Clinical Chemistry 50:3
`516 –521 (2004)
`
`Molecular Diagnostics
`and Genetics
`
`Downloaded from https://academic.oup.com/clinchem/article/50/3/516/5639778 by The Chinese University of Hong Kong user on 17 December 2020
`
`Interlaboratory Comparison of Fetal Male DNA
`Detection from Common Maternal Plasma
`Samples by Real-Time PCR
`Kirby L. Johnson,1* Kimberly A. Dukes,2 John Vidaver,2 Erik S. LeShane,1
`Idania Ramirez,2 William D. Weber,3 Farideh Z. Bischoff,4 Sinuhe Hahn,5
`Arun Sharma,6 Dianne X. Dang,4 Lisa M. Hire,3 Diana W. Bianchi,1
`Joe Leigh Simpson,4 Wolfgang Holzgreve,5 Sherman Elias,6 and
`Katherine W. Klinger3
`
`Background: Analysis of fetal DNA from maternal
`plasma by PCR offers great potential for noninvasive
`prenatal genetic diagnosis. To further evaluate this
`potential, we developed and validated a standard pro-
`tocol to determine whether fetal DNA sequences could
`be reproducibly amplified and measured across multi-
`ple laboratories in a common set of specimens.
`Methods: Each of five participating centers in a Na-
`tional Institute of Child Health and Human Develop-
`ment consortium collected 20 mL of peripheral blood
`from 20 pregnant women between 10 and 20 weeks of
`gestation. The plasma fraction was separated according
`to a common protocol, divided, and frozen in five
`aliquots. One aliquot was shipped to each participating
`laboratory, where DNA was extracted according to a
`standard protocol. All plasma samples (n ⴝ 100) were
`then analyzed blindly for the presence and quantity of
`total DNA (GAPDH) and male fetal DNA (SRY) by
`real-time PCR. Genomic DNA was isolated from female
`and male cells at one center, quantified, and shipped to
`
`1 Division of Genetics, Departments of Pediatrics, Obstetrics and Gynecol-
`ogy, Tufts-New England Medical Center, Boston, MA.
`2 DM-STAT, Inc., Medford, MA.
`3 Genzyme, Framingham, MA.
`4 Departments of Obstetrics and Gynecology, and Molecular and Human
`Genetics, Baylor College Medicine, Houston, TX.
`5 Department of Obstetrics and Gynecology, University Women’s Hospi-
`tal, University of Basel, Basel, Switzerland.
`6 Department of Obstetrics and Gynecology, University of Illinois at
`Chicago, Chicago, IL.
`*Address correspondence to this author at: Division of Genetics, Tufts-
`New England Medical Center, 750 Washington St., Box 394, Boston, MA 02111.
`Fax 617-636-1469; e-mail kjohnson@tufts-nemc.org.
`Received July 8, 2003; accepted December 17, 2003.
`Previously published online at DOI: 10.1373/clinchem.2003.024380
`
`the others to serve as calibrators for GAPDH and SRY,
`respectively.
`Results: The amplification of known quantities of DNA
`was consistent among all centers. The mean quantity of
`male DNA amplified from maternal plasma when the
`fetus was male ranged from 51 to 228 genome equiva-
`lents (GE)/mL. Qualitative concordance was found over-
`all among centers. The sensitivity of the assay for
`detection of male DNA when the fetus was male varied
`from 31% to 97% among centers. Specificity was more
`consistent (93–100%) with only four false-positive re-
`sults obtained across the entire study.
`Conclusions: All centers were able to consistently am-
`plify frozen and shipped DNA. The PCR procedure
`used here is reliable and reproducible. Centers that
`extracted and amplified more DNA per milliliter of
`maternal plasma had superior sensitivities of Y chromo-
`some sequence detection. The specificity of the assay
`was more consistent among centers. A robust and thor-
`oughly optimized protocol for the extraction of DNA
`from maternal plasma is needed to make testing of fetal
`DNA in maternal plasma a clinically relevant analytical
`tool.
`© 2004 American Association for Clinical Chemistry
`
`Since the first report in 1997, cell-free fetal DNA in the
`maternal circulation has become a primary target for
`noninvasive prenatal diagnosis (1 ). The detection of male-
`specific (Y chromosome) DNA sequences has been used
`for the assessment of X-linked disorders (2 ), the detection
`of unique gene sequences such as the RhD locus to
`determine fetomaternal blood group incompatibility (3 ),
`and the detection of dominantly inherited, paternally
`derived mutations for diagnoses of single gene disorders
`(1 ). The quantity of fetal DNA in maternal plasma or
`
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`517
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`serum has been used as a marker of genetic disorders and
`complications of pregnancy, including common trisomies
`(4 –7 ), preterm labor (8 ), and preeclampsia (9 ).
`Real-time PCR amplification of Y-chromosome se-
`quences from the plasma or serum of women pregnant
`with male fetuses has been used in many studies as a
`model for the detection and quantification of fetal DNA.
`Y-chromosome DNA has been detected as early as 5
`weeks of gestation (10, 11 ), before the time period in
`which invasive testing is typically performed. Thus, early
`detection of fetal DNA could have a profound effect on
`the way pregnancies are managed. Some investigators
`have reported ⬎95% sensitivity and specificity for real-
`time PCR (12, 13 ). However, these data are typically
`generated by a single laboratory under specific conditions
`and often include data from a small number of patients. In
`addition, confounders that could impact routine clinical
`application may be undetected in smaller studies. Indeed,
`it has been shown that methods of processing can drasti-
`cally affect the amount of DNA detected by real-time PCR
`(14 ). For PCR assays to become part of standard prenatal
`care, their accuracy and reproducibility must be improved
`and the underlying variables that affect performance must
`be better understood.
`In this study, we first established a common protocol
`for sample preparation, DNA extraction, and PCR analy-
`sis to be used by five independent laboratories that are
`part of the National Institute of Child Health and Human
`Development (NICHD) Fetal Cell Isolation Study. To test
`the potential clinical utility of fetal DNA analysis, a
`common set of samples was then evaluated at all sites by
`the standardized protocol. We report here the results of
`the first interlaboratory evaluation of fetal DNA detection
`in maternal plasma from a common sample set.
`
`Materials and Methods
`
`study design
`A prospective, repeated-measures design with five repli-
`cations for each individual was used to assess concor-
`dance in qualitative and quantitative PCR analysis of fetal
`DNA in maternal plasma. Before implementation, the
`study protocol, methods to ensure quality of results, and
`scoring were clearly defined as described below.
`
`patient enrollment/sample collection
`Institutional Review Board approval for this study was
`obtained by each of the five participating centers (Baylor
`College of Medicine, Genzyme Genetics, Tufts-New En-
`gland Medical Center, University of Basel, and University
`of Illinois at Chicago), which are hereafter denoted A–E.
`After receiving informed consent, we enrolled pregnant
`women in their late first or second trimesters (10 –20
`weeks of gestation) as study participants. Mean gesta-
`tional ages ranged from 13.75 to 18.13 weeks across the
`five sites. Prospectively, each participating center col-
`lected 20 mL of peripheral blood from 20 pregnant
`women (total of 100).
`
`sample processing
`Sample processing began within 24 h of blood collection.
`Blood was centrifuged at 800g for 10 min in the original
`collection tube. Plasma was removed, pooled if more than
`one tube of blood was collected, and divided into five
`1-mL aliquots. The 1-mL samples were then centrifuged
`for 10 min at 13 500g to remove all residual intact cells
`(14 ). Supernatant (900 ␮L) was removed from each ali-
`quot and stored at ⫺80 °C. One aliquot of each sample
`was shipped on dry ice to each of the four other partici-
`pating centers. Thus each center performed DNA extrac-
`tion and PCR analysis of the 20 samples that were
`collected on site and also on each of the 80 samples
`shipped from other centers.
`
`dna extraction
`After shipment, samples were thawed at room tempera-
`ture, and 800 ␮L of plasma was used for DNA extraction.
`The QIAamp DNA Blood Mini Kit was used as specified
`by the manufacturer (Qiagen Inc.), with minor modifica-
`tions. Reagents were increased proportionately to accom-
`modate the 800-␮L sample size. To elute DNA from the
`column, buffer AE prewarmed to 56 °C was used. After 50
`␮L of the buffer was applied to each column, the column
`was incubated at 56 °C for 5 min and then centrifuged at
`6000g for 1 min. This procedure was then repeated for a
`final elution volume of 100 ␮L. Samples were stored at
`4 °C pending analysis.
`
`pcr analysis
`PCR was performed with a Perkin-Elmer Applied Biosys-
`tems 7700 Sequence Detection System (Applied Biosys-
`tems). Extracted DNA was analyzed for both the GAPDH
`and SRY loci. The SRY sequence was used to measure the
`quantity of fetal DNA present in each sample from a
`patient bearing a male fetus, and the GAPDH sequence
`was used to confirm the presence and quality of DNA in
`each sample as well as measure the quantity of total
`(maternal and fetal) DNA in each sample. Primer and
`probe sequences were as follows:
`SRY forward primer: 5⬘-TCC TCA AAA GAA ACC
`GTG CAT-3⬘
`SRY reverse primer: 5⬘-AGA TTA ATG GTT GCT AAG
`GAC TGG AT-3⬘
`SRY TaqMan probe: 5⬘-CAC CAG CAG TAA CTC CCC
`ACA ACC TCT TT-3⬘
`GAPDH forward primer: 5⬘-CCC CAC ACA CAT GCA
`CTT ACC-3⬘
`GAPDH reverse primer: 5⬘-CCT AGT CCC AGG GCT
`TTG ATT-3⬘
`GAPDH TaqMan probe: 5⬘-AAA GAG CTA GGA AGG
`ACA GGC AAC TTG GC-3⬘
`Reactions were set up in a 50-␮L volume using 25 ␮L of
`PE-ABI Universal Mastermix and 5 ␮L of extracted DNA.
`Primers and probes were used at final concentrations of
`300 and 200 nM, respectively, with the exception of center
`
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`statistical methods
`Descriptive statistics were generated to assess quality
`issues (e.g., number of plates or samples needing to be
`rerun, calibration curve slopes, and correlations) and
`coding of outcomes. All analyses were stratified by center.
`To assess to what extent each of the five centers obtained
`the same qualitative PCR results for both the GAPDH and
`SRY loci, we estimated the concordance among centers by
`use of ␬statistics, which measure the degree of agreement
`beyond that expected by chance. Pairwise ␬statistics were
`estimated for each pair of participating centers, based on
`the 100 samples analyzed. The pairwise ␬ statistics were
`subsequently combined to produce an overall estimate of
`agreement among centers with respect to DNA detection.
`To compare quantitative values (i.e., amount of DNA
`detected), we estimated intraclass correlation coefficients
`(ICC) according to the same procedure as described for ␬.
`The ICC statistic that was chosen quantifies both the
`consistency and absolute agreement of DNA detection.
`All significance tests were two-sided and the level of
`significance was fixed at 0.05. Performance characteristics
`(i.e., sensitivity and specificity) were also estimated be-
`tween PCR qualitative results and definitive fetal gender
`detection obtained from all sites except center C, which
`did not have access to fetal gender. SAS, Ver. 8.0, was
`used to perform all statistical analyses.
`
`Results
`Each center ran between five and seven plates for the
`GAPDH locus and between six and eight plates for the
`SRY locus, and only center D needed to rerun an entire
`plate, because of a PCR instrument malfunction. We
`evaluated PCR efficiency at each center by comparing the
`results obtained for the initial concentration of the stan-
`dard female control DNA (GAPDH) supplied by center C
`(Table 1). For standard female DNA, the mean GAPDH
`concentration varied from 7626.71 to 8804.42 pg/reaction,
`in which 8515 pg was the approximate starting quantity
`that was distributed. The mean slopes and correlation
`coefficients over all plates by locus and center (Table 1)
`indicated that the quality and study criteria were met (i.e.,
`all slopes less than ⫺3.0 and all correlation coefficients
`⬎0.96).
`Of the 100 plasma samples analyzed, fetal gender was
`known for 63 (35 male and 28 female) at the time of data
`analysis. All centers were able to amplify GAPDH from all
`samples except center C, which successfully analyzed
`only 99 samples because of extraction failure. The mean
`quantity of GAPDH detected in these samples ranged
`from 5939 to 12 397 GE/mL (Table 2). Individual centers
`were able to amplify the SRY sequence from between 11
`and 34 of the 35 known male samples. The mean quantity
`of SRY detected in the known male samples with positive
`values ranged from 51 to 228 GE/mL.
`Rates of correct
`identification by PCR analysis of
`known fetal gender varied from 31% to 94% among
`centers and was, in general, directly related to the amount
`
`D, which used primer and probe concentrations of 100
`and 50 nM, respectively. Each sample was run in triplicate
`for both loci, and the mean of the values was used for
`further calculations. Each reaction plate was run simulta-
`neously with a duplicate calibration curve of titrated
`DNA, which was extracted and quantified from male and
`female cells by center C and then shipped to all centers for
`use. For GAPDH reactions, the calibration curve consisted
`of eight points at a 2⫻ serial dilution from 13 200 to 103.2
`pg. For SRY reactions, the curve consisted of eight points
`between 3300 and 25.8 pg. All reaction plates were run
`with three wells each containing 8515 pg of genomic
`female DNA. This functioned as a negative control for
`SRY reactions and as a positive control for GAPDH
`reactions. Three samples with no target DNA (i.e., no-
`template controls) were also included on each reaction
`plate. Cycling conditions for all reactions consisted of a
`2-min incubation at 50 °C to allow UNGErase activity, an
`initial denaturation step of 95 °C for 10 min, and then 40
`cycles of 95 °C for 15 s and 60 °C for 1 min. All samples
`were analyzed blindly with respect to fetal gender. The
`standard factor of 6.6 pg was used to convert the data to
`genome equivalents (GE).
`
`pcr analysis quality control and coding
`information
`Using Sequence Detection System software, we defined
`the baseline fluorescence as between cycle 3 and two
`cycles before initial amplification of the most highly
`concentrated calibrator or unknown point. Thresholds for
`determining threshold cycle values were set at 25 times
`the SD given by the software. Plates were considered
`valid when the calibration curve slope was less than ⫺3.0
`and the correlation coefficient of the calibration curve was
`⬎0.96. Plates were rejected when GAPDH DNA did not
`amplify in female controls, when SRY DNA did amplify
`in female controls, when no-template controls amplified,
`or when a center detected other problems with an assay.
`In these cases, the entire reaction was repeated. If the
`second run was considered valid, those results were used.
`Individual samples within a plate were considered in-
`valid if there was discordance between the three wells
`(e.g., positive amplification in only one or two wells) that
`could not be explained by technical problems. In the
`situation of individual sample discordance, the sample
`was rerun. Samples were designated as male if they had at
`least two replicates with positive amplification in the final
`validated assay for that sample. All other results were
`considered insufficient to detect male DNA and were thus
`designated as female.
`
`outcomes
`The primary outcome was qualitative and reflected
`whether fetal DNA was present, and was based on
`information obtained through PCR analysis. The second-
`ary outcome was quantitative and reflected the mean
`amount of DNA amplified.
`
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`Table 1. Center-specific PCR analysis of standardized control DNA.
`Calibration curve for GAPDHa
`Calibration curve for SRYa
`
`Mean (SD) GAPDH in female
`Mean (SD)
`correlation coefficient
`Mean (SD) slope
`controls, pg/reaction
`⫺3.63 (0.18)
`0.988 (0.007)
`7972 (1052)
`⫺3.62 (0.16)
`0.992 (0.004)
`8793 (990)
`⫺3.49 (0.05)
`0.989 (0.010)
`8194 (709)
`⫺3.80 (0.08)
`0.994 (0.003)
`8804 (646)
`⫺3.50 (0.11)
`0.994 (0.004)
`7627 (846)
`a Study criteria: slope less than ⫺3.0 and correlation coefficient ⬎0.96 to be valid.
`
`A
`B
`C
`D
`E
`
`Mean (SD) slope
`⫺3.95 (0.38)
`⫺3.51 (0.18)
`⫺3.48 (0.17)
`⫺4.15 (0.15)
`⫺3.55 (0.12)
`
`Mean (SD)
`correlation coefficient
`0.974 (0.009)
`0.978 (0.011)
`0.981 (0.010)
`0.981 (0.008)
`0.984 (0.009)
`
`of DNA amplified (Table 2). The rank ordering of rates of
`male gender detection and mean GAPDH were identical
`across centers; the centers with the highest and lowest
`concentrations of GAPDH amplified had the highest and
`lowest sensitivities, respectively. The relationship be-
`tween detection rates and mean GE of SRY was similar.
`The center with the lowest detection rate (31%) had the
`second-lowest mean concentration of amplified male
`DNA (64 GE/mL), whereas the center with the highest
`detection rate had the highest concentration of amplified
`male DNA. The specificities among centers were more
`consistent, varying from 93% to 100%. Male DNA was
`detected in only four samples from women carrying
`female fetuses in the entire study; a false-positive result
`was not obtained on any sample in more than one
`laboratory.
`The concordance of fetal DNA detection among centers
`is shown in Table 3. The overall ␬ value for all sites
`combined was 0.48 (P ⬍0.05), indicating statistically sig-
`nificant agreement among centers beyond that expected
`by chance. There was a stronger association (␬ ⬎0.61; P
`⬍0.05) between sites A and C, A and D, and C and D.
`These three sites (A, C, and D) had the highest mean
`concentrations of amplified DNA for both GAPDH and
`SRY (Table 2), and the best results with respect to perfor-
`mance characteristics (i.e., sensitivity and specificity of
`male gender detection). With regard to the concordance of
`total DNA detection (GAPDH), the overall ␬ value for all
`sites combined as well as between any two sites was 1.00.
`The concordance assessments for quantity of total
`DNA and fetal DNA detected between sites, expressed in
`terms of ICC, are shown in Tables 4 and 5, respectively.
`The ICC statistic signifies both consistency (i.e., agree-
`ment in terms of ordering) and reliability (i.e., agreement
`in absolute value). Entries in Tables 4 and 5 that are
`expressed in bold indicate statistical significance (P ⬍0.05)
`
`with respect to quantitative concordance between centers
`(e.g., A and C, B and C, B and E, C and D, and C and E all
`have significant concordance with respect to SRY detec-
`tion). A large but statistically insignificant ICC statistic
`(e.g., A and D and A and E for SRY detection) implies
`good consistency but poor reliability (i.e., high variability
`in absolute assessments).
`
`Discussion
`The discovery of nucleic acids of fetal origin circulating in
`the maternal plasma and their continuing characterization
`has raised the possibility of their use in noninvasive
`prenatal diagnosis. However, the eventual clinical appli-
`cation of circulating DNA technology will require thor-
`ough identification and understanding of the factors that
`may affect
`its performance in different
`laboratories.
`Through the processing of a common set of plasma
`samples by five laboratories using a standardized proto-
`col for DNA extraction and real-time PCR amplification,
`along with the blinded analysis of the raw data, we have
`addressed some of the issues required to translate this
`technology into clinical utility. Our results indicate that
`each of five sites could reproduce the PCRs and amplify
`previously extracted DNA of known quantity that had
`been frozen and shipped. However, not all centers were
`able to obtain this level of performance on maternal
`plasma samples that required DNA extraction to be
`performed. Differences in sensitivity among laboratories
`correlated strongly with the amounts of total and fetal
`DNA detected, suggesting that the extraction procedure
`was the most likely factor confounding the results.
`Some factors that may influence technical performance
`have been avoided or eliminated in this study. Each center
`used the same type of PCR instrument and reagents, and
`data generated from the common DNA calibrators were
`comparable among centers, suggesting that the instru-
`
`Table 2. Center-specific characteristics of fetal DNA detection (35 known male and 28 known female fetuses).
`Mean (SD) SRY, GE/mL
`Mean (SD) GAPDH, GE/mL
`Males detected, n
`Detection rate, %
`False positives, n
`Specificity, %
`33
`122 (88)
`94
`11 451 (36 803)
`0
`100
`11
`64 (38)
`31
`5939 (17 927)
`0
`100
`27
`154 (442)
`77
`9415 (31 491)
`2
`93
`34
`228 (145)
`97
`12 397 (34 587)
`1
`96
`15
`51 (27)
`43
`8337 (26 402)
`1
`96
`
`Center
`A
`B
`C
`D
`E
`
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`520
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`Johnson et al.: Interlaboratory Comparison of Fetal DNA Detection
`
`Table 3. Concordance of fetal DNA detection among
`centers: ␬ statistics.a
`Center
`
`Table 5. Concordance in amount of fetal DNA detection
`among centers: ICC.a
`Center
`
`Center
`
`A
`
`B
`
`C
`
`D
`
`E
`
`Center
`
`A
`
`B
`
`C
`
`D
`
`E
`
`Downloaded from https://academic.oup.com/clinchem/article/50/3/516/5639778 by The Chinese University of Hong Kong user on 17 December 2020
`
`A B
`
`0.23
`0.32b
`C
`0.67
`D
`0.55
`E
`a Overall ICC ⫽ 0.095.
`b Bold indicates significant agreement (P ⬍0.05).
`
`0.00
`0.11
`0.46
`
`0.35
`0.11
`
`0.35
`
`of DNA extraction on sensitivity. The center with the
`highest sensitivity (center D) had the highest mean rate of
`detection of both SRY and GAPDH sequences, whereas
`the center with the lowest sensitivity (center B) had the
`second lowest and lowest mean rate of detection of SRY
`and GAPDH sequences, respectively. This implies that the
`efficiency of DNA extraction correlates directly with the
`ability to detect fetal DNA sequences. This conclusion is
`further supported by the consistent results achieved
`among all centers when DNA was previously extracted
`and quantified by a single source, suggesting that real-
`time PCR performed at all of the centers did provide
`reasonable approximations of the amount of DNA present
`in each reaction. Although a standardized protocol was
`used, there are likely other factors confounding its repro-
`ducibility. These may include individual laboratory tech-
`niques and familiarity with the extraction protocol. One
`center (center D) used PCR primers and probes at con-
`centrations that were lower than the other sites. Although
`this center had the highest sensitivity of SRY detection,
`center A obtained similar results using the higher primer/
`probe concentrations. Therefore, this difference likely did
`not have a significant effect on outcome, although this
`variable should be considered for further studies of PCR
`optimization. These concerns can potentially be elimi-
`nated by a more in-depth standardized DNA extraction
`procedure, as well as increased experience with the pro-
`tocol. The centers with lower sensitivities are addressing
`these issues and are incorporating adjustments to their
`extraction procedures. Indeed, center B, by incorporation
`of a shaking heat block and vacuum manifold during the
`extraction process, now has achieved 97% sensitivity
`while maintaining 100% specificity on ⬎100 additional
`maternal plasma specimens subjected to blinded analysis
`using the described approach.
`The two sites with the highest sensitivities in gender
`detection also observed the highest quantities of DNA
`detected (centers A and D). In addition, the ICCs repre-
`senting these two sites were high, indicating similarity in
`differentiation (i.e., agreement in terms of ordering) of
`fetal DNA quantities detected. However, the ICC between
`these sites was not statistically significant, implying poor
`calibration (i.e., agreement
`in absolute value)
`in the
`amount of fetal DNA detected. Nevertheless, there was
`
`fair;
`
`A B
`
`0.39
`0.69b
`0.32
`C
`0.64
`0.86
`0.33
`D
`0.43
`0.37
`0.26
`0.51
`E
`a Overall ␬ ⫽ 0.48. ␬ ⫽ 0, poor; 0.01– 0.20, slight; 0.21– 0.40,
`0.41– 0.60, moderate; 0.61– 0.80, substantial; 0.81–1.00, high.
`b Bold indicates significant agreement (P ⬍0.05).
`
`ment’s performance was consistent in all laboratories. It is
`possible that instruments from other manufacturers may
`perform differently, and a future comparison of different
`systems would be useful. In addition, it is likely that
`analytic interpretation of raw PCR data may vary among
`different laboratories. However, this was not a concern in
`the present study because all raw data were sent directly
`to the statistical analysis center and analyzed in a consis-
`tent, unbiased manner to accurately calculate the amount
`of DNA present. Nevertheless, it is important to establish
`consistent guidelines for these analyses, such as the selec-
`tion of a threshold for determining the presence of a
`particular DNA sequence.
`Other factors that may affect performance but could be
`not eliminated from consideration in this study were
`shown to have little or no influence on the PCR results.
`For example, it is known that the amount of cell-free fetal
`DNA in the maternal circulation increases with gesta-
`tional age, peaking at the time of delivery (2 ). However,
`all centers processed a common set of samples of various
`gestational ages, and there was no apparent trend be-
`tween the sensitivity and specificity of fetal DNA detec-
`tion and gestational age. Delay in processing after blood
`drawing can also be a concern, especially when samples
`are shipped long distances. However, because all samples
`were processed within 24 h and it has been shown that the
`detection of fetal DNA is consistent for at least 24 h (15 ),
`this should not have been a concern in the present study.
`Of the factors shown to influence the detection of the
`DNA sequence of fetal origin, most notable was the effect
`
`Table 4. Concordance in amount of total DNA detection
`among centers: ICC.a
`Center
`
`Center
`
`A
`
`B
`
`C
`
`D
`
`E
`
`A B
`
`0.59
`0.94
`C
`0.82
`D
`0.86
`E
`a Overall ICC ⫽ 0.79.
`b Bold indicates significant agreement (P ⬍0.05).
`
`0.71
`0.83
`0.57
`
`0.88
`0.92
`
`0.70
`
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`Clinical Chemistry 50, No. 3, 2004
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`521
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`statistically significant qualitative agreement over all cen-
`ters as indicated by the ␬ statistic. Similar qualitative and
`quantitative agreement was found with respect to total
`DNA detection (GAPDH). Our findings suggest that al-
`though qualitative agreement is attainable, it may not be
`possible to reliably compare absolute quantitative values
`of fetal DNA in maternal plasma between sites even with
`a highly standardized protocol. Therefore, alternative
`statistical approaches, such as the development of site-
`specific analyses using multiples of the median, should be
`considered (16 ).
`
`In conclusion, the common protocol presented here is a
`robust PCR assay for the detection and quantification of
`fetal DNA sequences in maternal plasma. Variables that
`were found to be critical for accurate analysis have been
`identified, allowing adjustments to be incorporated to
`minimize or eliminate these concerns. This should facili-
`tate development of an optimized protocol that could
`provide a robust, clinically relevant platform for future
`applications.
`
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