1. Assoc. Prof. Ilona Hromadnikova Department of Molecular Biology and Cell Pathology, Third Faculty of Medicine, Prague

2.  EDTA tubes EDTA = ethylenediaminetetraacetic acid  Chelating agent, binds calcium → anticoagulated blood samples  Binds metal ions in chelation therapy (for mercury and lead poisoning)  For NIPD examination - 11 ml of anticoagulated blood

3.  Blood processing is crucial step, plasma is more suitable for the purpose of NIPD  Similar amount of cell-free fetal DNA in maternal plasma and serum  During blood coagulation – destruction of maternal cells and release of maternal DNA → up to 15x increase → decrease in sensitivity of analysis Lo et al., 1998

6.  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 1200g  Processing of blood till 24h Relative centrifugal force (RCF) – how many times is the mass of particles increased during centrifugation? ↑ radius of the rotor and speed → ↑RCF

7.  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 bodies of the trophoblast), mainly of 100-300 bp and 300- 500 bp length  Uses selective binding properties of silica-based membrane  Vacuum system, manually  Modified protocol

8. 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)

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

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

11. DNA/RNA isolation DNA amplification (amplification of RNA - 1. step is reverse transcription) Electrophoresis

12.  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

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

16.  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

17. www.appliedbiosystems.com

18.  Standard curve  Measurement of DNA concetration, using spectrophotometer

19.  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)

20.  From the 10th week  Pregnancies at risk of X- linked diseases or congenital adrenal hyperplasia (from the 6 th week because of therapy)  Conventional PCR – low sensitivity a specificity → real-time PCR

21.  Amplification of paternally inherited alleles using real-time PCR SRY gene (sex determining region) -1 copy DYS 14 gene (TSPY1- testis specific protein) – variable number of copies  Using SRY gene -100% sensitivity and 100% specificity

22.  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 5 th -9 th week of gestation

23.  Patient in the 10 th week of gestation  Children: boy, 6 years, CAH  Suspect CAH again SRY, GLO analysis SRY: 6/6+, It´s boy GLO: Isolation is OK End of Dexamethasone therapy GLO SRY

24.  Patient: carrier of haemophilia A  11+5 week of gestation SRY, GLO analysis SRY: 0/6+, It´s a girl GLO: isolation is OK Patient doesn´t have to undergo other procedures (AMC) GLO

25.  In the cases of anti-D alloimmunized pregnancies at risk of erythroblastosis fetalis or haemolytic disease of newborn  From the 10 th 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

26.  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%)

27.  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)

28.  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

29.  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 system RHD 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 GLO RhD exon 7 RhD exon 10

30.  RhD negative patient  Week of gestation: 20+6  Anti-D antibodies in maternal serum, titer 1:8+ RHD exon 7 a 10 testing, GLO system RHD 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

31.  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

32.  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

33. exon 2 (Rhc) intron 2 (RhC) exon 5 (RhE/Rhe)

34.  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

35.  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