1. UOG Journal Club: March 2013
Meta-analysis of second-trimester markers for trisomy 21
M. Agathokleous, P. Chaveeva, L. C. Y. Poon, P. Kosinksi and K. H. Nicolaides
Volume 41, Issue 3, Date: March 2013, pages 247–261
Journal Club slides prepared by Dr Asma Khalil
(UOG Editor for Trainees)

2. Meta-analysis of second trimester markers for trisomy 21
Agathokleous et al., UOG 2013
Second-trimester markers for
trisomy 21
• Ventriculomegaly
• Absent or hypoplastic nasal bones
• Increased nuchal fold thickness
• Intracardiac echogenic focus
• Aberrant right subclavian artery
• Echogenic bowel Trachea
• Mild hydronephrosis
ARSA
• Shortening of femur or humerus Spine

3. Meta-analysis of second trimester markers for trisomy 21
Agathokleous et al., UOG 2013
Calculation of risk for trisomy 21
Background risk x LR of each marker
Individual risk = a priori risk × LR1 × LR2 × LR3…
Normal Tr 21 LR+ LR- LRc*
Mild hydronephrosis 2.6% 17.1% 6.8 0.85 1.0
Echogenic foci 4.4% 30.3% 6.4 0.75 1.0
Short femur 5.2% 42.0% 7.9 0.62 1.5
Echogenic bowel 0.6% 17.3% 21.2 0.87 3.0
Nuchal fold >6 mm 0.6% 41.1% 53.1 0.67 10.0
Major defect 0.7% 21.4% 33.0 0.79 5.0
LRc* = LR isolated marker
Nicolaides UOG 2003, Nyberg et al. Ultrasound Med 2001

4. Meta-analysis of second trimester markers for trisomy 21
Agathokleous et al., UOG 2013
Objective
Examine the screening performance of second trimester
sonographic markers for the detection of trisomy 21

5. Meta-analysis of second trimester markers for trisomy 21
Agathokleous et al., UOG 2013
Methodology
Inclusion criteria Eligibility criteria
• Studies reporting on the 1. 2 × 2 tables for diagnostic
incidence of one or more performance could be
markers in trisomy 21 and constructed
euploid fetuses 2. Karyotype was unknown at
the time of ultrasound
• 1995 – September 2012 3. Chromosomal status was
confirmed by karyotype or
• GA at examination14-24 wk postnatal examination
• Prospective and retrospective cohort studies
• Case–control studies (for ARSA and absent/hypoplastic nasal bones)

6. Meta-analysis of second trimester markers for trisomy 21
Agathokleous et al., UOG 2013
Methodology
• Methodological quality of the studies: Newcastle–Ottawa scale
• Weighted independent estimation of DR, FPR, +ve LR
(sensitivity/(1−specificity)) and –ve LR ((1−sensitivity)/specificity)
• Heterogeneity between studies: Higgins’ I2 and Q-test
• Explore the effect of heterogeneity: analysis for the whole dataset
and in the subgroups (high risk and screening)

7. Meta-analysis of second trimester markers for trisomy 21
Agathokleous et al., UOG 2013
Results
Definitions of the markers
• Ventriculomegaly: ≥ 10 mm
• Increased nuchal fold thickness: ≥ 6 mm
• Echogenic bowel: equal echogenicity to that of bone
• Mild hydronephrosis: renal pelvis AP diameter varied from 3 to 4 or 5 mm
• Hypoplastic nasal bones: cut-off varied with gestation
• Short femur or humerus: cut-off varied with gestation

8. Meta-analysis of second trimester markers for trisomy 21
Agathokleous et al., UOG 2013
Marker DR FPR LR LR Isolated
+ ve – ve marker
Cardiac echogenic focus 24.4 3.9 5.8 0.80 0.95
Ventriculomegaly 7.5 0.2 27.5 0.94 3.81
Increased nuchal fold 26.0 1.0 23.3 0.80 3.79
Echogenic bowel 16.7 1.1 11.4 0.90 1.65
Mild hydronephrosis 13.9 1.7 7.6 0.92 1.08
Short humerus 30.3 4.6 4.8 0.74 0.78
Short femur 27.7 6.4 3.7 0.80 0.61
ARSA 30.7 1.5 21.5 0.71 3.94
Trachea
Absent or hypoplastic NB 59.8 2.8 23.3 0.46 6.58
ARSA
No markers LR 0.13 = 7.7 fold reduction Spine
Meta-analysis 47 studies 1995–2012

9. Meta-analysis of second trimester markers for trisomy 21
Agathokleous et al., UOG 2013
Results
Estimation of combined LR of multiple markers
•The LR for trisomy 21 of individual isolated markers is derived by multiplying the
+ve LR for the given marker by the –ve LR of each of all other markers
• The same approach when any combination of ≥ two markers are detected e.g.
with mild hydronephrosis (+ve LR 7.6) and ventriculomegaly (+ve LR 27.5), the
combined +ve LR is 209 (7.6 × 27.5). This must be multiplied by the combined -ve
LR of all other markers that were not present (0.8 × 0.8 × 0.9 × 0.8 × 0.7 × 0.5 =
0.2) to derive a final combined LR of 31.6

10. Meta-analysis of second trimester markers for trisomy 21
Agathokleous et al., UOG 2013
Results
Excel spreadsheet (online) allowing automated calculations of the LR for
any given combination of the presence and absence of markers
Present/Absent (choose from drop-down:
Marker Present/Absent/Not known) LR
Intracardiac echogenic focus Not known 1.00
Mild hydronephrosis Not known 1.00
Short femur Not known 1.00
Echogenic bowel Not known 1.00
Increased nuchal fold Not known 1.00
Aberrant right subclavian artery Not known 1.00
Absent or hypoplastic nasal bone Not known 1.00
Ventriculomegaly Not known 1.00
LR for combination: 1.00
http://onlinelibrary.wiley.com/doi/10.1002/uog.12364/suppinfo

11. Meta-analysis of second trimester markers for trisomy 21
Agathokleous et al., UOG 2013
Results
a) Echogenic foci
b) Ventriculomegaly
c) Nuchal fold thickness
d) Echogenic bowel
e) Hydronephrosis
f) Short humerus
g) Short femur
h) ARSA
i) Absent nasal bone
j) Absent or hypoplastic
nasal bone

12. Fetal fraction in maternal plasma cell-free DNA at 11–13 weeks
Ashoor et al., UOG 2013
Discussion
• Heterogeneity in results between the studies explained by differences in
design and focus of individual studies
• The problem of high heterogeneity was not overcome by sub-analysis of
data (screening versus high-risk populations)
• Studies published before 1995 were excluded (limited awareness)
• GA 14–24 weeks: potential effect of GA on the incidence of the markers
• The majority of the included studies only examined the value of individual
markers. There are no studies that systematically examined the possible
interrelationship between markers, and it is therefore assumed that they
are independent of each other, apart from short femur and humerus

13. Meta-analysis of second trimester markers for trisomy 21
Agathokleous et al., UOG 2013
Implications for practice
1. There is a 7.7-fold reduction in risk for trisomy 21 if a systematic
second-trimester ultrasound examination demonstrates the absence
of all markers
2. The detection of any one of the markers during the scan should alert
the sonographer to look for all other markers
3. The post-test odds is derived by multiplying the pre-test odds by the
+ve LR for each detected marker and the -ve LR for each marker
demonstrated to be absent
4. There is only a small effect on modifying the pre-test odds in the case
of most isolated markers (echogenic focus, echogenic bowel, mild
hydronephrosis and short femur)

14. Meta-analysis of second trimester markers for trisomy 21
Agathokleous et al., UOG 2013
Conclusions
• The data from this meta-analysis and their interpretation
could guide clinical practice.
• However, appropriate training, certification and regular audit
are essential.