recycling
Management of newborn with known HDFN once delivered
Risk of hyper bilirubin significantly increases once born –> this is because infants have a immature metabolic pathway unable to break down bilirubin (as when in utero placenta can clear this)
Interventions:
- phototherapy to oxidse unconjugated bilirubin to allow for urinary excretion
Exchange transfusions –> removes bili and maternal Ab
- Top up transfusions –> support oxygen carrying capacity to tissues
IVIG no longer approved for HDN
IUT requirements
ABO and RhD compatible
<5 days old
K negative
Negative for the antigen against which the maternal Ab is directed - and desirable to perform an extended maternal red cell phenotype
CMV negative
Irradiated
1-3% risk of fetal adverse events such as infection or rupture of membranes
NIPT vs NIPA
Limitations in FMH testing
Poorly standardized (user dependent)
Assumptions in formula (maternal weight and fetal cell size)
HbF increases in pregnancy
Hereditary persistence of HbF
Haemoglobinopathies
Reticulocytes resist acid hemolysis
FNAIT Abs
Commonest cause of significant thrombocytopenia in term neonates 1/1000 live births
HPA1b Abs most commonly implicated
HPA 5b and 15b antibodies
90% recurrence rate
DRB3**0101 allele –> presence increases immunization risk (33% compared to <1%)
Severe FNAIT when plts <25 –> occurs 1/10,000
~20% of these have an ICH
Fetus only begin to express plt Ags @ 16 weeks
IVIg antenatal management of FNAIT
IVIg should be offered to women whose pregnancies are at risk of FNAIT @ 20 weeks
If severe FNAIT in previous pregnancy –> then commence IVIg from 12 weeks and aim for Csection OR early NVD
Management of newborn with FNAIT
Aim plts >50
Plt transfusion –> HPA 1bb platelets
IVIg
Standard FNAIT testing that is always performed
Testing doesn’t happen in a stepwise fashion
- HPA genotyping – 1, 2, 3, 4, 5, 15 via PCR of all samples
- Maternal serum vs paternal platelets cross match by MAIPA assay
- Maternal HPA antibody screen against group O donor platelets by MAIPA assay
- Maternal HLA Class I Ab screen by luminex Single Antigen bead kit
PIFT –> standard test but not always reported
- Does the mum have an autoimmune process and IgG autoAbs herself
PIFT - platelet immunofluorescence test
- Whole cell flow cytometry assay -
- Is the ONLY assay where platelets are kept intact throughout. Most closely resembles the in vivo process
- Looking for Abs that bind to surface of the platelets
- Very sensitive BUT lacks specificity - platelets also express non HPA-specific antigens
MAIPA - monoclonal antibody immobilization of platelet antigens
ELISA test using color development to visualize the presence of an antibody
The only assay using platelets which can identify antibody specificity
Highly specific and sensitive
PAK-Lx bead kit
Faster to perform
Uses BEADS instead of platelets
Limited range of HPAs (as provided by the manufacturer)
cant use it for cross match
What interferes with HPA Ab identification?
- HLA Class I antigens
- any antigens against these will interfere in assay - Blood group antigens
- plts express low levels of blood group antigens with majority on HP IIb - IVIg interferes with ALL platelet Ab tests –> so ensure all samples are PRE IVIG
Presence of high titre maternal HLA Class I Abs and blood group Abs interfere with paternal platelet cross match results in flow and ELISA tests
MAIPA method
Intact platelets –> incubation of mums serum with platelets –> human IgG antibodies bind to plt antigens
Introduce a mouse antihuman monoclonal Ab which is specific to a SINGLE glycoprotein Ab –> binds at a DIFFERENT binding site of the same glycoprotein
Platelets are then LYSED –> leave a glycoprotein -antibody and MoAb triplex
this triplex binds to reaction well coated with a goat anti mouse capture antibody –> traps triplex
Then add enzyme linked anti-human IgG
Measure of optical density in reaction well
Platelet HPA genotyping method
Inhouse Taqman real time PCR
HPA genotypes are biallelic systems created by SNPs. (eg possible variations HPA 1aa, 1ab, 1bb)
HPA common antigens genotyped– 1, 2, 3, 4, 5 and 15
SNP variants are targeted using fluorescent labelled sequence specific proves and primers
Fluorescence is detected in real time after every PCR cycle and measured
This is then put onto an allelic discrimination plot to discriminate the aa, ab, bb reactions and where you would expect to see them.
How much anti-D does 625IU cover?
6ml of fetal red cells
