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Diagnosing Aneuploidy in Recurrent Miscarriage Products of Conception Using F.I.S.H

Diagnosing Aneuploidy in Recurrent Miscarriage Products of Conception Using F.I.S.H. Katie Camidge Nottingham Cytogenetics. Introduction.

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Diagnosing Aneuploidy in Recurrent Miscarriage Products of Conception Using F.I.S.H

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  1. Diagnosing Aneuploidy in Recurrent Miscarriage Products of Conception Using F.I.S.H Katie Camidge Nottingham Cytogenetics

  2. Introduction • In Nottingham, 62% of solid tissues received are products of conception (P.O.Cs) or placentas. Upon arrival P.O.C samples are examined to identify if any foetal parts are present, if not, as with placentas, CV tissue is identified for long term culture. • CV tissue is chosen for culture for its ease of identification and separation from maternal decidua to reduce the risk of maternal cell contamination.

  3. Maternal Cell Contamination • Nationally 2:1 (female: male) ratio in P.O.C derived CV. • The following audit suggested high levels of maternal cell contamination in early gestational age P.O.Cs (<13wks); with 40/41 normal karyotypes being female. • Only 4 trisomies, 1 triploid and 1 tetraploid were found giving a 13% abnormality rate, and a success rate of 59%. The success rate is the number of samples that grew sufficiently in long term culture to provide a result.

  4. Maternal Cell Contamination • Those at later gestational age (>14wks) showed a lower rate of maternal cell contamination with only 15/23 having a normal female karyotype. However success rate was only 39%. • In order to identify and overcome maternal cell contamination we decided to use uncultured F.I.S.H using vysis probes to identify the common abnormalities. Probes used were LSI 13, 18, 21, 22, CEP 15, 16 and CEPX SRY.

  5. Uncultured F.I.S.H Technique • 30 minutes in 1mg/ml collagenase at 37degrees Celsius. Agitate every 10 mins to encourage cell detachment. • The tube is centrifuged, supernatant poured off before Trypsin EDTA is added for 5mins to remove excess protein. • 4 mls of 0.056M KCl is added to the culture and then it is incubated for a further 20 mins at 37 degrees before adding 2mls cold fixative.

  6. Uncultured F.I.S.H Technique • 2 further fixatives prior to making slides. • F.I.S.H is carried out on interphase.

  7. Monosomy 21 LSI 13 LSI 21 LSI 13

  8. Trisomy 21 LSI 21

  9. Triploid cell LSI 15 LSI 16 LSI 13 LSI 21 CEP 15,16 LSI 13,21 LSI 22 LSI 22

  10. Results • F.I.S.H identified 45% abnormality rate compared to 13% identified by culture and karyotype. • 29/60 female therefore showing a lower rate of maternal cell contamination. Previously 40/41. • Success rate of 97% (58/60). Compared to 59% culture and karyotype.

  11. Abnormal Cases

  12. Conclusion • The low culture and karyotype abnormality rate indicates that the use of F.I.S.H for solid tissues provides more accurate results even with a limited range of probes being used. • Lower maternal cell contamination identified in uncultured technique compared to long term cultures. • Uncultured F.I.S.H is now routinely undertaken at Nottingham for all P.O.C samples received, coinciding with parental bloods to exclude any possible balanced rearrangements.

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