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ACC Spring Conference 2008 31st March to 2nd April Liverpool

UK audit of biallelic abnormal PCR results in CVS Jonathan Waters 1 , Kathy Mann 2 and Caroline Ogilvie 2 Gt Ormond St Hospital NHS Trust 1 and Guy’s Hospital Foundation Trust 2. ACC Spring Conference 2008 31st March to 2nd April Liverpool. The early embryo: late first trimester.

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ACC Spring Conference 2008 31st March to 2nd April Liverpool

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  1. UK audit of biallelic abnormal PCR results in CVSJonathan Waters1, Kathy Mann2and Caroline Ogilvie2Gt Ormond St Hospital NHS Trust1 and Guy’s Hospital Foundation Trust2 ACC Spring Conference 2008 31st March to 2nd April Liverpool

  2. The early embryo: late first trimester From Robinson et al., 2002

  3. Possible types of mosaicism within the fetal-placental unitFrom: Gardner RJM and Sutherland GR, 2004 oversimplification - cell-lineage specific mosaicism not considered

  4. chromomosomal mosaicism in CVS may be cell-lineage specific Bianchi DW et al., 1993 t mc Direct preparation: 46,XX – trophoblast LTC: 47,XX,+21 – mesenchymal core Amniocentesis: 47,XX,+21 – mesoderm/ectoderm Fetal tissue: 47,XX,+21 – mesoderm/ectoderm/endoderm Trisomy 21 conceptus with trisomy rescue in trophoblast cells

  5. QF-PCR should assay both cell lineages Bianchi DW et al., 1993 t mc Cardiff case: P07-2481 QF-PCR: disomy 21 (50%) / trisomy 21 (50%) – trophoblast + mesenchymal core Direct preparation: 46,XX[14] – trophoblast LTC: 47,XX,+21[44]/46,XX[1] – mesenchymal core Amniocentesis: 47,XX,+21[30] Postnatal blood: 47,XX,+21[30]

  6. UK data: PCR/Karyotype complete discrepancies as of 2006 Table (with modifications) from data supplied by Val Davison, Birmingham

  7. CVS double testing: QF-PCR and Karyotyping • test selectivity • different populations of cells are assayed • PCR – cytotrophoblast (ct) + mesenchymal (mc) cells • biological constraint: cytotrophoblast cells > or >> mc cells • LTC karyotype – in vitro selection for subset of mc cells • mosaicism within the biopsy sample • 1-2% CVS karyotypes are mosaic • >80% of this mosaicism is confined to the placenta • test sensitivity • QF-PCR abnormal results • Biallelic (AAB, AAC, etc) trisomies may represent mitotic errors and may rarely lead to discrepant results

  8. PCR/Karyotype complete discrepancy - practical steps • QF-PCR - sample representation • from whole fronds to use of minced, enzyme - digested whole sample mix • QF-PCR (biallelic) abnormal results • reported with a caveat that the result may represent post-zygotic non-disjunctional events and may not be representative of the fetus • suggested a UK audit might be helpful • Waters et al., 2007. Prenat Diagn 37: 332-9

  9. Results of audit – outline 10 laboratories providing a service responded 9 provided comprehensive data • Method of villus preparation for PCR assay • Complete discrepancies • trisomy 21 • trisomy 18 • trisomy 13 • biallelic results • conclusions - recommendations to Best Practice committee

  10. Results of audit • villus preparation for PCR assay • three fronds in one assay: 1 lab • three fronds in one assay and/or cellular aggregate: 1 lab • cellular aggregate (enzymatic digestion +/- finely chopped pretreatment): 7 labs • glass bead preparation: : 1 lab 6 labs have significantly changed their sample preparation approach from whole villi to cell aggregate 2 labs (Guy’s, TDL) provided overall discrepancy incidence data before and after change in sample preparation

  11. 1.20 0.93 frond 1.24 0.91 mush 1.66 digest 0.70 2.22 culture 0.64 Evidence for value of enzyme digestion method for PCR assayBiallelic trisomy 18 case – Liverpool (case 2) • Six informative markers all suggesting mosaic trisomy 18 at PCR • All diallelic (post-zygotic non-disjunction) • Cultured cells all showed +18 Data courtesy of Julie Sibbring, Regional Molecular Genetics Laboratory, Liverpool women’s Hospital See also Mann K et al 2007. Prenat Diagn 27:285-9

  12. Results of audit • trisomy 21 results • no of markers routinely used • 4 (2 labs), 5 (2 labs), 7 (1 lab), 8 (3 labs), 10 (1 lab) • 688 trisomy 21 samples • 71 showed biallelic results; average: 10.3% • with 10 markers: 6.7% • With 4/5 markers: 13.9% • 2 discrepant results from this data set (3 from previous data set) • 4 based on PCR analysis of individual villi • 1 based on glass bead sample

  13. Results of audit – trisomy 21 PCR/Karyotype complete discrepancy

  14. Results of audit • trisomy 18 results • no of markers routinely used • 4 (2 labs), 5 (1 lab), 6 (2 labs), 7,8 or 9 (1 lab each), 11 (1 lab) • 253 trisomy 18 samples • 43 showed biallelic results: average: 17.0% • with 11 markers: 14.1% • with 4/5 markers: 25% • 4 discrepant results • 2 based on PCR analysis of individual villi (x1 or x2) • 1 based on enzyme digest • 1 based on glass bead sample

  15. Results of audit – trisomy 18PCR/Karyotype complete discrepancy

  16. Results of audit • trisomy 13 results • no of markers routinely used • 4 (2 labs), 5 (3 labs), 6 (2 labs), 7 or 8 (2 labs) • 122 trisomy 13 samples • 43 showed biallelic results: average: 7.4% • 1 discrepant result • 1 based on enzyme digest

  17. Results of audit – trisomy 13 PCR/Karyotype complete discrepancy

  18. Results – sample preparation • use of mince/enzymatic digestion • experimental evidence from two laboratories – Liverpool and Guy’s (data not shown) suggests that this method enhances peak ratios for accurate analysis • two cases reported with complete discrepancies using this approach • use of glass bead approach should be monitored Relevant CVS data – incidence of complete discrepancy Guy’s : 3/4,025 (whole villi x 2) ¼,167 (mince, enzyme digest) TDL : 12/25,000 (whole villi X 1) 2/3,500 (glass bead)

  19. Results – biallelic trisomies • biallelic trisomic results • 9/10 discrepancies associated with biallelic trisomy • 3 labs distinguish between biallelic and triallelic results in QF-PCR report • proportion of biallelic trisomic results is as follows: trisomy 18: 17.0% > trisomy 21: 10.3% > trisomy 13: 7.4% -chromosome 18 markers less informative biallelic percentage dependent on number of markers used in assay • For trisomy 21 with 10 markers: biallelic incidence is 6.7% close to 4.5% for mitotic error rate previously reported in Down syndrome (Antonarakis SE et al., 1993)

  20. Summary • method of sample preparation • may help to ensure adequate representation of mesenchymal cells – usually a better predictor of fetal karyotype • use of glass beads should be evaluated • biallelic PCR results • in some cases might now be reported differently with greater experience • marker number dependent – minimum number? • report caveats • complete discrepancies • complete discrepancies are rare events • usually associated with biallelic trisomies • follow-up should be undertaken if at all possible Inform next ACC/CMGS QF-PCR rapid aneuploidy testing Best Practice meeting

  21. Acknowlegements • Sue Hamilton, QF-PCR User Group, Manchester • participating laboratories • Aberdeen • Birmingham • Bristol • Cardiff • Glasgow • Guy’s (with GOS, NWP, St George’s) • Liverpool • Oxford • Manchester • TDL

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