No results: significato e implicazioni

Francesca Romana GRATI, Ph.D., ErCLG
R&D Director
TOMA Advanced Biomedical Assays, S.p.A.
fgrati@tomalab.com
No results: significato e implicazioni

OUTLINE
Why cfDNA tests fail
Role of internal quality metrics
No results for low assay/cfDNA quality
Technical/statistical reasons
Biological reasons
No results for low FF%
Factors affecting FF%
Fetal fraction and no results in twins
No result rate @TOMA lab

Grati FR, Kagan KO. Ultrasound Obstet Gynecol. 2017 Jul;50(1):134-137
CFDNA TEST WORKFLOW
Submitted
initial
sample
population
Pre-
analytic
QC
Analysis of samples
cfDNA test
Post-
analytic QC
Samples
with a
report
Samples rejected:
• Administrative issues
• Handling issues (blood collection, maintenance and
transportation)
• Hematic plasma
• Insufficient blood/plasma amount
• Submitted sample are not suitable for the analysis
(e.g.: twin pregnancy when cfDNA test not validated for
twins; pregnancy obtained with heterologous egg donation
when cfDNA test not validated for heterologous
pregnancies, …)
• Low quality metrics of the extracted cfDNA
No results for assay quality issues
(low mat and/or fetal cfDNA quality)
QC metrics filter
Low quality QC metrics
(typically 4%)
Quality QC
metrics OK

Grati FR, Kagan KO. Ultrasound Obstet Gynecol. 2017 Jul;50(1):134-137; Jani et al.
Ultrasound Obstet Gynecol. 2015;46:515-517.
WHICH QC METRICS THRESHOLD?
QC metrics filter
Stringency too high:
•High rate of no results
•Complex follow-up and counseling
Stringency too low:
•FF not accurately measured
•Suboptimal samples included and
reported
•Higher FPs and FNs
•Unreliable results

Grati FR, Kagan KO. Ultrasound Obstet Gynecol. 2017 Jul;50(1):134-137; Jani et al.
Ultrasound Obstet Gynecol. 2015;46:515-517.
WHICH QC METRICS THRESHOLD?
Lowest rate of no
results
Highest performances,
reliability, accuracy and
quality of test results
Balance threshold is not unique and easy to determine
Dependent on the technology and laboratory quality metrics policy
BALANCE THRESHOLD

Studies where no results were not reported
VARIABLE RATE OF NO RESULTS – DEPENDING ON
TECHNOLOGY
Gil et al, Ultrasound Obstet Gynecol. 2017 Apr 11. doi: 10.1002/uog.17484

Studies where overall no results were reported
VARIABLE RATE OF NO RESULTS – DEPENDING ON
TECHNOLOGY

Studies where details for no results have been reported
VARIABLE RATE OF NO RESULTS – DEPENDING ON TECHNOLOGY
No result rate attributable to low FF% ranges from 0.1% to 6.1%
No result rate for reasons related to low quality assay ranges from 0.1% to 2.8%

NO RESULTS FOR LOW ASSAY QUALITY – POSSIBLE CAUSES
 Technical/statistical
• Assay failure
– Instrument/disposable issues
– Test is too complex for the lab
– Technology not enough validated
• Sample mix-up (air drop)
– FF is a minor component that must be
preserved with a specific lab set-up for
NIPT and automation
• Poor assay/cfDNA quality
– Intrinsic contaminants (proteins, salts, …)
– Sample variability
 Biological
• Contaminant DNA
– Dizygotic co-twin demise
– Wrong assumption about egg
donor
• Maternal and/or maternal-fetal
diseases
• Drugs?
• Borderline FF (4-6%)
• Issues in reference/normalizing
samples/chromosomes

 Biological
• Contaminant DNA
donor
diseases
• Drugs?
samples/chromosomes
LOW SAMPLE PURITY - cfDNA TESTS ANALYSING SNPs!
• Assay failure
NIPT and automation

NO RESULT FOR LOW SAMPLE PURITY
Detection of an unexpected additional genotype
Patient 1
Patient 2
G
C
G
SNPsA
T
A
T
C

 Biological
• Contaminant DNA
donor
diseases
• Drugs?
samples/chromosomes
LOW ASSAY CONFIDENCE VALUE
• Assay failure
NIPT and automation

DISTRIBUTION OF MEASUREMENTS
Countsvariability

Clinical conditions affecting both cfDNA fragmentation, amount and quality
Gestational diabetes
Pre-eclampsia
Maternal pernicious anemia
Maternal autoimmune diseases (SLE)
Intrahepatic cholestasis of pregnancy
Little is known about other possible concurrent metabolic processes affecting
cfDNA fragmentation and physiology during pregnancy
Inflammation? exercise-induced overtraining? Other immune and auto-immune
diseases? Vascular diseases? Cancer?...
Schuring-Blom et al, Prenat Diagn 2016:36:790–793; Chan et al, Proc Natl Acad Sci USA 2014;11:E5302–5311; Vlokova et al, Prenat
Diagn 2016;36:1156–1158; Dharajiya et al, Prenat Diagn 2015;35:990–993; Pavlidis NA. Oncologist 2002;7:279–287; Chan et al,
BJOG 2017; https://doi.org/10.1111/1471-0528.15006; Bianchi Genet Med. 2017 Dec 7. doi: 10.1038/gim.2017.219.

 Biological
• Contaminant DNA
donor
diseases
• Drugs?
samples/chromosomes
• Assay failure
NIPT and automation
• Poor assay quality

Aneuploidy of off-target normalizing chromosomes
DANSR likely fails: reference chromosomes at denominator are not euploid not
accurate risk estimation
counts (21)
counts (1/T2/3/…/12)
MPSS GW reports rare trisomy
Complex management, counseling burden, unecessary invasive procedure,
unecessary TOP
SNP-counting likely not affected: this technology does not use reference
chromosomes
NO RESULT DUE TO ISSUES IN REFERENCE CHROMOSOMES

Aneuploidies of normalizing/reference chromosomes
Dizygotic co-twin demise with an aneuploidy of normalizing chr
CPM for normalizing chr
Maternal malignancies involving normalizing chr - also benign
Uterine leiomyomas
http://atlasgeneticsoncology.org//Tumors/leiomyomID5031.html
Approximately 40% show abnormal karyotypes
Usually with single or few changes; rarely, they may show complex karyotypes
t(12;14), trisomy 12, deletions of portions of the long arms of chromosomes 3
or 7 and the short arm of chromosome 1, rearrangements of the short arm of
chromosome 6 and rearrangements of chromosomes 1, 3, 10, 13 and X.
Schuring-Blom et al, Prenat Diagn 2016:36:790–793; Chan et al, Proc Natl Acad Sci USA 2014;11:E5302–5311; Vlokova et al, Prenat
Diagn 2016;36:1156–1158; Dharajiya et al, Prenat Diagn 2015;35:990–993; Pavlidis NA. Oncologist 2002;7:279–287; Chan et al,
BJOG 2017; https://doi.org/10.1111/1471-0528.15006; Bianchi Genet Med. 2017 Dec 7. doi: 10.1038/gim.2017.219.

Grati FR, Kagan KO. Ultrasound Obstet Gynecol. 2017 Jul;50(1):134-137
CFDNA TEST WORKFLOW
Samples rejected:
• Administrative issues
• Handling issues (blood collection, maintenance and
transportation)
• Hematic plasma
• Insufficient blood/plasma amount
• Submitted sample are not suitable for the analysis
(e.g.: twin pregnancy when cfDNA test not validated for
twins; pregnancy obtained with heterologous egg donation
when cfDNA test not validated for heterologous
pregnancies, …)
• Low quality metrics of the extracted cfDNA
No results for assay quality issues
(low mat and/or fetal cfDNA quality)
QC metrics filter
Low quality QC metrics
(typically 4%)
Submitted
initial
sample
population
Pre-
analytic
QC
Analysis of samples
cfDNA test
Post-
analytic QC
Samples
with a
report
Quality QC
metrics OK

Palomaki et al, Genet Med 2011; 13: 913–920; Canick, et al. Prenat Diagn 2013, 33, 1–8
cfDNA results by MPSS are expressed in ‘z-score’ values
Distribution of Z-scores:
euploid fetuses: distribution is Gaussian, with a mean of 0 and a SD of 1
trisomic fetuses: the mean of the Z-score > 0 and increases with increasing fetal fraction
NO RESULTS FOR LOW FETAL FRACTION – WHY FF MATTERS

Wang E et al, Prenat Diagn. 2013 Jul;33(7):662-6; Ashoor et al, Fetal Diagn Ther 2012;31:237–243, 2012
Fetal fraction of cell-free DNA in maternal plasma
At 10GA FF is in average 10%

Courtesy of Thomas Musci; Wang E et al, Prenat Diagn. 2013 Jul;33(7):662-6; Ashoor et al, Fetal Diagn
Ther 2012;31:237–243, 2012
Fetal fraction of cell-free DNA in maternal plasma
22,384 samples
2% samples
< 4% fetal cfDNA

Courtesy of Thomas Musci (Ariosa); Wang E et al, Prenat Diagn. 2013 Jul;33(7):662-6.
Fetal fraction increases with gestational age
Post 21 wks GA:
fetal fraction
increases by
1.1% per week
Prior to 21 wks GA:
fetal fraction
increases by 0.11%
per week
10 15 20 25 30 35 40
0.00.10.20.30.4
GestationalAgeFractionalWeeks
FractionFetal

Courtesy of Thomas Musci (Ariosa); Wang E et al, Prenat Diagn. 2013 Jul;33(7):662-6, Ashoor et al, Fetal
Diagn Ther 2012;31:237–243, 2012
Fetal fraction decreases with increasing maternal weight
Maternal
Weight Bin
(kg)
Pregnancies with
≥ 4% fetal cell-
free DNA (%)
<50 >99
≥50 & <60 >99
≥60 & <70 >99
≥70 & <80 99
≥80 & <90 98
≥90 & <100 96
≥100 & <110 95
≥110 & <120 90
≥120 & <130 88
≥130 & <140 81
≥140 71
Increased apoptosis of cfmatDNA for a fixed amount of cffDNA
determined by the size of the placenta  dilution effect

Sarno et al Ultrasound Obstet Gynecol 2016; 47: 705–711
Global no result rate by cfDNA testing: risk factors
Increases with increasing maternal BMI

Wang E et al, Prenat Diagn. 2013 Jul;33(7):662-6; Canick, et al. Prenat Diagn 2013, 33, 1–8; Brar H et al., J Matern Fetal Neonatal Med.
2013 Jan;26(2):143-5; Poon et al, Fetal Diagn Ther 2013;33:215–223; Musci, Schmid et al, Oral presentation ISPD 2015; Ashoor et al, Fetal
Diagn Ther 2012;31:237–243, 2012; Ma et al, Ultrasound Obstet Gynecol; Ultrasound Obstet Gynecol 2018; 51: 274–277; Rolnik et al,
Ultrasound Obstet Gynecol. 2018 Jan 10. doi: 10.1002/uog.18993.
Other factors affecting FF%
↑ with maternal serum level of PAPP-A, ß-HCG*
↑ with CRL
↑ in women of Afro-Caribbean and East-Asian racial origin than in Caucasians***
↓ in smokers **
↓ in IVF pregnancies (probably related to PAPP-A, ß-HCG)
↓ in Twins
↓ in AMA
↓ chronic hypertension,
↓ type 1 or type 2 diabetes mellitus
↓ South Asian ethnicity
Use of low-molecular-weight heparin: heparin exerts a direct effect on the trophoblast by
reducing apoptosis
*** increased placental mass and/or activity in these racial groups reflected in the serum levels of free β-hCG and PAPP-A.
* the number of apoptotic cells would be proportional to the placental mass, reflected in the serum concentration of free
β-hCG and PAPP-A, which are also produced by the placenta
** impaired placental development and decreased placental mass in such patients

Supplement to JANUARY 2015, American Journal of Obstetrics & Gynecology, S45-Abstract 65; Pergament et al,
OBSTETRICS & GYNECOLOGY, 2014; Palomaki et al, Prenatal Diagnosis 2015; Norton et al, NEJM 2015; Musci, Schmid et
al, Oral presentation ISPD 2015; Revello et al, UOG 2016
Fetal fraction decreases in presence of a fetal trisomy
Samples with FF<4%: increased incidence, statistically significant, of chr
abnormalities compared with the overall cohort with FF≥4% (13.8% vs 2.4% - 2.7%
vs 0.4%)
↓ %FF trisomy 18-13-triploids-MX (Not in T21)
Median fetal fraction:
11.0% (IQR 8.3-14.4%) in the unaffected group
10.7% (IQR 7.8-14.3%) in trisomy 21
8.6% (IQR 5.0-10.2%) in trisomy 18
7.0% (IQR 6.0-9.4%) in trisomy 13
Via placental restrictionreduced cytotrophoblastic layerreduced ‘cfpDNA’
shedding:
Likely to involve any chr abn affecting placental growth

Rolnik et al, Ultrasound Obstet Gynecol. 2018 Jan 10. doi: 10.1002/uog.18993.
Fetal fraction as marker of placental dysfunction
Mean arterial pressure (MAP) and the mean uterine arteries pulsatility indices (UtAPI)
are negatively associated
PAPP-A and PlGF are positively associated with fetal fraction
Women at increased risk for PE and FGR and for placental disorders tend to have lower
fetal fraction and would be more likely to have reduced fetal fraction on cfDNA testing or
to receive a failed result report
Lower fetal fraction may be a consequence of smaller placental mass and even an
early sign of placental dysfunction.

Gil et al, Fetal Diagn Ther. 2014;35(3):204-11; Struble et al, Fetal Diagn Ther. 2014;35(3):199-203;
Bevilacqua, Ultrasound Obstet Gynecol. 2014 Oct 9. doi: 10.1002/uog.14690
Twins: risk assessment with cfDNA testing
Monozygotic:
• with rare exception concordant and
much like singleton
• Generally increased fetal cfDNA % (fetal
fraction)
Dizygotic:
• Different contributions of cfDNA in
maternal plasma: may vary by 2-fold
• DANSR technology proposes analysis
based on lower of the two fetal fraction
values
• Reduces possibility of ‘false negative’

Sarno et al Ultrasound Obstet Gynecol 2016; 47: 705–711; Lee et al, Hum Reprod. 2018
Feb 15. doi: 10.1093/humrep/dey033
Global no result rate by cfDNA testing: risk factors
IVF
Spontaneous
Singleton
---- Twins
Higher in twins than singleton (3-folds higher at first blood draw)
Higher in IVF than spontaneous
A repeated sampling provided a result in 51.3% of twin pregnancies and 63% of singletons

No results for low FF% in twins
The rationale for this choice is to avoid a false negative result in a dizygotic twin
pregnancy discordant for aneuploidy where the total fetal fraction is satisfactory but
the contribution of the affected fetus could be less than 4%.
An unavoidable consequence of such policy is that no result rate is higher with
those technologies measuring individual contribution from each zygote
MPSS cannot quantify the contribution from each twin as SNP genotyping is not
performed
a lower no result rate is expected at the cost of a higher FNR
Sarno et al Ultrasound Obstet Gynecol 2016; 47: 705–711; Grömminger et al, J. Clin. Med. 2014,
3, 679-692; doi:10.3390/jcm3030679

NO RESULT RATE (NRR) OF TOMA LAB
Overall NRR after 2nd redraw for T21,18,13: 0.65%
NRR for FF<4% at 2nd redraw: 0.1%
NRR for low cfDNA quality: after 2nd redraw: 0.55%
Partial NRR for SCA (all for quality metrics) after 1st draw : 0.85%
Unpublished data

NO RESULT FOR LOW QUALITY OF TOMA LAB
Unpublished data
• No results for quality issues increase at borderline FF% (4-6%)

No result rate does not always mean ‘lab failure’ rather ‘fail to meet quality
standards’ for a reliable result
If NRR is too high Technology is too complex for the lab or not enough
validated or high CQ metrics stringency
If NRR is too low or absent No/inaccurate FF measurement and/or low CQ
metrics stringency
There are not unique reasons for no results: different reasons for different
technologies
Strictly dependent on the laboratory performing the test
Medical providers
Should be aware of the reasons for no results of the chosen technology and
laboratory
Ensure appropriate technology-based pre/post test counselling is provided
CONCLUSION

No results: significato e implicazioni

No results: significato e implicazioni

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