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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