Assoc. Prof. Ilona HromadnikovaAssoc. Prof. Ilona Hromadnikova

Department of Molecular Biology and Cell Department of Molecular Biology and Cell Pathology, Third Faculty of Medicine, PraguePathology, Third Faculty of Medicine, Prague

EDTA tubesEDTA tubes

EDTA = ethylenediaminetetraacetic acid EDTA = ethylenediaminetetraacetic acid Chelating agent, binds calcium → anticoagulated Chelating agent, binds calcium → anticoagulated

blood samples blood samples Binds Binds metal ions in chelation therapymetal ions in chelation therapy ( (for mercury for mercury

and lead poisoningand lead poisoning)) For NIPD examination - 11 ml of anticoagulated For NIPD examination - 11 ml of anticoagulated

bloodblood

Blood processing is crucial step, plasma is more Blood processing is crucial step, plasma is more suitable for the purpose of NIPDsuitable for the purpose of NIPD

Similar amount of cell-free fetal DNA in maternal Similar amount of cell-free fetal DNA in maternal plasma and serumplasma and serum

During blood coagulation – destruction of maternal cells During blood coagulation – destruction of maternal cells and release of maternal DNA and release of maternal DNA → → up to 15x increase up to 15x increase → → decrease in sensitivity of analysisdecrease in sensitivity of analysis

Lo et al., 1998

Oddělení molekulární biologie a patologie buňky Gynekologicko-porodnická klinika 3. LF UK a FNKV

Ruská 87, 100 00 Praha 10, tel.: 267 102 170, 171

Žádanka na kvantifikaci extracelulárních nukleových kyselin z periferní krve těhotných žen pro určení možných placentárních dysfunkcí

10 ml nesrážlivé krve EDTA Nutno dopravit do laboratoře nejdéle 24 hodin po náběru. PROSÍME O PEČLIVÉ VYPLNĚNÍ ŽÁDANKY, JINAK BY MOHLO BÝT VYŠETŘENÍ ODMÍTNUTO (viz údaje pro pojišťovnu a akreditace JCI ) !!!!!!!

Jméno a příjmení: Adresa trvalého bydliště: PSČ: RČ: Pojišťovna: Žadatel (razítko): IČO žadatele: Týden gravidity: Diagnóza slovy: Kód(y) DG dle MKN: Datum náběru: Přesný čas náběru: Datum přijetí vzorku laboratoří: Přesný čas přijetí: Indikující lékař (jméno + podpis):

Oddělení molekulární biologie a patologie buňky Gynekologicko-porodnická klinika 3. LF UK a FNKV

Ruská 87, 100 00 Praha 10, tel.: 267 102 170, 171

INFORMOVANÝ SOUHLAS Neinvazivní RHD genotypizace plodu z periferní krve matky u těhotenství

s rizikem hemolytického onemocnění plodu (novorozence) v důsledku anti-D aloprotilátek.

Popis výkonu: Bude Vám odebráno 11 ml periferní (žilní) krve do EDTA zkumavek. Z žilní krve bude izolována DNA, která bude sloužit k neinvazivnímu vyšetření RhD faktoru plodu. V případě identifikace RHD negativního plodu není těhotenství ohroženo hemolytickým onemocněním novorozence. Vaše vzorky budou vystupovat pod kódem a tak budou chráněna Vaše osobní data a informace. Tento informovaný souhlas bude uložen ve Vaší lékařské dokumentaci. Na základě tohoto poučení prohlašuji, že souhlasím s uvedeným lékařským vyšetřením. Jméno, příjmení: …………………………………. Datum: …………………………………. Podpis: …………………………………..

Crucial step; using of higher centrifugal force could decrease concentration of total cell-free DNA and unfortunately also of fetal cell-free DNA

Centrifugation 2x at 1200gCentrifugation 2x at 1200g Processing of blood till 24hProcessing of blood till 24h

Relative centrifugal force (RCF) – how Relative centrifugal force (RCF) – how many times is the mass of particles many times is the mass of particles increased during centrifugation?increased during centrifugation?

↑ ↑ radius of the rotor and speed → radius of the rotor and speed → ↑↑RCFRCF

QIAamp DSP Virus kit (Qiagen) Commercial kit, CE-marked

For isolation of viral NA (DNA + RNA) from the human plasma and serum

cffDNA is fragmented (apoptotic cffDNA is fragmented (apoptotic bodies of the trophoblast), bodies of the trophoblast), mainly of 100-300 bp and 300-mainly of 100-300 bp and 300-500 bp length500 bp length

Uses selective binding properties of silica-based membrane

Vacuum system, manually Modified protocol

1. Lysis of the sample(AL buffer, protease, inactivation of RNases, at 56°C)

2. Silica-based membrane binding of nucleic acids during passing of lysate

3. Washing of membrane (removing residual contaminants)

4. Elution of nucleic acids from the membrane(60 μl of AE buffer)

Reaction mixture: dNTPs, PCR buffer with Mg2+, polymerase, primers, water

http://www.onkologickecentrum.cz/downloads/vysetreni/laborator-metody.pdf

DNA/RNA isolation

DNA amplification

(amplification of RNA -

1. step is reverse transcription)

Electrophoresis

Low sensitivity and specificity – crucial for NIPD Low resolution Laborious, work with ethidium bromide, special room necessary Analysis of final amplification product on electrophoresis →

targeted sequence is/isn´t present

BUT only approximate quantity

→ real-time PCR

Based on the principle of conventional PCR Enables quantification of target DNA sequence –

records each PCR cycle in real-time Using of probes (fluorescent probes), that

specifically or non-specifically binds amplified DNA

Flu

ores

cenc

e

PCR cycle

Ct = 1. significant increase of PCR product (increase of fluorescent emission, correlates with the initial amount of target sequence

Specific binding between probe and target sequence: TaqMan probes, MGB probes, Scorpion, Molecular beacons

Non-specific binding: SYBR Green Risk of false positive signals, not convenient

for NIPD

www.appliedbiosystems.com

Standard curve Measurement of DNA

concetration, using spectrophotometer

Example:42 ng/μl = 42 000 pg/μl : 6,6 = 6363

copies/μl How many copies/PCR well?Usually 3 concentrations, 10times dilutions Usage in NIPD:

Quantification of nucleic acids (SRY, GLO, RASSF1A) for diagnosis of pregnancies with placental insufficiency (PEP, IUGR)

From the 10th week Pregnancies at risk of

X-linked diseases or congenital adrenal hyperplasia (from the 6th week because of therapy)

Conventional PCR – low sensitivity a specificity

→ real-time PCR

Amplification of paternally inherited alleles using real-time PCR

SRY gene (sex determining region) -1 copyDYS 14 gene (TSPY1- testis specific protein) – variable number of copies

Using SRY gene -100% sensitivity and 100% specificity

X–linked diseases – female foetuses(SRY and DYS14 negative), no need for CVS and/or AMC, later ultrasound confirmation

CAH – male foetuses (SRY and DYS14 positive), termination of dexamethason therapy Dexamethason – prevents virilisation (abnormal

development of male sexual characteristics in a female), necessary from the 5th-9th week of gestation

Patient in the 10th week of gestation Children: boy, 6 years, CAH Suspect CAH again

SRY, GLO analysisSRY: 6/6+, It´s boyGLO: Isolation is OK

End of Dexamethasone therapy

GLO

SRY

Patient: carrier of haemophilia A 11+5 week of gestation

SRY, GLO analysisSRY: 0/6+, It´s a girlGLO: isolation is OK

Patient doesn´t have to undergo other procedures (AMC)

GLO

In the cases of anti-D alloimmunized pregnancies at risk of erythroblastosis fetalis or haemolytic disease of newborn

From the 10th week of gestation RhD negativity – 15% of Caucasian population

Gene deletion RHD gene variation (pseudogene RhD ψ, RHD-CE-D hybrid

gene)- stops expression of RhD protein, attention to false positive results in the cases of RhD negative individuals

RHD (pseudogene) Complete inactive RHD gene, 37-bp insertion in

exon 4 (PCR) + 1-2 stop codons in exon 6, earlier termination of translation, 0 HON

66% of Africans, 27,7% Japaneses and11% of Brazilian

Hybrid RHD-CE-D gene RhD negative phenotype: 3´ end of exon 3 a

exons 4-8 of RHCE gene RHD exon 10 +, exon 7 – (PCR)

Weak C, VS+, Africans (3%)

RHD genotyping– necessary to analyse more regions of RHD gene

Most often combination of exon 7 and 10 or exon 7 and 5

Interpretation of results together with ethnic group (incidence of RHD gene alterations)

Our laboratory – combination of exon 7 and 10

with 100 % specificity a 100 % sensitivity

RhD negative foetuses at alloimmunized pregnancies are not endangered by HDN, in the cases of RhD positive foetuses – important information for clinicians

RhD negative patient Week of gestation: 20+6 Anti-D antibodies in maternal serum, titer 1:32+

Testing of RHD exon 7 a 10, GLO systemRHD exon 7: 3/3+RHD exon 10: 3/3 +GLO: isolation is OK

Foetus is RhD positive, important information for clinicians

Foetus is endangered by erythroblastosis fetalis and HDN

GLORhD exon 7

RhD exon 10

RhD negative patient Week of gestation: 20+6 Anti-D antibodies in maternal serum , titer 1:8+

RHD exon 7 a 10 testing,GLO systemRHD exon 7: 0/3+RHD exon 10: 0/3 +GLO: isolation is OK

Foetus is RhD negative

Foetus is not endangered by erythroblastosis fetalis and HDN

GLO

Erythroblastosis fetalis and HDN can be caused by other antigens of Rh system

→ Fetal RHCE genotyping in the cases of anti-c, anti-E a anti-C alloimmunized pregnancies

Amplification of paternally inherited alleles using real-time PCR

C/c a E/e polymorphism - nucleotide substitutions and insertions in RHCE gene

Ser103Pro polymorphism (SNP in exon 2) for Rhc

Pro226Ala polymorphism (SNP in exon 5) for RhE

109 bp insertion in intron 2 for RhC

exon 2 (Rhc) intron 2 (RhC)

exon 5 (RhE/Rhe)

RHD exon 7 and exon 10, RHCE - C allele detection with 100 % specificity and 100 % sensitivity

RHCE - c allele and E allele genotyping (SNP) –

100 % specificity and 95 % sensitivity, more difficult – most of cell-free DNA is of maternal origin

RhcCE negative foetuses at alloimunized pregnancies – not endangered by HDN,positive foetuses – early information for clinicians

RhE negative patient Week of gestation: 20+6 Anti-E antibodies in maternal serum, titer 1:16+

RHE: 6/6+GLO: isolation OK

Foetus is RhE positive, special care by clinician

Foetus is endangered by erythroblastosis fetalis and HDN

GLO

RHE