Screening of Screening of DownDown syndromesyndrome

in pregnancyin pregnancy

Marta KalousováInstitute of Clinical Chemistry and Laboratory

Diagnostics,

1st Faculty of Medicine and General University Hospital, Charles University, Prague

Screening of Down syndrome Screening of Down syndrome

in pregnancyin pregnancy

• 1st trimester

• 2nd trimester

• integrated screening

Recommendations of experts for Recommendations of experts for

screening of Down screening of Down syndomesyndome

-- laboratory requirementslaboratory requirements

• At least 1 000 screening examinations per year in a lab

• Lab specialist responsible for screening and its quality control

• Quality has to be checked at least twice a year

• The lab has guidelines for sample collection, transport and

storage which are in line with preanalytical phase

requirements

• The lab co-operates with gynecologists and geneticians who are responsible for final evaluation of the screening results

• Results are provided by the lab within 3 days

• Results are given in absolute values as well as in multiples ofmedian for the gestational age

• Results are given to the requiring gynecologist who is responsible for further action

Recommendations of experts for Recommendations of experts for

screening of Down screening of Down syndomesyndome

-- laboratory requirementslaboratory requirements

Screening of Down syndrome Screening of Down syndrome

in pregnancyin pregnancy

1st trimester

• Determination of PAPP-A - pregnancy-

associated plasma protein A

• Determination of free ββββ-hCG

• US of the fetus – nuchal translucence (NT),

presence of the nasal bone

Screening of Down syndrome Screening of Down syndrome

in pregnancyin pregnancy

2nd trimester

• hCG

• AFP

• (unconjugated estriol) – triple test

• US of the fetus – in done earlier and

required for evaluation of the gestational

age

double test

Integrated test (Wald 99)Integrated test (Wald 99)

1/ PAPP-A ( 9th – 11th week)

2/ NT ( 11th -13th week) phase I

3/ Second trimester (15th -18th week)

double or triple test phase II

Integrated testIntegrated test

1st trimester

• PAPP-A

• US for determination

of gestational age

• NT

• Preliminary evaluation

by the medical doctor

2nd trimester

• AFP

• hCG

• Combined evaluation

with the results from

the 1st trimester

Required dataRequired data

• Exact evaluation of gestational age -

US

• Age and body weight of the mother

• Date of blood collection

• Fetus – how many

Markers Markers –– determination and determination and

evaluationevaluation

• Determination of absolute concetration in maternal serum

• Expression of the concentration in MoM (multiples of median), measured results has to be adjusted for body weight of the mother (dilution area) before calculation of MoM

• The same diagnostic kit has to be used – differences among various kits can be even 100 %!!!

• Influence of imprecise determination on the results of biochemical screening (immunochemical methods have intra-assay variability up to 10%, TRACE technology has variability 1-2%)

• The risk has to be evaluated by suitable software

PAPPsPAPPs

pregnancypregnancy--associated plasma proteinsassociated plasma proteins

• detected immunochemically in plasma of pregnant

women

• 4 types

PAPP-A – Zn-binding metalloproteinase, role in

cleavage IGFBP and subsequent activation of IGF

PAPP-B – unknown function

PAPP-C = SP1 protein

PAPP-D = human placental lactogen (hPL)

PAPPPAPP--AA

• Glycoprotein, MW 750 000, elfo α2, maximum at the end of the 3rd trimester, half-life after the birth 3-4 days

• Metalloproteinase, zinc-dependent (belongs to the metzincin superfamily of metalloproteinases), cleaves IGFBP-4, 2 and 5 (insulin like grown factor binding proteins), followed by release of IGF which play a role in regulation of local proliferative reactions – reproduction, wound heeling, atherosclerosis

• In pregnancy decreased in maternal serum in chromosomal anomalies (m.Down) in the 1st

trimester only!, at the end of pregnancy increased in preeclampsia, decreased in threatening abortion

• New marker of acute coronary syndrome

PAPPPAPP--A during pregnancy and A during pregnancy and

after the birthafter the birth

months of pregnancy days after the birth4 10 3 127 6 9

PAPP-A

mU/l

Concentration of Concentration of hCGhCG and AFP and AFP

during pregnancyduring pregnancy

week10 20 30 40

concentration

hCGAFP

Human chorionic Human chorionic gonadotrophingonadotrophin

((hCGhCG))• Glycoprotein produced by synciciotrophoblast of placenta, necessary for function of the yellow body (production of progesteron)

• MW 36 700, αααα and ββββ subunit, α subunit the same as in LH, FSH and TSH. β subunit (145 AMK) similar in hCG and LH, 24 amino acid difference.

• Physiological occurence: pregnancy

• Pathological occurence: tumour from the chorionic tissue – trophoblastic disease (chorioCAa mola), germ cells testicular and ovarian tumours, rare other tumours.

Human chorionic Human chorionic gonadotrophingonadotrophin

((hCGhCG))

β core

„free“β-hCG

β core – urinary excretion, very stable,

might be suitable for screening in the 2nd trimester

Determined Determined hCGhCG• „Total“ hCG – mostly using polyclonal antibodies,

sufficient for dg of pregnancy

• ββββ-hCG – „specific“ hCG – required as tumour marker

• „free“ββββ-hCG - demanding preanalytical phase (disintegration of hCG mainly due to high temperature and false increase of „free“β-hCG )

• ββββ-core hCG – suitable for determination in the urine, but not at the end of the 1st trimester due to high levels of hCGand insufficient cleavage of hCG

• Hyperglycosylated („acidic“) hCG – characteristic for immature placenta at the beginning of pregnancy and for chorioCA. According to some studies present with higher probability in the urine of women with fetus with m.Down.

hCGhCG

0.1 1 10 Log MoM

normal

Edwards Down

Risk of M.Down

MoM hCG>2,5

αααααααα ––11--fetoprotein (AFP)fetoprotein (AFP)

• Glycoprotein, structurally similar to albumin

• Function: binds estrogens, transport of fatty acids, immunosuppressive effects

• Physiological production: yolk sack and fetalliver

• Pathological production: liver tumours, germ cells testicular and ovarian tumours, less frequently GIT and other tumours

in pregnancy increased in maternal serum in neural cord defects, omphalocele, spontaneusabortion

AFPAFP

0.1 1 10 Log MoM

normal

Down

Edwards

Risk of M.Down

MoM AFP<0,5

Evaluation of results of biochemical Evaluation of results of biochemical

screening of chromosomal anomalies in screening of chromosomal anomalies in

pregnancy pregnancy –– „„likelyhoodlikelyhood ratioratio““(Knight and Palomaki. J Clin Immunoassay 1990)

0.2 1 5

MS AFP MoM

the ratio of the lenghts of

the sections is evaluated

/→→→→ risk

Down normal

Evaluation of Evaluation of atypicalityatypicality of results of of results of biochemical screening of chromosomal biochemical screening of chromosomal

anomalies in pregnancyanomalies in pregnancy(Wright et al. Ann Clin Biochem 1993)

0-0.3 0.3

0

-0.3

0.3

→ Log (MoM AFP).10-1

→Log (MoM

hCG).10-1

Edwards

NCD

Down

gemini

(trimini)

Down ?

Mahalanobis

distance –

from the centre

(normal)

healthy

EstriolEstriol in blood during pregnancyin blood during pregnancy

• Increases during pregnancy

• Produced by placenta, conjugated in the liver, excreted into the urine

• determined uE3 – unconjugated estriol

• Chylosity interfers with the determination

Problems in evaluation of the risk Problems in evaluation of the risk

of chromosomal anomaliesof chromosomal anomalies

• chorioCA and mola hydatidosa – high hCG and SP1, often various AFP – looks like m.Down or gemini

• Reduction of number of fetus in multiples pregnancies after IVF – often extremely high AFP and hCG, the concentrations change very quickly.

• Multiple pregnancies – elevation of biochemical markers is often non-symmetric, may look like NCD or m.Down

• Silent abortion or intrauterine fetus death – first elevation of biochemical markers, then decrease

• Donated ovulum – often from a younger donor – for evaluation of the risk – adjustment for body weight of the carrier, age adjustment for the biological mother

Positive findingsPositive findings

• optimal FPR (false positivity rate) – for risk 1:300 and higher genetic consultation recommended

• Collection of chorionic villi (11th -13th week)

• Amniocentesis (16th -18th week, possible from the 12th week)

Integrated testIntegrated test

30

40

50

60

70

80

90

100

0 2 4 6 8 10

Falešná pozitivita (%)

Dete

kce (%

)

Triple test

Double test

Integrovaný test

Integrovaný test - PAPP-A, NT, AFP, hCG

False positivity (%)

Intergrated test – PAPP-A, NT, AFP, hCG

Integrated test

Detection

(%)

Possibilities of screening

90-93%65%75-80%effectivity

uE3

(unstable)

hCG

AFP

Nuchal tranlucence(NT)

free β hCG

(unstable)

PAPP-A

Integrated2nd trimestr1st trimestr

LiteratureLiterature

• Zima T. : Laboratory diagnostics. 2nd Edition.

Galén Karolinum Praha 2007, 906 p. in Czech

• www.cskb.cz – recommendation for screening

• www1.lf1.cuni.cz/screeningDS