Maternal cell contamination (mcc) testing 2014 ecuador_draft

Andrea Puppio- Todos los derechos
reservados-

Líquido amniótico
(luego sem 16)
Vellosidad Coriónica
(sem 12 a 16)
reservados-

SANGRE O SALIVA U OTRA MUESTRA DE
LA MADRE, SANGRE EN EDTA, TARJETAS
FTA, WHATMAN
VELLOSIDAD O LÍQUIDO AMNIÓTICO
O
CORDÓN UMBILICAL
reservados-

 Depende el estudio
 Si es solo para STR con 15-20 ml. de líquido
amniótico en tubo estéril BD es suficiente. Si
requiere pruebas más complejas consultar al
laboratorio cantidad necesaria
 Si es solo para STR con 10-20 vellosidades limpias
(disgregadas con aguja estéril de posible sangre), y
llevada a volumen de 15-20 ml de solución
fisiológica estéril es suficiente. Si requiere pruebas
más complejas consultar al laboratorio cantidad
necesaria
reservados-

 Depende de cada paciente, y debe firmar un
consentimiento informado donde se le informa a
la paciente que hasta incluso esta practica puede
ocasionarle la pérdida del embarazo
 En general el riesgo es menor que el de una
cesárea
 En caso de haber problemas puede suspenderse
la practica o esperar una semana o dos mas, por
ejemplo en el caso que existan hematomas
reservados-

 Se informa en el consentimiento informado sobre los riesgos y
beneficios de la practica
 Se le informa que la practica será realizada por el equipo de
obstetricia bajo total control ecográfico 4D
 Se solicitan previamente ecografías anteriores, y se solicita
mandatoriamente los siguientes estudios serológicos de
Hepatitis B y C, HIV, Toxoplasmosis, VDRL (sifilis), rubeola, y si
es necesario otros estudios serológicos que puedan afectar al
embrión. Se solicita también grupo y factor de sangre (en ciertos
casos se receta aplicación de δ globulina)
 A los 7 días se le realiza una ecografía de control en la zona de
la toma de muestra
reservados-

ES MANDATORIA y no
se realiza habitualmente
en América Latina
reservados-

 Violación
 Incesto
 Hallazgo de bebé vivo o
muerto con el cordón
umbilical
 Etc.
 Determinar la paternidad
de un niño/a, donde hay
que saber si hay células
de la madre mezcladas
 Determinar si existe o no
contaminación materna
para un estudio con fin
diagnostico (cariotipo,
estudio molecular, etc.)
Forenses No forenses
reservados-

 Check for Maternal Cell Contamination
◦ Presence of the 2nd maternal allele in the fetal sample at all loci – seen
as a mixture with peaks of varying heights
 Compare multiple preps for
◦ Identity Testing (Compare 2 DNA preps, compare Amnio to CVS, etc)
◦ Zygosity Testing
 Demonstrate fetus/Mother relationship
◦ Allele sharing
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 Amniotic Fluid
 Amniocyte Cultures
 Chorionic Villi
 Cultured chorionic villi
 POC (Products of Conception, usually placenta)
 Externally extracted DNA from any of these sources
 + Maternal blood specimen, blood spot, or buccal mucosa
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 PCR (long final extension – fragments up to 450bp)
 Run products on ABI3500 8-cap or Beckman CEQ8000 for
fragment separation and sizing
 Review data
 Confirmations for unexpected results
◦ Sometimes even contaminated data is usable data!
 Written genotype analysis submitted to Second Review
reservados-

AmpFlSTR ID Direct also
contains:
D2S1338
D19S433
Combined DNA Indexing System – set of 13 loci with highly variable
Short Tandem Repeats, plus gender XY Andrea Puppio- Todos los derechos
reservados-

 Forward and reverse primers positioned at known locations
 Amplify entire sequence between, including the highly variable
short tandem repeats (TETRA-nucleotide rpts, here: TGAA)
 Software will calculate the number of repeats based on each
amplicon’s length (here: 6)
reservados-

This slide shows the 17 loci in the color of the fluorochrome attached to the primers. Each
locus has many common alleles, ALL of which are depicted in this artificial mix. A person
will have 1 or 2 alleles at each locus. AMEL (in red) is not highly polymorphic but is useful
as a gender test (next slide).
reservados-

AMELX (amp = 105 bp) vs AMELY (amp = 111 bp)
TGGGCTCTGTAAAGATAGTGTGTTGATTCTTTATCCC
TGGGCTCTGTAAAGATAGTGGGTGGATTCTTCATCCC
AGAT------GTTTCTCAAGTGGTCCTGATTTTACAG
AAATAAAGTGGTTTCTCAAGTGGTCCTGATTTTACAG
TTCCTACCACCAGCTTCCCAGTTTAAGCTCTGAT
TTCCTACCACCAGCTTCCCAGTTTAAGCTCTGAT
The copy of Amelogenin on X produces amplified DNA of 105 basepairs.
The copy of Amelogenin on Y produces amplified DNA of 111 basepairs.
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Percent of Specimens
Maternal cells
increase in CVS
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Amniotic Fluid Amniotic Fluid
Cultures
CVS CVS Cultures Products of Conception
1.8% 0.4% 2.2% 5.5% 20.5%
Maternal cells
decrease in amnio
cultures
* “Contaminated” = 15-100% of cells are maternal
cultures
Tissue collected after
fetal demise or abortion
often contains maternal
tissue.

Good Data without MCC – D3S1358
 When HET, peaks approx
equal heights
 Fetal sample shares 1 allele
with Mom at every locus
 Fetal sample must differ from
Mom for at least 1 locus
 No peak in fetal prep at
Mom’s UN-inherited allele
Fetus
Mom
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Good Data without MCC - AMEL
Fetus  Heterozygous peaks approx
equal height – especially
important for Amel-X and
Amel-Y
Mom
reservados-

Data with MCC – D7S820
 Peaks in the fetal prep for
ALL Maternal alleles
 Easiest to see when both
Fetus and Mom are
heterozygous
 Necessary for calculating
level of MCC!
Fetus
Mom
reservados-

Fetus
Mom
 Peaks in the fetal prep for
ALL Maternal alleles
 Easiest to see when both
Fetus and Mom are
heterozygous
 Necessary for calculating
level of MCC!
reservados-

Data with MCC – Which peak is which?
Fetus
Mom
 Which peak is the Mixed Peak?
 Inheritance from Mom PLUS
MCC…
 Which peak is the MCC peak?
 Mom’s 2nd Allele…
 Which peak is Fetus Only?
 “Dad’s” Allele…
 We’ll calc the level of MCC later…
reservados-

 Cannot quantify MCC when
either sample is HOMOZYGOUS
 Still visible though, but not
informative.
 Ex: Fetus is HOM and
Mom is HET
 IMPOSSIBLE to see at loci
where Fetus and Mom are both
HOMOZYGOUS
Fetus
Mom
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Calculating % MCC – D8S1179
 Marker must be informative:
 Fetus heterozygous
 Mom heterozygous
 Different HET
MCCarea
Fetusarea + MCCarea
x 100 = % MCC
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Calculating MCC Level (%MCC)
 Back to our previous example… D16S539
6692
5633 + 6692
x 100
= 54% MCC
Mixed
Fetus
MCC
 MCC peak will be bigger than the Fetus Only peak when
MCC level is 50% or more
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 Uninformative… Why???
 no Fetus Only (Paternal) peak…
833
? + 833
x 100
Calculating MCC Level (%)
= ? %MCC
Mixed
MCC
reservados-

Serendipitous Findings – non-MCC
 Trisomies detected (apparent*): 4, 8, 11, 13, 15, 16, 18, 21
 Some in Mosaic amounts
Fetus
Mom
*Apparent since we target only one locus or gene, not entire chromosomes – NOT
reportable based solely on MCC results
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Serendipitous Findings – non-MCC (con’t)
 XYY Syndrome (known)
Fetus
Mom
 Trisomy rescue???
 Uniparental heterodisomy
 Gain/Loss of a repeat length???
 Need Dad to solve...
 Either way – no imprinting of Chr21
Fetus
Mom
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 We can visually assess for Maternal Cell Contamination by
the presence of 2nd maternal alleles (informative)
 We can demonstrate Identity between fetal preps
 We can determine that it is highly probable that a fetal
sample is related to the Maternal sample indicated
 We can confirm certain findings from other platforms IF we
have an STR on the same chromosome
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Case 1: Hydrocephalus detected on sonogram. Sequencing of the X-linked gene
L1CAM gene was NEGATIVE on amnio cultures. It was important to make sure the
DNA was not the mother’s.
Amelogenin
Mother has X
Fetus has X
and Y in equal
amounts.
Fetus is male
and has no
contamination.
At this locus on
Chr 18,
Mother = 17, 18.
Fetus = 13, 18.
No contamination.
D18S51
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Case 1: Two other loci shown. Final interpretation was “Not
Contaminated”
D8S1179
D13S317
At this locus on
Chr 8,
Mother = 10, 13.
Fetus = 10, 13.
Not informative.
At this locus on
Chr 13,
Mother = 8, 10.
Fetus = 10, 11.
No contamination.
reservados-

Case 2: Test for Noonan Syndrome due to increased nuchal
translucency. CVS cultures were submitted.
D7S820
TH01
D8S1179
CSF1PO
All markers showed that the “fetal sample” was 100%
overgrown with maternal cells. The gene sequence for the
Noonan Syndrome genes could not be inteArpndrreeat ePudp.pio- Todos los derechos
reservados-

Case 3: Prenatal Diagnosis for Junctional Epidermolyis Bullosa
1. Baby with Junctional EB, died. DNA was saved.
2. Test hotspots in Lamin 5 genes --> no mutations
3. Sequence LAMB3 --> no mutations
4. Sequenced LAMC2 --> no mutations
5. Sequenced LAMA3 --> no mutations
6. Pregnant!
7. Sequenced COL17A1---> 2 different mutations found
8. CVS ---> Very small sample, uncultured, positive for maternal
contamination.
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Case 3: MCC results on the first CVS Sample.
C = CVS, M= Mother, F = Father*
C
Amelogenin X vs. Y D3S1358 D18S317 TH01
All markers show maternal DNA in the fetal specimen. There are
3 peaks, or, one peak is too high.
M
F
* Testing of fathers is not necessary and is not done now.
reservados-

Case 3: Prenatal Diagnosis for Junctional Epidermolyis Bullosa
9. We did not test the cultures that were available from the first CVS
procedure. They would be contaminated also. We requested they
perform another CVS procedure and obtain a larger specimen.
10. Repeat CVS --> Larger sample, uncultured, was free of maternal
contamination.
11. The COL17A1 result using the 2nd CVS specimen was:
Maternal COL17A1 mutation PRESENT
Paternal COL17A1 mutation ABSENT
9. This result is indistinguishable from the maternal genotype but we
know that it is the fetus’s genotype and he will be an unaffected
carrier.
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Utilización de Identifiler, Powerplex
Mínimo de 15 loci + amelogenina
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 Preguntas
 andreapuppio@gmail.com
 +5411-4778-1724
 Skype: andreapuppio
reservados-

Maternal cell contamination (mcc) testing 2014 ecuador_draft

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