Optimized criteria for using fluorescence in situ hybridization in the prenatal diagnosis of common...

PRENATAL DIAGNOSISPrenat Diagn 2008; 28: 313–318.Published online 27 February 2008 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/pd.1959

Optimized criteria for using fluorescence in situhybridization in the prenatal diagnosis of commonaneuploidies

Sandrine Leclercq1*, Aziza Lebbar1, Gilles Grange2, Vassilis Tsatsaris2, Dominique Le Tessier1 andJean-Michel Dupont1

1Hopital Cochin-Saint Vincent de Paul, Unite de, Cytogenetique, France2Hopital Cochin-Saint Vincent de Paul, Obstetrics, France

Objective To evaluate the medical and economic performance of three strategies for selecting patients eligiblefor interphase FISH in the prenatal diagnosis of common aneuploidies.

Methods We evaluated three protocols on the same population that was referred for prenatal diagnosisbetween June 2001 and December 2006. The number of aneuploidies detected by FISH and the relative cost(reagent and technical staff cost) are reported for each strategy.

Results 2707 women were referred for prenatal diagnosis either because of advanced maternal age over 38(48%), abnormal maternal serum screening (35%) or prenatal ultrasound anomalies (17%). A total of 4.8%chromosomal anomalies (balanced and unbalanced) were diagnosed after karyotyping. Theoretically, interphaseFISH should have detected 79.4% of the unbalanced anomalies. We observed a significant improvement in thetrisomy 21 detection by selecting the probes according to the reason for referral. The last protocol adopted,which offers a rapid test to 57% of women undergoing amniocentesis, presents the best aneuploidy detectionrate (68% of total aneuploidies, 87% of trisomy 21).

Conclusion Selecting probes according to medical criteria patients combined with a technical proceduremodification allows medico-economic improvement of interphase FISH in routine diagnosis. Copyright 2008 John Wiley & Sons, Ltd.

KEY WORDS: prenatal diagnosis; interphase FISH; aneuploidy

INTRODUCTION

Karyotyping of fetal cells cultured from amniotic fluidhas been the gold standard technique for the prenataldiagnosis of chromosomal disorders since the 1970s.This test is funded by the national health insurancesystem in France for specific cases: (1) the motherbeing greater than or equal to 38 years of age, (2) thecalculated risk of Down syndrome being greater than orequal to 1 in 250 after biochemical marker testing, or(3) ultrasound abnormality. However, a significant andwell-known limitation of this technique is that cells haveto be cultured, leading to a delayed result (commonlybetween 14 and 21 days).

Aneuploidy involving chromosomes 13, 18, 21 andsex chromosomes, account for 60–80% of abnor-mal fetal karyotype detected during prenatal diagnosis(Evans et al., 1999; Caine et al., 2005). Therefore, aspecific diagnosis for these aneuploidies through molec-ular techniques was proposed, allowing for a quickerturnaround time. Rapid detection of chromosome aneu-ploidy in uncultured amniocytes by interphase fluores-cence in situ hybridization (FISH) was the first molecu-lar method introduced in 1992 by Klinger, with a result

*Correspondence to: Sandrine Leclercq, Hopital Cochin-SaintVincent de Paul, Unite de, Cytogenetique, France.E-mail: [email protected]

usually available within 24–48 h (Klinger et al., 1992).A commercial kit assay became available in 1997 andwas validated by the FDA (AneuVysion assay kit) for thenumeration of chromosomes 13, 18, 21, X and Y, with99.9% sensitivity and 100% specificity. Various clin-ical trials have reported that the prenatal FISH assayhas a high level of reliability, reproducibility and accu-racy, and an extremely high concordance rate with stan-dard karyotype (Eiben et al., 1999; Bink et al., 2000;Pergament et al., 2000; Thilaganathan et al., 2000; Sawaet al., 2001; Tepperberg et al., 2001; Luquet et al., 2002;Wyandt et al., 2006; Shaffer and Bui, 2007). Diagnos-tic impairments are mainly due to sample contamina-tion by maternal cells, low-level mosaicism or size-polymorphism in the alpha-satellite region particularlyfor chromosomes Y and 18 (Skinner et al., 2001).

Interphase FISH is usually used in addition to kary-otyping because of the benefit of obtaining a resultwithin 1 or 2 days; this is beneficial for women pre-senting with a high risk of fetal chromosomal anomaly.However, this technique uses expensive reagents (thelabeled probes for each one of the tested chromosomes)and is labor intensive, leading to an increased cost forprenatal testing. This extra cost is charged to the patients,or private or public insurances, depending on the health-care policy of each country. This extra cost is an obviouslimiting factor for the wider use of interphase FISH inconjunction with standard cytogenetics, and reducing the

Copyright 2008 John Wiley & Sons, Ltd. Received: 19 September 2007Revised: 27 November 2007

Accepted: 4 January 2008Published online: 27 February 2008

314 S. LECLERCQ ET AL.

cost would offer a rapid diagnosis to a greater num-ber of women. This would reduce maternal anxiety andimprove the management of at-risk pregnancies.

In this study, we analyzed the medical efficacy andrelative cost of three protocols that use interphaseFISH for rapid diagnosis—these protocols have beensuccessively established in our laboratory. Results thatwould have been obtained with each strategy weresimulated on the same 2707 amniotic fluid samplesreceived in our laboratory over the last 51/2 years,between June 2001 and December 2006. We analyzedthe medical performance (the number of detected andundetected aneuploidies), and the cost in terms ofreagent and workload for technical staff for all threeprotocols.

MATERIALS AND METHODS

Patients

A retrospective study was conducted with the sam-ples received in our laboratory between June 2001 andDecember 2006. During these 51/2 years, 2777 womenunderwent prenatal diagnosis testing by standard kary-otyping on amniotic fluid. Amniocentesis was per-formed under ultrasound guidance with 20-gauge nee-dles. Transplacental sampling was avoided when pos-sible. The first 2 mL of amniotic fluid was used foralpha-fetoprotein quantification to avoid contaminationwith maternal cells. Then a 20 mL sample of amnioticfluid was taken for fetal karyotype and interphase FISH.

Our study focused on three main indications for pre-natal testing, as acknowledged by the French Ministryof Health: advanced maternal age (AMA) (maternalage ≥38 years on the day of the sampling), abnor-mal maternal serum screening (MSS) (calculated riskof Down syndrome ≥1/250 after measuring maternalserum markers) and abnormal ultrasound (AU) findings.These three indications represented 98.4% of the sam-ples referred to our laboratory (2733 samples of amnioticfluid). These indications were not mutually exclusive;therefore, if multiple indications were present, AU wasconsidered the major indication, followed by AMA, andthen MSS, which has the lowest predictive positive value(1/52 for women over 38 years and 1/87 for womenunder 38 years) (Muller et al., 2002).

Data relating to ultrasound or results of MSS were col-lected for each patient when available. Nuchal translu-cency measurements were collected for pregnanciesbetween 11 and 14 weeks of amenorrhea, when thecranio-caudal length measurements and ultrasound pic-tures were available.

Interphase FISH

FISH probes

The AneuVysion assay kit (Abbott) was used for testingall five commonly implicated chromosomes (trisomy 21,18, 13, and the two gonosomal aneuploidies). This kit

includes locus-specific probes for chromosome 13 (LSI13: retinoblastoma gene at 13q14) and chromosome 21(LSI 21 region 21q21.3 to 21q22), and alpha-satelliteprobes for chromosomes 18, X and Y.

However, for samples tested specifically for trisomy21, the LSI 21 (Abbott) probe was purchased separately.

Strategies for offering interphase FISH

The selection criteria for all three protocols are detailedin Table 1.

Interphase FISH is usually offered in cases of a highrisk for chromosomal abnormality, and is performedusing probes specific for the five most frequently impli-cated chromosomes: 13, 18, 21, X and Y. Decision-making protocol 1 was implemented in the course ofa French national prospective trial aimed at evaluatingthe overall performance of interphase FISH in prenataldiagnosis (Luquet et al., 2002). Selection criteria werechosen in order to obtain a chromosomal abnormalityrate of about 10% in the selected population. Our lab-oratory successively adopted two other protocols in theongoing improvement of our decision-making strategy.Protocol 2 was a refinement of the selection criteria, tar-geting FISH probes according to the reason for referral.

Protocol 3, which is currently in use in the laboratory,took advantage of an improved technical procedure(reduction of labeled probes used: 0.5 µL vs 1 µL inprotocols 1 and 2). Thus, interphase FISH could beoffered to an increased number of patients for a samereagent cost.

We evaluated these protocols on the same populationreferred for prenatal diagnosis between June 2001 andDecember 2006 to discover whether our current strategywas medically and economically more efficient.

Table 1—Interphase FISH selection criteria

Protocol 1 Protocol 2 Protocol 3

AneuVysion kit incase of:

LSI 21 assay incase of:

LSI 21 assay incase of:

maternal age≥43 years



maternal serumscreening≥1/25



ultrasoundanomaliesincluding heartdefect, digestivemalformation,associatedintrauterinegrowthretardation andmultiplecongenitalanomalies

selectedultrasoundanomalies:nuchaltranslucency>3 mm, minorsigns of Downsyndrome

selectedultrasoundanomalies:nuchaltranslucency≥95th centile,minor signs ofDownsyndrome

AneuVysion kit incase of major ormultipleultrasoundanomalies

AneuVysion kit incase of major ormultipleultrasoundanomalies

Copyright 2008 John Wiley & Sons, Ltd. Prenat Diagn 2008; 28: 313–318.DOI: 10.1002/pd

FISH IN PRENATAL DIAGNOSIS OF COMMON ANEUPLOIDIES 315

Economic evaluation

Economic evaluation was performed on two aspects:reagent cost and technical staff cost.

Relative reagent cost takes the AneuVysion kit cost asa reference (‘n’ is the cost of one test with this reagent).The price of the LSI 21 probe in France is about 40%that of AneuVysion, giving a relative reagent cost of0.4n for each LSI 21 test.

We assumed that the slide preparation time for an LSI21 investigation is the same as that for an AneuVysionone. We estimated that cost of the technical staff wasmostly dependant on microscopic analysis, which is themost time consuming part of the test. In our experience,a mean time of 30 min per patient is necessary foranalyzing the two slides of the AneuVysion test (thistime includes the 50 nuclei analysis on each slide by twoinvestigators). The cost of the technical staff referring tothis 30-min analysis is denoted ‘t’. LSI 21 analysis takesapproximately 15 min to complete; thus, its relativetechnical staff cost is 0.5t .

‘n’ and ‘t’ are used for estimating the cost per testand cost per aneuploidy detected. This method allows anindependent economic evaluation. This method removesdifferences in the cost of the AneuVysion kit and thedifferent labor costs per hour in various countries,making this analysis adaptable for any laboratory.

Aneuploidy detection rates using the three protocolswere compared using the Student’s t-test. A p-value ofless than 0.05% was considered significant.

RESULTS

A total of 2733 women underwent amniotic fluid sam-pling between June 2001 and December 2006 for oneof three selected indications: 1309 for AMA (48%),952 for MSS (35%), and 472 for AU (17%). MSSresults were available in 88% of cases for womenunder 38 years of age, compared with 23% available forwomen over 38 years of age. Ultrasound data (nuchaltranslucency measurements) were available in 48% ofcases for women under 38 years (vs 45% for womenover 38 years).

Cases with maternal blood contamination or withoutkaryotype result because of cell culture failure wereexcluded from our study. Therefore, the study wasperformed using data from 2707 samples (1297 forAMA, 943 for MSS and 467 for AU).

Karyotype results

After conventional cytogenetic analysis, 129 chromo-somal anomalies (4.8%) were identified (described inTable 2).

Balanced chromosomal rearrangement (inversion, bal-anced reciprocal translocation or Robertsonian translo-cation) was detected in 22 cases. Unbalanced anomalieswere detected in 107 cases (representing 83% of all thechromosomal rearrangements diagnosed). Aneuploidiesthat should have been detectable by interphase FISHwere detected in 85 cases (85/107 cases, 79.4%) includ-ing trisomies for 13, 18, 21 (69 cases), sex chromosomeaneuploidies (13 cases) and triploidy (3 cases).

The chromosomal rearrangement distribution variedaccording to the reason for referral (Table 3). The rate ofchromosomal rearrangement was 10.1% if an ultrasoundanomaly was present (vs 3.1 and 2.1%, respectively forAMA and MSS). Increased nuchal translucency waspresent in 34% of samples with ultrasound anomaliesin our population. Overall, rapid FISH testing detected79.4% of unbalanced cytogenetic anomalies, but theefficacy of interphase FISH was dependent on theclinical indication for prenatal diagnosis (85% for AU,80% for AMA, and 65% for MSS). The residual riskof unbalanced cytogenetic abnormality was 0.63% for

Table 2—Summary of the 129 chromosomal rearrangements

Balanced chromosomalrearrangement

n = 22 (17%)

Robertsonian translocation 6Reciprocal translocation 6Inversion 10Unbalanced chromosomalrearrangement

n = 107 (83%)

Trisomy 13 7Trisomy 18 9Trisomy 21 53Sex chromosomesaneuploidy

13

Sex chromosomesmosaicism

5

Triploidy 3Unbalanced translocation 1SMC, ring 5Deletion, duplication,isochromosome

7

Mosaic trisomy 4

SMC, Supernumerary marker chromosome.

Table 3—Chromosomal rearrangement diagnosed in terms of indication

a: indicationfor testing (n)

b: cases withunbalancedcytogeneticresult (%)

c: unbalancedcytogeneticallyabnormal cases

detectable by FISH

(a–c): cases withnormal

FISH result

d: cytogeneticallyabnormal cases notdetectable by FISH

d/(a–c): residual risk forunbalanced cytogenetic

abnormalities (%)

AMA (1297) 40 (3.1%) 32 (80%) 1265 8 0.63MSS(943) 20 (2.1%) 13 (65%) 930 7 0.75AU (467) 47 (10.1%) 40 (85%) 427 7 1.64

Results are expressed as number and (%) of total cases karyotyped for each indication group.AMA, advanced maternal age (≥38 years old); MSS, maternal serum screening risk calculation ≥1/250; AU, abnormal ultrasound.



AMA, 0.75% for MSS, and up to 1.64% for AU, in thecase of a normal FISH result being issued.

Medico-economic evaluation of thedecision-making protocols

The medico-economic efficiency of protocols 1, 2 and 3was evaluated by simulating their use in 2707 samples.

The number of FISH tests that would have been per-formed on this population according to their respectiveselection criteria would have been 360, 740 and 1543for protocols 1, 2 and 3, respectively.

The aneuploidy detection rate and relative cost ofFISH analysis are summarized in Table 4.

The total aneuploidy detection rate was not signifi-cantly different between protocols 1, 2 and 3. However,analysis of the simulated results shows that protocol3 significantly improved the trisomy 21 detection rate(46/53 cases, 87% of trisomy 21 cases rapidly diag-nosed).

We separately analyzed the total reagent cost, theoverall time of analysis, the cost per test and the cost peraneuploidy detected for the relative cost to a laboratory.

The total reagent cost was roughly the same forthe three protocols. This result was obtained eventhough there was a 200% increase in FISH experimentsperformed between protocols 1 and 3 (360 × 2 vs 1543slides). Only 13% of women would have been offereda rapid FISH testing with protocol 1 compared to 57%with protocol 3.

By contrast, overall technical staff costs increasedbecause many more slides were analyzed with protocol3 than with protocols 1 and 2.

We noticed an important reduction between the threeprotocols after analyzing the cost per test for eachprotocol: protocol 3 allowed the greatest reduction inreagent cost.

Evaluation of the cost per abnormality is less clear-cut because of the increased analysis time, making theoverall cost analysis dependant upon the respective pric-ing of the reagent and the technician’s salary per hour.

Despite the greatest workload required with protocol 3,we noticed a reduction in terms of cost per abnormalitydetected with this protocol, after applying French figuresfor technician’s salary and reagent cost.

Undetected aneuploidies

We collected data from samples with aneuploidies notdetected by rapid FISH testing using protocols 1, 2 or 3in order to improve our selection criteria (Table 5).

Undetected cases were all related to unselected amni-otic fluid with clinical variables outside the respectivecut-off levels for protocols 1, 2 or 3. No interphase FISHfailures or false negatives were attributed to these unde-tected cases.

Lowering the maternal age and serum screening cal-culated risk cut-off levels for protocols 2 and 3 led toa significant increase in trisomy 21 detection. However,testing only for trisomy 21, if no ultrasound abnormalityindicates whether trisomy 13 or 18 is present, leads toa poorer overall detection rate for the other aneuploi-dies (mainly trisomy 18 and gonosomal aneuploidies).Indeed, the majority of X and Y aneuploidies are notassociated with ultrasound abnormality (with the excep-tion of Turner syndrome), and would go undetected withprotocol 3 used in this study. Some trisomy 18 can alsogo undetected at the first echographic examination andbecome an unexpected finding in amniotic fluid referredfor AMA.

Finally, three cases required interphase FISH becauseof fetal demise (1 case of IUFD and two cases ofamniocentesis practised at MTP).

DISCUSSION

This study was designed to evaluate medical and eco-nomic performances of three strategies for selectingpatients eligible for interphase FISH. Our improvementstrategy focused on the best use of the FISH tests thatcould be performed with a given amount of funding for

Table 4—Protocol evaluation


Total FISH tests (n) 360 740 1543- AneuVysion global test 360 103 103- LSI 21 test 0 637 1440

Aneuploidy (13,18,21,X,Y) detectable by FISH 85 85 85- detected by FISH 46 47 58- 95% confidence interval [34–58] [35–59] [43–73]

Trisomy 21 (including cases of MTP and IUFD) 53 53 53- trisomy 21 detected 29 35 46- 95% confidence interval [19–39] [24–46] [33–59]

Laboratory costTotal reactive cost (5 chromosomes = n;LSI 21 = 0.4n) 360n 357.8n 339.5nGlobal analysis time (hours) 180 211 411Technical staff cost (5 chromosomes = t, LSI21 = 0.5t) 360t 421.5t 823tCost by test n + t 0.48n + 0.57t 0.22n + 0.53tCost by anomaly detected 7.8n + 7.8t 7.6n + 8.9t 5.8n + 14.1t


FISH IN PRENATAL DIAGNOSIS OF COMMON ANEUPLOIDIES 317

Table 5—Cases of aneuploidies undiagnosed by interphase FISH in the three decision-making strategies


Trisomy21

Other aneuploidies(involving

chromosomes 13,18, X and Y) Total

Trisomy21



Trisomy21



Maternal ageunder protocolcut-off level

14 10 24 12 13 25 4 13 17

MSS overprotocol cut-offlevel

9 0 9 5 0 5 2 0 2

Isolated minorUA

0 2 2 0 4 4 0 4 4

IUFD 1 0 1 1 0 1 1 0 1Amniocentesisat MTP

0 3 3 0 3 3 0 3 3

Total 24 15 39 18 20 38 7 20 27

MSS, maternal serum screening risk calculation; UA, ultrasound abnormality; IUFD, intrauterine fetal demise; MTP, medical termination ofpregnancy.

reactions and technical staff, while maintaining similarefficacies at diagnosing aneuploidy.

Our study is consistent with interphase FISH beingaccurate for the prenatal diagnosis of common aneu-ploidies involving chromosomes 13, 18, 21 and sexchromosomes. In this 51/2-year retrospective study, wereported a total of 4.8% of chromosomal anomaliesdiagnosed after karyotyping; this is close to the over-all rate of 4% reported in a national French survey ofprenatal cytogenetic activity (Dupont and Carles, 2004).Almost 80% of the unbalanced chromosomal abnormali-ties could be detected by interphase FISH in this cohort.This result is consistent with previous reports (Evanset al., 1999; Lewin et al., 2000; Homer et al., 2003).Hence, our studied population is roughly representativeof usual prenatal cytogenetic activity, and our resultsrelated to decision-making strategies may be used as abasis for improvement in other laboratories.

The medical performance of interphase FISH wasenhanced by these new decision-making criteria (onlychromosome 21 testing when there are no major echo-graphic findings, or when there is only increased nuchalthickness). This was demonstrated by the significant dif-ference in numbers of trisomy 21 cases rapidly diag-nosed between protocols 1 and 3. This finding wasconsistent with previous observations in a retrospectivestudy of 5049 samples (Witters et al., 2002)—this studyadvised performing complete FISH testing only on ultra-sonographic indications.

The main drawback of protocols 2 and 3 was thelower efficacy for diagnosing aneuploidies other thantrisomy 21 and 13. Gonosomal aneuploidies, without theinclusion of Turner syndrome, are not usually associatedwith significant echographic abnormality, and are notdetected with our new selection criteria, whereas theywere with the first protocol. However, the geneticcounseling offered in these cases is difficult and nomajor therapeutic developments have been reported formost of these abnormalities; therefore, there may be less

clinical benefit from obtaining a rapid prenatal FISHresult. Some cases of trisomy 18 may go undetectedat the first echographic screening even though it isusually associated with major fetal malformations. Thus,using only the LSI 21 probe when normal echographicreports are issued leads to a reduced sensitivity fortrisomy 18 by interphase FISH (this does not apply totrisomy 13, which is almost always associated with earlymajor ultrasound abnormalities). The respective benefitof protocols 2 and 3 versus protocol 1 in the managementof at-risk pregnancies is therefore a matter of debate. Thebenefit related to the rapid detection of three times moretrisomy 21 cases may be worth leaving 1 or 2 trisomy18 cases and most gonosomal aneuploidies undetectedby FISH since these abnormalities will be diagnosed bystandard karyotype. Another major consequence is thatthere is a 200% increase in the number of women whoare given early reassurance through a normal FISH assayreport.

Using protocol 3, which results in an importantdecrease in the cost per test, interphase FISH can beoffered to more than half the referred patients fora constant total reaction cost. However, the overallanalysis time, hence cost of technical staff, is greaterwith this protocol, and it appears that staff workload isthe major concern for further increasing the number ofanalyzed samples by FISH.

Analyses of the reasons for referral of aneuploi-dies undetected by interphase FISH show that mostwere associated with echographically normal pregnan-cies (either AMA or MSS). Thus, increasing interphaseFISH sensitivity would require lowering the selectioncriteria further, but this would in turn lead to a largeincrease in the number of samples to be analyzed. There-fore, each laboratory should delineate the selection cri-teria correlating the number of interphase FISH testswith the maximum workload and reagent cost that canbe afforded. Ultimately, a significant step forward in theclinical management of at-risk pregnancies will be based



on the systematic use of rapid testing along with thestandard karyotype. FISH testing would require furtherreduction in reagent and staff costs, which has been fore-cast through the use of new automated hardware. Staffmember workload (human part of the overall cost) canbe cut down through automation of the hybridizationprocedure or more efficient use of the microscopic anal-ysis. Automated microscopic devices are now emerging,which are reliable and decrease the workload for tech-nical staff (Lev et al., 2005; Evans et al., 2006; Wauterset al., 2007). Reducing the cost of the interphase FISHprocedure is an important step toward using it as a sys-tematic complement to fetal karyotype. Indeed, the mainimpact of this test is to provide rapid results, giving earlyreassurance in most cases, hence improving the medicalmanagement of at-risk pregnancies.

Finally, some authors have suggested that rapid pre-natal testing molecular tests could efficiently replacestandard karyotype (Thein et al., 2000). However, it hasbeen demonstrated that omitting karyotype analyses willlead to a significant number of false negative resultsrelated to other unbalanced abnormalities (Caine et al.,2005). In our population, switching from karyotype torapid aneuploidy testing alone would have misdiagnosed20% of clinically significant anomalies (including unbal-anced structural rearrangements, marker chromosomesand mosaic aneuploidies). Even the decision to main-tain karyotyping only in women presenting with AUleads to a significant number of undiagnosed clinicallyrelevant anomalies (15/107 cases, 14%). This is bestdemonstrated by the residual risk of unbalanced abnor-mality after a normal FISH result, which was calculatedin our samples to be of 0.63, 0.75 and 1.64% in thecase of AMA, MSS and UA, respectively. These figuresare higher than the usually accepted risk for counselingan invasive prenatal diagnosis procedure; they illustratethat interphase FISH (or any other molecular techniqueallowing only specific testing) must still be consideredas a complement to standard karyotyping, at least untila noninvasive replacement procedure for amniocentesisbecomes available.

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