Case 1:20-cv-01644-RGA Document 1-13 Filed 12/03/20 Page 1 of 7 PageID #: 844
`Case 1:20-cv-01644-RGA Document 1-13 Filed 12/03/20 Page 1 of 7 PageID #: 844
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`EXHIBIT 13
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`EXHIBIT 13
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`Case 1:20-cv-01644-RGA Document 1-13 Filed 12/03/20 Page 2 of 7 PageID #: 845
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`PRELIMINARY
`COMMUNICATION
`
`Methods to Increase the Percentage
`of Free Fetal DNA Recovered
`From the Maternal Circulation
`
`Ravinder Dhallan, MD, PhD
`Wei-Chun Au, PhD
`Subhendra Mattagajasingh, PhD
`Sarah Emche, PhD
`Philip Bayliss, MD
`Marian Damewood, MD
`Michael Cronin, PhD
`Victoria Chou, MS
`Michelle Mohr, MS
`
`PRENATAL DIAGNOSIS IS USEFUL
`
`for managing a pregnancy with
`an identified fetal abnormal-
`ity and may allow for plan-
`ning and coordinating care during de-
`livery and the neonatal period.1 A
`variety of prenatal diagnostic tests are
`available but have limitations. Nonin-
`vasive tests such as maternal serum
`marker testing and ultrasound can be
`used to screen for the presence of chro-
`mosomal abnormalities but are not de-
`finitive.2-5 On the other hand, invasive
`diagnostic tests (eg, amniocentesis, cho-
`rionic villus sampling, percutaneous
`umbilical blood sampling) for fetal
`chromosomal abnormalities are highly
`reliable, but the procedure used for each
`test carries a risk for loss of preg-
`nancy.6,7 Many patients who are can-
`didates for these tests decline them be-
`cause of the risk of pregnancy loss.
`An alternative to existing methods for
`prenatal diagnosis is to use fetal cells
`
`See also pp 1127 and 1135.
`
`Context Noninvasive prenatal diagnostic tests using free fetal DNA provide an alterna-
`tive to invasive tests and their attendant risks; however, free fetal DNA exists in the ma-
`ternal circulation at low percentages, which has hindered development of noninvasive tests.
`Objective To test the hypothesis that using formaldehyde to reduce cell lysis could
`increase the relative percentage of free fetal DNA in samples of maternal blood.
`Design, Setting, and Patients The first phase of the study was conducted from
`January through February 2002 at a single US clinical site; 2 samples of blood were
`collected from each of 10 pregnant women, and the percentage of free fetal DNA in
`formaldehyde-treated and untreated samples was determined. The second phase of
`the study was conducted from March 2002 through May 2003, and measured the
`percentage of free fetal DNA in 69 formaldehyde-treated samples of maternal blood
`obtained from a network of 27 US clinical sites in 16 states.
`Main Outcome Measure Percentage of free fetal DNA in samples of maternal blood.
`Results In the first phase of the study, the mean percentage of free fetal DNA in the
`untreated samples was 7.7% (range, 0.32%-40%), while the mean percentage of free
`fetal DNA in the formaldehyde-treated samples was 20.2% (range, 1.6%-40%) (P = .02
`for difference). In the second phase, a median of 25% (range, 3.1% to ⬎50%) free
`fetal DNA was obtained for the 69 formaldehyde-treated maternal blood samples. Ap-
`proximately 59% of the samples in this study had 25% or greater fetal DNA, and only
`16% of the samples had less than 10% fetal DNA. In addition, 27.5% of the samples
`in this study had 50% or greater fetal DNA.
`Conclusion Addition of formaldehyde to maternal blood samples, coupled with care-
`ful processing protocols, increases the relative percentage of free fetal DNA, providing a
`foundation for development of noninvasive prenatal diagnostic tests to distinguish fetal
`DNA from maternal DNA in the maternal circulation.
`JAMA. 2004;291:1114-1119
`
`www.jama.com
`
`and fetal DNA that exist in the mater-
`nal circulation.8-15 Circulating fetal DNA
`has been used to determine the sex of
`
`the fetus through detection of se-
`quences present on the Y chromo-
`some.13 In addition, several studies have
`
`Author Affiliations: Ravgen Inc, Columbia, Md (Drs
`Dhallan, Au, Mattagajasingh, Emche, and Cronin
`and Mss Chou and Mohr); Department of Obstetrics
`and Gynecology, York Hospital, York, Pa (Drs Bayliss
`and Damewood). Dr Bayliss currently is affiliated
`with the Department of Maternal Fetal Medicine,
`Lancaster General Women & Babies Hospital, Lan-
`caster, Pa.
`Financial Disclosures: Dr Dhallan is the founder, chief
`executive officer, and chairman of the board of and a
`stockholder in Ravgen Inc. Drs Au, Mattagajasingh,
`Emche, and Cronin and Ms Chou are employed
`
`by and have options to purchase stock in Ravgen. At
`the time of her participation in the study, Ms Mohr
`was employed by Ravgen, and she currently is a stock-
`holder in Ravgen. Dr Bayliss is a member of the board
`of directors of and has options to purchase stock in
`Ravgen. Dr Damewood is an unpaid member of the
`advisory board of and has options to purchase stock
`in Ravgen. Ravgen has filed for patents for the meth-
`odology described in this article.
`Corresponding Author: Ravinder Dhallan, MD, PhD,
`Ravgen Inc, 9241 Rumsey Rd, Columbia, MD 21045
`(rdhallan@ravgen.com).
`
`1114 JAMA, March 3, 2004—Vol 291, No. 9 (Reprinted)
`
`©2004 American Medical Association. All rights reserved.
`
`

`

`Case 1:20-cv-01644-RGA Document 1-13 Filed 12/03/20 Page 3 of 7 PageID #: 846
`
`FREE FETAL DNA IN MATERNAL CIRCULATION
`
`attempted to use free fetal DNA to
`screen for chromosomal abnormali-
`ties in the fetus.16-21 However, the use
`of free fetal DNA for detecting chro-
`mosomal abnormalities has been lim-
`ited by the seemingly low percentage
`of free fetal DNA in the maternal cir-
`culation. Lo et al13 reported a mean of
`3.4% free fetal DNA in maternal plasma
`in the late first to the mid second tri-
`mester and a mean of 6.2% free fetal
`DNA in the late third trimester. Any
`method that can increase the relative
`percentage of free fetal DNA in the
`sample would make it easier to distin-
`guish fetal DNA from maternal DNA.
`Noninvasive prenatal diagnostic tests
`that are DNA-based would benefit from
`higher percentages of free fetal DNA in
`the samples.
`We hypothesized that inhibiting cell
`lysis during sample collection, ship-
`ping, handling, and processing would
`permit the recovery of a larger percent-
`age of free fetal DNA. By decreasing the
`amount of maternal cell lysis, and thus
`the amount of free maternal DNA, the
`relative percentage of free fetal DNA
`likely can be increased.
`
`METHODS
`Blood samples were collected from
`women carrying a male or a female fetus;
`however, the majority of samples (81
`of 85 [95.3%]) analyzed were obtained
`from women carrying a male fetus. The
`Y chromosome is the accepted marker
`for quantitating percentages of fetal
`DNA. Each clinical site received insti-
`tutional review board approval for par-
`ticipation in this research protocol. All
`women were aged 18 years or older, had
`a singleton pregnancy, and provided
`written informed consent prior to
`enrollment.
`
`Collection of Blood Samples
`First Phase. The first phase of this study
`was conducted from January through
`February 2002, and recruited women
`pregnant with a male fetus (identified
`by ultrasound). Blood samples were col-
`lected from the women at a single clini-
`cal site prior to an amniocentesis pro-
`cedure. Two tubes of blood (9 mL in
`
`each tube) were collected from each of
`10 women. One tube was treated with
`0.225 mL of a 10% neutral buffered so-
`lution containing formaldehyde (4%
`weight per volume) (Sigma, St Louis,
`Mo), a chemical that stabilizes cell
`membranes and impedes cell lysis. The
`other tube was left untreated. The tubes
`were assigned a numerical code and
`hand-delivered to our facility for analy-
`sis. Laboratory personnel were blinded
`as to which specimens contained form-
`aldehyde.
`Second Phase. For the second phase
`of the study, conducted from March
`2002 through May 2003, a network of
`27 clinical sites, operating in 16 states
`in the United States, was established to
`collect formaldehyde-treated blood
`samples from pregnant women prior to
`amniocentesis or chorionic villus sam-
`pling. Seventy-one samples from women
`carrying a male fetus and 4 samples from
`women carrying a female fetus were ana-
`lyzed in this phase of the study. Fetal sex
`was confirmed by amniocentesis or cho-
`rionic villus sampling report. Each
`sample was coded so that laboratory per-
`sonnel did not know whether it was ob-
`tained from a woman carrying a male or
`a female fetus.
`All samples collected in the second
`phase of the study were treated with
`formaldehyde. The clinical sites were
`provided with a kit used for the veni-
`puncture procedure, which included
`21-gauge needles, 9-mL EDTA blood
`collection tubes, a syringe for each tube
`containing 0.225 mL of a 10% neutral
`buffered solution containing formal-
`dehyde (4% weight per volume), an ice
`pack, and a shipping container. The
`clinical sites were instructed to add the
`formaldehyde to the tubes and gently
`invert them immediately after blood was
`drawn. The specimens were shipped by
`commercial carrier for overnight de-
`livery to our facility.
`
`Isolation of Plasma and DNA
`The protocols for isolation of plasma
`were optimized to reduce cell lysis.
`Tubes were centrifuged at 200g for 10
`minutes with the brake and accelera-
`tion powers set to zero. Tubes then were
`
`centrifuged at 1600g for 10 minutes
`with the brake and acceleration pow-
`ers set to zero. The supernatant (ie, the
`plasma) of each sample was trans-
`ferred to a new tube and spun at 1600g
`for 10 minutes with the brake and ac-
`celeration powers set to zero. The
`plasma was transferred carefully to a
`new tube and stored at −80°C. Approxi-
`mately 0.5 mL of supernatant was left
`in the tube to ensure that the buffy coat
`was not disturbed.
`DNA was isolated from plasma
`samples using the QIAamp DNA Blood
`Midi Kit (Qiagen, Valencia, Calif) for
`purification of DNA from blood cells,
`according to the manufacturer’s in-
`structions. DNA was eluted in 100 μL
`of distilled water.
`
`Primer Design
`Two sets of primers were used: 1 set am-
`plified the sex-determining region Y
`gene (SRY), which is located on the Y
`chromosome and is thus representa-
`tive of fetal DNA, and the other set am-
`plified the cystic fibrosis gene (CYS),
`which is present on both maternal tem-
`plate DNA and fetal template DNA.
`Unique regions of the SRY gene and the
`CYS gene were identified by sequence
`searches using the Blast program avail-
`able from the National Center for Bio-
`technology Information (http://www
`.ncbi.nlm.nih.gov).
`The following primers were designed
`to amplify the SRY gene: upstream
`primer: 5⬘ TGGCGATTAAGT-
`CAAATTCGC 3⬘; downstream primer:
`5⬘ CCCCCTAGTACCCTGACAATG-
`TATT 3⬘. The following primers were
`designed to amplify the CYS gene:
`upstream primer: 5⬘ CTGTTCTGT-
`GATATTATGTGTGGT 3⬘ ; down-
`stream primer: 5⬘ AATTGTTG-
`GCATTCCAGCATTG 3⬘.
`
`Gene Amplification
`The SRY gene and the CYS gene were
`amplified from plasma free DNA by
`polymerase chain reaction (PCR) us-
`ing the HotStarTaq Master Mix kit (Qia-
`gen). Each PCR reaction used 8 μL of
`template DNA (diluted or undiluted),
`1 μL of each primer (5 μM), and 10 μL
`
`©2004 American Medical Association. All rights reserved.
`
`(Reprinted) JAMA, March 3, 2004—Vol 291, No. 9 1115
`
`

`

`Case 1:20-cv-01644-RGA Document 1-13 Filed 12/03/20 Page 4 of 7 PageID #: 847
`
`FREE FETAL DNA IN MATERNAL CIRCULATION
`
`Table 1. Effects of Formaldehyde on Percentage of Free Fetal DNA Recovered From
`Maternal Plasma Samples
`
`Free Fetal DNA, %
`
`Sample No.
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`
`Formaldehyde-Treated Sample
`8.0
`8.0
`8.0
`8.0
`40.0
`1.6
`40.0
`40.0
`40.0
`8.0
`
`Untreated Sample
`1.6
`8.0
`1.6
`0.32
`8.0
`1.6
`40.0
`8.0
`8.0
`0.32
`
`Difference
`6.4
`0
`6.4
`7.7
`32.0
`0
`0
`32.0
`32.0
`7.7
`
`of HotStarTaq mix. The following PCR
`conditions were used: (1) 95°C for 15
`minutes, (2) 94°C for 30 seconds, (3)
`54°C for 15 seconds, (4) 72°C for 30 sec-
`onds, (5) repeat steps 2 through 4 for
`45 cycles, and (6) 72°C for 10 minutes.
`
`Quantification of Fetal DNA
`The percentage of free fetal DNA in the
`maternal plasma sample was deter-
`mined by PCR using serially diluted
`plasma DNA, which accurately quan-
`tifies the number of genomes that har-
`bor the amplified gene. For example,
`if the blood sample contains 100% male
`fetal DNA, and 1:2 serial dilutions are
`performed, then on average the SRY sig-
`nal will disappear 1 dilution before the
`CYS signal, since there is 1 copy of the
`SRY gene and 2 copies of the CYS gene.
`The percentage of free fetal DNA in
`the maternal plasma was calculated us-
`ing the following formula: percentage
`of free fetal DNA=(No. of copies of SRY
`gene ⫻2 ⫻100)/(No. of copies of CYS
`gene), where the number of copies of
`each gene was determined by observ-
`ing the highest serial dilution in which
`the gene was detected. The formula
`contains a multiplication factor of 2,
`which is used to normalize for the fact
`that there is only 1 copy of the SRY gene.
`In the first phase of the study, 6 se-
`rial dilutions (1:5) were performed for
`each sample (1:5 to 1:15625). Pro-
`vided that there is at least 1 copy of the
`SRY gene and that the CYS gene is de-
`tected in the sixth serial dilution, the
`lowest possible value is 0.0128% free
`
`fetal DNA ([1 ⫻2 ⫻100]/15625). All
`other values increase by multiples of 5
`from 0.0128, depending on the num-
`ber of dilutions that were positive for
`the SRY and CYS genes (eg, 0.064, 0.32,
`1.6, 8, and 40).
`In the second phase of the study, 10
`serial dilutions (1:2) were performed for
`each sample (1:2 to 1:1024). Provided
`that there is at least 1 copy of the SRY
`gene and that the CYS gene is detected
`in the 10th serial dilution, the lowest
`possible value is 0.1953% free fetal DNA
`([1 ⫻2 ⫻100]/1024). All other values
`increase by multiples of 2 from 0.1953.
`The number of fetal genomes per mil-
`liliter of plasma was calculated using the
`following formula: No. of genomes/mL
`of plasma=(No. of copies of SRY gene/
`volume of DNA in reaction [μL]) ⫻
`(volume of DNA eluted [μL]/total vol-
`ume of plasma through column [mL]).
`
`Statistical Analysis
`The primary outcome in the first phase
`of the study was the difference in the
`percentage of free fetal DNA between
`formaldehyde-treated and untreated
`samples. The nonparametric Wil-
`coxon signed rank test, which as-
`sumes that there is information in the
`magnitude of differences, was used to
`analyze the data from the first phase of
`the study. All analyses were per-
`formed using Analyse-it General &
`Clinical Laboratory Statistics, version
`1.71 (Analyse-it Software Ltd, Leeds,
`England); P⬍.05 was used to deter-
`mine statistical significance.
`
`RESULTS
`First Phase
`The results from the first phase of the
`study are summarized in TABLE 1. Analy-
`sis of the untreated samples revealed a
`mean of 7.7% (range, 0.32%-40%) free
`fetal DNA. The formaldehyde-treated
`samples had a mean of 20.2% (range,
`1.6%-40%) free fetal DNA.
`Several of the untreated samples
`(from participants 1, 3, 4, and 10) con-
`tained percentages of free fetal DNA that
`were substantially below the median.
`Even with these samples, the addition
`of formaldehyde increased the relative
`percentage of free fetal DNA. For in-
`stance, in untreated samples from par-
`ticipants 4 and 10, the percentage of free
`fetal DNA was 0.32%, whereas in for-
`aldehyde-treated samples collected from
`the same women the percentage of free
`fetal DNA was increased to 8%.
`In 3 of the samples (from partici-
`pants 2, 6, and 7), there was no mea-
`surable effect of formaldehyde on the
`percentage of free fetal DNA. However,
`analysis of the paired samples from the
`first phase of the study using the Wil-
`coxon signed rank test revealed that,
`overall, the addition of formaldehyde sig-
`nificantly increased the percentage of
`free fetal DNA (P=.02; W=28 for y=7).
`
`Second Phase
`Because analysis of the samples from
`the first phase of the study revealed
`that the effect of formaldehyde was
`statistically significant, the second
`phase of the study was designed to
`evaluate the percentage of free fetal
`DNA in formaldehyde-treated samples
`in a larger patient population from
`multiple clinical sites. A total of 75
`samples from pregnant women were
`collected in this phase of the study.
`Seventy-one samples were collected
`from women who carried a male fetus.
`However, 2 samples were excluded
`from analysis because these samples
`were not received within 24 hours
`after collection.
`Four samples were obtained from
`women who carried a female fetus. For
`each of these samples, a robust signal
`was observed for the CYS gene; as ex-
`
`1116 JAMA, March 3, 2004—Vol 291, No. 9 (Reprinted)
`
`©2004 American Medical Association. All rights reserved.
`
`

`

`Case 1:20-cv-01644-RGA Document 1-13 Filed 12/03/20 Page 5 of 7 PageID #: 848
`
`FREE FETAL DNA IN MATERNAL CIRCULATION
`
`pected, no signal was detected for the
`SRY gene, which is specific for the Y
`chromosome.
`Analysis of the 69 formaldehyde-
`treated samples revealed a median of
`25% (range, 3.1% to ⬎50%) free fetal
`DNA (TABLE 2). Approximately 16.0%
`of the samples (11/69) had less than 10%
`free fetal DNA; approximately 59% of the
`formaldehyde-treated samples had 25%
`or greater free fetal DNA and 27.5% of
`the samples had 50% or greater free fe-
`tal DNA (4 [5.8% of total] samples had
`3.1% free fetal DNA; 7 [10.1%] had
`6.2%; 17 [24.6%] had 12.5; 22 [31.9%]
`had 25%; 8 [11.6%] had 50%; and 11
`[15.9%] had ⬎50%). Although many of
`the formaldehyde-treated samples con-
`tained high percentages of free fetal
`DNA, there was variability in the per-
`centages obtained.
`Analysis of the formaldehyde-treated
`samples also revealed a mean of 66.1 fe-
`tal genomes/mL of plasma, with a range
`of 3.0 fetal genomes/mL to 533 fetal ge-
`nomes/mL (Table 2). Some of the
`samples (eg, sample 12) had a limited
`number of fetal genomes but a high per-
`centage of fetal DNA. Conversely, some
`samples had an ample number of fetal ge-
`nomes but a lower percentage of fetal
`DNA. For instance, sample 40 had 112.5
`fetal genomes/mL but the percentage of
`fetal DNA was 6.2% (Table 2).
`
`COMMENT
`We have shown that the relative per-
`centage of free fetal DNA recovered from
`maternal blood samples can be in-
`creased. Addition of formaldehyde to
`maternal blood samples, coupled with
`careful processing protocols, resulted in
`an increase in the percentage of free fe-
`tal DNA recovered from the maternal cir-
`culation. This increase in the relative per-
`centage of free fetal DNA likely resulted
`from a combination of factors.
`First, formaldehyde stabilizes cell
`membranes, thereby preventing cell ly-
`sis and the release of DNA. Prior to the
`venipuncture procedure, the amount of
`free maternal DNA in the maternal cir-
`culation likely is low. However, the
`maternal cells may lyse during sample
`collection, shipping, handling, and
`
`Table 2. Number of Fetal Genomes and Percentage of Free Fetal DNA in
`Formaldehyde-Treated Blood Samples Collected at Various Weeks of Gestation
`From Women Carrying Male Fetuses
`Sample
`Weeks of
`No.
`Gestation
`1
`16
`2
`19
`3
`17
`4
`22
`5
`32
`6
`19
`7
`18
`8
`17
`9
`16
`10
`17
`11
`16
`12
`16
`13
`16
`14
`17
`15
`17
`16
`17
`17
`17
`18
`17
`19
`19
`20
`20
`21
`15
`22
`11
`23
`18
`24
`18
`25
`16
`26
`17
`27
`14
`28
`11
`29
`18
`30
`19
`31
`19
`32
`16
`33
`16
`34
`11
`35
`16
`36
`11
`37
`16
`38
`18
`39
`17
`40
`18
`41
`17
`42
`28
`43
`17
`44
`18
`45
`16
`46
`17
`47
`15
`48
`16
`49
`17
`50
`17
`
`Fetal Genomes
`per Milliliter
`40.0
`533.0
`26.0
`83.0
`228.0
`200.0
`400.0
`50.0
`25.0
`12.5
`47.4
`17.3
`11.2
`25.0
`13.2
`22.5
`47.4
`14.1
`14.1
`5.6
`5.6
`8.3
`6.6
`6.2
`56.2
`62.1
`45.0
`50.0
`116.0
`313.0
`56.2
`211.8
`211.8
`52.9
`24.8
`5.9
`60.0
`171.4
`25.7
`112.5
`200.0
`90.0
`10.2
`12.9
`34.3
`109.1
`37.5
`20.3
`50.0
`7.0
`
`Free Fetal
`DNA, %
`25.0
`⬎50.0
`50.0
`25.0
`50.0
`⬎50.0
`⬎50.0
`50.0
`25.0
`12.5
`12.5
`50.0
`25.0
`12.5
`12.5
`25.0
`⬎50.0
`6.2
`25.0
`12.5
`12.5
`12.5
`25.0
`6.2
`⬎50.0
`25.0
`50.0
`⬎50.0
`⬎50.0
`⬎50.0
`⬎50.0
`⬎50.0
`25.0
`25.0
`3.1
`12.5
`25.0
`⬎50.0
`25.0
`6.2
`12.5
`25.0
`12.5
`25.0
`3.1
`25.0
`6.2
`3.1
`25.0
`12.5
`(continued)
`
`©2004 American Medical Association. All rights reserved.
`
`(Reprinted) JAMA, March 3, 2004—Vol 291, No. 9 1117
`
`

`

`Case 1:20-cv-01644-RGA Document 1-13 Filed 12/03/20 Page 6 of 7 PageID #: 849
`
`FREE FETAL DNA IN MATERNAL CIRCULATION
`
`Table 2. Number of Fetal Genomes and Percentage of Free Fetal DNA in
`Formaldehyde-Treated Blood Samples Collected at Various Weeks of Gestation
`From Women Carrying Male Fetuses (cont)
`Sample
`Weeks of
`Fetal Genomes
`No.
`Gestation
`per Milliliter
`51
`22
`11.2
`52
`15
`14.1
`53
`17
`25.0
`54
`18
`29.0
`55
`14
`50.0
`56
`16
`29.0
`57
`16
`6.8
`58
`16
`12.5
`59
`20
`22.5
`60
`16
`11.8
`61
`18
`3.0
`62
`15
`14.1
`63
`17
`25.0
`64
`16
`180.0
`65
`16
`12.5
`66
`16
`24.3
`67
`16
`23.9
`68
`14
`13.2
`69
`17
`62.1
`Mean, 17
`Mean, 66.1
`(SD, 3.1)
`(SD, 96.3)
`
`Free Fetal
`DNA, %
`12.5
`12.5
`3.1
`50.0
`25.0
`25.0
`12.5
`6.2
`25.0
`12.5
`6.2
`6.2
`25.0
`50.0
`12.5
`25.0
`12.5
`50.0
`25.0
`Median, 25.0%
`(interquartile range, 12.5-50)
`
`processing. For example, during cen-
`trifugation the cells are exposed to
`gravitational forces, which may rup-
`ture cells. The presence of formalde-
`hyde protects the cells from lysis.
`Second, the addition of formalde-
`hyde may allow a larger recovery of free
`fetal DNA by inhibiting enzymes that
`destroy DNA, such as DNases. For the
`samples analyzed in the second phase
`of the study, a mean of 66.1 fetal ge-
`nomes/mL was obtained, which repre-
`sents a 2.6-fold increase over the mean
`reported in the literature (25.4 fetal ge-
`nomes/mL).13 Inhibition of enzymes
`that destroy DNA would permit a larger
`recovery of DNA (including free fetal
`DNA) already present in the sample.
`Also, the addition of formaldehyde may
`stabilize and preserve the structure of
`DNA, which may increase the amount
`of DNA recovered.
`Third, in conjunction with the addi-
`tion of formaldehyde to the maternal
`blood samples, sample-processing pro-
`tocols designed to minimize cell lysis
`likely contributed to increases in the per-
`centage of free fetal DNA. A centrifuga-
`
`tion protocol was designed to minimize
`gravitational forces imposed on the cells.
`Samples initially were spun at a low
`speed, which allowed the majority of cells
`to separate from the plasma under mini-
`mal forces. In addition, all centrifuga-
`tion steps were performed with the ac-
`celeration and brake powers set to zero.
`This reduced the formation of a vortex
`during centrifugation, minimizing mix-
`ing of the plasma and the buffy coat,
`which contains maternal cellular mate-
`rial. Also, when removing the plasma
`sample, care was taken to ensure that the
`buffy coat was not disturbed.
`A larger study would be useful to de-
`lineate factors contributing to the re-
`sults presented in this study, and to un-
`derstand why some samples have a
`higher percentage of free fetal DNA than
`others. For instance, in 3 samples from
`the first phase of the study, there was
`no measurable effect of formaldehyde
`on the percentage of free fetal DNA. It
`is possible that formaldehyde was not
`added or mixed properly with the
`treated samples or may have been added
`to both treated and untreated samples.
`
`Also, in some samples there may be
`a greater amount of free maternal DNA
`already present in the maternal circu-
`lation. While the addition of formal-
`dehyde will impede cell lysis that oc-
`curs during sample collection, shipping,
`handling, and processing, it likely will
`not reduce the concentration of free ma-
`ternal DNA already present in the
`sample. A larger study comparing the
`percentage of free fetal DNA in for-
`maldehyde-treated and untreated
`samples will help to address these
`issues.
`Furthermore, several controllable
`factors likely contribute to the varia-
`tion in the percentages of free fetal
`DNA. One such factor is the time in-
`terval between the venipuncture pro-
`cedure and sample processing. The
`shorter this time interval the more likely
`it is the integrity of the sample will be
`preserved and cell lysis kept to a mini-
`mum. Similarly, the length of time be-
`tween the venipuncture procedure and
`the addition of formaldehyde is thought
`to be critical. If formaldehyde is not
`added shortly after the venipuncture
`procedure, cells may lyse and release
`DNA. Also, it is important that the
`formaldehyde is gently mixed through-
`out the tube to allow maximum expo-
`sure to the reagent.
`With an increased percentage of free
`fetal DNA in the maternal blood samples,
`the sequence of fetal DNA can be dis-
`cerned from maternal DNA using natu-
`ral genetic markers, such as single
`nucleotide polymorphisms. For ex-
`ample, at certain genomic sites, the ma-
`ternal genome will be homozygous for
`allele A, while the paternal genome is ho-
`mozygous for allele B, which means the
`fetal genome will be heterozygous at this
`genomic site. Allele B represents a dis-
`tinct fetal signal in the maternal blood
`sample. The detection and quantita-
`tion of fetal DNA, in this case allele B,
`is more attainable with an increased per-
`centage of fetal DNA, and can be used
`to diagnose single-gene disorders and
`chromosomal abnormalities.
`A ratio for alleles A and B can be quan-
`titated and used to detect chromosomal
`disorders. When samples have a high
`
`1118 JAMA, March 3, 2004—Vol 291, No. 9 (Reprinted)
`
`©2004 American Medical Association. All rights reserved.
`
`

`

`Case 1:20-cv-01644-RGA Document 1-13 Filed 12/03/20 Page 7 of 7 PageID #: 850
`
`percentage of free fetal DNA, the differ-
`ence between the expected ratio of the
`chromosomes for a healthy fetus and that
`for an abnormal fetus is greater, which
`makes it easier to diagnose chromo-
`somal abnormalities. Thus, the meth-
`ods described herein for increasing the
`percentage of free fetal DNA provide a
`solid foundation for the development of
`a noninvasive prenatal diagnostic test.
`
`Author Contributions: Dr Damewood had full ac-
`cess to all of the data in the study and takes respon-
`sibility for the integrity of the data and the accuracy
`of the data analyses.
`Study concept and design: Dhallan, Bayliss.
`
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`Acquisition of data: Au, Mattagajasingh, Emche,
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`Analysis and interpretation of data: Dhallan, Au,
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`Drafting of the manuscript: Dhallan, Au, Mattagajas-
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`tellectual content: Dhallan, Bayliss, Damewood, Cronin.
`Statistical expertise: Dhallan, Damewood.
`Obtained funding; study supervision: Dhallan.
`Administrative, technical, or material support: Dhallan,
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`Funding/Support: This study was financed in its en-
`tirety by Ravgen Inc.
`Role of the Sponsor: Ravgen Inc designed and ex-
`ecuted the study and drafted and submitted the manu-
`script.
`Disclaimer: Participation in the study is not an en-
`dorsement of any service or product offered by Rav-
`gen Inc.
`
`Acknowledgment: We thank the following physi-
`cians and their associated staff who took the time
`and effort to participate in this study: Jeffrey Boyle,
`MD, Robert H. Debbs, DO, Eric Dellinger, MD,
`Adam J. Duhl, MD, Andrew Garber, MD, Sheri
`Hamersley, MD, Gina Hanna, MD, Cathleen Harris,
`MD, MPH, Kimberly Heller, MD, James W. Hole,
`DO, Brian K. Iriye, MD, Jon Katz, MD, Carolyn
`Kline, MD, MPH, Janet Larson, MD, Harvey Leder-
`man, MD, Sanford M. Lederman, MD, Glenn
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`Neubert, MD, William Polzin, MD, Uma Reddy,
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`Joseph Wax, MD. We thank King-Wai Yau, PhD,
`for his analysis of the data, and for his thoughts
`and guidance regarding the presentation of the
`data. We thank Susan Higgins for administrative
`support, and Amartya Basu, BS, for assistance with
`data analysis.
`
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`©2004 American Medical Association. All rights reserved.
`
`(Reprinted) JAMA, March 3, 2004—Vol 291, No. 9 1119
`
`