Transcript Prenatal diagnosis service in Hong Kong

Prenatal diagnosis service in HK
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Introduction
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Statistics
Prenatal screening tests
Prenatal diagnostic tests
Prenatal Diagnosis laboratory service
Three-/four-dimensional ultrasound
Intrauterine therapy
Preimplantation genetic diagnosis
Clinical and laboratory service
• Antenatal clinic in Queen Mary Hospital
(QMH) and Tsan Yuk Hospital (TYH)
• Prenatal diagnosis clinic and laboratory
(referral centre) in TYH
• DNA Diagnostic Laboratory in QMH, under
Department of Medicine, HKU
• IVF laboratory in Medical Faculty Building
• Genome Centre under HKU
Statistics of prenatal diagnosis
service in 2003
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Total referral: 1629
Ultrasound examinations: 3414
Down syndrome screening: 1018
Thalassemia screening: 3155
Invasive diagnostic procedure : 515
In-utero therapy: 8
Chromosomal studies
Amniotic fluid
3138
Chorionic villi
334
Tissue
225
Blood
323
Total
4020
Down syndrome screening
7899
Haematological studies
Mean Cell Volume
3792
Hb electrophoretic pattern
259
Haemoglobin A2 quantitation
170
Haemoglobin F quantitation
134
Haemoglobin H quantitation
242
Red Cell Morphology
106
Kleihauer's test
42
Fetal blood haemoglobin study
9
Fetal-maternal curve
12
Referral indications
Number (%)
Maternal age (=35)
749 (46.0%)
Fetal abnormality detected on USG
128 (7.9%)
Haematological disorder
87 (5.3%)
Maternal anxiety
14 (0.9%)
Past history of chromosomal abnormality
25 (1.5%)
Family history of chromosomal abnormality
25 (1.5%)
Parental chromosomal abnormality
1 (0.1%)
Chromosomal abnormality of present pregnancy
8 (0.5%)
Congenital abnormality in previous pregnancy
69 (4.2%)
Abnormal serum screening result
126 (7.7%)
Drug intake
214 (13.1%)
Miscellaneous
183(11.3%)
Total
1629
Prenatal screening for abnormalities
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Routine ultrasound
Down syndrome screening
Universal thalassemia screening
Counselling
Ultrasound examination
• If women booked before 20 weeks, we offer a
routine anomaly scan around 18-20 weeks
• If they come before 16 weeks, we will offer an
early dating scan 8-16 weeks in addition to the
routine anomaly scan.
• If they opt for Down syndrome screening, scan
will be done 10-14 weeks to measure nuchal
translucency
• Additional ultrasound examinations will be done if
there are special indications
Dating USG
• Done on the date of the booking clinic
• To determine gestational age accurately
• To diagnose multiple pregnancy and
determine chorionicity and amnionicity
• To confirm fetal viability before 12 weeks
Routine anomaly scan
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Done around 18-20 weeks
To determine gestational age
To diagnose multiple pregnancy
To screen for major congenital anomalies
To determine placental location: repeat
around 30 weeks if low lying
Routine anomaly scan
• Explain the objectives to women and give
information sheet
• Detection rate for major congenital abnormalities
is 75% and may of the minor abnormalities are not
detectable.
• Limited by thick abdominal wall, abdominal scars,
unfavourable fetal position or ultrasound machine
with poor image quality.
Down syndrome screening
• Maternal age at expected date of
confinement
• Age >=35: amniocentesis, CVS, or
screening test
• Age<35: screening test, optional, need to
pay
Down syndrome screening test
• First trimester combined test: nuchal translucency,
materanl serum free beta hCG and PAPP-A at 1113 weeks 6 days
• Integrated test: nuchal translucency, maternal
serum alpha fetal protein and hCG at 16 to 19
weeks and 6 days
• Maternal serum alpha fetal protein and hCG alone
for those who come late
• Nuchal tranlucency alone for multiple pregnancy
and those who do not want blood taking
Workflow for Down syndrome
screening
• Video show and information pamphet to all
women who book before 20 weeks
• A nurse specialist will give a short talk and answer
any queries.
• Book screening test for those who opt for it
• All negative reports will be mailed to women
within two weeks
• All positive results will be referred to PDC for
management
Universal prenatal screening for thalassemia
• Maternal blood samples were collected at the antenatal
booking clinics and forwarded to the Laboratory where
haemoglobin (Hb) and mean corpuscular volume (MCV)
were measured using a Cell Dyn 1400 or Cell Dyn 1700
(Abbott Laboratories Ltd).
• If maternal MCV was 80fL or above, her fetus not at risk of
severe thalassaemia.
• If maternal MCV was found to be low (<80 fL), call the
partner for blood taking and arrange for an iron testing
(serum iron, percentage of saturation, and ferritin levels) for
the wife.
• Test the wife’s blood sample for Hb H by incubation of red
blood cells with brilliant cresyl blue, while waiting for
husband’s blood sample to arrive.
• If the partner’s MCV was also low, determine the
couple’s Hb A2 by micro-column chromatography
and test the husband’s blood sample for Hb H.
• HbA2 was estimated by micro-column
chromatography (Isolab before 1999 and
BioSystems from 1999 because there was no more
supply from Isolab).
• β-thalassaemia trait was diagnosed by the finding of
raised Hb A2 level (>=3.5%).
• The presence of Hb H inclusion bodies was almost
diagnostic of α0-thalassaemia.
• If her partner’s MCV was normal (>=80 fL), their
fetus not at risk of severe thalassaemia.
• If Hb A2 level was normal, Hb H inclusion bodies
were absent, and iron deficiency was detected, they
would be treated with iron therapy first, followed by
repeat MCV four weeks later
• If MCV is low and iron status is normal, a normal
Hb pattern would not necessarily exclude thalassaemia and we manage as suspected αthalassaemia trait.
Thalassemia couple
• Couples with confirmed or suspected
thalassaemia traits were counselled at the Prenatal
Diagnostic and Counselling Clinic.
• DNA analysis would be performed to confirm
suspected cases.
• If both couples were α0-thalasaemia or βthalassaemia minor, their fetuses would be at a 1
in 4 risk of developing homozygous α0thalassaemia or β-thalassaemia major respectively.
Discordant thalassaemia traits
• If the couple had discordant thalassaemia traits,
their fetus would not be at risk for serious
thalassaemia unless one parent who carried βthalassaemia trait had co-inheritance of αthalassaemia.
• ζ-mapping was performed to exclude αthalassaemia, the prevalence of which was
reported as 7% in one study (Lam et al., 1997).
• If the couple had β/ E thalassaemia traits, their
fetus would be at 1 in 4 risk of having severe
thalassaemia.
Different cut-off points
MCV 75fL Miss 3.4% SEA deletion, and 3.6% β
thal carrier
78fL Miss 0.9% SEA deletion
80fL Detect all SEA deletion, and β thal
carrier
MCH 25fL Miss 0.9% SEA deletion
SK Ma Haematological 2001
Partner’s MCV >=80fL
Not at risk of severe thal
Raised Hb A2 level (>=3.5%) β-thal
Hb H inclusion bodies
α0-thal
Iron deficiency and other
findings normal
Iron deficiency +/thalassaemia
Low MCV but normal iron
status and Hb pattern
? 0-thal or +-thal
DNA needed
(normal A2 β-thal- very rare)
Increased Hb F
δβthal
Hereditary persistence of fetal
hemoglobin
Iron deficiency but normal Hb pattern
• Risk of concomitant thalassaemia trait
• In the presence of iron deficiency, Hb A2 production
may be depressed and the brilliant cresyl blue for
detection of Hb H inclusion bodies may be affected
• However, Hb A2 level did not reduce below the
diagnostic range
• Sonographic features of Hb Bart’s disease
• Iron therapy for four weeks and recheck MCV
(Fleming and Lynch, 1969, Ghosh et al., 1985, Li, 1990).
Does MCV>80fL mean ‘no thal’?
• Hb E, α+-thal (LC Chan, J Clin Pathology, 2001)
• Fetus at risk of βE or Hb H disease if her
partner has β thal or α0-thal
• αβ thal, silent β thal (not documented in
Chinese)
Hb E
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Mutation of β gene
Hb E synthesized at a slightly reduced rate
Homozygous Hb E resembles β carrier
But βE can be severe, and variable
MCV can >80fL, increase in Hb A2
Cambodian (25%), Thai (11%), Laotian (10%),
Vietnamese (3%), Asian Indian, Filipino (0.5%),
Chinese (0.4%)
• Rule out β in partner and coinheritance of αthal
At risk couple
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Low MCV couple
α couple
β couple
αβ couple: DNA study of β individual to
rule out 7% risk of coinheritance of both α
and β
(Lam et al., 1997)
• β E couple
Prenatal diagnosis of alpha thal
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Direct invasive vs Indirect (USG monitoring)
DNA diagnosis: PCR vs Southern Blotting
2D US: CT ratio vs placental thickness
Early detection: 3D US or maternal blood?
Ultrasound prediction of Hb H disease?
Direct method
• CVS: 10-12 wk
• Amnio: 18 wk
• 0.5 to 1% miscarriage
Indirect approach at TYH
• Serial USG examinations at 12 wk, 18wk
and 30 wk
• CTR (>=0.50, 0.52 and 0.59) and placental
thickness (>18mm)
• Invasive test if cardiomegaly and/or
placentomegaly
Results of USG monitoring in a
Guangzhou hospital
• Intensive training 5 wk in HK and then regular
supervision
• 350 at risk women and 90 affected
• USG monitoring reduced the need for
cordocentesis by 77.7%
• Performance similar to TYH, but mean age at
diagnosis (14 vs 19 wk)
Leung et al UOG (in press)
DNA analysis
• Southern Blotting
• PCR: rapid report within 1-2 days (Chan V
et al., 2001)
• PCR is less expensive
• Misdiagnosis of homozygous α0thalassaemia may occur if PCR alone is
used in prenatal diagnosis (Ko et al., 1997).
Early prediction
• Limitation of 2D US monitoring: 12 wk
• 3D ultrasonographic measurement of
placental and fetal volume
• Immunofluorescence staining of Fetal
erythrocytes in maternal blood
Prenatal diagnosis of ß thal
• Direct invasive methods: CVS or amnio
(time needed to work out mutation esp
Indian or other ethnic groups), rarely FBS
• Fetal HLA typing
Fetal HLA typing
• If a mother had already an affected baby (ß
major), and PDC showed a normal fetus,
check HLA identity, consider cord blood
saving and transplant (less GVH disease, no
need for marrow harvest).
(J Ped Haematology/oncology 2000)
Preimplantation Genetic Diagnosis
• Biopsy of one or two cells from single
blastomere on D3 (8 cells stage)
• Using PCR and micromanipulation
techniques, able to amplify and study
specific DNA sequences of globin gene
• Problems: contamination and allele dropout
• Multiplex fluorescent PCR (Prenat Diagn 2001)
HLA typing in PGD
• Use PGD to select an embryo not only
free of β major but also, if possible,
HLA compatible with their sick child
Cost-effectiveness of prenatal screening for
thalassaemia in Hong Kong
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From 1998 to 2002, 18,936 women were screened
10.3% of them had a low MCV.
Referral: 153 (TYH), 238 (tertiary), and 157 (self).
Of 84 fetuses at risk of β-thal major syndrome, 19
affected and 18 aborted.
• Total expenditure ($10.0 million) < Costs of
postnatal management for 18 β-thal major fetuses
if they were born (40.8 million).
• Cost effective in area where βand αare prevalent.
(Leung et al, Prenat Diagn, 2004)
Cost-effectiveness of prenatal
screening program in HK
• 86.2% of 361 women opted for USG monitoring
• Effectively reduced an invasive test by 75.5%
• 92.8% of affected pregnancies were terminated
before 14 wk.
• CVS +PCR instead of cordocentesis to reduce risk
• Cost saving: $2,812 (indirect) vs $5,463 (direct)
• Relatively small in comparison to the whole
screening program
(Leung et al, Prenat Diagn, 2004)
Thalassaemia in multiple pregnancy
• 1 in 4 risk in each fetus
• CVS vs amnio: early detection, feasible
• USG prediction for α thal works on twin
(Leung et al UOG, in press)
• Selective feticide
Referral to prenatal diagnostic
clinic (I)
• For chromosomal abnormality
• Advanced maternal age
• Previous child with chromosomal
abnormality
• Family history of Down syndrome
• Either parent is a translocation carrier
Referral to PDC (II)
• For fetal anomalies
• Sonographic abnormalities during the
present pregnancy
• Previous child with congenital abnormalites
Referral to PDC (III)
• For genetic and metabolic disorders
• Previous child with Hb Bart’ disease or
beta-thalassemia major.
• Couples who are diagnosed to be alpha or
beta-thalassemia carrier
• Family history of haemophilia
• Family history of metabolic or genetic
disorder
Prenatal diagnostic tests
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Detailed anomaly scan
3D/4D ultrasound
Fetal MRI
Amniocentesis
Chorionic villus sampling
Cordocentesis
Indications for 3D/4DUS
• When fetal abnormalities are documented or
suspected on 2DUS examinations
• When the pregnancies are at high risk of
having fetal abnormalities
• When 2DUS scans are suboptimal due to
technical factors such as fetal positioning
• When measurement of the volume of an
object is required
3D/4D Ultrasound (I)
• When fetal abnormalities are documented or
suspected on 2DUS examinations, 3DUS can
prove a useful tool in appreciating the severity of
the abnormalities.
• In the study by Dyson et al, the 3DUS images
provided additional information in 51% of 103
anomalies. Overall, 3DUS demonstrated six
anomalies not seen on 2DUS imaging: four cases
of micrognathia, one neural defect, and one
hypoplastic scapulae..
3D/4DUS examination (II)
• When the pregnancies are at high risk of
having fetal abnormalities, 3DUS can
provide more convincing evidence of a
normal fetus than conventional 2DUS.
3D/4DUS examination (III)
• When 2DUS scans are suboptimal due to
technical factors such as fetal positioning,
the use of 3D multiplanar analysis may
allow the assessment of the planes that are
difficult or impossible to obtain with
2DUS3.
3D/4DUS examination (IV)
• When measurement of the volume of an
object is required, it is generally accepted
that 3DUS volumetry gives more precise
results than 2DUS volume calculations, in
particular, of irregularly shaped objects5
Fetal MRI
Check list before amnio or CVS
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Indications
Gestational age
MCV
Rh status
Anomaly scan
Consent form
Amniocentesis
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Clean with iodine and alcohol
Ultrasound monitor
Free hand technique
22 G needle
Avoid contamination with USG gel
Fundal or upper part of uterus
Avoid placenta, blood vessels and fetus
Discard first few ml of amniotic fluid
Chorionic villus sampling
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Transabdominal
Clean with iodine and alcohol
Ultrasound monitor
Local analgesia: few ml lignocaine
18 G needle (flush with heparin beforehand)
Avoid blood vessels and amniotic cavity
10 ml syringe with 1-2ml culture medium
Aspiration using negative pressure as the handle is
withdrawn
• The handle will not fall back after withdrawal.
• Move the needle and handle up and down
Consultation
• Mainly for counselling, sometimes
diagnosis or in-utero therapy
• Paediatrician
• Paediatric cardiologist
• Paediatric surgeon: surgical problems e.g.
diaphragmatic hernia
• Plastic surgeon: facial cleft
• Rhematologist: heart block and antibodies
Prenatal diagnosis of alphathalassemia (I)
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<18 weeks
CVS and DNA study at 10-12 wk
Amnio and DNA study at 18 wk.
Serial USG at 12, (14), 18 and 30 wk to
measure the fetal cardiothoracic ratio,
middle cerebral artery peak systolic velocity
and placental thickness.
Prenatal diagnosis of alphathalassemia (II)
• >=18 weeks:serial USG on referral and at
30 weeks
• Invasive procedure if abnormal USG
findings. PCR for rapid results.
• No invasive tests if USG findings are
normal.
Prenatal diagnosis of beta
thalassemia
• If DNA study of couple shows informative
gene mutations (95%), CVS and DNA study
10-14 wk or amniocentesis and DNA study
18 wk.
• If mutations cannot be identified,
cordocentesis and globin chain synthesis
Hb E/ beta-thalassemia couple
• If the couple had β/ E thalassaemia traits,
their fetus would be at 1 in 4 risk of having
severe thalassaemia.
• Variable manifestation
• In most sever form, affected children can be
transfusion dependent
• Prenatal diagnosis performed by CVS or
amniocentesis and DNA study
Couples discordant for alpha and
beta thalassemia
• If the couple had discordant thalassaemia traits, their fetus
would not be at risk for serious thalassaemia unless one
parent who carried β-thalassaemia trait had co-inheritance
of α-thalassaemia.
• Routine haematological tests cannot distinguish pure betathalassemia from coinheritance of both alpha- and betathalassemia
• ζ-mapping was performed to exclude α-thalassaemia, the
prevalence of which was reported as 7% in one study (Lam
et al., 1997).
• Serial USG to rule out Hb Bart’s disease
Fetal cardiothoracic ratio
• A four-chamber view was obtained and the fetal
transverse cardiac diameter was taken at the level
of the atrioventricular valves between the
epicardial surfaces.
• The value was expressed as a CTR against the
transverse thoracic diameter.
• The cut off value for normal was 0.50, 0.52, and
0.59 at 12-14 weeks, 18 weeks and 30 weeks of
gestation respectively.
Abnormal USG findings
• If there were fetal cardiomegaly and/or
placentomegaly (the placental thickness being >=
18mm at 12 weeks of gestation), we would offer
CVS or cordocentesis before 18 weeks of
gestation, and amniocentesis or cordocentesis after
18 weeks.
• Cordocentesis followed by haemoglobin analysis
would be a more rapid, less expensive, but more
invasive diagnostic method than CVS or
amniocentesis for fetal DNA analysis.
DNA diagnosis of homozygous
alpha 0 thalassemia
• Southern Blotting is used for α-thalassaemia
DNA testing.
• Although Southern Blotting is much more
expensive than polymerase chain reaction
(PCR), the misdiagnosis of homozygous α0thalassaemia may occur if PCR alone is
used in prenatal diagnosis (Ko et al., 1997).
Noninvasive prenatal diagnosis of alpha
thalassemia
• 311 women (85.9% of all women at risk) opted for
the noninvasive approach.
• Fetal cardiomegaly and/or placentomegaly were
present in 24.4%. All accepted an invasive test, and
90.1% of them had an affected fetus.
• Of all the affected pregnancies, 64 (92.8%) were
terminated before 14 weeks’ gestation.
• The sensitivity and false positive rate of the
noninvasive approach was 100.0% and 2.9%
respectively.
Laboratory techniques
• Karyotyping
• Quantitative fluorescence Polymerase Chain Reaction (QFPCR) was used for rapid detection of aneuploidy involving
chromosomes 13, 18, 21 and XY.
• In order to avoid homozygous uninformative results,
additional small tandem repeating markers were used.
• Fluorescence in situ hybridization (F.I.S.H.) was used for
detection of DiGeorge microdeletion, confirmation of 45,X
and mosacism, and investigate rare chromosomal
abnormalities such as marker chromosome.
• Comparative genome hybridization (CGH) was used to
assess gain or loss of chromosomal material
Preimplantation genetic diagnosis
• PGD for aneuploidy screening was offered
to women with recurrent miscarriages,
previous aneuploidy, and couples with
chromosomal translocation.
Molecular testing for genetic
diseases
• Sequencing for common mutations in the
fibroblast growth factor receptor-3 gene
(FGFR3) was performed in 3 fetal samples
where thanatophoric dysplasia or
achondroplasia was suspected.
First trimester
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Viability
Fetal parameters
Nuchal translucency
Nasal bone
Twin: chorionicity and amnioncity
Alpha thalassemia: fetal cardiothoracic ratio
and placental thickness
Early second trimester anomaly scan
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Overall
Systematic
Heart
All structures
Four limbs
Routine anomaly scan ~18 wks
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Fetal parameters
Fetal anatomy
Liquor
Placenta
Uterus and adnexa
cervix
Indications for fetal echo
• Maternal and familial: congenital heart
disease, DM, SLE, infections, alcohol, drug
• Fetal anomaly, IUGR
• Increased nuchal translusency
• Only 30% of CHD have risk factors
Screening techniques
• 4 chamber view: sensitivity (25-81%)
• Outflow tracts: better sensitivity (78%), but
not easy, time consuming
• 3 vessels view: MPA, AA, SVC, easily
taught (UOG 1997)
• Color Doppler of DA and aortic arch (UOG
2002)
Three vessel view
• MPA, AA, SVC in a straight line from left
ant to right post, decreasing diameter
• Abnormal vessel size: left or right outflow
tract obstruction
• Abnormal alignment or arrangement:
conotruncal lesions
• Abnormal vessel number: truncus, PA,
bilateral SVC
Fetal echocardiography
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Real-time gray scale: the gold standard
5-7MHz
High frame rate
Magnify
Cine-loop
Approach (1)
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Sequential
left and right side of fetus
Visceral situs
Four-chamber
Left and right outflow tract
Aortic arch, ductus arteriosus
Superior and inferior vena cava
Pulmonary veins
Approach (2)
• M-mode: arrhythmia
• Color: assess heart defect, regurgitation and
reverse flow; facilitate Doppler
• Doppler: stenosis, regurgitation
• Power Doppler: small VSD, orientation of
great vessels