Haemolytic Disease of the Foetus/Newborn Flashcards
What is the history on HDFN?
In 1892 a disease of icterus neonatorum and edema was first described
In 1938 Maternal-foetal alloantibodies to RBCs were found to be involved by Ruth Darrow
In 1929 RhD was identified as a causative antigen
In 1939 Levine and Stetson were the first to demonstrate a foetal/maternal blood group incompatibility as a possible mechanism for the disease
Who won the Nobel for discovery of HDFN
Levine and Stetson - demonstrated in 1939
Who was Ruth Darrow?
She was the first to suggest maternal/foetal alloantibodies to RBCs were a thing
She had three miscarriages herself
Talk about the ability of antibodies to travel across the placenta
The maternal transfer of IgG antibodies to a foetus is a normal event -> confers resistance for the first few months of life until baby develops its own immune sysstem
Following birth this transfer of antibodies no longer occurs and the baby can be cured if treatment is prompt and complete enough to offset complications caused by gross red cll haemolysis
What two methods of treatment are available for babies born with HDFN
Exchange transfusion
Photolight therapy
How does HDFN affect a baby?
Causes antibody mediated destruction of foetal rbcs
Causes foetal anaemia
Enlarged liver and spleen
Erythroblastosis foetalis
Hydrops foetalis
Elevated bilirubin levels - kernicterus and jaundicce
Disabilities or death
What is hydrops foetalis
Oedema, swollen and pale babies (when first born)
Low oncotic pressure
Low albumin production
Why does HDFN not occur in first pregnancy
Sensitisation only occurs in first, reaction in second
Why does HDFN not occur in first pregnancy
Sensitisation only occurs in first, reaction in second
How does sensitisation occur in first pregnancy
During birth Rh+ fetal cells leak into maternal blood after breakage of the embryonic chorion which normally isolated the fetal and maternal blood
Maternal B cells are activated by the Rh antigen and produce large amounts of nti-Rh antibodies
What might put a mother at incerased risk of sensitisation in her first pregancy?
if she has had a transfusion prior
How does second exposure in second pregnancy occur
(3)
Rh antibody titer in mothers blood is elevated after first exposure
Rh antibodies are small enough (IgG) to cross the embryonic chorion and attack the fetal cells
Through pinocytosis of syncitiothrophoblast cells the mother thus transfers to the foetus the variety of IgG she has previously synthesised
What cells are responsible for transfer of IgG across placenta
syncitiothrophoblast
What is RhoGam and what effect does it have?
RhoGAM prevents a Rh- expectant mother from making antibodies during pregnancy that could cause HDFn in future pregnancies
As long as the Rh negative mother received RhoGAM appropriately during every pregnancy her babies are at very low risk of developing HDFN
How does Rho-GAM work
We dont know how it works exactly
It is a human made product - unable to make monoclonal antibody
We know it binds and removes any RhD positive foetal cells in mothers circulation before mother is able to become sensitised but we dont know how the body clears these complexes etc
Comment on the frequency of HDFN, trends etc
In the 1960s HDFN accounted for 10% of perinatal deaths in the UK
Now we only see 15-20 deaths per year in the UK
Significant drop of in freuency and deaths since Rh prophylaxis introduced in 1970s
What problems can anti-D cause in the BT lab
Women come up anti-D positive due to past RhD prophylaxis treatment (RAADP)
It is difficult to tell if this anti-D is true anti-D or not
Patient will still need RAADP
Talk about the use of IgG anti-Rh(D) Ig
Its been in use since 1968
It is 99% eeffective at preventing Rh disease
- combined pre/ante-netal and post-natal use
If women have access to this it can prevent HDFN
How does Ireland’s RAADP compare to the UK
The UK gives anti-D to any Rh- pregnant lady regardless of the babies type
In Ireland we are required to molecularly type te babies of Rh- pregnant women - only if foetus is Rh+ will we give RAADP
What percentage of women dont have access to RAADP?
50% of pregnant women who need RAADP dont reeive it
This is about 2.5million women a year
Why is HDFN still a problem?
Only reduced burden by 50% due to poor access
160,000 perinatal deaths/year
100,000 diabilities/year
Mostly in resource-poor countries
In modern world cases occur where there is failure to take appropriate preventative measures - in Fabians experience HDFN occured where patient just wasnt given anti-D due to miscommunication/doctor changeovers etc etc
Why is HDFN not as bbig a problem in China?
Only 0.5% of women in China/Japan and Indonesia are RhD-
Why is HDFN not as bbig a problem in China?
Only 0.5% of women in China/Japan and Indonesia are RhD-
Why is HDFN not as bbig a problem in China?
Only 0.5% of women in China/Japan and Indonesia are RhD-
How prevalent are RhD- women across the world, why is this significant?
19% in Brazil
15% in White/Europeans
3-15% in Africa
4% in India
0.5% in China/Japan/Indonesia
When you look at the amount of anti-D actually administered in these countries you will see that there is a lot less prescribed then there should have bee
What three factors are essential in the pathogenesis of HDFN
Maternal red cell alloatnibodies which cross the placenta
Foetal rbcs which express antigens against which the antibodies are directed
- if antigen not expressed on foetal cells then no disease
Antibodies which are able to mediate red cell distruction
- plenty of antibodies cross the placenta and dont cause HDFN
What is the most common cause of HDFN?
Anti-D HDFN
What percentge of women suffer from anti-D HDFN
Over 50% of HDFN is caused by anti-D
What percentage of pregnant women have clinically significant antibodies
Approximately 1% of all pregnant women
- anti-D is the most common 50%
- ABO very common but doesnt cause true HDFN
- Many other IgG blood group antibodies have caused HDFN but not to the same extent e.g. Dfy and Kidd
- Prevention is focused on D and K due to IgG class 1 and 3 being actively transferred
How are ABO alloantibodies involved in HDFN?
ABO antibodies in ABO incompatible pregnancy cause an untrue form of HDFN
There will be no sign of foetal haemolysis
This is because ABO antigens are expressed in way lower amount than RhD -> this is thought to be one of the reasons why this expression is so low
The baby should be monitored for signs of haemolysis but usually they are fine
Why might ABO antibodies cause confusion in query HDFN
They might appear in an O- mother with an A- baby
It might appear as if the baby is suffering from the beginning stages of HDFN -> might look like baby is RhD+
Can be a cause of concern, might think mother has become immunised -> will go and type baby then
When might maternal immunisation occur
Normally doesnt happen in first pregnancy
There may be immunisation if there is a foetal red cells entering maternal circulation i.e. trauma/bleed etc
Danger period for immunisation = 72hours after birth of an RhD positive baby (arbitrary rule = in general you produce antibodies against somethine within 3 days of exposure to it)
This risk of immunisation is increased if mother has been previously transfused at any point by either red cells or platlets (both potentially immunising agents)
How does HDFN affect the baby while stil in utero?
Coated foetal rbcs are removed extra-vascularly ->FC receptors in spleen
Increased production of rbcs by foetal haematopoietic tissues (liver if early in development or bone marrow if later)
Late pregnancy = premature release of nucleated cells from marrow - erythoblastosis foetalis
Early pregnancy = increased erythropoiesis by liver with severe anaemia and oedema brought on by hypoproteinaemia/hypoalbuminaemia - hydrops foetalis
How does HDFN reduce rbc survival
RBCS are sensitised by antibodies
Antibodies bind to rbcs by Fab fragment -> Fc portion sticking out
Sensitised rbcs travel to spleen and liver in circulation
Macrophages in spleen and Kupfer cell in liver recognise and bind Fcs and either take bites out of rbcs or engulf them entirely
This causes hepatosplenomegaly
What is erythroblastosis foetalis?
This is the premature release of nucleated cells from bone marrow of baby suffering from HDFN
What is hydrops foetalis
In early stages of development erythropoesis occurs in a foetus’ liiver
If there is severe anaemia there is increased erythropoiesis from the liver, erythropoiesis process under so much pressure to put out cells that it cannot move to the bone marrow
This puts the liver under pressure causing hepatic dysfunction - liver fails to make enough proteins, particularly albumin as it tries to maintain sufficient erythropoiesis
Hypoalbuminaemia/hypoproteinaemia causes oedema
=> baby is born with oedema, hepatosplenomegaly, and anaemia (palor)
Why are HDFN babies born pale and not jaundiced if rbcs are being destroyed?
The breakdown products of the red cells destroyed by the liver and spleen go straight into the mothers circulation - absorbed across
These products are cleared quite quickly by mothers much larger system -> we can upregulate this as a way of treating HDFN
Hence why babies only become jaundiced after birth -> a lack of albumin which binds bilirubin in these babies also causes this
What can a build up of bilirubin cause in a newborn
Kernicterus - due to high affinity of bilirubin for lipid in myelin of CNS
Why exactly can a baby no longer excrete excess bilirubin after birth?
After delivery the newborns liver stops producin glucuronyl transferase
This meants the baby can no longer convert bilirubin into an excretable
This results in bilirubin build up in tissues - kernicterus
What percentages of HDFN babies are born with pallor vs kernicterus, frequency of symptoms?
25-30% of HDFN babies are born pale - jaundice ensues
If not treated, brain damage occurs within 3-4 days
Death occurs in 90% of untreated, remaining 10% have severe brain damage
Death due to respiratory arrest
Many of these babies are miscarried and dont make it to term
How exactly does bilirubin build up cause kernicterus
Nerve axons are insulated by myelin which conducts electrical impulses
Myeline is composed of lipis
Unconjugated bilirubin i.e. bilirubin not bound to albumin (low in HDFN babies due to hepatic dysfunction) binds to myelin
Bilirubin impregnates the myelin sheat impairing nerve conduction leading to short circuits
When do we carry out investigation of our pregnant women for risk of HDFN
On first visit to hospital
Follow up 28 weeks later
When do we carry out investigation of our pregnant women for risk of HDFN
On first visit to hospital
Follow up 28 weeks later
What initial laboratory investigation do we do on pregnant women to predict their risk of HDFN
Transfusion and pregnancy history
ABO and Rh group - ie. are they D-
Antibody screening -> do they already have antibodies
IgG or IgM? if screen positive
What follow up laboratory investigation do we do on pregnant women at 28 weeks to predict their risk of HDFN
Titration/quantiication of anti-D if its been developed
Blood grouping of partner DD or Dd or dd etc
Blood grouping of foetus using cffDNA
Amniocentesis, CVS orPUBS - measure bilirubin
Doppler ultrasound - predicter of anaemia
Why do we carry follow up testing at 28 weeks?
This is the last trimester of pregnancy
Mothers are at highest risk of sensitising events at this time
What antibodies do we quantify/do titres for if we detect them?
anti-D and anti-c
What is cffDNA, what do we use it for??
Cell free foetal DNA
this is foetal DNA that is always present in the mothers circulation
We can detect this in the IBTS using molecular methods
i.e. if we detect RhD gene in the circulation of an Rh- mother then we can determine that the foetus is Rh+ and therefore mother should get RAADP
What was done before Dopler ultrasound for haemoglobin measurement, why did we switch over?
Amnioscentesis (collection of amniotic fluid) for bilirubin measurement
Chorionic Villus Sampling = biopsy of placental cells
Percutaneous Umbilical Blood Sampling/Cordocentesis = foetal blood straight from umbilical cord
*- risk of spotaneous abortion 1/100
What is Dopler Ultrasound
Ultrasounography of middle cerebral artery to measure peak systolic velocity (PSV)
Used for prediction of foetal anaemia
What quantities of anti-D if detected are unlikely indicatve of HDFN, moderate risk or high risk of hydrops?
<4 IU/ml = HDFN unlikely
4-15 IU/ml = moderate risk of HDFN
>15 IU/ml = high risk of hydrops fetalis
What quantities of anti-c if detected are unlikely indicatve of HDFN, moderate risk or high risk of hydrops?
<7.15 IU/ml = continue to monitor
7.5-20 IU/ml = risk of moderate HDFN, refer to FAU
>20 IU/ml = risk of severe HDFN
Why do we carry out titres for anti-D and anti-c but not anti-K
Anti-K titre does not correlate with risk of anaemia
Anti-D and anti-c quantification is used as a predicter of anaemia - used to determine if Intra-uterine transfusion necessary or delivery induction necessary etc
What does a large peak systolic velocity on Dopler ultrasound indicate?
The greater the peak the greater the anaemia
How sensitive and specific is PSV for prediction of foetal anaemia, how do we use PSV to predict anaemia
88% sensitive
82% specific
We map PSV against gestational age (weeks)
- rising will mean pregnant woman will have to come in more often to have titres checked more frequently especially near end stage of pregnancy
Why does ABO incompatible anti-D HDFN cause a less severe form of HDFN
If mom is O- and baby is A+, mother will naturally already have anti-A antibodies
These anti-A antibodies will clear out any A+ foetal cells relatively quickly meaning moms immune system will have less time to mount an immune response against the RhD+ antigen on cells
Cells destroyed immediately, less immunisation, less severe HDFN if any
How is anti-D product made?
Injection of D+ cells into D- males
Good responders i.e. those that produced high titre anti-D were selected to donate plasma
Who is responsible for developing anti-D product and when
Developed in mid 1960s by Clarke et al
What exactly is anti-D product
A blood product
Virus inactivated
From fractionation of plasma
How does anti-D administration vary from country to country?
In the USA 1500IU (300ug)
In Europe 500IU (100ug)
Some countries give prenatal administration, others do not
Usually given 72 hours after birth of RhD+ baby
Only recently Ireland has brought in prophylaxis anti-D at 28weeks
Generarily how do we detect if there has been a foetal bleed?
We carry out flow cytomettry for foetal cells in mothers circulation
We give another dose of anti-D if we detect a large bleed
How many mls of red cells do you normally get in a bleed
Youll never get like 10mls of foetal blood as this is 1% of babies blood, going to get more like 1ml or less
How many IU of anti-D is needed to counteract 1ml of RCC
125 IU - 1ml RCC or 2mls of blood
What are two examples of anti-D used in Ireland
Rhesonative
Rhophylac
How manu IU is in a single vial of anti-D and how much of a bleed does this counteract?
1500 IU or 300ug
Equivalent of 24ml whole blood
What do we do with a RhD- pregnant woman within the first 28 weeks?
Send sample for foetal RHD screening >11weeks gestation
The woman is eligible for anti-D Ig administration for PSEs pending foetal RHD screen result - referred to RAADP midwife
Why cant we test for feotal DNA before 11 weeks?
There isnt enough foetal DNA in circulation before week 11
Why cant we test for feotal DNA before 11 weeks?
There isnt enough foetal DNA in circulation before week 11
What do we do with a D- pregnant woman at 28 weeks gestation?
if foetus is predicted RhD through molecular methods then the woman is now eligible to receive targeted RAADP
We can blood group confirm and antibody screen check at this time aswell
What do we do with an RHD- mother at delivery of a RhD+ baby?
Cord bloods are tested to confirm the infants blood group by serological methods
Postnatal administration of anti-D Ig and FMH estimation through flow cytometry - more anti-D given if large bleed
How did RAADP affect HDFN deaths/epidemiology?
Before RAADP there were 46/100,000 perinatal births
With introduction of RAADP mortality fell to <1.6/100,000 births
What percentage of women become sensitised at childbirth, how much of a bleed?
Fetal cells are found in 50% of women at normal delivery
3mls of bleed or more in 1% of women
10mls of bleed or more in 0.3% of women
What can increase chance of foetal cells entering mothers circulation at birth?
The frequency and volume of bleeds is increased by caesarean section and manual removal of placenta
Talk about the kinetics of anti-D Ig
Intramuscular injection takes 2-4 days to reach peakconcentration
anti-D has a half life of 3-5 weeks
The quantity needed is dependent on size of the mother
How many doses of anti-D do we give in Ireland, why is this the preferred method?
We do a single dose at 28 weeks
A ddouble dose is used in some countries but thiere is really poor compliance with this
- mother wont come for second dose if she feels unwell after first etc
How many doses of anti-D do we give in Ireland, why is this the preferred method?
We do a single dose at 28 weeks
A ddouble dose is used in some countries but thiere is really poor compliance with this
- mother wont come for second dose if she feels unwell after first etc
What are the three main methods of FMH estimation?
Kleihauer-Betke acid elution
Rosetting technique
Flow Cytometry
What is the Kleihauer-Betke acid elution test?
A technique for estimation of FMH
It relies on the resistance of HbF to be eluted out of the rbc by an acid buffer solution
Slid is dipped in acid - foetal haemoglobin remains stained but adult haemoglobin fades resulting in ghost stains
Gives % foetal cell in maternal circulation
Can give some semi-stained cells which are difficult to interpret but is a very simple test that can be done in any lab
High counts are usually then sent for flow cytometry
What is the rosetting technique of estimation of FMH
Used in America
Used to demonstrate small numbers of D positive cells in a predominantly D- population
very simple but not specific or sensitive - needs a large amount of fietal cells >10mls - BT labs always have reagents for this
Incubate D-antisera with maternal blood sample, add indicator cells which will agglutinate if foetal cells are present
How do you calculate a FMH using Kleihauers
Count number of foetal cells per 2000 maternal cells
- 30/2000 = 1.5%
Assume mothers circulation = 5000ml => 2500ml = red cells
1.5% x 2500ml = 38ml foetal haermorrhage
15ml covered per vial
=> 38/15 = 2.53 doses = 3 doses + 1 extra = 4 vials or 1200ug
When issuing anti-D what is always the rule if not sure how many to give?
always roung up and add another extra vial
What might be some indicators that HDFN is occuring?
Low foetal Haemoglobin
Raised foetal bilirubin
DAT + mothers blood -> DAT + may also be due to ABO antibodies
Ultrasound - Doppled
What are some treatment options for HDFN?
Intrauterine transfusions
Exchange trasfusion on mother
What was Fabians experience with exchange transfusions?
Mother suffering from ITP - antibodies affecting babys rbcs
Anti-platelet antibodies were crossing the placenta and coating the babies cells
This required mother to come in twice a week for exchange transfusions
These take about 4 hours each time
What was Fabians experience with exchange transfusions?
Mother suffering from ITP - antibodies affecting babys rbcs
Anti-platelet antibodies were crossing the placenta and coating the babies cells
This required mother to come in twice a week for exchange transfusions
These take about 4 hours each time
If baby is very anaemia what is the cut off point for an intrauterine transfusion vs inducing delivery
If baby is less than 36 weeks along - consider transfusion
If baby is over 36 weeks they would probably just induce
What is the DAT
Direct antiglobulin test
Blood sample from a patient with immune mediated haemolytic anaemia: antibodies are attached to antigens on the rbc surface
The patients washed RBCs are incubated with antihuman antibodies
RBCs agglutinate: antihuman antibodies form links between RBCs by binding to the human antibodies on the RBCs
How is HDFN managed in the infant
Before birth:
- IUT
- Exchange Transfusion
After birth:
- Photo therapy and IVIg
- Top-up transfusion
Talk about photolight therapy for babies
Ultraviolet blankets are used and not lamps
- issues with baby wearing eye protection with use of lamps
What is IVIg?
IVIg is a way of blocking the transfer of antibody from mother to foetus
How are top-up transfusions used for treatment of HDFN
Used if baby is born anaemic
Actually quite frequently carried out - There is a peak in transfusion in babies less than 1 year old
Why is anaemia in babies common?
Babies can be slightly anaemic as HbF is converted to HbA
Why is anaemia in babies common?
Babies can be slightly anaemic as HbF is converted to HbA
When would anti-D administration be pointless?
If anti-D is already present ie if mother already sensitised
What treatment is there for severe HDFN while in utero
Intra-uterine transfusion >20 weeks, IPT or IVT i.e. severe cases
Intravenous Ig therapy
Plasma exchange
Phobarbital - to increase uptake of bilirubin by mothers liver
compatible exchange trasfusion after birth
phototherapy
Why are intra-uterine transfusions so dangerous
The blood volume of the baby is normal i.e. high pressure
We have to inject blood into an already full circulatory system
Have to get as many cells in as possible to give the baby as long as possible before having to retransfuse
Want to try the baby over till birth
What would you do with an anti-U case of HDFN
Plasma exchange would be far more likely as treatment then IUT as we dont have U blood to give baby
What blood is used in IUTs what are the requirements of blood for IUT?
O or ABO compatible with infant and mother
If group O then must be haemolysin negative (low titre)
Must lack the causative antigen
Blood must be less than 5-7 days old
Partially packed red cells Hct 0.75/l
Leucocyte depleted
HbS neg
CMV- and Irradiated
In CPD/A not SAGM
Small volume - approx 50-100mls
Crossmatched against mother
prewarmed
What additive is used for IUT red cells, why not SAGM?
CPDA - not SAGM
SAGM contains mannitol which in large proportions is toxic to the brain and kidney ie. toxic in small children and newborns
SAGM is only ever used for transfusing tiny amounts e.g. top-up transfusions and never exchange transfusions for this reason
What are partially packed red cells?
Red cells that contain less plasma
Centrifuge a red cell pack and squeeze of 50% of the plasma and then seal the paack again
Talk about ABO HDFN
Group O mothers with a group A or B baby
1st pregnancy can be affected
Mild disease
Naturally present anti-A or anti-B is causative, IgG
Action not usually required until birth - photolight therapy
DAT can be pos or neg
Anti-A or anti-B can be eluted of the foetal cells to confirm disease
What is HDFN of platelets?
Neonatal alloimmune thrombocytopenia NAITP
How frequent is NAITP
1/1000 births affected
Affects the 1st born in 50% of cases
Great clinical variation in anti-platelet antibodies
Tends to run in families, mother, aunt, sister will have experienceed it etc -> baby is born with a lot of bruising -> baby will often need a top-up transfusion
We currently have no product availabl to stop it from happening
Hence it is a common cause of IUts
