FISH for aneuploidy

o Ex. AneuVyision

§ Probes:

· CEP X,Y,18 probe cocktail

· LSI 13 and 21 cocktail

§ Reporting criteria (Mt Sinai)

· Normal - >= 80% of nuclei show normal number of signals for each probe

· Abnormal - >= 70% of nuclei show the same abnormal number of signals for a probe

· Inconclusive (any one of the below criteria met):

o neither of above conditions are met

o inadequate number of nuclei

o slide quality

o failure to meet slide quality assessment criteria (clear signals, low background)

o bloody sample that contains only XX nuclei

o Generally cutoff of 60% abnormal nuclei for a reliable call of aneuploidy

o Informative test rate:

§ 97.2% (Tepperberg et al, 2001)

§ Reasons for uninformative test:

· Inadequate volume of amniotic fluid

· Maternal cell contamination

· Other

· Hybridization failure (8 / 5348)

o Sensitivity:

§ 67 to 94% (ref in Wyandt et al, 2006)

§ 83.8% (Wyandt et al, 2006)

§ 99.2% overall (Hulten et al, 2003)

§ 99.7% (0.024 false negative rate) 7 false negatives in 30,000 total cases false negative (Tepperberg et al, 2001)

§ 94% (Weremowicz et al, 2001)

§ 99% (Thilaganathan et al, 2000)

· Older single probe technique and the stricter analytical criteria used by older studies are the most likely cause of lower sensitivities and higher failure rates

o Specificity:

§ 100% (Wyandt et al, 2006)

§ 99.98% (1 in 30,000 false positive) (Tepperberg et al, 2001)

§ 99.96%

o Mosaicism is not reliably detected

o Possible reasons for false positive:

§ Trisomy:

· Cross-hybridization with other chromosomes

o 13/21 to 22 (acrocentric to acrocentric)

§ Reciprocal translocation with a breakpoint at the centromere, with exchange of short arm material is most likely for acrocentric chromosomes (Thangavelu M et al, 1998)

o 13/21 to 14 (acrocentric to acrocentric)

o 18 to 22 (submetacentric to acrocentric) (Thangavelu M et al, 1998)

§ May be due to an interchromosomal rearrangement involving 18 and 22

· DNA from chromosome 18 was inserted into chromosome 22

§ Or a mutation involving the alpha-satellite DNA on 22

· Mutation would have to be repeated multiple times for this to account for the signal

· Less likely than the first explanation

o X to 19 (Winsor et al, 1999)

o 18 to heterochromatic region of 9 (9 qh) (Wei et al, 2007)

§ Most likely mechanism is insertion of chromosome 18 a-satellite DNA into the heterochromatic region of 9

· Both reciprocal translocation and mutation are unlikely

· Constitutionally abnormal chromosome

o t(Y;15)

§ X (XXY)

§ Y (XYY)

· Unexplained

o Rare cases using LSI 21

· Contamination with X,Y,18 cocktail (Ycen probe has efficacy even at very low dilutions)

o Experimentally induced (Wang et al, 2007)

§ Monosomy:

· Pericentromeric deletion

o 21

o Y

· Centromeric deletion

o 21

· Heteromorphism in centromeric region

o 21

· Polymorphism

o 21

o 18

o Particularly X, Y, 18 alpha satellite regions

· Variation in signal intensity (difficult to differentiate normal and abnormal signals)

o 21

· High background fluorescence / autofluorescence

o 21

o 18

· Maternal cell contamination

o 21

o 13

o Y

· Poor hybridization

o 21

o 13

o Y

o 18

o X

· Reduced copy number of alpha satellite sequences

o 18

o Y

· Abnormal chromosome

o der(Yp)

o Possible reasons for false negative trisomy:

§ Deletion:

· 13 (RB1 deletion)

§ Poor sample quality (late gestational age, dark brown colour)

· Degraded, weak signals

§ Maternal cell contamination

§ Polymorphisms

· Particularly with alpha satellite regions of chromosomes X, Y, and 18 may affect signal size and intensity resulting in absence of a signal

o Significant polymorphisms of repetitive DNA sequences can influence signal size and lead to discrepant results

o Lab procedures to avoid false positive results (Mt Sinai):

§ Slide quality assessment :

· Probe signal intensity should be bright, distinct, easily seen

· Background should be dark, free of excessive fluorescent particles or haziness

· Watch for duplicate weaker signals indicative of cross-hybridization

§ Criteria for assay rejection:

· Probe signals consistently weak (hard to visualize even with signle band filters)

· Cross-hybridization in the majority of nuclei resulting in inconsistent scoring

· Excessive background noise (signals) that cannot be distinguished from “real” signals

· Excessive diffuse (spiderlike) signals which tend to span the nucleus

· Suspicion of probe or patient cross-contamination

· Inadequate controls

§ Procedure for any interphase FISH result of loss of X or Y regions:

· Rerun the assay on a fresh slide, with LSI Kallmann and LSI SRY

· Normal male control

§ Check the aqua filter on the LSI 13/21 target to check for any aqua signals

· Suggests contamination with CEP X,Y,18(aqua) cocktail

· Could result in a false positive result

§ Check for mixture of XX and XY nuclei

· If present, score only XY to avoid maternal cell contamination

§ Interphase testing is always followed up with conventional chromosome analysis

· Inconsistency of the two results is reviewed

o

References:

1. Wyandt HE, Tonk VS, Huang XL, Evans AT, Milunsky JM, Milunsky A. Correlation of abnormal rapid FISH and chromosome results from amniocytes for prenatal diagnosis. Fetal Diagn Ther. 2006;21:235-240.

2. Hultén MA, Dhanjal S, Pertl B. Rapid and simple prenatal diagnosis of common chromosome disorders: advantages and disadvantages of the molecular methods FISH and QF-PCR. Reproduction (2003);126:279-297.

3. Tepperberg J, Pettenati MJ, Rao PN, et al. Prenatal diagnosis using interphase fluorescence in situ hybridization (FISH): 2-year multicenter retrospective study and review of the literature. Prenat Diagn (2001);21:293-301.

4. Weremowicz S, Sandstrom DJ, Morton CC, Niedzwiecki CA, Sandstrom MM, Bieber FR. Fluorescence in situ hybridization (FISH) for rapid detection of aneuploidy: experience in 911 prenatal cases. Prenat Diagn (2001);21:262-269.

5. Tepperberg J, Pettanati MJ, Rao PN, et al. Prenatal diagnosis using interphase fluorescence in situ hybridization (FISH): 2-year multi-center retrospective study and review of the literature. Prenat Diagn (2001); 21: 293–301.

6. Thangavelu M, Chen PX, Pergament E. Hybridization of chromosome 18 alpha-satellite DNA probe to chromosome 22. Prenat. Diagn (1998);18:922–925.

7. Thilaganathan B, Sairam S, Ballard T, Peterson C, Meredith R. Effectiveness of prenatal chromosomal analysis using multicolour fluorescent in situ hybridization. BJOG (2000);107:262-266.

8. Wei S, Siu VM, Decker A et al. False-positive prenatal diagnosis of trisomy 18 by interphase FISH: Hybridization of chromosome 18 alpha-satellite DNA probe (D18Z1) to the heterochromatic region of chromosome 9. Prenat Diagn (2007);27:1064-1066.

9. Winsor EJT, Dyack S, Wood-Burgess EM, Ryan G. Risk of a false-positive prenatal diagnosis using interphase FISH testing: hybridization of alpha-satellite X probe to chromosome 19. Prenat Diagn (1999);19:832-836.