nipt ( noninvasive prenatal testing ) technologies rely on massively parallel sequencing or single nucleotide polymorphismbased analysis of cellfree dna ( cfdna ) isolated from maternal plasma . the fetal fraction ( cellfree fetal dna , cffdna ) , which represents approximately 10% of the cfdna 1 , has been found to be derived primarily from placental trophoblastic cells , rather than from the fetus 2 . multiple reports have demonstrated that discrepancies between fetal genotype and nipt results can occur as a consequence of cpm ( confined placental mosaicism ) for the detected aneuploidy 3 , 4 , 5 , 6 , 7 . each commercial laboratory has independently defined the statistical measures by which it classifies patient results . in this case , the maternal sample was sent to verinata ( now illumina ) for the verifi prenatal aneuploidy test . sensitivity and specificity for this test are reportedly high : > 99.9% and > 99.8% for chromosome 21 , and > 97.4 and > 99.6% for chromosome 18 , respectively ( verinata ) . as a matter of company policy , verinata reports only the qualitative result and does not release quantitative information about the thresholds for its tests or the patient 's test values relative to the thresholds . we present a case of trisomy 21 with cpm for a cell line with both trisomy 18 and trisomy 21 , in which nipt detected both abnormalities . a 32yearold primigravida was referred by her local obstetrician at 17 weeks ' gestation regarding positive second trimester quad screen results , in which the risk of trisomy 21 in the fetus was estimated to be 1/130 . the results indicated no increased risk for either trisomy 18 or an open neural tube defect . ultrasound examination identified a single intracardiac echogenic focus , increasing the risk 8 of trisomy 21 to 1/49 . the patient was counseled regarding prenatal genetic testing options and declined both amniocentesis and nipt . , the patient was referred for further evaluation after a third trimester ultrasound revealed a fetal growth lag . a result of  aneuploidy detected  was returned for chromosomes 18 and 21 and interpreted as consistent with trisomy for both chromosomes . amniocentesis was declined . at 38 weeks , oligohydramnios was identified by ultrasound , and labor was induced . a male infant was delivered with apgars of 7/7 and weighing 2450 g. the newborn 's facial features , including upslanting palpebral fissures , epicanthal folds , and flat nasal bridge , were consistent with trisomy 21 . there were no features concerning for trisomy 18 by physical examination . at 7 months of age , a cord blood sample and eight placental biopsies ( four from the fetal side , four from the maternal side ) were taken for routine cytogenetics and fish ( fluorescence in situ hybridization ) analyses . an extensive workup was performed to screen for evidence of fetal or placental mosaicism ( table 1 ) . the cord blood sample was positive for trisomy 21 in all 50 cells analyzed by routine cytogenetics . fish analysis was performed using probes for chromosomes 21 and 18 at the runx1 and bcl2 loci , respectively , in conjunction with a chromosome 12 probe ( etv6 ) to control for the ploidy level ( abbott molecular , abbott park , illinois , usa ; fig . a total of 500 interphase cells from the cord blood sample was examined , and a fluorescence pattern consistent with trisomy 21 was observed in all . cell counts from interphase fish and karyotype analysis of cord blood and placental biopsies interphase fish performed on direct cytotrophoblast preparations ; karyotyping performed on cultured whole villi . fish ( fluorescence in situ hybridization ) probes for chromosomes 21 ( red signal ) , 18 ( yellow or fused red / green signal ) , and 12 ( green signal ) were used to identify cells with trisomy 21 ( left ) and both trisomy 18 and trisomy 21 ( right ) . independent direct cytotrophoblast preparations were established from the eight placental biopsies . from each biopsy , 100 interphase cells were examined by fish for evidence of aneuploidy for chromosomes 18 and 21 . a fluorescence pattern consistent with double trisomy for chromosomes 18 and 21 ( 48,xy,+18,+21 ) was observed in 49/800 cells distributed among only three of the eight biopsies . the majority ( 46/49 ) two cells with double trisomy were identified in an additional biopsy from the fetal side , and only one cell with double trisomy was identified among the 400 cells examined from the four independent biopsy preparations from the maternal side of the placenta . all 140 metaphase cells examined by routine cytogenetics from the eight biopsy sites had a trisomy 21 karyotype . here , we report a pregnancy with two trisomies identified by nipt , which were subsequently found to be due to true fetal trisomy 21 and cpm for trisomy 18 . this case represents a uniquely complicated scenario for diagnosis and counseling . nonmosaic double trisomy for chromosomes 18 and 21 has been reported in cases of early spontaneous abortion 9 , but to our knowledge never in a third trimester pregnancy as in this case . the clinical information available suggested trisomy 21 was more likely to be confirmed than trisomy 18 . however , in the absence of prenatal diagnostic testing , it remained possible that either trisomy , both trisomies , or neither would be present in the fetus . potential outcomes also included true fetal mosaicism and cpm involving one or both of the trisomies . each of these possibilities and their likely effects on the health and development of the fetus were discussed with the patient . this report adds to the growing body of literature documenting cpm as a common cause of discordant nipt test results . in most previously reported cases , nipt detected aneuploidy for a single chromosome , while the fetus was found to have either a normal chromosome complement 3 , 4 , or an entirely different aneuploidy 5 , 7 . in the present case , nipt results for chromosomes 18 and 21 were both reported as  aneuploidy detected .  given the average proportion of cells with evidence for trisomy 18 in the eight placental biopsies was quite low ( 49/800 cells , or 6.1% ) , but all eight samples were overwhelmingly positive for trisomy 21 , it is of interest that the nipt report did not distinguish the true trisomy 21 from the lowlevel cpm for trisomy 18 . assuming the relative excess cffdna derived from chromosomes 21 and 18 is in proportion to the respective levels of trisomy identified in the placenta , the identical results for the two chromosomes may be due at least in part to the dual threshold method verinata uses to detect aneuploidy . that is , even if the score for chromosome 18 were only marginally greater than the abnormal threshold and the score for chromosome 21 well above it , by definition both chromosomes would be reported positive for trisomy . alternatively , recent data suggest that cffdna may not represent the average chromosome complement across all trophoblastic cells , but instead derives preferentially from trophoblastic cells in discrete regions of the placenta . mao et al . 7 reported a case of fetoplacental mosaicism in which the fetus was nonmosaic for trisomy 18 , and the placenta contained three cell lines : trisomy 18 , trisomy 21 , and euploid . analysis of multiple placental biopsies showed highly variable proportions of the three cell lines , and the authors concluded that the cffdna may have arisen primarily from regions of the placenta with a relative excess of trisomy 21 cells in the trophoblast . the current case is similar in that there was a highly uneven distribution of placental cells with double trisomy ( ranging from 0% in five biopsies to a maximum of 46% in a single biopsy ) . thus , it seems plausible that our patient 's nipt results reflect a disproportionate contribution of cffdna from placental regions with a relatively high fraction of double trisomy cells . although it has been demonstrated that cpm is a relatively common contributor to discordant nipt results , it is difficult to establish on a casebycase basis . in the present case , trisomy 21 was present in all of the cells examined from cord blood and eight placental biopsy sites , but dual trisomy , 18 and 21 , was observed in only 49 of the 940 cells examined from the eight biopsy sites and in none of the 550 cells examined from cord blood . furthermore , the percentage of cells demonstrating dual trisomy was variable and ranged from 1% to 46% within the three positive placental biopsy sites . while it is typically not possible to assay multiple placental sites for an ongoing pregnancy , and it would be cost prohibitive to do so as extensively as done here after each birth , a thorough clinical evaluation and followup karyotyping and/or fish studies should be attempted . in summary , this report illustrates the necessity of considering the possibility of cpm following any positive nipt result , but especially in rare cases of highly unusual findings , such as the double trisomy described here . the complicated nature of this case underscores the importance of discussing with patients the possibility of both atypical and discordant results during pretest counseling and consent .
key clinical message 
 nipt ( noninvasive prenatal testing ) detected trisomy for two chromosomes . 
 one trisomy reflected the fetal karyotype , and the other resulted from cpm ( confined placental mosaicism ) . 
 this case illustrates that extensive cytogenetic analysis can be required to identify cpm , and that patients should be counseled regarding the possibility of discordant nipt results .